Microbiome in Human Health and Disease: Pallaval Veera Bramhachari Editor

Pallaval Veera Bramhachari Editor
Microbiome
in Human
Health and
Disease
Microbiome in Human Health and Disease
Pallaval Veera Bramhachari
Editor
Microbiome in Human
Health and Disease
Editor
Department of Biotechnology
Krishna University
Machilipatnam, Andhra Pradesh, India
ISBN 978-981-16-3155-9 ISBN 978-981-16-3156-6 (eBook)

https://doi.org/10.1007/978-981-16-3156-6
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Foreword
The microbial world is largely invisible to the human eye, but it is almost beyond
imagination. There are hundreds, thousands of different kinds of bacteria (leaving
aside other kinds of microbes: archaea, viruses, fungi, and protists), living in every
possible environment including deep seabed, high in the clouds, and in the boiling
hot springs. Multicellular organisms created an entirely new set of habitats, in and on
all those animals and plants.
Research data suggested that during the last two decades, extensive research has
been carried out on endophytic fungi and several biologically active compounds
have been isolated from endophytic fungi. This book makes all the readers generally
conversant in the language of microbiomes and metagenomics. It also provides
excellent examples of how microbial communities affect health and cure diseases
and dole out typical practical examples of how medical interventions interact with
the microbiome and change outcomes.
Understanding the Host–Microbiome Interactions in Human Health. The volume
published by the Springer Nature Biomedicine is an important volume, and I
strongly believe that it will attract readers working in the field. The present volume
has 14 chapters contributed by diversified academicians and scientists working on
microbiome research throughout the world. I must congratulate the editor of this
book for bringing out this volume with excellent contributions from the most
talented scientists working on microbiome research and their applications in under-
standing the host microbiome interactions in human health.
Krishna University K. B. Chandra Sekhar

Machilipatnam, India
v
Preface
The human body is a beautifully complex system, hosting trillions of microbial cells
that colonize epithelial surfaces such as those present in the mouth and gut are
included. There is growing evidence that these microbiomes, which play important
roles in human physiology and organ development, do more than just lie on tissues.
Indeed, relative to our human genome, there are 100 times more genes in our
microbiome and these microbial genes code for proteins that affect different pro-
cesses such as digestion, immunity, and development. The purpose of this book is to
provide a summary of human microbial diversity and to explain attempts to connect
microbial communities to human beings.
The Human Microbiome Project (HMP) projects in the USA, Canada, Europe,
and Asia have provided a boost to attempt to understand human-associated microbial
populations. More than $200 million has been invested by the NIH in the Western
World to fund these studies, which are rising now with the introduction of evidence
and a flurry of publications. This is a time of extraordinary breakthroughs, and the
area is sufficiently mature, while still new to merit a book highlighting advancement.
Responses to several issues are appearing now. How do various microbial
populations vary across locations in the body? What is the heterogeneity of micro-
bial composition at the same specific location in stable and diseased humans?
To maximize well-being and mitigate disease risk, how can microbial populations
be manipulated? Over the course of human evolution, how do microbial populations
change? What are the internal factors (genetic, anatomical, hormonal, and physio-
logical) and external environmental factors that form human-associated microbial
communities (diet, sexual behavior, and hygiene)? Many additional concerns regard-
ing the personal interactions between human and microbial cells in the body come
with partial answers to these questions. The researchers continue to make notewor-
thy and exhilarating contributions to our understanding of the basic biology of
human health in the area of microbiome.
Yet the practical translational applications of this fascinating and enthralling area
of science are outstanding. The book also discusses that research on microbiomes
provides a more comprehensive view of the genetics of humans and other species,
and how it can include innovative human health treatments and new techniques. For
the beginner and microbiome enthusiasts, this book may be an essential reading of its
importance with existing applications in medicine, agriculture, and climate. With
vii
viii Preface
these aims in mind, the material of this textbook has been structured from basic to
more advanced topics in a sequential progression. Finally, this book also reviews
advancement from fundamental research to relationships between immune–
microbiomes and human health microbiomes: clinical applications.
We hope that your creativity is inspired by this book and wish you luck in your
experiments. This book illustrates astonishingly the urgency with which the numer-
ous scientific brains are committed to the welfare of the scientific world. I am
immensely grateful to the contributors for consistently paying attention to my
request and expressing confidence in my skills. I will still be forever highly obliged
to all the contributors forever. The worthlessness of their efforts cannot be explained
by these terms.
Because of the heartfelt interest and painstaking effort of many other well-wishers
whose names are not listed, but they are already in our hearts, we have effectively
compiled our innovative and reflective research work. So, the reward for their
sacrifices is definitely worth it. I want this book to be devoted to my mum,
S. Jayaprada (late). From the bottom of our souls, I and the contributing authors
hope this book will be a good guide and guidance for scientific studies to understand
the host microbiome relationships in human health.
Machilipatnam, India Pallaval Veera Bramhachari

Acknowledgements
My sincere thanks are extended to all the academicians and scientists who have
contributed chapters and happily agreed to share their work on Understanding the
Host–Microbiome Interactions in Human Health in this volume.
This book is a stunning reflection of the seriousness with which several scientific
minds are dedicated from the scientific community. I am extremely thankful to the
contributors for paying continuous attention to my request and showing faith in my
competencies and capabilities. I shall always remain highly obliged to all of them
forever. These words cannot justify the worthiness of their efforts. We appreciate the
excellent work of the authors and co-authors who were invited to contribute chapters
to this book. The credit for making this book a reality goes to them. As an editor and
the review team for the chapters especially appreciate sharing expertise with the
contributors. Each chapter is informative and written as a stand-alone, so the reader
can begin anywhere in the book depending upon his/her interests and needs.
At the same time, I also express my deepest gratitude to my family members
especially my wife (Ramadevi Ramaswamy) and my kids (Ruthvik and Jayati) for
their kind support which has prompted me to complete the assignment on time. I am
also thankful to the Department of Biotechnology, Krishna University, for the
support. I am equally thankful to the Springer Nature Publishing group for their
full cooperation during the peer review and production of the volume.
I am thankful to my beloved teachers and mentors, for their constant support and
motivations at all stages of the progress.
ix
About the Book
The book provides an overview on how the microbiome contributes to human health
and disease. The microbiome has also become a burgeoning field of research in
medicine, agriculture and environment. The readers will obtain profound knowledge
on the connection between intestinal microbiota and immune defense systems,
medicine, agriculture and environment. The book is addressed to several researchers,
clinicians, and scholars working in biomedicine, microbiology, and immunology.
The application of new technologies has no doubt revolutionized the research
initiatives providing new insights into the dynamics of these complex microbial
communities and their role in medicine, agriculture and environment shall be more
emphasized. Drawing on broad range concepts of disciplines and model systems,
this book primarily provides a conceptual framework for understanding these
human–microbe, animal–microbe, and plant–microbe interactions while shedding
critical light on the scientific challenges that lie ahead. Furthermore, this book
explains why microbiome research demands a creative and interdisciplinary think-
ing—the capacity to combine microbiology with human, animal, and plant physiol-
ogy, ecological theory with immunology, and evolutionary perspectives with
metabolic science.
This book provides an accessible and authoritative guide to the fundamental
principles of microbiome science, an exciting and fast-emerging new discipline
that is reshaping many aspects of the life sciences. These microbial partners can
also drive ecologically important traits, from thermal tolerance to diet in a typical
immune system, and have contributed to animal and plant diversification over long
evolutionary timescales. Also, this book explains why microbiome research presents
a more complete picture of the biology of humans and other animals, and how it can
deliver novel therapies for human health and new strategies.
xi
Contents
Part I Introduction to Microbiomes

1 Microbiomes and Its Significance with the Current Applications
in Human Health and Disease: Goals and Challenges
of Microbiome Research Today . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Part II Microbiome for Human Health: From Basic Science

to Immune-Microbiome Interactions
2 Modulation of Systemic Immune Responses Through Genital,
Skin, and oral Microbiota: Unveiling the Fundamentals
of Human Microbiomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Pavani Sanapala and Sudhakar Pola
3 A Systematic Review on Crosstalk Between Microbiome and
Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Y. Aparna, K. Anuradha, S. Anju, and J. Sarada
4 Diversity and Dynamics of the Gut Microbiome and Immune
Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Prerna Pathak
5 Overview on Human Gut Microbiome and its Role in
Immunomodulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Sudhakar Pola and Dhana Lakshmi Padi
6 Cancer Microbiome and Immunotherapy: Understanding the
Complex Responses Between Microbes, Immunity, and Cancer . . . 83
Kishore Kumar Godisela, Badithala Siva Sai Kiran, and Pallaval
Veera Bramhachari
7 Dynamics of Respiratory Microbiome Profiles Contributes to
Imbalance and Lung Dysbiosis in the Respiratory Tract . . . . . . . . . 101
Harish Babu Kolla, Prakash Narayana Reddy,
and Pallaval Veera Bramhachari
xiii
xiv Contents
8 Understanding the Interplay Between the Host

Immune–Microbiome Interactions: A State of the Art Review . . . . 123
Kuraganti Gunaswetha, Edla Sujatha, and Pallaval Veera Bramhachari
Part III Microbiome for Human Health: Clinical Applications

9 Intestinal Microbiome–Macromolecule Signaling That Mediates
Inflammation and Immune System Interaction . . . . . . . . . . . . . . . . 145
S. Anju, Y. Aparna, K. Anuradha, Pallaval Veera Bramhachari,
and J. Sarada
10 Microbiome Diagnostics and Interventions in Health
and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Nandini Dasgupta, Alka Srivastava, Amrita Rao, Vrishali Murugkar,
Ravi Shroff, and Gautam Das
11 Microbiome Therapeutics: Emerging Concepts and Challenges . . . 217
J. Sarada, S. Anju, Y. Aparna, and K. Anuradha
12 Recent Advancements in Microbiome–Immune Homeostasis
and their Involvement in Cancer Immunotherapy . . . . . . . . . . . . . . 239
Anusha Konatala, Fain Parackel, and Pola Sudhakar
13 Insight into the Animal Models for Microbiome Studies . . . . . . . . . 259
Chanda Vikrant Berde, P. Salvi Sagar, V. Kajarekar Kunal,
A. Joshi Suyoj, and B. Berde Vikrant
14 Bioinformatics Algorithms and Software for Predicting
Microbiomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Shrikant Pawar, Yong Chiang Tan, and Chandrajit Lahiri
Editor and Contributors
About the Editor
Pallaval Veera Bramhachari is currently a Head and

Senior Faculty member of the Department of Biotech-
nology at Krishna University, Machilipatnam, India. He
has earlier served as Postdoctoral Research Scientist at
the Bacterial Pathogenesis Laboratory, Queensland
Institute of Medical Research (QIMR-Berghofer
Research Center), Brisbane, Australia and DBT Post-
doctoral Fellow at the Department of Microbiology and
Cell Biology, Indian Institute of Science (IISc),
Bangalore. He has completed Ph.D. in Microbiology
from Goa University, India. His major research interests
are applied researches in microbiology and cell biology
including biochemical mechanism of bacterial–biofilm
formations, structure–function relationship of bacterial
rhamnolipids, basic and translational cancer research.
He has published more than 140 research articles in
Scopus-indexed and web of science journals, authored
and co-authored 6 international books, and scores
of invited book chapters in prestigious peer-reviewed
international edited volumes and presented 47 abstracts
at various national and international conferences. He has
served as editorial board and reviewer for a number of
national and international journals. He is a member of
numerous international scientific societies and
organizations, most importantly, Indian Science Con-
gress and Society of Biological Chemists, India. He
has successfully completed two major research projects.
He was awarded with Travel scholarship from
QIMR-2007, Australia for attending Fourth
Indo-Australian Biotechnology Conference at
Queensland, Brisbane, Australia, and young scientist
travel fellowship (2007) from the DST, Govt. of India
xv
xvi Editor and Contributors
for attending XVII Lancefield International Symposium

at Porto Heli–Greece-2008. He was conferred with vari-
ous prestigious awards, notably, Science Education
Research Board (SERB), Government of India-Young
Scientist award (2011) with a research project and
Nominated as Associate Fellow of Andhra Pradesh
Academy of Sciences (APAS)-2016, MASTER
TRAINER: Andhra Pradesh English Communications
Skills Project. British Council and APSCHE 2017,
Andhra Pradesh State Best Scientist (APCOST) award-
2017 by State Govt of Andhra Pradesh, Dr.V.
Ramalingaswamy Memorial award for Biomedical
Sciences-2019 by Science city of Andhra Pradesh. He
also obtained two Indian patents in 2017. He has more
than 13 years of teaching and research experience at the
university level.
List of Contributors
S. Anju Bhavan’s Vivekananda College of Science, Humanities, and Commerce,

Secunderabad, Telangana, India
K. Anuradha Bhavan’s Vivekananda College of Science, Humanities, and Com-
merce, Secunderabad, Telangana, India
Y. Aparna Bhavan’s Vivekananda College of Science, Humanities, and Com-
merce, Secunderabad, Telangana, India
Chanda Vikrant Berde Marine Microbiology, School of Earth, Ocean and Atmo-
spheric Sciences (SEOAS), Goa University, Taleigao Plateau, Goa, India
B. Berde Vikrant Department of Zoology, Arts, Commerce and Science College,
Lanja, Maharashtra, India
Pallaval Veera Bramhachari Department of Biotechnology, Krishna University,
Machilipatnam, Andhra Pradesh, India
Gautam Das miBiome Therapeutics LLP, Flexcel Park, C Wing, Mumbai,
Maharashtra, India
Nandini Dasgupta miBiome Therapeutics LLP, Flexcel Park, C Wing, Mumbai,
Maharashtra, India
Kishore Kumar Godisela Department of Biotechnology, SRR Govt. Arts & Sci-
ence PG College, Karimnagar, Telangana, India
Kuraganti Gunaswetha Department of Microbiology, Kakatiya University,
Warangal, Telangana, India
Editor and Contributors xvii
A. Joshi Suyoj Gogate Jogalekar College, Ratnagiri, Maharashtra, India

V. Kajarekar Kunal Gogate Jogalekar College, Ratnagiri, Maharashtra, India
Badithala Siva Sai Kiran College of Pharmaceutical Sciences and Research,
Krishna University, Machilipatnam, Andhra Pradesh, India
Harish Babu Kolla Department of Biotechnology, Vignan’s Foundation for
Science Technology and Research (Deemed to be University), Guntur, Andhra
Pradesh, India
Anusha Konatala University of Windsor, Windsor, ON, Canada
Chandrajit Lahiri Department of Biological Sciences, Sunway University,
Petaling Jaya, Selangor, Malaysia
Vrishali Murugkar miBiome Therapeutics LLP, Flexcel Park, C Wing, Mumbai,
Maharashtra, India
Dhana Lakshmi Padi Department of Biotechnology, Andhra University,
Visakhapatnam, Andhra Pradesh, India
Fain Parackel University of Windsor, Windsor, ON, Canada
Prerna Pathak Jawaharlal Nehru University, New Delhi, India
Shrikant Pawar Yale Center for Genome Analysis (YCGA), Yale University,
New Haven, CT, USA
Sudhakar Pola Department of Biotechnology, Andhra University,
Amrita Rao miBiome Therapeutics LLP, Flexcel Park, C Wing, Mumbai,
Maharashtra, India
Prakash Narayana Reddy Department of Biotechnology, Vignan’s Foundation
for Science Technology and Research (Deemed to be University), Guntur, Andhra
Pradesh, India
P. Salvi Sagar Gogate Jogalekar College, Ratnagiri, Maharashtra, India
Pavani Sanapala Department of Biotechnology, Andhra University,
J. Sarada Bhavan's Vivekananda College of Science, Humanities, and Commerce,
Secunderabad, Telangana, India
Ravi Shroff miBiome Therapeutics LLP, Flexcel Park, C Wing, Mumbai,
Maharashtra, India
Alka Srivastava miBiome Therapeutics LLP, Flexcel Park, C Wing, Mumbai,
Maharashtra, India
xviii Editor and Contributors
Pola Sudhakar Department of Biotechnology, AUCST, Andhra University,

Edla Sujatha Department of Microbiology, Kakatiya University, Warangal,
Telangana, India
Yong Chiang Tan Department of Biological Sciences, Sunway University,
Petaling Jaya, Selangor, Malaysia
Abbreviations
ACT Adoptive cell treatments

AI diseases Autoimmune diseases
AID Activation-induced cytidine deaminase
ALT Alanine aminotransferase
AMPK Adenosine monophosphate-activated protein kinase
AMPs Antimicrobial peptides
Ang4 Angiogenin-4
AvrA Avirulence protein A
BAL Bronchioscopic alveolar lavages
BB Bio-breeding
BB-DP Bio-breeding diabetes-prone
BCG Bacillus Calmette-Guerin
BD Bayesian redistribution
BIOM Biological Observation Matrix
BLAST Basic local alignment search tool
BOS Bronchiolitis obliterans syndrome
BURST Based upon related sequence types
c.f.u. Colony-forming units
CagA Cytotoxin associated gene A
CAPD Continuous ambulatory peritoneal dialysis
CAR Chimeric antigen receptor
CD Celiac disease
CD Crohn’s disease
CDI Clostridium difficile infection
CF Cystic fibrosis
CFTR gene Cystic fibrosis transmembrane conductance regulator
gene
CIA Co-inertia analysis
CIA Collagen induction arthritis
CKD Chronic kidney disease
CLA Conjugated linoleic acid
CNS Central nervous system
COPD Chronic obstructive pulmonary disease
xix
xx Abbreviations
CRC patients Colorectal cancer patients

CRDs Chronic respiratory disorders
CRP C-reactive protein
CTLA-4 Cytotoxic T lymphocyte-association protein 4
CVDs Cardiovascular diseases
CYP Cytochrome P450
DAG Directed acyclic graph
DC Dendritic cells
DIAMOND Double Index Alignment of NGS Data
DM Diabetes mellitus
DMARDs Disease-modifying anti-rheumatic drugs
EC Enzyme Commission
ED Enteric dysbiosis
eHOMD Human Oral Microbiome Database
EPS Exopolysaccharides
ESRD End-stage renal disease
EVs Extracellular vesicles
FadA Fusobacterium nucleatum effector adhesin A
FDA Food and Drug Administration
FGID Functional gastrointestinal disease
FIT Fecal immunochemical test
FMT Fecal microbiota transplantation
FOBT Fecal occult blood test
FODMAPs Fermentable oligo-, di-, and monosaccharide and polyol
FOS Fructo-oligosaccharides
FUT2 Galactoside 2-alpha-L-fucosyltransferase2
FXR Farnesoid X receptor
GALT Gut-associated lymphoid tissue
GF animals Germ-free animals
GF mice Germ-free mice
GF Green fluorescent
GIT Gastrointestinal tract
GLP-1 Glucagon-like peptide-1
GMO Genetically modified organisms
GO Gene ontology
GOS Galactooligosaccharides
GPCRs G-protein-coupled receptors
GUI Graphical user interface
HDAC inhibitors Histone deacetylase inhibitors
HFGP Human Functional Genomics Project
HM Hematological malignancies
HMOS Human milk oligosaccharides
HMP Health Human Microbiome Project
HRMS High-resolution mass spectrometry
IARC International Agency for Cancer Research
Abbreviations xxi
IBD Inflammatory bowel disease

IBS-C IBS with constipation
IBS-D IBS with diarrhea
IBS-M IBS with mixed bowel habits
ICIs Immune checkpoint inhibition
ICIs Immune checkpoint inhibitions
IECs Intestinal epithelial cells
IELs Intraepithelial lymphocytes
IgA Immunoglobulin A
IGC Integrated human gut microbial Gene Catalog
iHMP Research Network Integrative HMP Research Network
ILCs Innate lymphoid cells
ILFs Isolated lymphoid follicles
ILFs Lymphoid follicles
IPF Idiopathic pulmonary fibrosis
IS Indoxyl sulfate
ISCs Intestinal stem cells
ITFs Inulin-type fructans
JNK pathway Jun N-terminal kinase (JNK) pathway
KO KEGG ortholog
LCA algorithm Last-common ancestor (LCA) algorithm
LCA Lowest common ancestor
LCMS Liquid chromatography-mass spectrometry
LGG Lactobacillus rhamnosus GG
LHMP Lung HIV microbiome project
LoD Limit od detection
LP Lamina propria
LPS Bacteria lipopolysaccharide
LPS Lipopolysaccharide
LTi Lymphoid tissue-inducer
MALTs Mucosa-associated lymphoid tissues
MAMPs Microbe-associated molecular patterns
MCP-1 Mmonocyte chemoattractant protein-1
MDP Marker Data Profiling
MDP Muramyl dipeptide
MLH Mesenteric lymph hubs
mLNs Mesenteric lymph nodes
MNS Microbiome novelty score
MOS Mannan oligosaccharide
MP toxin Metalloproteinase toxin
MPA MetaProteomeAnalyzer
MRSA Methicillin-resistant Staphylococcus aureus
MS Mass spectrometry
MS Multiple sclerosis
mTOR Mechanistic target of rapamycin
xxii Abbreviations
MyD88 protein Myeloid differentiation primary response 88 protein

NADPH Micotinamide adenine dinucleotide phosphate
NAFLD Non-alcoholic fatty liver disease
NASH Non-alcoholic steatohepatitis
NCDs Non-communicable diseases
NGS Next-generation sequencing
NIH Human Microbiome Project
NK cell Natural killer cell
NLRs Nod-like receptors
NMDS Non-metric multidimensional scaling
NO Nitric oxide
NOD 1/2 Nucleotide-binding oligomerization domain 1/2
NOD mouse Non-obese diabetic mouse
NSAF Normalized spectral abundance factors
ONOO Peroxynitrite
ONT Oxford Nanopore Technology
OTU Operational taxonomic units
PA approaches Procrustes analysis (PA) approaches
PacBio Pacific biosciences
PCA Principal component analysis
PCA Protocatechuic acid
PCD-1 Programmed-cell-death protein 1
PCDB ProteoClade Database
PCoA Principal-coordinate analysis
PPD Projection with Public Data
PRRs Pattern recognition receptors
PSM Peptide-spectrum match
QoL Quality of life
R value Correlation coefficient
RA Rheumatoid arthritis
RCT Receptive cell treatment
RF Random forest
RNS Reactive nitrogen species
ROS Reactive oxygen species
RSAI Respiratory system associated infections
SCFA-GPCR G-protein-coupled receptors
SCFAs Short-chain fatty acids
SDP Shotgun Data Profiling
SFB Segmented filamentous bacteria
SG Sleeve gastrectomy
SIBO Small intestine bacterial overgrowth
SLE Systemic Lupus Erythematosus
SODs Superoxide dismutase
SPF Specific-pathogen free
SR-A1 Scavenger receptor A1
Abbreviations xxiii
SRM Selected reaction monitoring

SVM Support vector machine
T1DM Type 1 diabetes
Th17 T helper 17
TLR2 Toll-like receptors 2
TLRs Toll-like receptors
TMA Trimethylamine
TMAO Trimethylamine oxide
TME Tumor microenvironment
TNF-α Tumor necrosis factor-α
TPL The monophosphoryl lipid A
TPP Trans-proteomic pipeline
Tregs T regulatory cells
TSEA Taxon Set Enrichment Analysis
TSLP Thymic stromal lymphopoietin
UC Ulcerative colitis
WNT Wingless-related integration site
Part I
Introduction to Microbiomes
Microbiomes and Its Significance
with the Current Applications in Human 1
Health and Disease: Goals and Challenges
of Microbiome Research Today
Abstract
Human beings are home to a massive invisible microbial community, which

powers nearly all processes in the body. Bacteria, archaea, viruses, protozoa,
and fungi are the most common organisms in or on our bodies. These fascinating
microbial species are called our microbiota collectively. The human body
includes a wide number of bacteria, both inside and outside. In particular, it is
the microbial genome set that leads to an overall human genetic picture. We know
very little about how the pendulum between health and illness swings in our
microbiome. The diversity and balance of our microbiome and vulnerability to
diseases are inextricably linked. Here, we try to consolidate our existing knowl-
edge about microbiome evolution and ecology in future studies, as well as to
consider the relationships between host microbiomes and human health and
disease. Finally, we highlight modern methods and technology to human health
advancement. The present book on microbiomes is therefore intended to give
readers a broad understanding and encouragement for future research and multi-
disciplinary cooperation on the goals and challenges of microbiome research
today.
Keywords
Microbial communities · Microbiome’s diversity · Microbiomes · Human health
P. V. Bramhachari (*)
Department of Biotechnology, Krishna University, Machilipatnam, India
# The Author(s), under exclusive license to Springer Nature Singapore Pte 3

Ltd. 2021
P. V. Bramhachari (ed.), Microbiome in Human Health and Disease,
https://doi.org/10.1007/978-981-16-3156-6_1
4 P. V. Bramhachari
1.1 Introduction
The set of microbiomes inside and within the human body can be classified as
microbiome items. Scientists begin to recognize that microbes play a significant role
in our human health. A standard microbiome has ten times as many cells as humans.
Indeed, our body is a complex microbial ecosystem; a broad variety of bacteria,
fungi, viruses, and microeucaryotes is contained here. The unique microbial combi-
nation that exists at each level and we sound like we have created a very significant
and precise task along with our microbial partners in every niche. The human
microbiome makes up more than 50% and it can influence our mood, appetites,
and immune responses to a range of biological functions. Human beings host a huge
invisible microbial environment, which affects almost every system of the body.
Bacteria, archaea, viruses, protozoa, and fungi are the most common microbes that
live within or on our bodies. This fascinating microbial group is known as our
microbiota collectively.
1.2 The Complexity of the Microbiomes
The abundance and complexity of the microbiome are staggering with more than one
million genes versus 23,000 in the human genome; microbiome communities have
unique profiles in different body sites, as do each organism, affected by diet,
medications, and other environmental factors (Coyte et al. 2021). The overwhelming
proliferation of microbial organisms means that a supra-organism is the human
body. Microbiomes are not microorganisms living alongside each other, but instead,
form highly regulated complex structurally and functionally organized communities
attached to the surfaces as biofilms that contribute to their ecological stability
through interspecies and interspecies collaboration. Disease prevention is not the
future, but health is the future, free of essential diseases (Shanahan et al. 2021).
Multifaceted illness like autoimmune disorders, the human body is an extremely
complex system in humans. It is getting more and more difficult to find medications
today. It is time to alter the paradigm shift so that scientists across the globe begin
focusing on the common theme that microbiomes in combinations of virus bacteria
fungi and bacteriophage are related to any disease. In the microbes that we bring, we
are each unique, yet they reflect another molecular fingerprint present in each
individual. We deal with good and poor microbes in such a way that we know
about dental caries caused by bacteria in the mouth, aches, or body odor in the skin
and now we have a chance to interact with good bacteria. What these microbial
species are doing within the human body is a fascinating issue, and there is a simpler
way to cope with tooth cavities, aches, and digestive disorders to treat more serious
health problems such as obesity, type 2 diabetes, irritable bowel syndrome, and
depression. Given the pros and cons, we need to naturally achieve better health by
using microbes that have co-evolved with us.
Otherwise, the human microbiome could be referred to as an undiscovered world
that gets benefited in many respects viz. (a) Synthesizes and excretes vitamins
1 Microbiomes and Its Significance with the Current Applications in Human. . . 5
Vitamin B12 and Vitamin K. (b) Prevents contaminants that are difficult for attach-
ment sites or essential nutrients. (c) From colonizing. Could probably alienate in the
production of substances that inhibit or destroy nonindigenous species with other
bacteria (nonspecific fatty acids, peroxides, bacteriocins). (d) Stimulate the growth
of certain tissues, such as intestines, lymphatic tissues, capillary density. (e) The
production of cross-reactive antibodies is enkindled. In addition to the ability
to extract nutrients, microbiomes generate extra energy that is otherwise unavailable
to the host, produce vitamins, metabolize trivial xenobiotics, and provide resistance
to cancer and tumor-causing neoplasms, and help grow a mature immune system
(Parida and Sharma 2021). Instead of researching the relationship between the
microbiota, health, and disease, several studies have shown a connection between
various microbial consortia and certain disease states; however, there is still little
evidence that a specific disease is triggered by some sophisticated microbial group.
The quest was subjugated by the early history of microbiology to discover the
microbes responsible for disease and uncover ways to impede them. The first step
in setting up ways to prevent and cure infectious diseases was the discovery and
analysis of causative agents (Honda and Littman 2012).
1.3 Co-Evolution of Microbiomes with Humans
It looks like we have co-evolved with our microbial partners with very critical and
complex tasks that exist in any given section. We are each special in the microbes we
bear, but in each individual, they constitute another molecular fingerprint (Hooks
and O’Malley 2020). Without any exception, we may map the microbes on the
fingerprints and microbes on the machine keyboards to map the keyboard owner and
verify how special these microbial signatories are. Microbiome habitats with high
diversity are more robust and more resistant and able to return to their safe state of
perturbation. We know that over the past few decades, there has been a rising
prevalence of allergies, hypersensitivity disorders, and asthma in children. Changes
in eating patterns, access to packaged food, decreased interaction with the natural
world, the usage of antibacterial soaps, and the possibility of cleansers living in a
sterile environment should be taught.
Unfortunately, we have pushed the development of our microbial species to a less
complex and inherently less secure condition. Strikingly, microbiota modifications
are known to be associated with different diseases, i.e., diabetes, obesity, cardiovas-
cular diseases, carcinogenesis, host anatomy, metabolism, irritable bowel syndrome,
and immune dysfunction (Tang et al. 2017). An increasing body of evidence
indicates a strong correlation with changes in various disease states in microbiomes.
To decipher the deeper understanding of the interactions of microbe disease, multi-
ple studies are ongoing. This poses the exhilarating possibilities that we might think
of different therapeutic goals if illnesses are correlated with changes in microbiota
and begin to consider ways to transition back to a healthier state.
In multiple environments, microbiomes live and display remarkable differences
within and between individuals. With different health states and phenotypes,
6 P. V. Bramhachari
variability is associated. In several roles, the microbiome is involved viz. production

of vitamins, metabolic rate, digestion, odor, behavior, parasite or pathogen defense
and Immune Regulation (Costello et al. 2009). Nevertheless, within the human body,
hundreds of beneficial microbes reside and are critically important for sustaining
human health. They are very functional in educating our immune system in the early
years of life to identify them like themselves, but not themselves. They harvest
energy from all of the food sources we consume in the (Gastrointestinal) GI Tract.
Strikingly, we do not break down all the polysaccharides that we consume, for
example. In the gut, microbes develop synthetic vitamins, metabolites, nourish the
cells that line the GI tract, and most likely in all environments in the human body.
Researchers are now exploring circumspectly what our microbial inhabitants are
doing and how they are contributing to or defending against disease. Research on
microbiomes is evolving pretty rapidly to test how we see ourselves as humans. The
body harbors at least as many microbial cells as human cells, and there are more than
a million genes in our microbial gene database. Nevertheless, we still know very
little about the function of most of the second genome and how it affects human
health. Besides, microbiome types preserve innate immunity and adaptive immunity
by building up the host’s metabolic capacity to digest plant carbohydrates, milk
products (glycans), vitamin endowments (e.g., B2, B12, K, and folic acid), protect
against pathogenic bacteria colonization, and create resistance to colonization
(Thaiss et al. 2016). Microbiota metagenome may also function with rapid modifi-
cation of various strains, exchange of genetic elements, and occurrence of mutations
in response to multiple environmental stimuli. The following variables have a vital
effect on the microbiota (Nayfach et al. 2019). Host biology, individual lifestyle,
diseases, antibiotic exposure, at-time colonization, and birth delivery type,
respectively.
1.4 Microbiome Dysbiosis
The proliferation of our beneficial thriving microbes holds in check the pathogenic
microbes and maintains a harmonious equilibrium. However, this equilibrium is
disrupted when pathogenic microbes govern and we reach a state of dysbiosis.
Dysbiosis of the microbiome has been associated with a large number of health
problems and is causally concerned with metabolic, immunological, and develop-
mental disorders, as well as susceptibility to the development of infectious diseases.
Several illnesses, including cancer, inflammatory bowel disease, obesity, and
asthma, are associated with dysbiosis. The composition of the microbiome can be
affected by our lifestyle choices, our diet, our use of antibiotics and drugs, and the
climate in which we live.
Microbiome dysbiosis has been associated with surplus health problems and has
been associated with metabolic, immunological, and developmental disorders as
well as susceptibility to infectious disease growth. Genetic and environmental
factors causing impaired barrier function, overgrowth of pathogenic bacteria, and
subsequent inhibition of defensive bacteria are proposed mechanisms that lead to
dysbiosis. Translocation of bacteria and bacterial products into cells, immune acti-
vation and development of proinflammatory cytokines, chronic inflammation, the
leaky gut term, leads to tissue destruction and complications. In the human body, the
gut comprises the largest, densest, and most diverse microbial group. As a crucial
determinant of nutrient uptake, energy regulation, and eventually, weight gain and
metabolic disorders, recent research has also implicated the gut microbiota. In the
future, gut flora modulation could be an important part of weight loss services and
various therapies for illnesses. Some metabolites processed by gut microbes that
drive the progression of many cardiovascular pathologies, such as atherosclerosis,
hypertension, heart failure, and type 2 diabetes, have recently been identified by
researchers (Lee and Hase 2014). “These findings suggest that by generating bioac-
tive metabolites that can directly or indirectly affect host physiology, the gut
microbiome functions like an endocrine organ” (Tang et al. 2019). Besides, in cancer
development affecting predisposing conditions, particularly initiation, progression,
response to therapy, microbiota also plays an imperative role (Matson et al. 2021).
The complexity of the fecal microbiota is actively being established and recent
studies have shown that microbiota-related dysregulation results in the pathogenesis
of several diseases. In the future, gut flora modulation could be an important part of
weight loss services and various therapies for illnesses. A healthy intestinal
microbiota is restored by fecal microbiota transplantation (FMT) and results in
remarkable cure rates for many diseases and is likely to achieve widespread thera-
peutic advantage for several diseases in the future (Kelly et al. 2021). To work at the
petri dish, genomics, and clinical results stage, such studies need translational
science.
1.5 Significance of Microbiome Research.
The considerable quantity of research on the microbiome has led to a better under-
standing of the microbiome and its function in the human, urban, and natural
environments in recent years (Cullen et al. 2020). Host-microbe studies such as
interactions between the gut and diet offer a major insight into how the microbiome
reacts over time to the introduction of new microbes and changes and may poten-
tially serve as the roadmap for microbiome-based intervention and diagnostic
technology.
The future microbiome diagnostics and therapeutics armamentarium provide
broad and deep possibilities for the monitoring and treatment of a range of
diseases-personalized diets, prebiotics, postbiotics, microbiota transplantation,
engineered bacteriophages, microbial metabolites, precision editing of microbiota,
and modulation of the intestinal barrier. Great data from such microbiome
populations have led to the advent of computational genomics that helps to explore
the enduring uncultured microbes attributable to current developments in
metagenomic sequencing technologies and other omics platforms. Microbiome
8 P. V. Bramhachari
research has been catapulted into an exciting new frontier in medicine and human
health by enormous developments in next-generation sequencing and Omics
technologies. Promising knowledge indicates that the microbiome, the rich ecosys-
tem of more than 100 trillion bacteria, fungi, and viruses in and on the human body,
is important to all aspects of human health and disease, and that 1 day microbiome
analysis may play an imperative role in clinical practice.
In recent years, microbiome research has improved noticeably, powered by
developments in technology and a substantial reduction in study costs. Such research
has unlocked a wealth of data that has provided significant insight into the existence
of microbial communities, including their interactions and consequences, both as
part of an ecological community within a host and in an external environment.
Understanding the function of microbiota, including its complex interactions with its
hosts and other microbes, will make it easier for new diagnostic techniques and
interventional methods to be established that can be used in a variety of fields, from
ecology and agriculture to medicine and forensics to exobiology.
It would also be very amazing to understand what microbes harbor each tissue
and organ, what they encode, what they create as communication signals and how
they transform over time, and how human health could be advanced by manipulating
these signals. In order to facilitate tailored preventive and therapeutic approaches,
systematic understanding and application of microbiome heterogeneity hold great
promise. Increasing our experience in this area will afford proof of concept and
implement new therapeutic pathways. The ultimate objective should facilitate to
reinstate the status quo, using probiotics to replace vital missing and/or extinct
microbiome species and strains that respond to essential developmental pathways,
likely with accompanying prebiotics.
1.6 The Way Forward
To decide whether there are sets of microbes common to each person, several
challenges are still underway. To learn whether modifications to our microbiota
result in various health or disease states. New technologies for the study of complex
microbial systems and the study of complex microbial systems within their natural
environments should be developed (Carr et al. 2020; Galloway-Peña and Hanson
2020). Therefore, therapeutically targeting the makeup of the microbiota will put
forward new methods for disease prevention and treatment. The unidentified taxa of
bacteria and the analogous genetic levels at which they function are still the greatest
impediment. For the next scientific frontier of human health that needs a lot of study,
this may be resolutely indispensable (Liu et al. 2020). In addition, the convergence
of multiple scientific disciplines and the use of innovative technical methodologies
in microbiome research are expected to pave the way for the conception of evidence-
based clinical treatments for the health problems of modern life (Methé et al.
2012; New and Brito 2020).
An overview of how the microbiome contributes to human health and illness is
given in the book. The microbiome has also become a flourishing field of medical,
agricultural, and environmental study. Readers can gain in-depth knowledge of the
connection between microbiota, medicine, agriculture, and the environment, and
immune defense systems. A number of researchers, physicians, and scholars work-
ing in biomedicine, microbiology, and immunology will be discussed in the book.
There is no question that the execution of emerging technologies has revolutionized
research initiatives by bringing new insights into the complexities of these complex
microbial communities and their role in the fields of medicine, agriculture, and the
environment. Centered on a wide variety of discipline principles and model systems,
this book mainly offers a conceptual outline for understanding these interactions
between human microbes, animal microbes, and plant microbes, while flaking
fundamental light on the scientific challenges ahead. In addition, this book discusses
why microbiome study warrants the opportunity to combine microbiology with
human, animal, and plant physiology, evolutionary perspectives with metabolic
science, ecological theory with immunology, and innovative and interdisciplinary
thought (Berg et al. 2020).
This book offers an accessible and definitive guide to microbiome science’s
fundamental concepts, an exciting and rapidly evolving new field that transforms
many aspects of life sciences. From thermal tolerance to diet in a typical immune
system, these microbial partners can also drive ecologically essential traits and have
led to animal diversification over long evolutionary timescales. This book also
discusses why microbiome research offers a more comprehensive image of human
and other animal biology, and how it can produce innovative human health
treatments and new strategies.
Acknowledgments DR. PVBC is grateful to Krishna University, Machilipatnam for the support
extended.
Conflict of Interest The author declares that they have no competing interests.
References
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definition re-visited: old concepts and new challenges. Microbiome 8(1):1–22
Carr V, Shkoporov A, Hill C, Mullany P, Moyes D (2020) Probing the mobilome: discoveries in the
dynamic microbiome. Trends Microbiol 29(2):158–170
Costello EK, Lauber CL, Hamady M, Fierer N, Gordon JI, Knight R (2009) Bacterial community
variation in human body habitats across space and time. Science 326:1694–1697
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microbiome communities. PLoS Biol 19(2):e3001116
Cullen CM, Aneja KK, Beyhan S, Cho CE, Woloszynek S, Convertino M et al (2020) Emerging
priorities for microbiome research. Front Microbiol 11:136
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(3):674–685
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Immunol 30:759–795
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microbiome research. J Eukaryot Microbiol 67(2):279–295
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Kelly CR, Yen EF, Grinspan AM, Kahn SA, Atreja A, Lewis JD et al (2021) Fecal microbiota
transplantation is highly effective in real-world practice: initial results from the FMT National
Registry. Gastroenterology 160(1):183–192
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Chem Biol 10(6):416–424
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commensal microbiota on cancer, immune responses, and immunotherapy. Gastroenterology
160(2):600–613
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Part II
Microbiome for Human Health: From Basic
Science to Immune-Microbiome Interactions
Modulation of Systemic Immune Responses
Through Genital, Skin, and oral Microbiota: 2
Unveiling the Fundamentals of Human
Microbiomes
Pavani Sanapala and Sudhakar Pola
Abstract
The human system is encountered by several microbes within and on the external
surface of the cells. The prevalence of microbes is more compared to the body
cells. The microbial organisms have both beneficial and harmful effects on the
system. Studying the microbiome of the system is an accessible and escalating
field of research. These organisms eventually involve in modulation. Many
studies reported the presence of microbes in the gut, vaginal, oral, skin, urinary,
and on the lining of the respiratory tract. Factors such as external environment,
internal modulation, and epidemiological factors signify the distinct function and
composition of microbes. It is necessary to understand the topography, mecha-
nism, and action of microbes so that it would be beneficial to known the
pathophysiology, alteration of metabolic events, and progression of many
diseases which may pave a pathway for biomarker discovery or drug delivery
that can cure or treat or manage the disease with clinical practices.
Keywords
Microbiota · Genital microbiome · Skin · Topography · Oral cavity · Modulation
2.1 Introduction
Human beings are super organisms consisting both microbial cells and their cells.
The resident of microbes on the body is reported to be ten folds higher than the body
cells (Turnbaugh et al. 2007). These organisms colonizing modulate the immune
P. Sanapala · S. Pola (*)

Department of Biotechnology, Andhra University, Visakhapatnam, Andhra Pradesh, India
e-mail: [email protected]

Ltd. 2021
https://doi.org/10.1007/978-981-16-3156-6_2
14 P. Sanapala and S. Pola
system and maintain homeostasis in the human system (He et al. 2015). Microbiota
of the human body is classified into core and variable microbiome. The core is the
usual and universal familiar for all the individuals, while the variable is the distinct
microorganism unique to each person depending on their physiological and lifestyle
character variations. According to the literature, the body habitat, the skin, the oral,
the gastrointestinal, the urinary and the genital tracts, and the lining of the respiratory
tract (mucosal membrane) often have discrepancy bacterial communities. The micro-
bial floras often present are usually referred to as healthy flora; however, indigenous
flora is expressed alternatively.
Over thrice a 100 million symbiotic microbes exist such as archaea, bacteria,
viruses, and fungi inhabit within and on human beings. Over a decade, the role of
microbiomes in the health and disease of an individual has been strongly evident
(Ley et al. 2006). The human microbiota or microbiomes affect the host bodily
processes to an enormous level basing on various mechanisms. Foremost, the
microbiota can boost energy extraction from food sources (Turnbaugh et al. 2006)
after that increases the nutrient harvest (Gill et al. 2006; Roberfroid et al. 1995) and
later altering the appetite (Perry et al. 2016; Cani et al. 2004), besides it benefits the
host in metabolic processes in either xenobiotic processing or in gaining nutrients.
Next, the human microbiomes act as a barrier in protecting the host against foreign
pathogens in the course of competitive eliminations and the production of antimi-
crobial material (Cash et al. 2006; Hooper et al. 2003; Schauber et al. 2003).
Moreover, lastly, the microbiota is crucial in the progress of intestinal mucosa and
boosts the immune system (Cash et al. 2006; Bouskra et al. 2008). The human
system consists of no less than a thousand different species of already identified
bacteria and known to cart 150 times added microbial genes compared to the whole
genome of humans (Ursell et al. 2014).
The function and composition of microbiota vary as per age, gender, race, and
diet. The composition of the microbiome is distinct in each individual, with the
difference being more substantial in individuals than the biochemical differences
that occur within a human being eventually (Lax et al. 2014). Interactions of humans
with the surrounding environment or even other individuals build up possible
microbes on the body sites either indulged in influencing the immune system or as
a source to other organisms that can take over the human system (Gilbert et al. 2018).
The human microbiome track specific body sites adapting pathway during the body
growth, maturity, and development so that each site develops defined biogeography,
for example, skin, colon, vaginal, esophagus, urogenital tract, and the oral cavity
where the uniqueness of an individual is characterized and confounded by the
differing microbiota (Fig. 2.1).
Here we present the up-to-date status of comprehension relating the microbiomes
in modulation to the human oral, vaginal, and skin. A detailed note of the function,
diversity of species colonized with the human, factors for growth and development
of microbes, and the different modulation process is helpful in improving the health
of the human population.
2 Modulation of Systemic Immune Responses Through Genital, Skin, and oral. . . 15
Fig. 2.1 Represents distribution of various microorganisms; that defines uniqueness of human's
microbiome
2.2 Genital Microbiome
The ecosystem of the genital refers to the vagina that includes the uterine cervix, the
periurethral area, and the preputial pocket of uncircumcised men. Being the signifi-
cant parts of the reproductive system in both men and women, they are more
accessible for infectious microbes during sexual intercourse.
The female genital system (FGT) is divided into upper and lower tracts. The
lower FGT constitutes vaginal and ectocervix. The FGT plays a significant role as a
protective physical and immunological barrier and as structural support with multi-
ple leukocytes present beneath the epithelial-stromal fibroblasts. The upper FGT
comprises the endocervix, uterus, fallopian tube, and ovaries (Wira et al. 2005).
Vagina has a unique microbiota, the region is heavily loaded with microbes that as a
complex, varied, and eventually active ecosystem that decides the vaginal health.
The feminine genital system keeps up a well-maintained immune response that
poises tolerance against infections during reproduction. Though inflammatory

responses are useful in eradicating many STDs, the occurrence of elevated inflamma-
tion in women might increase the risk of disease acquirement (Lajoie et al. 2012;
Morrison et al. 2014). The microbial community colonizing the FGT are mainly
bacteria, and the most common species be anaerobic and microaerophilic bacteria.
Studied till the nineteenth century constitute the species Lactobacilli for the vaginal
pH, whereas Gardnerella vaginalis as imprecise vaginitis. In the pre-menarchal child,
the microbiota colonized is both anaerobic and aerobic, which is similar to that of skin
and periurethral area; however, the change of microbial community is reported gradu-
ally in increase with the levels of estrogen hormone. Many studies reported that
pathogens Chlamydia trachomatis, Neisseria gonorrhoeae, HSV-2, and Trichomonas
vaginalis are more related to the onset of inflammation. A study report by Delaney and
Onderdonk exemplified the role of cervicovaginal bacteria modulating an immune
response in the female genital tract (Delaney et al. 2001). Bacterial vaginosis (BV) is a
complex polymicrobial vaginal disorder with diverged microbiota. BV is exemplified
by the alternate of lactobacillus predominance over pathogenic anaerobes.
The difference in the number of microbial colonize of the male genital system
mainly depends on circumcised or uncircumcised. The circumcised are less prone to
infections with preferably a low number of microbes than the uncircumcised
(Wiswell and Roscelli 1986; Spach et al. 1992). M. hominis anaerobic bacteria
have been reported to colonize the preputial pocket of uncircumcised males, which
are associated with bacterial vaginosis (Serour et al. 1997).
2.2.1 Modulation of the Genital System
Vaginal microbiota (VMB) and endometrial microbiota can modulate inflammation.

Cultures of endometrial cells in combination with Neisseria gonorrhoeae (patho-
genic bacteria) provoke proinflammatory mediators, whereas L. crispatus and
G. vaginalis colonizing reproduction did not show proinflammatory activity
signifying endometrial microbes modulating inflammation in the host (Łaniewski
et al. 2017). Additionally, the epithelial cells of lower FGT do modulate leukocyte
function by fabricating cytokine and chemokines, which in turn induce inflammation
(Fahey et al. 2005).
Studies showed endogenous sex steroid hormones (Estradiol) playing a role in the
modulation of FGT immunity. Estradiol modifies the down streaming gesture of
receptors as well as the NκF-β function. However, modulation by human
contraceptives of VMB has also been explored. These modifications are vulnerable
to HIV-1 in women (Ghisletti et al. 2005). Facultative anaerobes, namely
Gardnerella vaginalis, Prevotella, Mobiluncus, and Atopobium are linked with
reproductive health outcomes (Atashili et al. 2008; Zevin et al. 2016). The process
of vaginal drying correlating through an increased bacterial variety and also
improved inflammatory marks shows significance for HIV risk. Modulation in
vaginal microbiota through vaginal drying might cost complications on the mucosal
barrier and promotion of cellular inflammatory practices (Anahtar et al. 2015).
The vaginal microbiota plays an essential role as a mutualistic relationship with

the host system and involves modulating the threat of acquiring and transmitting
sexually transmitted infections (STI). STIs are usually caused by Chlamydia
trachomatis. Nowadays, these infections are prevalent worldwide, especially in the
USA (Newman et al. 2015).
Studies reported the role of lactic acid in modulating the infection caused by
C. trachomatis (Gong et al. 2014). Nevertheless, the species Lactobacillus inhibits
C. trachomatis and intracellular development (Mastromarino et al. 2014). Both the
forms D and L isomers institute the microbiome of the vagina. The ratio of the two
forms depends on the Lactobacillus that predominates vaginal microbiota (Boskey
et al. 2001). But still, the mechanism is unclear; however, the vaginal microbiome
imparts confrontation to infection through modulating host cellular function.
2.3 Skin Microbiome
Skin, the largest organ of the human body, acts as a surface barrier protecting against
invading pathogens or foreign bodies. The human skin a trillion of microbiota that
constitutes mainly of bacteria, fungi, and viruses in various proportions. The skin
microbes play significant roles such as protection (defense mechanism), enhancing
immune response once in a while, and helps in the breakdown of innate products
(Scharschmidt and Fischbach 2013; Belkaid and Segre 2014; Grice 2015). The skin
is the first line of defense in opposition to pathogens, while concurrently wharf a
diverge surroundings of commensalism, including bacteria, fungi, and viruses.
These organisms take part in vital roles in lipid metabolism, colonization resistance
to transient organisms, and edification of the immune system (Scharschmidt and
Fischbach 2013; Belkaid and Segre 2014; Grice 2015). Genetical strategies show
more considerable variation in the organism. The phyla Actinobacteria, Firmicutes,
Bacteroidetes, and Proteobacteria are the most common inhabitants on the skins.
The species Propionibacterium is dominant in sebaceous areas, while species Cory-
nebacterium and Staphylococcus reside in moist sites. However, most of the diver-
sification of microbiomes is observed in dry sites with gram-negative organisms
more likely to colonize the skin (Gao et al. 2007). In contrast to the colonization of
bacterial communities on the skin, non-bacterial microorganisms fungi and
arthropods have been isolated from the skin. The most predominant fungal genus
is Malassezia at sebaceous sites, whereas Aspergillus colonized other sites, Crypto-
coccus, Rhodotorula, Epicoccum, and many more (Findley et al. 2013). The pres-
ence of Demodex folliculorum and Demodex brevis microscopic arthropod mites on
the skin illustrates that these arthropods are also among the healthy skin flora. These
mites colonize sebaceous areas of the face and also nourish on epithelial cell lining of
the pilosebaceous unit. Viruses also occupy a place in the flora of skin microbiota,
but studies have not yet reported its promising role (Costello et al. 2009). The
microbiota of the skin falls under two groups: transient or resident. The residents
are the healthy commensal fixed flora that is homeostatic with the host and are not
harmful but are beneficial to the host where transient are microbes from the
environment that lives for the time being (Chiller et al. 2001). The assortment of
transient and resident microbes on the skin depends on topographical sites of the
human body, specific characteristics such as pH, temperature, humidity, sebum
content, intrinsic factors (age, sex, genome), and many extrinsic factors such as an
epidemiological parameter that is lifestyle, domicile and occupation and antidrug,
and many more (Fierer et al. 2010). Both the groups are non-pathogenic in normal
conditions such as proper hygiene, healthy immune response, and functioning
properly yet, these groups proliferate, colonize, and cause disease after perturbation.
2.3.1 Skin Architecture
Skin is self-possessed into two distinct layers: the epidermis and dermis. The layer
outmost is known epidermis, which constitutes distinct keratinocyte layers. The
stratum corneum, the top layer consists of mortally differentiated enucleated
keratinocytes that act as a bridge to strengthen the skin barrier (Segre 2006). Besides
these structural layers, the locations of the body make available to diverge
microenvironments that differ in UV light exposure, temperature, moisture, pH,
sebum content, and topography (Grice and Segre 2011). Specific skin sites have
specific habitats of the microbiome; knowing the composition of the microbiota is
useful to define the etiology of the skin disorders, which will be a boon to the
dermatologist in analyzing the infections. For instance, hairy, moisture underarms
that are distant from the smooth, dry forearms have a different ecological niche that
is habitant for several devoid microbial communities (Costello et al. 2009). Factors
persuading the composition of the skin microbiome are divided into intrinsic and
extrinsic. The intrinsic factors are age, immunity, and genetic manipulation, while
extrinsic factors climate and hygienic conditions within the surroundings do affect
the microbial community. Basing on these characteristics, human skin is typified into
three types; that is, it may be oily (sebaceous), moist or dry. The site’s face, chest,
and back are composed of oily or sebaceous while bending of the elbow, knee back
and groin make up the moist, and forearm and palm fall under dry. All these sites are
predisposed by appendages, such as sweat glands, hair follicles, and sebaceous
glands. The role of sweat glands in moist areas is thermoregulation by water
evaporation that acidifies the skin that makes it unsuitable for the growth and
colonization of microorganisms (Grice and Segre 2011).
2.3.2 Association of Microorganisms and Skin Diseases
Skin diseases occur either if the barrier of the skin is damaged or if any stability
flanked by commensalism and pathogen is disturbed. The skin is populated by a vast
number of assorted microbes, which have either beneficial or harmless (Grice et al.
2009). Over time the composition of species becomes moderately stable (Kong et al.
2012). Nevertheless, skin disorders, namely acne vulgaris (Lomholt and Kilian
2010), eczema (Kong et al. 2012; Kobayashi et al. 2015; Chng et al. 2016; Myles
et al. 2016), psoriasis (Alekseyenko et al. 2013), or dandruff (Wang et al. 2015;
Clavaud et al. 2012) are linked with alteration in the microbiome.
Skin disease with modified microbiota is termed dysbiosis that is determined by
commensal species (Iebba et al. 2016). Acne vulgaris is the common inflammatory
skin disorder seen in teenagers and healthy adults, caused mainly by the presence of
species P. acnes (Leyden et al. 1975; Fitz-Gibbon et al. 2013). Other disorders such
as atopic dermatitis (AD) and chronic eczema alter several factors of skin most likely
worsening the inflammatory disease, impairment of epidermal barrier, immune cell
activation, and alteration in inhabitant of skin microbes. The disease AD has nearly
30 mutations in the host genome along with filaggrin, a barrier protein, and the genes
associated with immune response (Palmer et al. 2006). The species S. aureus is
colonized with AD (Paternoster et al. 2015). Studies reported microbes to instruct the
immune system, one such example is primary immunodeficiency (PID). PID, when
investigated for colonization, showed opportunistic fungi Candida and Aspergillus
species and also bacteria Serratia marcescens that have not appeared in controls.
Besides the classical skin diseases, microbes were shown to affect the curing of
chronic wounds in the adult population, diabetic and obese people. The study of a
diabetic foot ulcer (DFU) is an example, DFU occurs in every 15–20% of individuals
having diabetic nephropathy. Sequencing of 16S rRNA reported bacterial species,
namely Staphylococcus sps and Proteobacteria sps showed shorter and longer
duration for shallow and deeper ulcers, respectively (Valensi et al. 2005; Ramsey
et al. 1999; Gardner et al. 2013).
2.3.3 Topography of Skin
Studies based on cultures illustrated variation of skin microbiota due to different

topographical regions, and the variation shows distinct sets of microbes on the skin
site. Sites such as the groin, axillary vault, and toe web are partly occluded. The
microbes that can withstand temperate and humid conditions such as gram-negative
Bacilli, Coryneforms, and S. aureus grow in these areas of moist conditions (Roth
and James 1988). Highly sebaceous sites such as the face, chest, and back facilitate
the growth of lipophilic microbes, especially Propionibacterium spp. and
Malassezia spp. (Roth and James 1988), whereas other regions, arms, and legs
show fluctuation in temperature and hence experience lower habitats for microbial
growth (Marples 1965).
Nevertheless, the topography of skin differs equally at microscopic and macro-
scopic points. Microscopically uneven skin surface, thickness, folds, and mass of
hair follicles present and density of glands exemplify the skin microbiota.
Macroscopical levels comprise plateaus of the forearm and back, elbow, shoulder,
and cervices. Besides, skin appendages that are ununiform are also staged under the
macroscopic level (Kong 2011). The mass of the epidermis and dermis lining also
varies which affects skin texture. Other factors such as ambient humidity, seasonal
variations, clothing, usage of cosmetics, antidrug, and other environmental surfaces
affect the topography of microbes (Fierer et al. 2010).
The distinct changes in the skin at the time of birth in an infant is that it switches
to gaseous or environmental microbes interface from a sterile condition marking the
habitant for several microorganisms. As soon as few days from birth, the infant
undergoes quick changes with surface colonization that plays a vital role as a skin
barrier. The changes usually notified are water loss and gain and change in pH and
sebaceous activity, which characterize the colonization of microbiota (Chiou and
Blume-Peytavi 2004). At the time of development, the structure of skin shows the
difference from that of adult function and biochemical composition (Stamatas et al.
2010). Recent studies reported that over time the point of stability poses topographi-
cal and temporal differences (Peterson et al. 2009).
2.3.4 Skin Modulation
Additionally, acting as a physiological barrier, the skin plays a vital role as an

immunological barrier (Borkowski and Gallo 2011). The immune response of the
skin modulates the commensal microbiota that inhabits the skin. Specific receptors,
namely PRRs (pattern recognition receptors) colonize the skin incessantly by
keratinocytes. PRRS, especially toll-like receptors (TLRs), mannose, and
NOD-like receptors, recognize pathogen-associated molecular models with flagellin
and nucleic acids, LPS (lipopolysaccharides) of gram-negative bacteria, mannan and
zymosin of fungal cell walls, peptidoglycan and teichoic acid of gram-positive
species. Cytokines, chemokines, and antimicrobial peptides aid in the activation of
keratinocyte PPRs. Afar affecting the adaptive immune response, AMPS slaughter
the growth of microbes (Braff et al. 2005). For that reason, the continuous
interactions of keratinocytes, immune cells, and microbes modulated by AMPs,
cytokines, chemokines, and peptides. Differentiating between transient and resident
microbes is still unclear; however, TLRs interact on protracted exposure to com-
mensal microbes. A commensal bacterium S. epidermidis has reported modulating
the host immune response by inhibiting the skin pathogens such as S. aureus and
Streptococcus and also assist the host AMPs with alcoholic soluble modulins (Cogen
et al. 2010a; Cogen et al. 2010b). Another modulin lipoteichoic acid from
S. epidermidis modulates restrain inflammation by TLRs 2 and 3 (Lai et al. 2009).
The complement system modulates the cutaneous microbiome and inflammatory
response. The complement system is triggered by either of the three pathways,
classical, lectin, or alternative. Upon activation, the active proteins trigger the
defense mechanisms, for instance, opsonization, lysis of microbial cells through
membrane attack complex, production of effector molecules that arbitrate activation
of inflammatory cells, and phagocytosis (Ricklin et al. 2010). The activation of
inflammatory cells by the complement components C3a and C5a fragments
modulates the innate and acquired immune responses through C5a receptor and
pattern recognition signaling (Hajishengallis and Lambris 2010). A study by
Chehoud et al. through a culture-based sequencing method suggested C5aR signal-
ing directs essential changes in the skin microbiota eventually, as well as reducing
variety and distorted taxonomic composition (Chehoud et al. 2013).
Studies recently reported that the application of healthy flora into the skin lowers
the pH and enhances the moisture withholding activity (Nodake et al. 2015). Mixing
different microbial components of the skin microbiota and altering the composition
of the beneficiary unlock the probability of expanding probiotic solutions that aid or
modulate the skin reverting the microbiome in a healthy person (Paetzold et al.
2019).
Vitamin B12 biosynthesis in Propionibacterium acnes showed significant down-
regulation in acne patients. Vitamin B12 is known to modulate the activity of the
skin microbiome and put in acne pathogenesis. Analysis suggested that supplemen-
tation reticent the expression of the vitamin B12 pathway in P. acnes that modified
the transcriptome of the skin microbiome (Kang et al. 2015).
2.4 Oral Microbiome
The oral microbiome is one of the critical components among other parts of the
human system, and the second most common complex microbiota is followed to
colon (Wade 2013). The oral microbiome refers to the population of microbes
residing in the oral cavity (Dewhirst et al. 2010). Over 700 varieties of microbes
are present in the human mouth. Studies manifest the co-relation of oral microbiota
with systemic diseases, besides rheumatoid arthritis (RA), cardiovascular disease,
and poor pregnancy effects (Graves et al. 2019; Chen et al. 2018; Cobb et al. 2017;
Ray 2017). Several microbes enter the downstream digestive tract from the mouth
through saliva, causing diseases related to the digestive system (Dewhirst et al.
2010). Nevertheless, microbiota aids in curing oral and systemic diseases. The oral
cavity is dispersed under two types of facades: the hard and the soft tissues. Hard
tissue implies the teeth, while oral mucosa falls under soft tissue (Zaura et al. 2014).
The diversity of microorganisms at different sites of the oral cavity is reported.
Bacteria usually plethora are coated onto the surface, forming a biofilm (Zhao et al.
2017). Temperature range around 37 C without significant changes is apt for
bacterial growth; also, pH of 6.5 to 7 serves as the growth factor, which is equivalent
to that of saliva pH where the medium aids in carrying of nutrients to microbes (Lim
et al. 2017). Influencing factors of the oral microbiome are time (Costello and
Relman 2014), age (Anukam and Agbakoba 2017), diet (Lassalle et al. 2018),
exterior environment (Brown et al. 1976), and other factors such as gender, educa-
tion, community, breastfeeding in infants (Galvão-Moreira et al. 2018).
2.4.1 Ecology of the Oral Microbiome
The cavity of the mouth is a composite location that covers different and minutes
microbial locales with diversifying heterogeneous ecological systems on parts of the
oral cavity that are teeth, buccal mucosa, hard and soft palate, and tongue (Kilian
2018). All varieties of phylum reside or inhabit the mouth, of which the major are
bacteria, fungi, and viruses. Table 2.1 lists the various microbiota in association with
Table 2.1 Microbiota in association with oral diseases

Site of Related microbial
Organisms colonization Type of disorder species References
Bacteria Dental plaque, Caries Streptococcus (Dzidic et al.,
teeth mutans, 2018; Gomez
Staphylococcus, et al., 2017;
Prevotella spp, Hujoel et al.,
Dialister spp, 2018)
Filifactor spp,
Veillonella,
Lactobacillus,
Bifidobacterium,
Propionibacterium,
Actinomyces spp., and
Atopobium spp.
Periodontal Fall of teeth losing Porphyromonas (Gomez et al.,
gums gingivalis 2017)
Saliva Hyposalivation Firmicutes (genus (Keijser et al.,
Streptococcus and 2008)
Veillonella) and
Bacteroidetes (genus
Prevotella)
Tongue dorsal Halitosis Streptococcus Scully and
salivarius, Rothia Greenman
mucilaginosa (2008)
Supragingival Dental decay on Corynebacterium and Keijser et al.
plaque occlusal Actinomyces (2008)
Subgingival Prostatic Obsidian Pool OP11, Marsh (2006)
plaque inflammation and TM7,
periodontitis Deferribacteres,
Spirochaetes,
Fusobacteria,
Actinobacteria,
Firmicutes,
Proteobacteria, and
Bacteroidetes
Fungi Periodontal Oral candidiasis, Candida, Ghannoum
and gingival periodontitis, Cladosporium, et al. (2010)
plaque gingivitis Aureobasidium,
Saccharomyces,
Aspergillus,
Fusarium, and
Cryptococcus
Archaea Periodontal Periodontitis Thermoplasmatales, Dridi et al.
Methanobrevibacter, (2011), Lepp
Methanobacterium, et al. (2004),
Methanosarcina, and Nguyen-Hieu
Methanosphaera et al. (2013)
(continued)
Table 2.1 (continued)

Site of Related microbial
Organisms colonization Type of disorder species References
Viruses Gingival Benign-like oral Herpes simplex virus Woo and
plaque papillomas, oral Challacombe
condylomas, and (2007)
focal epithelial
hyperplasia
Gingival Oral hairy Human Reznik (2005)
plaque, leukoplakia, linear immunodeficiency
periodontal gingival erythema, viruses (Retrovirus)
necrotizing
ulcerative
periodontitis, and
Kaposi’s sarcoma
oral diseases. Of all the bacteria, species of Bacillus, Firmicutes, Proteobacteria, and
Actinomycetes are more likely to colonize the oral cavity (Welch et al. 2016).
Distinct to the gut microbiota, the bacteria of the oral cavity act no change consider-
ably. Factors such as diet and environment have a negligible effect on the composi-
tion of bacteria. Nearly 85 species of fungi reside in the human mouth of which fungi
Candida the most common and significant (Baker et al. 2017). The activity of fungi
is standard at regular times, but it may cause infections or harass oral tissues in case
of imbalance in the composition. Studies reported nearly 101 fungal species of
which each individual ranged between 9 and 23 in number (Ghannoum et al.
2010). The pathogenicity of Candida is shown up in combination with Streptococcal
species (Wang et al. 2012). Viruses’ especially phages, act on oral microbiota, which
is stable all through the stages of life (Wang et al. 2016; Dudek et al. 2017). Other
viruses, namely HIV and mumps, often show in the composition of the oral
microbiome (Sällberg 2009; Presti et al. 2018).
2.4.2 Association of Microbiota and Oral Diseases
Microbiota of the oral cavity generates metabolites that influence the development of
a series of oral diseases. Oral diseases such as dental caries, periodontal diseases,
oral tumors, and recurrent Aphthous Stomatitis are the most common diseases
caused by microbiota and are discussed elaborately in the sections below.
2.4.2.1 Caries
Dental caries is the most prevalent infection in the mouth, causing oral pain and tooth
loss (Bowen et al. 2018; Selwitz et al. 2007). The rate of incidence is high, occurring
from children to adult ages. However, caries are reported more in children than
adults. In children with caries, the root cause was recommended to be eating sweets
at bedtime (Cao et al. 2017). Species, namely Prevotella spp., Lactobacillus spp.,
Dialister spp., and Filifactor spp are likely to colonize and show progression and
pathogenicity for dental caries. Comparing the healthier and caries individuals, the
results had shown increased complexity and lower diversity in caries due to acidic
nature (Lu et al. 2019). This feature is noticeable in the microbiota of saliva, which
shows a considerable number of S. acidophilus in caries. Nutrients in the form of
glycoproteins are supplied to microbiota through saliva and gingival crevicular fluid
when people are under fast or starvation conditions (Cao et al. 2017). Glycoproteins
are broken down to saliva and peptide which are under the neutral condition when
the host is starving, later after ingesting sugars or starch the acid-generating bacteria
reign. Weak acid decay the teeth. The rate of corroding depends on the rate of teeth
regeneration and the repair activity they trigger by themselves, which finally results
in caries (Lu et al. 2019).
A study by Aas et al. stated the species of Propionibacterium, Lactobacillus,
Veillonella, Bifidobacterium, and low-pH non-S. mutans streptococci, and
Atopobium spp and Actinomyces spp. genera take part in the progression of caries
(Aas et al. 2008). In contrast, a study investigation revealed Selenomonas, Strepto-
coccus mitis, and Neisseria, and high frequency of Propionibacterium FMA5 is seen
in younger individuals with severe caries progression (Coventry et al. 2000). Apart
from these species, S. pneumoniae, S. infants group, Eubacterium IR009 strain,
Corynebacterium matruchotii, Streptococcus cristatus, Capnocytophaga gingivalis,
Streptococcus gordonii, and Lachnospiraceae sps. C1 strain and Campylobacter
rectus showed to lower in number as the disease progressed (Gross et al. 2010).
2.4.2.2 Periodontal Diseases

Periodontitis and gingivitis are the two periodontal diseases of which, gingivitis is
the most common, affecting 90% of adults (Coventry et al. 2000). Gingivitis is a
reversible inflammatory disease rooted by a local bacterial plaque that occurs at the
gingival lining. Gingivitis plaque is caused by Veillonella, Leptotrichia, Prevotella,
Streptococcus, Haemophilus, Selenomonas, TM7 strain, and Lautropia. If gingivitis
is unrestrained at the earliest, it may develop into periodontitis.
Periodontitis is a chronic irreparable inflammatory disease for the period in which
infiltrates of immune cells penetrate persuading obliteration of connective tissue,
vascular propagation, and alveolar bone devastation (Pihlstrom et al. 2005). A study
by Costalonga et al. reported that the development and progression of periodontitis
are related to Treponema denticola, Porphyromonas gingivalis, and Tannerella
forsythia (Socransky et al. 1998). The development of periodontal pockets happens
in predisposed individuals at times when the connection between the gingivae and
the teeth is lost where anaerobic bacteria colonize (Darveau 2010). Recent studies
with sequencing technologies revealed few more organisms associating with peri-
odontal diseases. They are Peptostreptococcus stomatis, Filifactor, Prevotella,
Desulfobulbus, Megasphaera, and Synergisters (Kumar et al. 2003; Kumar et al.
2005). Candida albicans was practically declared to be highly allied with chronic
periodontitis (Canabarro et al. 2013). However, viruses such as Herpes simplex,
Human cytomegalovirus, and HIV are described to biofilm periodontitis.
2.4.2.3 Oral Tumor and Recurrent Aphthous Stomatitis

Recurrent aphthous stomatitis is the oral mucosal disease affecting 20% population
and is a common disease (Akintoye and Greenberg 2014). The disease features a
painful ulcer in the mouth and is also associated with dysbiosis of mucosa and saliva
(Marchini et al. 2007; Seoudi et al. 2015; Hijazi et al. 2015). Microbiota associated
with RAS was found to be Prevotella (Akintoye and Greenberg 2014),
Actinobacteria sps., (Seoudi et al. 2015), Streptococceae (Kim et al. 2016).
Oral tumors termed oral squamous cell carcinoma sited from the epidermis of the
oral cavity (Markopoulos 2012). Aerobes and anaerobes are the general species
infecting the cavity (Nagy et al. 1998). Association of Streptococcus salivarius,
Peptostreptococcus stomatis, Gemella haemolysans, Streptococcus gordonii, Strep-
tococcus parasanguinis, Gemella morbillorum, and Johnsonella ignava was more
prevalent in tumor sites where Granulicatella adiacens was seen in non-tumor sites
(Hooper et al. 2007; Pushalkar et al. 2012).
2.4.2.4 Association with Systemic Diseases

Diabetes and periodontitis are interrelated; both are reversible mechanisms. Peri-
odontitis is one of the complication factors for uncontrolled diabetes; likewise,
diabetes is one of the root causes of periodontitis (Preshaw et al. 2012; Hintao
et al. 2007; Casarin et al. 2013). Diabetes coupled with supragingival plaque is
reported to be colonized by Streptococcus Intermedius, Prevotella nigrescens,
Streptococcus sanguinis, T. denticola, and Streptococcus oralis (Hintao et al.
2007). Few other studies revealed Veillonella, Eikenella, Fusobacterium, Gemella,
Actinomyces, Neisseria, Capnocytophaga, Aggregatibacter and Streptococcus,
Selenomonas and TM7 genera and lower levels of Filifactor, Synergistetes,
Porphyromonas, Tannerella, Eubacterium and Treponema genera, Eikenella
corrodens, Fusobacterium nucleatum, Veillonella parvula, and V. dispar (Casarin
et al. 2013).
The microbiota plays an influential role in cardiovascular diseases; these species
are closely related to that of periodontal pathogens (Ramirez et al. 2014). Genera
Veillonella and Streptococcus were associated with atherosclerotic plaques that are
similar to the oral cavity (Koren et al. 2011). Besides these genera, Aggregatibacter
actinomycetemcomitans, F. nucleatum, Campylobacter rectus, P. gingivalis,
T. forsythia, and E. corrodens were also detected in plaque (Figuero et al. 2011;
Pucar et al. 2007).
Rheumatoid arthritis (RA) is a systemic autoimmune malady. Both RA and
periodontitis share a common pathogenic mechanism that is bone loss and inflam-
mation. Patients with periodontitis and RA are enriched with anaerobic bacteria
species such as Atopobium, Cryptobacteriumcurtum, Lactobacillus salivarius,
Prevotella, and Leptotrichia with high frequency, where Corynebacterium is present
in reduced levels. Individuals with only RA are associated with Prevotella periodon-
titis bacteria (Corrêa et al. 2016).
2.4.3 Modulation of the Oral Microbiome
The function of oral microbes in the causation and pathogenesis of oral and systemic
diseases is crucial to improve the protection of oral pathogens so that it sustains
homeostasis. Understanding the mechanism of oral microbiome is necessary to

reduce oral pathogen development and progression.
Disproportion in the oral microbiota is said to be associated with condensed CVD
and metabolic health. The relation between oral microbiota and health is probably
the nitrate, nitrite, and nitric oxide pathways that lower the three compounds
regulating vascular endothelial function and after that increased blood pressure.
Inorganic nitrate, the natural source to humans through dietary vegetables is itself
purely inert. Human cells lack nitrate reductase ability yet, commensal bacteria in the
oral cavity use the inorganic nitrate as an energy source for the synthesis of ATP
where the nitrate is reduced to nitrite with an electron transfer. The nitrite obtained is
further reduced to nitric oxide, a potent vasodilator (Larsen et al. 2006). This
pathway highlights the findings that dietary nitrate supplementation through nitrate
salts or vegetables lowers blood pressure in old and adolescents (Webb et al. 2008).
Sequencing 16S rRNA genes of the bacterial species detailed the correlation of oral
microbiome and nitrate mechanism. Neisseria and Rothia, Veillonella, and
Prevotella are present in high and low abundance, respectively, were correlated
with a more substantial raise in plasma in response to nitrate supplementation. The
finding of Vanhatalo et al. demonstrated these findings in his study (Vanhatalo et al.
2018).
Pro and prebiotics are served to modulate the oral microbiome. The probiotic
methods are reported to treat caries that intrusive with the oral microbiota of
cariogenic pathogens. Species of Lactobacillus (L. rhamnosus) (Näse et al. 2001),
L. paracasei (Holz et al. 2013), Lactobacillus reuteri (Caglar et al. 2006),
B. animalis (Cildir et al. 2009), Bifidobacterium (Busscher et al. 1999) have the
inhibiting ability in vivo in reducing carcinogenic bacteria preventing dental caries.
L. reuteri is reported to reduce gum bleeding and gingivitis (Cildir et al. 2009).
Probiotics, namely inulin, lactose, galactose, xylo, and fructooligosaccharides, are
common probiotics used as modulates. Prebiotics majorly xylitol, xylose, and
arabinose are the most potent that suppress the activity of S. mutans (Busscher
et al. 1999).
Antibiotics are the targeted drugs designed especially for pathogenic bacteria in
mammals (Krasse et al. 2006). Studies described that administration of amoxicillin
and metronidazole drugs before scaling and rooting significantly enhanced periodon-
tal factors and lowered the levels of P. gingivalis, P. intermedia, and T. forsythensis
(Kojima et al. 2016). A study by Haffajee and his colleagues detailed the reduction
of periodontal microbiota at subgingival through antibiotic administration (Haffajee
et al. 2006). Knowing the mechanism of antibiotics and genome analysis may
identify the response of the treatment which is necessary to reduce the development
and progression of oral diseases (Zarco et al. 2012).
Certain mechanical debridement such as self-cleaning that is brushing teeth may
improve or control the level of plaque (Petersilka et al. 2002). Professional practices
such as scaling, rooting, and surgeries probably reduce the number of pathogenic
bacteria and maintain ecological balance. Pathogens causing periodontal disorders,
namely Tannerella forsythia, Treponema socranskii, P. gingivalis, and Tannerella
denticola are marked to vanish after mechanical debridement (Sakamoto et al. 2004).
2.5 Conclusion
In recent years, the expansion of molecular methods in identifying the composition

and function of the microbiome in health and disease is of huge interest. Exploring
and understanding the interaction between the species are a highly complex chore.
However, several studies have drawn many conclusions on the microbiome interac-
tion with the human system, it is still in an emerging stage. Further research with vast
sample sizes and novel techniques is essential to get reliable models to create
concrete data. This will further engage in the discovery of novel drugs, biomarkers
and assist in targeted therapies and customized medicines for healthier management
in clinical applications.
Acknowledgments The authors Pavanai Sanapala and Sudhakar Pola are grateful to the
authorities of Andhra University Visakhapatnam, India for providing the necessary facilities to
carry out the research work and for extending constant support.
Conflict of Interest The authors declare that they have no competing interests.
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A Systematic Review on Crosstalk Between
Microbiome and Immune System 3
Y. Aparna, K. Anuradha, S. Anju, and J. Sarada
Abstract
The microbiome includes a diverse group of microorganisms that inhabit a

particular niche. The microbes that inhabit humans and exhibit beneficial or
symbiotic association are termed as normal flora, most of which secrete
metabolites like vitamins and other growth factors which supplement the basic
nutritional requirements for the body. The interspecies balance that is observed
among the diverse groups of microbes is termed eubiosis and any deviation from
this state is termed dysbiosis. It was hypothesized that there exists a crosstalk
between the immune system and the microbiome of the host. These organisms
play an important role in the induction and education of immune cells. This
bilateral interaction plays a crucial role in maintaining human health by striking a
fine balance between tolerances exhibited to normal flora and evoking responses
against invading pathogens. Recent studies on the discovery of Pattern recogni-
tion receptors (PRRs), Toll-like receptors (TLRs), C-type lectin receptors, etc.
expressed by a variety of cells are known to play a central role in immune
surveillance mechanisms and are involved in maintaining homeostasis between
the microbiome and immune responses. Many studies have proven that disruption
of this balance paves the way for the development of pathogenesis in many
disease conditions like allergy, autoimmune disorders, asthma, etc. The present
review highlights the role of the human microbiome and its interaction with the
immune system concerning its beneficial aspects and disease pathogenesis.
Keywords
Microbiome · Immune system · Eubiosis · Dysbiosis · Autoimmunity
Y. Aparna (*) · K. Anuradha · S. Anju · J. Sarada

Department of Microbiology, Bhavans Vivekananda College of Science, Humanities, and
Commerce, Sainikpuri, Telangana, India

Ltd. 2021
https://doi.org/10.1007/978-981-16-3156-6_3
36 Y. Aparna et al.
3.1 Introduction
Humans are made up of both microscope and macroscopic structures. It is estimated

that the total number of microbes far exceeds the human cells inside the body. They
are known to express unique genes which are far different from the host genome and
exist as complex communities in humans. Microbes that inhabit humans include
bacteria, virus, fungi, and protozoa which are known to control host physiology.
A recent development in immunology has paved the way for better understanding
the role of microorganisms in induction, education, and functioning of the immune
system in humans. It is understood that commensals in the human body play a
fundamental role in educating and training the immune system. Many studies prove
that microbes play a remarkable role in controlling many disease conditions in the
body. Different parts of the body inhabit different types of microflora depending on
the type of environment they are exposed to. The highest microbial load was
observed in the colon. The type of microbes varies from human to human. Despite
the variation, 90% of gut microflora belong to Gram-Negative bacteria like
Bacteroides and Gram-Positive Bacteria like Firmicutes (Sender et al. 2016).
Microbiota forms a symbiotic relationship with their host. This mutualistic
relationship between the microbes and the host co-evolved thereby integrating
signaling and sensing pathways to ensure its survival in a microbial predominated
world. This dynamic interaction is necessary to maintain homeostasis in individuals.
If this homeostasis is disturbed, it leads to dysbiosis causing diseases in humans. The
microbiome composition in an individual is greatly influenced mainly by two
factors, termed as genetic factors and immunological factors. They are also
influenced by environmental diet and hygiene. All these factors together shape the
microbiome in an individual (Mezouar et al. 2018). Recent advances in identifying
and characterizing the microbial flora reveal the importance of microbes in
maintaining a healthy steady state in humans.
The term microbiome includes not only bacteria but also fungal communities.
Both bacteria and fungi form essential parts of the microbiome. It is estimated that
nearly 400 species are found in humans (Halwachs et al. 2017). A shift in these
bacterial and fungal compositions results in disease conditions like inflammatory
bowel disease, cystic fibrosis in humans (Kim et al. 2015).
It was believed earlier that host-microbe interaction has evolved for metabolic
and nutritional requirements which led to a mutualistic interaction between
microorganisms and humans. It was observed now that there is an interaction
between the microbiome and the immune system. The natural interface between
microorganisms and the immune system is the gastrointestinal tract and the mucosal
cell surfaces. The immune system can differentiate between the invading pathogens
and the commensals residing in the gut. This led to the evolution of the immune
system. The commensals protect the body from invading pathogens by consuming
the nutrients that are required for the growth of pathogens. Also, the commensals
secrete secondary metabolites which inhibit the growth of invading pathogens. This
competitive exclusion plays an important role in the immunomodulatory mechanism
(Corr et al. 2007). Many studies suggest the role of commensals in shaping the
3 A Systematic Review on Crosstalk Between Microbiome and Immune System 37
immune system. It was identified that they play a major role in the maturation of
Mucus-Associated Lymphoid Tissue (MALT).
On the other hand, the immune system controls the microbiome by spatial
segregation which is noted especially in the intestine. This type of stratification is
essential to protect the beneficial microbiome in the body. For example in the colon,
a load of microbes is more. A thick impermeable mucus layer made of
O-glycosylated MUC2 mucin which is rich in lectin-like proteins such as ZG
16 and beta-defensins keeps away the microbiome (Bergstorm et al. 2016).
3.2 Role of Microbiota in Hematopoiesis
Though there is a clear anatomical separation between the microbiota and the human
immune system, there is very clear evidence which marks the dispersion of detect-
able commensal metabolites in the tissues following the colonization by microbes.
These metabolites reach circulation and show a profound impact in tuning the host
immune system. Few experimental pieces of evidence state that commensals in the
bloodstream help in bringing the steady-state hematopoiesis (Maslowski et al. 2009;
Shi et al. 2011). Recognition of commensal-derived products by TLRs is important
to maintain bone marrow myeloid cells. TLR components like MyD88 and TICAM1
specifically are known to mediate steady-state granulopoiesis, though it is not very
clear which microbial product is responsible for this activation. Such results indicate
the coevolution of the host and the normal flora, where the host is dependent on the
microbial-derived signals to maintain vigilance on invading pathogens (Balmer
et al. 2014).
The process of hematopoiesis and education of hematopoietic stem cells are
influenced by the products of bacterial metabolism in mammals. The gut
microbiome helps in breaking down of dietary components like fiber which are
indigestible. The net result of this digestion is the production of short-chain fatty
acids (SCFA). These products are produced uniquely by bacterial fermentation in the
intestine (Cummings et al. 1987). These SCFAs serve as energy sources for
enterocytes. They not only can activate G-protein-coupled receptors which are
expressed on hematopoietic cells and epithelial cells but also inhibit Histone
deacetylase (HDAC) in the gut leading to altering the gene expression of local
macrophages (Macia et al. 2015; Maslowski et al. 2009; Chang et al. 2014; Singh
et al. 2014). Research on SCFA which is produced by the metabolites derived from
commensals led to alterations in the process of hematopoiesis which are identified by
increased production of macrophages and dendritic cell precursors (Trompette et al.
2014). Also, neutrophil aging is known to be promoted by commensal microbiota by
tonic sensing of TLR ligands (Zhang et al. 2015).
Several findings state that Gut microbiota is involved in promoting the process of
hematopoiesis and controlling central immunity. The key findings of this study are
gnotobiotic and oral antibiotic-treated mice were susceptible to Listeria
monocytogenes due to defects in myelopoiesis. But when they are re-colonized by
complex microbiota, it restores the defects in myelopoiesis thereby resisting the
38 Y. Aparna et al.
infection with Listeria monocytogenes. These findings are promising as they dem-
onstrate the evolutionary connection between the microbes and the host. They are
known to promote and maintain both embryonic-derived myeloid cells and
hematopoietic stem cells thereby striking a perfect balance under steady-state
conditions. The absence of commensals also reduced the population of neutrophils,
monocytes, and macrophages in germ-free animals. This study states that regulation
of hematopoiesis and shaping of immunity are largely mediated by gut microflora
(Khosravi et al. 2014).
Commensal bacteria are reported to influence T-helper type 2 (TH2) cytokine-
dependent inflammation leading to allergic disorders. Alteration of commensal
bacteria by oral antibiotic treatment in mice resulted in elevated serum IgE which
in turn exaggerated the basophil production resulting in hyperimmunoglobulinemia
E syndrome in mice. This clearly states commensal bacteria-derived signals play a
vital role in regulating basophil hematopoiesis (Hill et al. 2012).
Nevertheless, it was clearly understood that microbial signals that are transmitted
to the neonate during gestation, through the placenta, breast milk, and during the
early developmental stages shape the immune repertoire in the bone marrow.
Components of microbiota like SCFAs, MAMPs, LPS, Peptidoglycans, etc. reach
circulation, access the distal sites like bone marrow, and regulate the proliferation
and differentiation of HSCs. These responses protect the neonate from invading
pathogens by reducing the susceptibility and modulating the inflammatory responses
(McCoy and Thomson 2018).
Dysbiosis, a term that is generally referred to as imbalance of gut microbiome is
associated with suppression of hematopoiesis. This is observed in conditions like
inflammatory bowel disease (IBD), obesity, anemia, nutritional disorders, and
altered hematological abnormalities both in humans and in mice. A model was
proposed on how microbial products regulate the cascade of reactions and regulate
hematopoiesis. Briefly, MAMP’s and other microbial products activate the TLR
pathway while Diaminopimelic acid (DAP) activates the NOD1 pathway in bone
marrow stromal cells which are MyD88-dependent. These two pathways have a
common signaling molecule, i.e., Tumor necrosis factor receptor-associated factor
3 (TRAF3) which in turn signals IRF 3. This signaling cascade induces type I
interferon which activates STAT1, which is known to induce the process of hema-
topoiesis (Yan et al. 2018) (Fig. 3.1).
3.3 Microbiome and Innate Immune Responses
The immunity that is conferred by an individual right from birth is termed innate
immunity. The understanding of host-microbial interactions has revolutionized the
field of immunology. The past two decades have brought two Paradigm shifts that
enable us to understand the role of microbes in conferring immunity. The major
discoveries that led a strong impact in this field include the discovery of Pattern
Recognition Receptors (PRR’s) present in the host which are involved in identifying
the conserved molecular structures in microorganisms. Some of the main families in
Fig. 3.1 Mechanism describing the activation of cascade of events for regulating the process of
hematopoiesis induced by microbial components
PRR’s include Toll-like receptors (TLR’s), Nucleotides-binding oligomerization

(NOD)-like receptors, C-type lectin receptors, RIG-I-like receptors, AIM2-like
receptors, OAS-like receptors. These receptors act as continuous surveillance
systems for invading microorganisms. Another breakthrough in the past two decades
is the characterization of a culture-independent microbiome.
The different innate barriers include Anatomical barriers like skin and mucous
membrane that line the gastrointestinal tract, respiratory tract, genitourinary tract,
physiological barriers, cellular barriers, inflammatory barriers, etc. The diversity of
microorganisms far exceeds the total number of cells in the body. The immune
system plays a crucial role in shaping the microbial communities that are tolerated by
the host. This bilateral interaction plays a pivotal role in maintaining human health.
Innate immunity and microbiota are interdependent. Let us look at the processes in
detail.
40 Y. Aparna et al.
3.4 Cutaneous Immunity and Microbiome Interactions
Skin acts as the first line of defense mechanism protecting the body from invading
foreign pathogens and toxic substances. Skin is colonized by a diverse group of
microorganisms, which includes bacteria, fungi, viruses, mites, etc. These organisms
live as symbionts on the skin protecting the body from harmful pathogenic
microorganisms. They are also involved in educating the T-cells thereby striking a
delicate balance between the host immunity and the microorganisms. Disruption of
this balance leads to skin infections and skin disorders. Cutaneous immunity is
brought both by innate and adaptive immune responses which are modulated by
the microbiota.
Skin acts as a strong physical barrier because of its top layer epidermis, the
Stratum corneum. This layer consists of enucleated cells known as Keratinocytes
which are also known as squames. These squames are embedded in the lipid layer
forming bricks and mortar structures in the epidermis. Cutaneous invaginations and
appendages in the skin include sweat glands, sebaceous glands, and hair follicles,
which attract many microorganisms. The characteristic body odor is due to the
presence of different types of microbes inhabiting the skin. Sebaceous glands
support the growth of facultative anaerobes like Propionibacterium acne, which is
a commensal bacteria. These bacteria hydrolyse triglycerides present in the sebum
and release fatty acids which contribute to the acidic pH on the skin. Many
pathogens like Staphylococcus aureus and Streptococcus pyogenes are inhibited
because of this acidic pH. Host factors like age, location, and gender significantly
contribute to the variation of microbial flora on the skin. Environmental factors like
occupation, usage of antibiotics, and clothing choice also significantly influence the
type of skin microbiota. The use of cosmetics, soaps, and moisturizers are the most
potential factors that influence the diversity of microbes in an individual. Molecular
analysis and genomic approaches revealed a much greater diversity of organisms.
Four predominant phyla on most of the human skins are identified as Actinobacteria,
Firmicutes, Bacteroidetes, and Proteobacteria. They are also found in the inner
mucosal surfaces underlying the skin. Metagenomic analysis revealed Staphylococ-
cus and Corynebacterium spps to inhabit mostly in the moist areas like umbilicus,
axillary vault, side of the groin, sole inner elbow, etc. Temporal variation of the skin
microbiome depends on the sampled site (Grice and Segre 2011).
Skin not only acts as a physical barrier but it also acts as an immunological
barrier. The immune responses generated during infectious conditions significantly
modulate the commensal microbiota colonizing the skin. Pattern Recognition
Receptors (PRR’s) such as Toll-Like Receptors (TLR’s), Mannose receptors, and
NOD-like receptors are continuously involved in recognizing Pathogen-Associated
Molecular Patterns (PAMP’s) which include LPS layer, nucleic acids, mannans, a
peptidoglycan layer, teichoic acids, etc. This identification results in the activation of
keratinocytes which initiates the innate immune response resulting in the secretion of
cytokines, chemokines, and antimicrobial peptides. They also assist in lysing bacte-
ria, fungi, and enveloped viruses. Recent shreds of evidence state that Langerhans
cells, a set of dendritic cells found in the skin promote tolerance to self-antigens and
Fig. 3.2 Microbiome interactions with skin and their role in cutaneous immunity
commensal bacteria on the skin by inducing regulatory T cells in a steady-state

(Seneschal et al. 2012; Romani et al. 2012). Though the mechanism is not clear they
are known to be involved in the induction of immune tolerance which is achieved by
the combined recognition of PAMP’s by PRR’s (Strober 2004; Fukao and Koyasu
2003). Many reports specify the significant role played by microbes in inducing
immune responses, for example, Staphylococcus epidermidis is known to modulate
the innate immune response by producing phenol soluble modulins (PSM’s) that
selectively inhibit skin pathogens like Staphylococcus aureus and Group A Strepto-
coccus. They are also known to assist the host AMP’s by enhancing lytic activity.
Recent studies also reported that commensal-induced TLR signaling is essential to
repair the damaged cells postinfection. Lipoteichoic acid-mediated crosstalk
between TLR2 and TLR3 inhibits skin inflammation (Fig. 3.2).
On the other hand, dysregulation of skin immune response leads to several skin
disorders like Atopic Dermatitis (AD), Psoriasis, and other disorders. Skin disorders
are known to be associated with microorganisms inhabiting the skin like Seborrhoeic
dermatitis, a hyperproliferative disorder that affects the scalp. The causative agent is
42 Y. Aparna et al.
Malassezia spp, cultured from infective sites. They are also found on healthy skin.
The factors responsible for turning it into a pathogen depend on age, genetic,
environmental factors and need to be studied in more detail (Gupta et al. 2004).
Another example is Propionibacterium acne which is associated with acne espe-
cially in teenagers. This organism secretes lipases, proteases, and hyaluronidases
which injure the tissue lining. It damages the pilosebaceous unit, activates classical
and alternate complement pathways, induces proinflammatory cytokines, and also
activates many neutrophil chemotactic factors (Webster and Leyden 1980; Kim
2005). Atopic Dermatitis (AD) is also known to be associated with pathogens like
Staphylococcus aureus. Sometimes the commensals like Staphylococcus
epidermidis can turn into opportunistic pathogens in immunocompromised
individuals. Most frequent cases related to this are nosocomial and increased levels
of antibiotic resistance which are confined specifically to the host (Uckay et al. 2009;
Otto 2009).
3.5 Microbiome–Mucosal Cell Surface Interactions
Mucous linings form another anatomical barrier apart from the skin. They are found
mainly in the gastrointestinal tract, genitourinary tract, and respiratory tract. These
linings act as a protective shield from microorganisms entering the host tissue
(McGuckin et al. 2011). The intestinal mucosal system mainly comprises three
lymphoid structures, which include Peyer’s patches, lamina propria, and epithelial
cells. Respiratory mucosal linings include submucosal cartilage and adventitia. The
gut microbiome is influenced by various factors which include diet, environmental
factors, exogenous substrates which are the key regulatory factors. Changes in the
diet like consumption of high fat and high sugar diet changed the composition and
diversity of normal flora in the intestine.
Mucosal epithelial cells produce peptides that have an antimicrobial property that
limits the growth of commensal bacteria by exerting their enzymatic action on cell
walls (Hooper and Macpherson 2010). An example of the mucosal antimicrobial
peptide is RegIIIγ (lectin) which is produced soon after birth. This lectin protein
forms a “demilitarized zone” forming a physical barrier between microbiota and host
intestine. The production of this protein is controlled by the MyD88 pathway which
is highly regulated (Cash et al. 2006; Vaishnava et al. 2011).
In addition to these inflammasomes, a special type of cells perform pleiotropic
immune functions and activate inflammatory caspases. NLRs (NOD-like receptors)
assemble to form multiprotein complexes and are found more in these
inflammasomes. One such studied example is NLRP6 inflammasome. It regulates
microbiome composition and is involved in maintaining homeostasis. The signaling
of this NLRP6 is modulated by metabolites derived from microbiota and is involved
in regulating epithelial IL 18 secretion and AMP expression (Levy et al. 2015).
The majority of antimicrobial peptides were produced by Paneth cells, which are
secretory cells present in small intestine mucosa (Bevins and Salzman 2011). In
addition to this pancreatic acini secretions are also vital in maintaining intestinal
homeostasis which is demonstrated with increased mortality in mice due to reduced

secretions of pancreatic acini (Ahuja et al. 2017). Bacteria like Bacteroides fragilis is
a well-studied commensal as it produces Polysaccharide A (PSA), a single molecule
involved in promoting symbiosis and educating the host immune system. PSA is
identified by TLR2/TLR1 in association with Dectin-1 and regulates intestinal
immunity by controlling Treg cell differentiation thereby conferring intestinal
immunity and bringing homeostasis.
Recent reports state that in a steady state, commensal microbiota present in the
intestine signals for the production of Interleukin-17 (Th 17) and Interferon-γ (Th 1)
(Gaboriau-Routhiau et al. 2009). The constant signaling by microbes is helping in
maintaining homeostasis. The categorization of intestinal bacteria also depends on
secretory immunoglobulin, i.e., IgA. IgA specific for the intestinal microbiome is
produced by B cells with the help of dendritic cells and T-cell interactions. This IgA
is specifically produced against commensal-derived antigens. If these commensals
cross the intestinal barrier, the phagocytic cells like macrophages and dendritic cells
engulf them, transport them to MALT tissue, induce B cells for IgM production.
These reactions prevent the adherence of commensals to epithelial cell surfaces
thereby striking a balance between commensal community and host immune
responses. However, it was noted that IgA lacks classical memory and hence
changes with antigen encounters (Hapfelmeier et al. 2010).
Commensal containment is attributed to discrete pathways in the host in addition
to nonspecific and broad modes. For example, bacteria belonging to the Alcaligens
genus will proliferate more in the presence of IL_22 which is produced by Intestinal
Lymphoid cells. These works state that microbes and host immunity might have
coevolved together to promote the maintenance of selective species in an ecological
niche (Qiu et al. 2013; Sonnenberg et al. 2012).
3.6 Microbiome and Adaptive Immunity
In addition to innate immune responses, recent advances in research demonstrated

the role of commensals in promoting adaptive immunity. They are involved in
training the immune cells and acting as adjuvants in whole. GALT
(Gut-Associated Lymphoid Tissue) with Lymph Nodes act as major sites of antigen
priming in adaptive responses. This is well studied in a process termed “homeostatic
immunity”, which is defined as a process in which the development and establish-
ment of adaptive responses with the response to microbiota in absence of inflamma-
tion. The best-studied example of homeostatic immunity is IgA responses. This
isotype is the most abundant secretory form which is produced in mammals and
plays a pivotal role in shaping the initial interactions with microbes. Secretory IgA is
referred to as both innate and adaptive as it is produced by both T-independent and
T-dependent mechanisms (Kawamoto et al. 2014; Sutherland et al. 2016). IgA
produced by the T Cell-dependent mechanism plays a significant role in shaping
the microbiome. The mutualistic relationship between IgA and microbiota results in
44 Y. Aparna et al.
maintaining a balanced and diversified microbiome. This facilitates the expansion of

Foxp3+ T cells which are involved in maintaining a regulatory loop.
Advances in sequencing techniques revealed that the microbiome contains nearly
3X10(Bevins and Salzman 2011) genes which indicate that human microbiota has
coevolved with the host (Qin et al. 2010). Many bacteria are known to exhibit
mutualism and aid in maintaining homeostasis. In an experiment, gnotobiotic mice
were monocolonized with Bacteroides thetaiotaomicron. This bacteria colonizes the
intestines of both humans and mice and is involved in altering host genes which
shown an impact on the absorption of nutrients, angiogenesis, maturation of immune
cells, etc. while monocolonization with other bacteria like E.Coli and
Bifidobacterium showed different effects. These data reveal that different bacterial
species colonizing the human body have varying functions and need to be
investigated in detail for their role in host immunity (Hooper et al. 2001).
3.7 Microbiome and Cell-Mediated Immunity
Recent studies demonstrated the role of the microbiome in cell-mediated immune

responses fostering the development of different T-cell subsets. Nearly more than
half of genes of microbiome colonized are known to be associated with immune
responses when experimented with GF mice. This data reveals the role of microbes
in shaping immunity. Monocolonization with Segmented filamentous bacteria
(Clostridia-related organism) produced multiple T-cell lineages specifically in the
induction of T17 cells. They augmented mRNA transcripts, RegIII-γ in ileal cells.
Up-regulation of RegIII-γ increased IL-17 production. The SFB monocolonized
mice are resistant to infections caused by Citrobacter rodentium by increasing the
production of cytokines like IL-22, IL-17, and IL-23 which are associated with
increased Th17. This data suggests the protective role of SFB in maintaining the
health of epithelial cells (Gaboriau-Routhiau et al. 2009).
Many studies have reported the role of bacteria in the induction of Regulatory T
cells like CD4 + Foxp3+ and CD4+ IL10+ in the intestine. In an experiment,
46 different strains of Clostridium are reconstituted in GF mice, which induced
production of CD4+ Foxp3+ cells through TGF-β activator production only in the
colon and cecum where the introduced clostridium strains inhabit more. They also
induced the production of IL-10 under inflammatory conditions. When tested with
Lactobacillus and SFB, the levels of Treg production were minimal which indicate
the specific responses to clostridia alone. Interestingly they are also resistant to DSS
and oxazolone-induced colitis indicating the significant role of clostridia in
maintaining mucosal homeostasis (Atarashi et al. 2011).
Also, Effector T-cell production and Regulatory T-cell production were
dynamically maintained by commensal bacteria in the gut by TLR9-dependent
pathway. The process is known to be mediated by production by SCFAs, coloniza-
tion of segmented Filamentous bacteria, and other related organisms through signal
recognition by TLR5 and TLR9 (Hall et al. 2008).
3.8 Microbiome and Humoral Immunity
Mucosal IgA binds to epithelial serves and confers protection to the host against any
invading bacteria. It also regulates the composition of the microbiota, controls the
expression of genes by microbes inhabiting the intestine. For instance, the commen-
sal bacterium Bacteroides thetaiotaomicron will not trigger the process of inflam-
mation in the gut. IgA after affinity maturation and somatic hypermutation selects for
specific components in microbes which results in increasing the diversity of the
microbiome and bringing mutualism between commensals and host. IgA-producing
plasma cells are generated by T-dependent and T-independent mechanisms along
with help of epithelial cells, macrophages, dendritic cells, and innate lymphoid cells
(ILCs). Levels of IgA-producing plasma cells from Peyer’s patches and lamina
propria showed significant variation in production when comparative studies are
done with normal mice and germ-free mice which is very less in later cases. In an
interesting study bacteria, Sutterella species inversely correlated with IgA produc-
tion in feces in contrast to the above statement. In Rheumatoid arthritis, an autoim-
mune disorder, these bacteria degrade IgA and J chain peptide in IgA that is required
for its stability in the lumen. This study states that the anatomical location of bacteria
also determines the stability of IgA responses.
Concerning T Cell-dependent responses, the bacteria SFB and Mucispirillum in
intestinal epithelium elicits T Cell-dependent IgA responses. SFB induces Th17 and
follicular T-helper cells resulting in high-affinity IgA responses (Fig. 3.3). Mice
lacking TCR chains β and δ also produce IgA specific to commensal bacteria
indicating T cell-independent mechanisms (Fig. 3.4). IgA-producing B Cell clones
persist for longer periods. The repertoire of IgA changes dynamically with the
increasing diversity of commensal bacteria. The composition of microbiota changes
concerning various factors like diet, exercise, environmental conditions, etc. (Honda
and Littman 2016).
3.9 Microbiome and Immune System Dysbiosis
In general, genetically susceptible individuals’ dysbiosis between the microbiome

and immune system leads to the development of different types of immune-mediated
diseases. Two research studies state that dysregulation of microbes and immune
system results in many multifactorial diseases like neurodegenerative diseases, some
of the well-studied diseases are highlighted in the study (Table 3.1).
Inflammatory bowel disease (IBD) is a chronic inflammatory disorder. Many
studies have shown evidence of the role of the gut microbiome in the pathogenesis of
inflammatory bowel disease. This may be due to a reduction in the number of various
bacteria taxa like Firmicutes, Lactobacillus, Clostridia, etc., and increased diversity
of Enterobacteriaceae members. The main reasons identified were alterations in the
microbes-associated metabolites, breakdown of the intestinal mucosal barrier, tissue
injury, mutations in NOD2 gene, autophagy-related ATG16L1, suppression of
pro-inflammatory bacteria like Bacteriodes vulgatus, enhanced colonization of
46 Y. Aparna et al.
Fig. 3.3 T Cell-dependent pathway
bacteria like Akkermansia muciniphila, etc. However, the detailed mechanism was
yet not clear (Zheng et al. 2020).
Rheumatoid arthritis (RA) which involves joints, Synovial inflammation, bone
cartilage destruction is also known to be implicated in the microbiome along with
environmental and genetic factors. Different bacteria like Prevotella copri,
Collinsella, Eggerthella, faecalibacterium, Lactobacillus salivarius are proved to
be associated with Rheumatoid arthritis condition. Microbe-derived metabolites like
short-chain fatty acid derivatives are observed to be interacting with immune
pathways which are implicated in RA condition. Further research is required to
understand the details of microbiome alterations in RA condition (Zheng et al.
2020).
Another disease identified with microbiome dysbiosis is cardiometabolic disease.
This is characterized by chronic low-grade inflammation, which is found to be the
root cause of many metabolic disorders like diabetes mellitus, obesity, nonalcoholic
fatty liver disease, atherosclerosis, etc. Recent pieces of evidence stated that
microbiome-derived metabolites are fueling the process of inflammation by crossing
the gut barrier. It was noted that TLRs present in the liver identify bacterial ligands
initiating the inflammatory cascades. Activation of these cascades contributes to
NAFLD and nonalcoholic steatohepatitis (NASH). In the case of obesity, tryptophan
metabolites produced by microbes are known to modulate adipose tissue
Fig. 3.4 T cell-independent pathway
inflammation which is mediated by the miR-181 family of microRNAs. In athero-

sclerosis heart disease metabolite TMAO produced by gut microbiota up-regulates
macrophage scavenger receptors CD36 and SR-A1 which results in the accumula-
tion of cholesterol (Virtue et al. 2019; Truax et al. 2018; Koeth et al. 2019; Wang
et al. 2011).
A direct link is also found between gut microbiota and cancer immune surveil-
lance mechanisms. Presence of Fusobacterium nucleatum in colorectal cancers is
involved in the inhibition of NK cells which has tumor suppressor function. Another
example is the Pancreatic adenocarcinoma (PDAC) study in which
Gammaproteobacteria and intratumor microbiota promote carcinogenesis in both
humans and mice via TLRs and T-cell energy. In contrast, presence of more
commensals like Clostridium, Bifidobacterium longum, Collinsella aerofaciens,
and Enterococcus faecium favor T cell-mediated responses to anti PD 1 therapy in
metastasized melanoma (Zheng et al. 2020).
3.10 Concluding Remarks
The present review is focused on the current knowledge of crosstalk between the
microbiome and immune reactions with a special focus on eubiosis and dysbiosis.
As discussed in the paper, majority of immune responses are controlled either
48 Y. Aparna et al.
Table 3.1 Microorganisms associated with some major chronic diseases

S.
No. Disease Microorganisms associated with disease References
1 Inflammatory Increased diversity of Enterobacteriaceae, Zheng et al. (2020),
bowel disease Akkermansia Muciniphila, etc. and decreased Devkota et al.
number of Firmicutes, Lactobacillus, (2012)
Clostridia, Bacteroides vulgates, Bilophila
wadsworthia, E.Coli, Bacteroidetes
2 Cystic fibrosis Pseudomonas,Staphylococcus Blainey et al.
(2012)
3 Rheumatoid Prevotella copri, Collinsella, Eggert Hella, Scher et al. (2013)
arthritis faecalibacterium, Lactobacillus salivarius
4 Cardiovascular Candida, campylobacter, Shigella sps Jin et al. (2019)
disease
5 Cancer Fusobacterium nucleatum, Helicobacter pylori Atherton and Blaser
in colorectal cancers; Gammaproteobacteria in (2009)
Pancreatic adenocarcinoma, Bacteroides
fragilis in tumorogenesis
6 Type II E.Coli, clostridia Karlsson et al.
diabetes (2013), Qin et al.
(2010)
7 Obesity Firmicutes Turnbaugh et al.
(2006)
8 Crohn’s disease Adherent invasive E.Coli, Yersinia, Issa et al. (2008)
Clostridium difficile
directly or indirectly by microbiome. The discovery of PAMPs, TLRs, and other

novel molecular determinants helped in expanding the knowledge on interactions of
microbes with immunity. Microbes play a crucial role right from the production of
immune cells by hematopoiesis to a well-developed acquired immunity in humans.
There is growing interest in the field as is evidenced by the findings. Though there
are many research works focused on studying interactions between microbiome and
immunity, the study is still in its infancy as many mechanisms remain unexplored
concerning their regulation via integration of environmental, diet, microbiology, and
immunological approaches. Moreover, less is known about the interactions of
commensal bacteria, virus, fungi, and protozoa cooperate among themselves and
influence each other in bringing homeostatic immunity. Many diseases known today
are known to be associated with microorganisms, their products, and metabolites
which are termed as dysbiosis. Modulation of microbial derivatives or molecules
may provide a novel way for combating antibiotic resistance and also inflammation-
induced diseases in humans.
Acknowledgments There is no financial support for the present review paper. The authors thank
the Principal and Management of Bhavan’s Vivekananda College of Science, Humanities, and
Commerce for their support and encouragement.
Conflicts of Interest The authors claim that there are no conflicts of interest.
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Diversity and Dynamics of the Gut
Microbiome and Immune Cells 4
Prerna Pathak
Abstract
Human microbiome consists of multiple species out of which most of them reside
in gut. Gut microbiota is most complex and dynamic in terms of species diversity
and therefore regulates the host homeostasis. The intricate relation between the
gut microbiota and host is crucial for host functioning. Dysbiosis in microbiota
affects myriads of processes which result in multiple diseases such as IBD, type
1 diabetes, and rheumatoid arthritis, etc. This chapter highlights the role of gut
microbiota in innate and adaptive immune system development and further
explains how alteration in microbiota leads to dysbiosis which makes host
susceptible to several diseases.
Keywords
Gut microbiota · Autoimmunity · Innate immunity · Adaptive immunity · T1DM ·

Rheumatoid arthritis · Systemic lupus erythematosus
4.1 Introduction to Gut Microbiota
Microorganisms are the part of normal human microbiota, over the period of time a
symbiotic relationship leads to their colonization in the nasal tract, oral cavity, skin,
respiratory, and genitourinary tract (Opazo et al. 2018). Apart from microorganisms,
human microbiota comprises protozoans, fungi, archaea as well as viruses (Neish
2009; Sekirov et al. 2010; Sonnenburg and Bäckhed 2016), this collective coloniza-
tion is termed as gut microbiota. The gastro intestinal tract (GIT) of humans is an
intricate open system which harbors 1014 microorganisms (Seksik and Landman
P. Pathak (*)
Jawaharlal Nehru University, New Delhi, India

Ltd. 2021
https://doi.org/10.1007/978-981-16-3156-6_4
54 P. Pathak
2015). Recent advances in deep sequencing technology bring insight about the
genome of gut microbiome, it encodes 3.3 billion genes; this surpasses the number
of human gene by 100-fold (Dwivedi et al. 2017), therefore it is also termed as
“human second genome” (Xu et al. 2019). Human health is governed by the
microbes in the gut microbiota which have both favorable as well as pathogenic
effects. GIT is the largest region which gets exposed with the external habitat and
comprises two-third of the total human microbiome (Virili et al. 2018).
The diversity and the dynamics of the gut microbiota are governed by several
factors like age, gender, health alignments, immunity, genetics of the host, geo-
graphical changes, lifestyle, and treatments (Ardissone et al. 2014; De Martino et al.
2004). Metagenomics study reveals that the most of the species of the microbiome
are missing in the same person at a same time frame; however, microbiota of healthy
individuals shows abundant of some species over others (Human Microbiome
Project Consortium 2012; Qin et al. 2010). In fact, there is diversity in the microbiota
of the gut on the bases of the types of cells; cells of mucus layer, intestinal lumen,
and epithelial cells show diverse microhabitats (Sekirov et al. 2010).
Development of human microbiome starts even before the birth as it is revealed
by the study on microbiome composition of the placenta (Aagaard et al. 2014).
Moreover the study performed on the first stool of the infants shows the presence of
30 genera which are normal inhabitant of amniotic fluid, oral, and vaginal cavity
(Ardissone et al. 2014; Clemente et al. 2012; De Martino et al. 2004). It is believed
that this is occurred due to mother to child transmission at the time of pregnancy
(Lagier et al. 2012). The microbiota of the infants depends on the mode of delivery
such as caesarean section born infants have microbiota similar to that of skin while
vaginally born infants have vaginal microbiota (Dominguez-Bello et al. 2010).
Pregnancy period and the initial few months after the delivery are crucial for the
development of the microbiota which further influences immune homeostasis
(Gordon Jeffrey et al. 2012).
Breast feeding is another decisive factor that links with microbiome and immu-
nity development (Stewart et al. 2018). Human milk contains ~109 bacterial cells/L
(Endesfelder et al. 2014), apart from its nutritional value it possesses various
bioactive and immunological molecules which govern the microbiome and intestine
maturation of the infants. Studies illustrate that immunological components of the
human milk such as sIgA, lysozyme, complex lipids, alpha lactalbumin, and
lactoferrin impart protection to the infants (Gordon Jeffrey et al. 2012). Initiation
and development of microbiota during infant stage impact the health and immunity
during adulthood (Ranucci et al. 2017), any perturbation in this development may
lead to negative consequences (Fulde et al. 2018). The gut microbiome keeps on
evolving from infants to early childhood in a phased manner (Xu et al. 2019).
The mutual relationship between host and gut microbiota plays significant bene-
ficial role. It impacts the development of the gut by influencing proliferation of
epithelial cells and host cells apoptosis. Short-chain fatty acids which are the
by-product of polysaccharide fermentation mediate interactions between host cells
and gut microbiota (Lazar et al. 2018). Apart from maintaining gastrointestinal
homeostasis, gut microbiota play role in the development of components of immune
4 Diversity and Dynamics of the Gut Microbiome and Immune Cells 55
Fig. 4.1 Gut microbiota in normal and diseased conditions
system, synthesis of vitamins like B-complex, folic and biotin, detoxification of

xenobiotic compounds, maintaining nutritional homeostasis (Gérard 2013). Among
all the known members of gut microbiota, bacteria are most explored. They are
classified into three categories, aerobic, facultative anaerobic, and obligate anaerobic
bacteria out of which obligate anaerobic dominates them all (Fig. 4.1).
The interspecies balance is pivotal for the proper functioning of the body; this
balance is termed as eubiosis. Any imbalance to eubiosis is termed as dysbiosis
could lead to plethora of diseases which further affects multiple organs (Clemente
et al. 2012). Hereafter this chapter will focus on the role of gut microbiota in the
development of immune system and disease.
4.2 Interactions Between Gut Microbiota and Immune System
The symbiotic relationship between the microbiome and the human gut is beneficial
for both of them as human gut acts as nutrient source as well as provides breeding
habitat to microflora; in return gut micobiota helps in vitamin synthesis, gut devel-
opment, and forms mucosal barrier as a defense mechanism (Berg et al. 2015). The
human mucosa is the largest and the most exposed component to the external
environment. Eubiosis is pivotal for maintaining host homeostasis and its defense.
The relevance of gut microbiota came into light after the studies performed on germ-
free (GF) mice revealed that it produces relatively reduced amount of Intraepithelial
lymphocytes (IELs) (Bandeira et al. 1990), IgA-secreting plasma cells (Crabbé et al.
1968), Tregs cells (Ostman et al. 2006), and Angiogenin-4 (Ang4) (Hooper et al.
2003).
Moreover in GF mice, the germinal center of Peyer’s patches is smaller compare
to conventional mice (McDermott and Huffnagle 2014). Another study illustrates
56 P. Pathak
that concentration of IgA in the feces enhanced reasonably after prebiotics treatment;
however, expression of Peyer’s patches and pro-inflammatory factor of mesenteric
lymph nodes reduced considerably (Carasi et al. 2015). All together these results
pinpoint the role of gut microbiota in immunity development as well as eubiosis.
Immune system works basically by recognizing and eliminating the pathogen
from our system. The intestinal immune homeostasis is crucial for both host as well
as trillions of microbes within the system. This immune homeostasis is a combined
effect of innate and adaptive immunity. Several innate and adaptive responses which
are crucial in shaping the intestinal microbiota are explained in next section.
4.3 Innate Immunity and Gut Microbiota
Gut-Associated Lymphoid Tissues (GALTs) are present throughout the intestine;

they are part of mucosa-associated lymphoid tissues (MALTs) (Brandtzaeg et al.
2008). The innate immune cells of the GALTs are involved in presenting antigen to
activate adaptive immune response after recognizing pathogens in a nonspecific
manner (Jiao et al. 2020). GALTs have dual function of immune tolerance and
immune homeostasis. GALTs include following components; Peyer’s patches,
isolated lymphoid follicles (ILFs), crypt patches, M cells, appendix, and mesenteric
lymph nodes (mLNs) (Brandtzaeg et al. 2008; Mowat 2003). M cells are involved in
delivering intestinal antigen to GALTs (Mabbott et al. 2013). Several studies pin
point the role of gut microbiota in shaping GALTs. Lymphoid tissue inducer (LTi)
cells are involved in formation of secondary lymphoid organs of gut such as Peyer’s
patches, mLNs, and ILFs (Adachi et al. 1997; Mebius et al. 1997).
Pattern-recognition receptors (PRRs) are crucial for innate immune response;
they sense pathogen through specific structures. The pathogen-associated molecular
patterns (PAMPs) of the intestinal microorganisms are recognized by PRRs and lead
to the development of ILFs. Mouse deficient in PRRs shows defects in ILFs
development. PRRs are of several types depending on their location, ligand speci-
ficity, and functions, several PRR-related molecules are involved in mechanism like
toll-like receptors 2 (TLR2) (Round et al. 2011), myeloid differentiation primary
response 88 protein (MyD88) (Medzhitov et al. 1998; Wesche et al. 1997),
nucleotide-binding oligomerization domain 1/2 (NOD 1/2) (Bouskra et al. 2008;
Clarke et al. 2010; Petnicki-Ocwieja et al. 2009), and TIR domain-containing
adaptor protein inducing interferon-β (TRIF) (Bouskra et al. 2008). PRR-PAMP
recognition plays pivotal role during host defense response as well as structural
development of GALTs.
Toll-like receptors (TLRs) are another crucial member of innate immune system,
they recognize specific region in pathogens and start immune response (Rakoff-
Nahoum et al. 2004). They are also involved in balancing microbiota composition
(Larsson et al. 2012; Wen et al. 2008). Several studies report that TLR5-deficient
mice show compositional changes in microbiota (Vijay-Kumar et al. 2010). These
changes may further lead to the development of spontaneous colitis, metabolic
syndrome, and obesity; this highlights the role of TLR5 in maintaining gut
microbiota composition (Carvalho et al. 2012; Chassaing et al. 2014; Chassaing

et al. 2014; Vijay-Kumar et al. 2010).
The nucleotide-binding oligomerization domain-like receptors or Nod-like
receptors (NLRs) are intracellular stress sensors of pathogen-associated molecular
patterns (PAMPs) associated with cellular stress. Like TLRs, they are also involved
in maintaining microbiota composition as it is shown by the study where NOD1/2-
deficient mice show altered composition of microbiota (Bouskra et al. 2008;
Couturier-Maillard et al. 2013; Petnicki-Ocwieja et al. 2009). Paneth cells which
are present in small intestine have enhanced expression of NOD2 protein; upon
getting exposed to pathogens this protein induces multiple responses which include
cytokines production, autophagy initiation, generation of antimicrobial peptides, and
intracellular vesicle trafficking, thus impacts the composition of the microbiota
(Couturier-Maillard et al. 2013; Nigro et al. 2014; Ramanan et al. 2014). These
members of NLR family elicit immune response after recognizing bacterial
peptidoglycans, viruses, and parasites.
Innate immune system functions by initiating response upon sensing the meta-
bolic state of the gut microbiota. Evidence from all the studies reveals the role of
innate immune system in governing composition of microbiota (Levy et al. 2015), as
mice deficient in NOD2 (Couturier-Maillard et al. 2013; Petnicki-Ocwieja et al.
2009; Ramanan et al. 2014), NLRP6 (Elinav et al. 2011), and TLR5 (Vijay-Kumar
et al. 2010), leads to dysbiosis. It is therefore believed that sensors of innate immune
system work by promoting the growth of beneficial microorganisms as well as
maintaining the stable microbiota. Additionally, there are several other molecules
which are involved in maintaining composition of microbiota such as any alterations
in paneth cells, which produce antimicrobial peptides (AMPs) lead to dysbiosis
(Salzman et al. 2010; Salzman and Bevins 2013). Moreover altered AMP expression
brings about alterations in spatial organization of microbiota; RegIIIγ deficiency
causes colonization of microorganism in the inner mucus layer, which is devoid of
microorganism in normal condition (Vaishnava et al. 2011).
4.4 Adaptive Immunity and Gut Microbiota.
4.4.1 T cells
T cells are the crucial member of adaptive immune system; they are of two types
CD4+ T cells and CD8+ T cells. CD4+ T cells are present in lamina propria of
intestine and upon activation it differentiates into following subtypes T helper
1 (Th1), Th2, Th17, or regulatory T cell (Treg). The balanced expression of these
subtypes is important determinant factor of human health. Each subtype has different
functions like Th1 cells play important role during host defense against microorgan-
ism, while Th2 cells remove parasitic infections (Wu and Eric 2012). Unregulated
Th responses lead to autoimmune diseases and allergic reactions. CD8+ T cells are
present in intraepithelial compartment of the gut. GF mice show less number of
CD8+ T cells with reduction in their cytotoxicity indicates the role of microbiota in
58 P. Pathak
monitoring CD8+ T cells and its function (Helgeland et al. 2004; Imaoka et al. 1996;
Kawaguchi-Miyashita et al. 1996).
Several studies revealed that mice deficient in adaptive immune system display
changes in their microbiota, this suggest the role of adaptive immune system in
balancing microbiota composition (Kato et al. 2014; Zhang et al. 2015). Mice
lacking T cells also show altered microbiota, it is believed that T cells regulate
microbiota by triggering expression of AMP; however, there is no direct evidence to
this. Rather the prime mechanism by which T cells regulate microbiota is by
influencing B cells to produce secretory IgA (Kato et al. 2014). Tregs and Th17
cells are known to be involved in intestinal IgA production, there are reports which
suggest that Tregs assist B cells in IgA production (Tsuji et al. 2009). Another study
points out the role of Th17 cells in antigen-specific IgA production upon immuniza-
tion with cholera toxin (Hirota et al. 2013). All together these studies brought insight
about the role of T cells in development of microbiota.
4.4.2 B Cells
Another crucial molecule of adaptive immune system which governs the composi-
tion of intestinal microbiota is immunoglobulin A (IgA). It is produced by plasma
cells into the intestinal lumen where it attaches to microbes as well as microbial
components. This generates a physical barrier which averts detrimental interactions
with immune system (Pabst 2012). IgA maintains the eubiosis by two proposed
mechanism, first by inhibiting the growth or inflammatory effects of microorganisms
and secondly by preserving the diversity of healthy microbiota (Palm et al. 2015).
Mice deficient in Activation-Induced Cytidine Deaminase (AID) show defect in
class switching or somatic hypermutation, this defect is reversed by substituting IgA;
this highlights the role of IgA in forging microbiota (Fagarasan et al. 2002; Suzuki
et al. 2004), IgA known to play crucial part in forging microbiota during develop-
ment, as its deficiency fails to curb proteobacteria during microbiota maturation
(Mirpuri et al. 2014). IgA is also known to suppress the inflammatory response there
by promoting the mutualism between host and microbiota (Peterson et al. 2007). The
phenomenon of immune exclusion is mainly involved in suppressing inflammatory
response by prohibiting microorganisms from approaching mucosal epithelium
(Corthésy 2013). Moreover alternate mechanism by which IgA directly suppresses
inflammatory responses is by coating microorganisms gene expression (Cullender
et al. 2013). Studies are still at their infancy about how IgA arbitrates these
responses.
4.5 Role of Gut Microbiota in Disease Development
The impact of gut microbiota on innate and adaptive immune system is already
discussed above, any dysbiosis in microbiota leads to critical diseases. The modifi-
cation in the eubiotic state of microbiota triggers myriad of diseases such as type I
diabetes, nonalcoholic fatty liver disease, rheumatoid arthritis, obesity, cancer, etc.,
however how these alterations cause these diseases is still ambiguous. The symbiosis
between host and microbiota is crucial for homeostasis. Together, this chapter
reveals the crosstalk between host, microbiota, and environmental cues which
leads to these pathophysiologies.
4.6 Type 1 Diabetes
Type 1 diabetes (T1DM) was previously called as juvenile-onset diabetes; it is a

chronic disease which is linked with high mortality at premature stage (Lazar et al.
2018). It is occurred due to inability of pancreatic β cells to produce insulin due to
autoimmune obliteration (Aathira and Jain 2014). Normally this disease occurs
during early stage of life but there are reports which reveal that 50% of T1DM
occurs in individuals older than 20 year. Several factors govern the development of
this disease such as diet, genetics, and gut microbiota (Pociot and Lernmark 2016;
Rewers and Ludvigsson 2016; Todd et al. 2007). Data from various studies show
that the composition of gut microbiota varies between healthy individuals and
individuals with T1DM. Bio-Breeding (BB) rat and nonobese diabetic (NOD)
mouse bear alike attribute with that of human disease (Pearson et al. 2016). The
composition of gut microbiota in Bio-Breeding diabetes-prone (BB-DP) rats is
altered strikingly before and after the outbreak of T1DM (Brugman et al. 2006).
Consistent with this, the composition of gut microbiota is altered between healthy
and T1DM individuals (Han et al. 2018).
Large amount of research in this direction highlights the role of gut microbiota in
TIDM development by regulating immune responses. The outer membrane compo-
nent of gram-negative bacteria lipopolysaccharide (LPS) or endotoxin is crucial in
enhancing the proinflammatory cytokines and damaging the function of pancreatic β
cells (Allin et al. 2015), which further leads to diabetes (Pussinen et al. 2011).
Another study showed that circulating LPS is higher in T1DM individuals compared
to that of healthy individuals (Devaraj et al. 2009). Additionally it is considered that
LPS is derived from gut microbiota. Therefore, it is considered as a link between gut
microbiota and TIDM (Han et al. 2018). Any change in gut microbiota causes LPS
and fatty acids leakage by damaging the mucosal barrier, this leads to simultaneous
induction of TLR4 which results in metabolic inflammation (Velloso et al. 2015).
Studies performed in NOD mouse lacking TLR4 show the increased rate of T1DM
development (Gülden et al. 2013). Moreover, a study in NOD mice lacking MyD88
reveals that the T1DM development is not their when raised under specific-
pathogen-free (SPF) condition; however, NOD mice lacking MyD88 shows
increased development of T1DM under GF conditions (Wen et al. 2008). MyD88
deficiency changes the composition of gut microbiota and leads to T1DM by
regulating host immune response (Wen et al. 2008).
60 P. Pathak
4.7 Rheumatoid Arthritis
Rheumatoid arthritis (RA) is an autoimmune disorder which is caused by oblitera-

tion of bone and cartilage which leads to development of pain and swelling in and
around the joints of body. It is very frequent disease affecting 1% of total population
and is quite common among females.
RA correlates with the inflammatory responses caused by CD4+ Th1 and Th17
cells and any variation in these responses leads to advancement of RA (Xu et al.
2019). Study on collagen induction arthritis (CIA) mice model shows that gut
microbiota plays crucial role in impacting susceptibility to arthritis. The gut
microbiota of CIA-susceptible and CIA-resistant mice was altered (Liu et al.
2016). It is also reported that the GF mice having microbiota of CIA-susceptible
mice show increased initiation of RA than those having microbiota of CIA-resistant
mice (Liu et al. 2016).
4.8 Inflammatory Bowel Disease (IBD)
IBD is a gastrointestinal disorder in which structure of mucosa gets altered, compo-

sition of gut microbiota changes along with some systemic deformity (Mulder et al.
2014). Depending on the symptoms and intestinal localization it is mainly divided in
two forms, Crohn’s disease (CD) and ulcerative colitis (UC) (Mulder et al. 2014;
Wijmenga 2005). IBD is a progressive disease and its frequency increased world-
wide (Chow et al. 2009; Kaplan 2015; Wang et al. 2010). Recent research points out
the role of gut microbiota in development of IBD, Th17, and Treg cells harmony is
crucial for intestinal homeostasis. Study on segmented filamentous bacteria (SFB) in
mice reveals the inflation of Th1 and Th17 cytokines (Gaboriau-Routhiau et al.
2009; Ivanov et al. 2009; Lee and Mazmanian 2014).
Several studies suggested that IBD patients with dysbiosis show alteration in their
stool microbiome as well as loss of beneficial microorganisms compared to that of
healthy individuals (Moustafa et al. 2018). The cause and development of IBD is
linked with dysbiosis in various reports (Abu-Shanab and Quigley 2010; Casén et al.
2015; Huttenhower et al. 2014; Marchesi et al. 2007; Tamboli et al. 2004; Wright
et al. 2015; Zhang et al. 2007). In IBD patients, number of commensal bacteria such
as Firmicutes and Bacteroides are comparatively less in number; however, bacteria
of family Enterobacteriaceae are higher in number (Bien et al. 2013; Hedin et al.
2014; Li et al. 2015; Mondot et al. 2011; Nguyen 2011). Another study reports the
interaction between reduced gut diversity and disease onset in individuals with CD
(Gevers et al. 2014). The studies performed in CD and UC individuals reveal
decrease in Clostridium groups of bacteria and increase in Proteobacteria (Frank
et al. 2007; Macpherson et al. 2000; Sartor 2008), as well as compelling reduction of
commensal bacterial species belong to genera Bacteriodes, Lactobacillus, and
Eubacterium (Nemoto et al. 2012; Sha et al. 2013).
4.9 Celiac Disease (CD)
It is a chronic disorder of the digestive system. Gut microbiota plays pivotal role in
onset of this disease, any alteration in the composition of gut microbiota leads to the
CD (Festi et al. 2014). It is hypothesized that gut microbiota have pathogenic role in
CD development (Collado et al. 2007). Studies show that species like Streptococcus
mutans and Streptococcus anginosus were present in less number in CD patients
compared to that of healthy individuals (Lazar et al. 2018). Galactoside 2-alpha-L-
fucosyltransferase2 is an enzyme which is encoded by FUT2 gene; monitors the
expression of ABH blood group antigens in intestinal mucus as well as other
secretions. Study conducted in Fut2-deficient mice illustrates higher susceptibility
to Candida albicans colonization compared to that of control mice, this leads to
induction of CD (Lazar et al. 2018). Bifidobacterium spp. of bacteria are commensal
of gut and provide protection against pathogens, any changes in the microbiome due
to mutation in FUT2 gene lead to development of CD (Nagao-Kitamoto et al. 2016).
4.10 Systemic Lupus Erythematosus (SLE)
SLE as the name suggests is a systemic, chronic, and inflammatory autoimmune

disease of ambiguous mechanism mainly defines by inflammation at multiple site of
the body (Paglia et al. 2017). Manifestation of this disease is indicated by upsurge of
Bacteroides phyla and reduction in Firmicutes (Hevia et al. 2014). The composition
of microbiota is believed to be crucial, as any modification in microbiota is
associated with onset of SLE. There are several reports which explained the link
between dysbiosis and SLE development. Recent study shows that microbiomes of
SLE patients of northeastern China have higher number of Proteobacteria and lesser
number of Ruminococcaceae (Wei et al. 2019).
4.11 Cancer
The major factor which is pivotal for cancer development is chronic inflammation.
Inflammation accelerates the tumor development and hastens invasion and metasta-
sis. Inflammatory cytokines cause damage in the DNA, any changes in the methyla-
tion of DNA induce inflammation-associated cancers (Nagao-Kitamoto et al. 2016).
Development of cancer is not associated with change in single entity; however, it is
linked with dysbiosis of entire microbiome. During dysbiosis, there is alteration in
the bacterial populations with upsurge in tumor-inducing species and decrease in
commensal species (Lazar et al. 2018). During inflammation, there is increased
alteration of microbiota which assists bacterial translocation into the neoplastic
tissue and leads to expression of inflammatory cytokines which in turn causes
tumor growth (Grivennikov et al. 2012). The microbiota of the colon induces
colorectal cancer by triggering immune response of Th17 cells (Wu et al. 2009).
62 P. Pathak
The balance between microbiota and host defense mechanism is crucial for the
development of colorectal cancer.
4.12 Conclusion and Future Perspectives
The diversity and dynamics of human microbiome are intriguing and therefore there
is increased research in this direction in the past decade. Eubiosis of the microbiome
is crucial for the host functions. The role of the gut microbiota in the development of
the host immune system and autoimmune disorder is already evident from the
research. Host genetics and environmental cues are pivotal in shaping the gut
microbiota. Any modulation in gut microbiota increases the prospect of autoimmune
disease.
This chapter highlights the association between the gut microbiota with immune
system and disease development. The identification and characterization of crucial
microorganism and their mechanism of action will allow us to understand their
contribution in disease development and progression, as well as lead the way for the
development of novel strategies which can prevent disease development. Apart from
this, human microbiome can also be used to identify gut-associated disease as
change in gut microbiota is a hallmark in various gut-linked diseases. Considering
the fact that microbiome can change upon dietary changes this could be used to
customize diets which can reshape microbiota and its function in order to prevent
disease. This knowledge can be used in future for accurate and efficient treatment of
patients. Further recognition of unique symbiotic microorganism which prevents
aggregation of disease causing bacteria and boosts host immunity will pave a way in
development of medicine which can reverse the defects caused by dysbiosis.
Acknowledgments I gratefully acknowledge the University Grants Commission (UGC), Govern-

ment of India for awarding Junior and Senior Research Fellowship.
Conflict of Interest Author has no conflict of interest.
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Overview on Human Gut Microbiome
and its Role in Immunomodulation 5
Sudhakar Pola and Dhana Lakshmi Padi
Abstract
Microbes are first acquired from mother to child during pregnancy and they are
involved in programming fetal immunity. Proper maintenance of these microbes
in early infancy aids in adult life and, therefore, can be achieved by a healthy diet,
exercise, and clean environment as it plays an essential role in shaping gut
microbes. Microbes reside within different tissues and organs, and thus, they
form a mutualistic relationship with the human body. The effects of microbes
on the human body can be both beneficial and harmful. As definite changes in
the microbiome of some specific microorganisms and alteration in the
tumor-promoting and suppressing genes may lead to the development of cancer.
Contrarily, they are also involved in boosting the immune system to fight against
cancer. Therefore microbes are involved in modulating the immune responses
and this can be triggered by microbial products (polysaccharides and formyl
peptides), metabolites (short-chain fatty acids), immunotherapies (TLR agonists
and immune checkpoint inhibitors), and drugs.
Keywords
Microbiome · Mutualistic relationship · Infancy · Cancer · Immunotherapy ·
Immunomodulation
S. Pola (*) · D. L. Padi

Department of Biotechnology, Andhra University, Visakhapatnam, Andhra Pradesh, India

Ltd. 2021
https://doi.org/10.1007/978-981-16-3156-6_5
70 S. Pola and D. L. Padi
5.1 Human Gut Microbiome
There are trillions of microorganisms inhabited in the human body and they included
bacteria, viruses, archaea, protists, and fungi (Backhed et al. 2005). Commensal
microorganisms do not harm the human body even though they form a mutualistic
relationship. Therefore, they colonize and form composite ecosystems in the areas
such as our skin, mouth, and gut. In contrast to the human body, microorganisms
encode many numbers of genes (Bhatt et al. 2017) and thus, their function and
interaction with the host can be studied by many advanced genome sequencing
technologies.
The human gut microbiome (Shreiner et al. 2015) plays a vital role in an
individual’s well-being; therefore, they help the host in maintaining many functions
like homeostasis, metabolism, regulating nutrition and development and function of
the innate and acquired immune system (Thaiss et al. 2016). A favorable environ-
ment and a proper diet are required for the remarkable growth of microbes. The right
conditions for microbes help in the better development of good colonies; however,
an uncertain environment may lead to the death of good microbes and cause the
formation of harmful colonies.
Initially, human beings obtained microbes from their mothers during pregnancy;
the maternal microbes from different sites such as the gut, skin, breast milk, and
vagina help the infant in improving the immune system. In early infancy, if these
microbes are nurtured properly, it helps in shaping up gut microbes, which in turn
boosts the immune system. The alteration in the infant’s gut microbiota may cause
many disorders related to autoimmunity and inflammation (Sekirov et al. 2010;
Amon and Sanderson 2017).
The microbes present in the human gut play a crucial role, as they interact with all
the cells present in the human body. Therefore, gut mucosa has 60% of immune
cells; thus, the mucosal immune system aid in resisting the attacks of harmful
microbes and inflammatory reactions (Molloy et al. 2012), hence it acts as a defense
barrier of our body. Pattern recognition receptors (PPRs) such as toll-like receptors
(TLRs) help the innate immune system in recognizing the self and non-self-antigens
and in turn, these TLRs recognize the pathogen-associated molecular patterns
(PAMPs). These interactions activate the signaling cascade pathways, stimulate
the effector responses, and thus produce cytokines, apoptotic factors, AMPs, and
chemokines. Therefore, this mechanism is constructive in maintaining homeostasis
and disease pathogenesis.
The human diet is linked with metabolic health; thus, gut microbes act as
intermediator, which involves the conversion of metabolites. Gut microbiota helps
the host in digesting dietary products such as non-digestible polysaccharides, com-
plex proteins, aromatic amino acids, lipids, choline, and vitamins; hence the colonic
microbes are involved in the degradation and fermentation of food particles. The
main by-product produced after bacterial fermentation is short-chain fatty acids
(SCFAs). Therefore, the metabolites are involved in the strengthening of the host-
cell barrier against pathogens, maintaining controlled inflammation, and also
regulating the mucosal immune responses (Belkaid and Hand 2014).
5 Overview on Human Gut Microbiome and its Role in Immunomodulation 71
Sometimes, the toxic metabolites which are released into the human body may
cause the onset of cancer (Garrett 2015). As many microbes may relocate to other
parts of the body, there will be many chances of spreading these tumors (Rajagopala
et al. 2017). The complications in the metabolic pathways may induce many
diseases. For example, the difficulty in choline metabolism may lead to cardiovas-
cular heart disease (Wang et al. 2011).
Homeostasis in the intestine is maintained by the gut microbial commensals. The
homeostatic relationship between the host–microbe is generally disturbed due to the
changes in food habits and the environment. The action of some medicines and
antibiotics, exposure to pathogens, and also psychological disturbances may lead to
the overall changes in the structure and activity of the gut microbes, which is known
as dysbiosis (Shui et al. 2020). Some diseases like obesity, diabetes, liver problems,
cancer, and even neurodegenerative diseases lead to modifications in the gut
microbiota. The alterations in the gut microbes and their interaction with the
human body may result in some illnesses and cause cancers. The gut microbes
play a dual role in both tumor development and also in anti-cancer therapies (Cani
2018).
There are many numbers of research papers and articles were published on human
gut microbes, as many studies were carried out on the host–gut microbiota interac-
tion. Among these studies, the interactivity of human gut microbiota with the
immune system and the discovery of novel immune therapeutics is one of the
exciting topics in the last couple of years. Some immunotherapeutic treatments
were in use to combat cancers such as immune checkpoint inhibitors (ICIs), activa-
tion and maturation of T- cells and B-cells, adoptive cell therapy (ACT), and
vaccines. There are also some immunomodulatory drugs present that can elicit the
anti-cancer immunity to kill the cancer cells.
5.2 Gut Microbes During Pregnancy and Early Infancy
Gut microbes are initially acquired from mother to child during delivery through
breastfeeding, skin-to-skin contact, and delivery through the vaginal canal (Chu and
Englund 2014). Gut microbial commensals in the early infants help in defining and
anticipating the human’s health status.
During pregnancy, a woman undergoes many physical and mental changes like
gaining weight, hormonal, metabolic, and immune changes, and even mood swings.
These changes may alter the gut microbes present in a pregnant woman; this may
lead to some difficulties in the course of pregnancy. The poor maternal diet and
alterations in the maternal gut microbiota throughout pregnancy may affect the
infant’s gut microbiota (Schwartz et al. 2012) and adult life (Kumbhare et al. 2020).
Maternal microbes present in the gut, breast milk, and vagina are involved in the
development of fetal immunity before delivery. The gut microbes in the fetal
intestine undergo colonization; however, the diet, antibiotics, and mode of birth
during pregnancy impact the microbial colonization (La Rosa et al. 2014). The
infant’s gut microbial flora which is obtained from the mother helps in the
Fig. 5.1 Different mechanisms are showing the impact of maternal microbiota on a fetus during
pregnancy
progression of the mucosal immune system; if that gut microbial environment gets
disturbed, it may lead to mucosal infection and inflammation in childhood, and also
its adverse effects continue to adulthood (Welliver and Ogra 2008) (Fig. 5.1).
Maternal microbial metabolites such as short-chain fatty acids (SCFAs) (Koh
et al. 2016) help develop the fetal immune system. In the course of pregnancy,
maternal bacterial metabolites translocate from gut to mammary glands and there the
bacteria present in the breast milk are coated with immunoglobulins, IgG or IgA;
therefore, this helps the neonate to get the proper immune protection. Maternal
immunoglobulin G is also actively moved across the placenta and hence provides
passive immunity to the neonate.
The uterus has been considered sterile, but likely susceptible to be affected by
vaginal bacteria, therefore microbes are most probably seen at the endometrium.
Thus these endometrial microbes help in assessing the success/failure of implanta-
tion during pregnancy (Moreno et al. 2016). Infants delivered vaginally have
valuable maternal vaginal microbes such as Bifidobacterium and Lactobacillus,
although cesarean section delivered infants are composed of Staphylococcus spp.
(Dominguez-Bello et al. 2010) and also, they are more prone to type I diabetes by
20% (Charbonneau et al. 2016).
5.3 Shaping of Maternal Gut Microbes with Proper Diet during

Pregnancy
The intake of fiber, prebiotic, and probiotic foods in the diet (De Filippo et al. 2010)
during pregnancy helps in the increase of good microbes. The prenatal gut
microbiota is stable and can grow better when good dietary food is introduced
(Dawson et al. 2019). Not merely diet, but also external environmental factors
play an essential role in shaping up gut microbes. High consumption of a fiber-
rich diet assists the gut microbial commensals in maintaining the equilibrium in the
intestine (Shui et al. 2020). Diets including high amounts of fruits, vegetables, and
fibers are suitable for the enrichment of gut microbes, in contrast with a diet rich in
fats, sugars, and animal protein (Gali 2015). Improper diet may cause many meta-
bolic diseases like type 2 diabetes, thus focus on microbes that are linked to
metabolism should be increased to overcome metabolic disorders (Sonnenburg and
Backhed 2016).
Breastfeeding for the newly born child for at least four months to six months
helps in the proper maintenance of gut microbes, as it contains carbohydrates,
proteins, fats, nutrients, immunoglobulins, and endocannabinoids, thus it gives a
complete supplement to the infant. Nevertheless, the early introduction of solid
foods to the newly born child may lead to obesity (Koleva et al. 2015) and other
disorders (Differding et al. 2020). Therefore, this leads to a drastic change in the
microbiota composition in an infant’s gut.
However, various prenatal, neonatal, and postnatal factors affect the infant’s gut
microbes, which are obtained from the mother during pregnancy and also affect the
early infancy and childhood (Fig. 5.2). Therefore, the various factors are maternal
diet, vaginal infections, psychological effects, gestational period, host genetics,
mode of delivery (vaginal/cesarean), diet (milk consumption/solid food introduc-
tion), and environmental factors (Christian Milani et al. 2017).
5.4 Gut Microbes: Onset of Cancer
Despite commensals, some pathogenic microbes are linked to human carcinogene-

sis. Gene mutation and alteration in the oncogenes and tumor suppressor genes and
inflammation, which provides the tumor-promoting environment, may lead to the
beginning of cancer (Dzutsev et al. 2015). The modifications in the gut microbiota
and their interaction with the human body may cause many health implications,
including cancers like colon cancer, pancreatic cancer, and lung cancer (Zheng et al.
2020).
There are a high number of microorganisms located in the human colon and they
play a significant part in colorectal carcinoma (CRC). The patients suffering from
adenoma or adenocarcinoma have a relatively high number of some specific
microbes and thus they adhere to the colon cells, inhibit the tumor suppressor
genes, and activate the oncogenes that cause colon cancer (Abu-Ghazaleh et al.
2020). Some studies, like metagenomics and transcriptomics, are practical to know
Fig. 5.2 Different prenatal, neonatal, and postnatal factors that are affecting the composition of gut
microbes
the relation between Fusobacterium nucleatum and colon cancer (McCoy et al.
2013). The toxic metabolites produced during the degradation and fermentation of
dietary products may also cause cancer. For instance, the fermentation of proteins
releases toxic by-products like ammonia, amines, and branched-chain fatty acids,
which causes the onset of colon cancer to the host.
The gut microbiota may also play a crucial role in pancreatic ductal adenocarci-
noma (PDAC), human pancreatic diseases, pancreatitis (Akshintala et al. 2019), and
lung cancers. The geographical and environmental factors affect the lung
microbiome (Mao et al., 2018); therefore, the microbes present in the lungs are
involved in the onset of lung cancer and lung diseases. Chronic obstructive pulmo-
nary disease (COPD) is an inflammatory disease, which is persistent and causes
when the respiratory tract is colonized by pathogenic microbes (Budden et al. 2016),
whereas cystic fibrosis (CF) is caused by a gene mutation (Chmiel et al. 2014).
5.5 Immunomodulation
5.5.1 Immunomodulation by Microbial Components
The immune cells present in the gastrointestinal (GI) tract are continuously exposed
to microbial antigens and thus the epithelial cells in the mucosa transcribe the
requisite information to the immune cells which activates the host immune system.
The PPR-mediated mechanisms through microbial components are helpful in the
modulation of the immune system. The pathogen-associated molecular patterns

(PAMPs) such as lipopolysaccharides, peptidoglycans, lipoteichoic acid, flagellin,
and formyl peptides are recognized by pattern recognition receptors (PPRs) such as
toll-like receptors (TLRs) and nod-like receptors (NLRs) (Osamu and Shizuo 2010).
TLRs have a significant role in maintaining homeostasis in the intestine and
stimulating the inflammatory responses during pathogenesis (Valentini et al. 2014).
These interactions activate the antigen-presenting cells (APCs) and dendritic cells
(DCs) and then the priming of B- and T-cells occurs when they mediate with APCs
at mesenteric lymph nodes. The naive T-cells are differentiated into CD4+ T-cells
and gut-associated lymphoid tissue (GALT) is stimulated by commensal bacteria,
which induce B-cells to differentiate and also activate the production of IgA. The
activity of the immune system can also be altered by circulating cytokines via
soluble immunomodulatory factors (Khan et al. 2020).
The commensal microorganisms modulate epithelial immunity and thus it leads
to the maturation and function of the mucosal immune system. Mucosal immune
response arose from the three layers (epithelium, lamina propria, and muscularis
mucosae) of the mucosa (Fig. 5.3a), as each layer composed of mucus proteins,
mucins, immunoglobulins IgA, and lymphocytes (T- and B-cells and DCs), which
helps in triggering the mucosal immune system to fight against pathogens (Shui et al.
2020).
Microbial products like polysaccharides and formyl peptides influence the
PRR-mediated responses; this study helps to understand the host–microbe homeo-
stasis. Polysaccharides and formyl peptides influence the innate and adaptive
immune system (Fig. 5.3b). Therefore, they are useful immunomodulatory
PAMPs, which are effective in treating autoimmune diseases, inflammation, neuro-
degenerative diseases, and cancers (Michelle and Wendy 2016).
5.5.2 Immunomodulation by Metabolites
Human dietary intake includes carbohydrates, proteins, lipids, and vitamins, which
are degraded by gut microbes to produce metabolic products. Metabolism of dietary
products yields energy and also maintains gut health. The main by-product, which is
produced after microbial degradation, is SCFAs, which involve in modulating the
immune system (Fig. 5.4).
The immune cells which are present in the epithelial layer of the mucosal
interface when coming in contact with microbial metabolic products impact the
immune responses and disease risks (Michelle and Wendy 2016). In this process, the
involvement of host and microbes plays a critical role, as they permeate the meta-
bolic products to get access to the epithelial layer of mucosa, which has immune
cells.
Humans take carbohydrates as a diet; the majority of them include non-digestible
polysaccharides, oligosaccharides, unabsorbed sugars, and plant fibers. These are
resistant to the action of amylase; thus, colonic microbes play a vital role in
degrading these substrates by producing various hydrolytic enzymes. Initially,
Fig. 5.3 (a) and 3(b)

Different layers of the
gastrointestinal tract (GI) and
the modulation of the host
immune system by microbial
products
complex carbohydrates are degraded to polysaccharides and then polysaccharides to

oligosaccharides; thus, these oligosaccharides undergo fermentation and produce
by-products such as gases (like hydrogen, carbon dioxide, methane) and intraluminal
solute (such as short-chain fatty acids (SCFAs).
SCFAs (such as acetate, butyrate, and propionate) act as immunopotentiators that
enhance the production of antibodies. These metabolic products communicate with
various immune cells present in the epithelial layer and they activate the immune
system through many immune responses such as plasma B-cell proliferation, Treg
development, macrophages, and dendritic cells (DCs) activity and the release of
cytokines which involves reducing inflammation (Ziying Zhang et al. 2019). Buty-
rate strengthens the barrier between the blood–brain by connecting the cells, i.e., the
5
Overview on Human Gut Microbiome and its Role in Immunomodulation
Fig. 5.4 Gut microbes are associated with the degradation of the dietary products such as carbohydrates, proteins, lipids, and vitamins by anaerobic degradation
77
and fermentation; therefore, they maintain gut health by triggering the host immune system
gut–brain axis (Smith 2015). Lactate, another metabolite commonly found in the
milk diet possesses many metabolic and immune properties. Lactic acid secreted by
tumor cells helps in the development of pro-tumor immunity (Yan et al. 2018).
Lactate creates a pro-tumor microenvironment, which helps in the increasing effi-
cacy of tumor therapy (Feichtinger and Lang 2019).
Firstly, complex proteins are breakdown by microbes into short peptides and
amino acids by various peptidases and proteases. After this, short peptides and
amino acids undergo fermentation and produce by-products such as hydrogen,
carbon dioxide, organic acids, branched-chain fatty acids (such as 2-methyl butyrate
and isobutyrate), and trace amounts of amines, ammonia, and phenols. Aromatic
amino acid metabolism is also carried by intestinal microbiota, as they produce
various types of metabolites like phenols and indoles by transamination, decarbox-
ylation, dehydrogenation, and deamination. These indoles help in strengthening the
host-cell barrier, increasing the resistance to pathogens, and maintaining inflamma-
tory responses (Monika et al. 2017).
Lipids are synthesized in the liver into primary bile acids; furthermore, these
primary bile acids are degraded into secondary bile acids by gut microbes. Thus,
secondary bile acids are involved in the activation of many receptors present in the
host and it also influences immunomodulation (Ridlon et al. 2014). Moreover, a
wide variety of vitamins such as vitamin-B, vitamin-K, riboflavin, pyridoxine,
nicotinic acid, pantothenic acid, cobalamin, and folate are synthesized by gut
microbes. Therefore, these are helpful in the maintenance of host physiology.
5.6 Tumor Immunotherapy
Tumor immunotherapy is an effective way to treat cancer as they directly target the
immune system. Tumor microenvironment (TME) and chemotherapeutic drugs,
which are metabolized by the gut microbiota, are helpful in the improvement of
immunotherapies (Fig. 5.5). However, some immunosuppressive and anti-
inflammatory factors such as arginase, TGF-B (Transforming growth factor-beta),
and IL-10 (interleukin 10) are produced by macrophages, other myeloid and Treg
cells are involved in preventing anti-cancer immune response (Dzutsev et al. 2015).
Many immunomodulatory drugs enhance anti-cancer immunity, namely, metfor-
min, angiotensin receptor blockers (ARBs), anthracyclines, thalidomides, and statin
(Maiko and Kawaguchi 2018). They modulate the anti-cancer immunity by
strengthening the immune system to kill the cancer cells. Some chemotherapeutic
drugs like cyclophosphamide (Viaud et al. 2013; Shui et al. 2020) and gemcitabine
(Zhang et al. 2020; Banerjee et al. 2018) are also used for immunomodulation.
Some tumor immune-targeted treatments are useful to treat cancers, such as toll-
like receptor (TLR) agonists, immune checkpoint inhibitors (ICI), adoptive T-cell
therapy (ACT), and vaccines. The synthetic toll-like receptor (TLR) agonists are
used as a vaccine for cancers. TLRs play a critical role in connecting adaptive and
innate immunity. PPRs such as TLRs when recognizes pathogens via dendritic cells
Fig. 5.5 Different types of tumor immune-targeted treatments to treat cancer by directly targeting
the immune system
(DCs) involve boosting the T-cells and B-cells immune responses (Lazar et al.
2018).
PD-1 and its ligands PD-L1 and PD-L2 (Lee et al. 2016) are immune checkpoint
inhibitors, which play a crucial role in T-cell exhaustion (Schildberg et al. 2016).
These are usually expressed on antigen-presenting cells (APCs), which convert
naive CD+ T-cells to regulatory T-cells.
Adoptive cell therapy (Cohen et al. 2017) includes the modification of cancer
cells in vitro to make it more efficient for treating cancers by increasing their immune
response. TCR-engineered T-cells or CAR-engineered T-cells are used to elevate the
anti-tumor response.
Vaccines such as DNA vaccines, peptide vaccines, heterologous whole-cell
vaccines, and immunotherapeutic drugs are used to treat some cancers (Thomas
and Prendergast 2016). Vaccines are generally composed of antigens and adjuvants;
they activate antigen-presenting cells, which in turn elevate the immune responses
(Banchereau and Palucka 2018).
5.7 Conclusion
Gut microbes and their association with the immune system is one of the primary
subjects to be focused. Gut microbes obtained from mother to child in the course of
pregnancy play a critical role in an individual’s entire life. Thus, shaping these
microbes in early infancy is essential in combating many health issues and also
cancers. Proper diet and environmental factors are essential in keeping gut microbes
healthy. Focus on dietary intake should be increased as health is linked with the diet
and this is the root cause for many metabolic diseases. Microbial dietary products,
metabolites, and immunotherapies are involved in the modulation of the immune
system and thus fight against cancers. Therefore, immunotherapy is an exciting and
significant theme that is very useful for future studies and it has considerable scope
for new researches.
Acknowledgments The authors Sudhakar Pola and Dhana Lakshmi Padi are grateful to the
authorities of Andhra University Visakhapatnam, India for providing the necessary facilities to
carry out the research work and for extending constant support.
Conflict of Interest The authors declare that there is no potential conflict of interest.
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Cancer Microbiome and Immunotherapy:
Understanding the Complex Responses 6
Between Microbes, Immunity, and Cancer
Kishore Kumar Godisela, Badithala Siva Sai Kiran, and

Abstract
The microbiome is the inherited substance of the numerous microscopic

organisms that exist on and within the human body, including bacteria, protozoa,
fungi, and viruses. The human gut microbiota is dominatingly made out of four
groups of microbial phyla: Firmicutes, Bacteroides, Actinobacteria, and
Proteobacteria; this entire microbiome assumes a significant job to the improve-
ment of immunity. However, disruption of this homeostatic host–microorganism
relationship can promote disease pathogenesis, such as in autoimmune diseases
and cancer. Current investigations additionally show with the intention of the gut
microbiome might influence the reaction to cancer treatment, by balancing the
host cell inflammatory reaction. As the investigation of the microbiome is
growing, various endeavors are being prepared to incline the range on the
“ideal” microbiome region. In this regard, this chapter extensively discusses
various types of cancers and the role of the microbiome in their treatment.
Keywords
Microbiome · Cancer · Microbial metabolites · Gastric malignancy · Fecal
microbial transplantation
K. K. Godisela (*)
Assistant Professor of Biochemistry, SRR Govt. Arts & Science College, Karimnagar, Telangana,
India
B. S. S. Kiran
College of Pharmaceutical Sciences and Research, Krishna University, Machilipatnam, Andhra
Pradesh, India
P. V. Bramhachari
Department of Biotechnology, Krishna University, Machilipatnam, Andhra Pradesh, India

Ltd. 2021
https://doi.org/10.1007/978-981-16-3156-6_6
84 K. K. Godisela et al.
6.1 Introduction
Humans are by most part microorganisms, more than a hundred trillion of them.
Microscopic organisms predominate our human cells ten to one. The lion’s share
lives in our gut, particularly in the stomach-related organ. The microbiome is the
inherited substance of the significant number of microscopic organisms—bacteria,
protozoa, fungi, and viruses—that exist on and within the human body. The number
of genes in all the microbes in one person’s microbiome is two hundred times the
number of genes in the person genome. The commensal microbiota in the host
coexist in a symbiotic interaction, with every beneficial advantage and the wellness
of the host could be considered as a superorganism. Much late research has centered
on the bacterial part of the microbiota. By and large, a strong human body is
contained roughly thirty trillion cells and is possessed by around thirty-nine trillion
microscopic organisms (Sender et al. 2016). Microbial cells in the microbiome help
us absorb nutrients, guide our immune system, protect us from other pathogenic
microbes that cause infection, and manufacture supplements such as hormones,
essential vitamins (riboflavin, thiamine, and B12), and other bioactive compounds
(Vitamin K, which is required for blood clotting) that the host cannot obtain.
(McFall-Ngai and Ruby 1991; Hooper et al. 2001; Lupp et al. 2012; Lepage et al.
2013). However, disturbance of this homeostatic host–microorganism relationship
can promote disease pathogenesis, such as various autoimmune diseases (Frank et al.
2007; Paulos et al. 2007; Jenq et al. 2012).
6.2 Microbiome for Well-Being
The microbiome is fundamental for a person’s turn of events, immunity, and

nourishment. The microscopic organisms existing in and on us are not intruders
except rather gainful settlers. Autoimmune disorders like hypoglycemia, rheumatoid
arthritis, muscular dystrophy, multiple sclerosis, and fibromyalgia are related to
brokenness in the microbiome. Infection-causing microorganisms build up over
time, modifying gene activity and metabolic operations and ensuing in anomalous
immune reactions contrary substances and tissues typically exiting in the body.
Autoimmune disorders radiate an impression of being transferred in families not by
DNA inheritance yet by gaining the family’s microbiome.
6.3 The Microbiome and Immunity
The human gut microbiota is dominatingly made out of four groups of microbial
phyla: Firmicutes, Bacteroides, Actinobacteria, and Proteobacteria; this entire
microbiome assumes a significant job in the improvement of immunity
(Dominguez-Bello et al. 2019). The microbiome is defined as the collective genomes
of microorganisms within a network, whereas the term microbiota refers to the
organisms as a whole. Inside a person, nearby trillion of microorganisms as various
6 Cancer Microbiome and Immunotherapy: Understanding the Complex Responses. . . 85
as human cells–which associate among the host continually at various locales

(counting the epidermis and mucosal coverings, for example, the gastrointestinal
tract) all through the turn of events (Fig. 6.1). In this manner, it is not amazing that
they expect such a huge job in various host capacities including resistance (Morgan
and Huttenhower 2012).
The crosstalk among microbiota and the immune framework at the level of the gut
is basic and not just takes into consideration the resistance of commensal micro-
scopic organisms and oral food antigens, yet furthermore enables the immune
framework to perceive and assault shrewd microorganisms in this way forestalling
bacterial intrusion and disease. Notwithstanding impacting restricted immune
reactions, this microbiota likewise has more extensive impacts adding to intrinsic
and adaptable immunity at different levels. This idea is upheld in preclinical models;
germ-free mouse that needs intestinal microbiota is renowned to contain extreme
imperfections in immunity, with a missing mucous coat, modified immunoglobulin
A discharge, and diminished dimension and usefulness of Peyer’s patches and
depleting mesenteric lymph hubs (Johansson et al. 2015; Spiljar et al. 2017).
Considering this, retarded immunomodulatory effects are surrendered by transfer-
ring the gut microbiome of wild-type mice to laboratory mice, and considerably after
a few generations of reproduction, the effects stay. In this manner, it is favorable for
combating viral diseases merely as mutagenic and inflammation-induced
tumorigenesis (Rosshart et al. 2017).
The arrangement of the gut, which holds a mucosa involving a single epithelial
layer comprised of intestinal epithelial cells (IEC) and intraepithelial lymphocytes,
energizes this coordinated effort with the immune framework. The IECs have Paneth
cells that discharge anti-bacterial polypeptides and goblet cells that emit mucus,
which thus overlie the epithelial layer. Underneath the mucosal sheet conceals the
lamina propria, a connexion tissue film enclosing Peyer’s patches, and a large group
of additional immune cells comprising antigen-presenting cells and native lymphoid
cells, just as CD4+ and CD8+ B plus T cells. This gut-related lymphoid tissue speaks
to the biggest segment of the immune organization inside the body and impacts
immune reactions jointly local and systemically (Fig. 6.2).
Native immunity is fostered via appreciation of pathogen-linked molecular
patterns (PAMPs) (namely, lipopolysaccharide as well as flagellin) by pattern
recognition receptors (such as toll-like receptors existing on IECs as well as native
immune performers inside the gut). Metabolites delivered by microorganisms per-
haps likewise influence native immunity via the manufacture of short-chain fatty
acids (SCFAs), which, within various main exercises, being appeared to expand
immunity through IgA making by plasma cells (Pabst 2012). IgA works by effec-
tively affecting bacterial virulence by freezing microbial attachment to epithelial
cells; adhesion, trapping, and freezing microbial attachment to epithelial cells (Pabst
2012).
The role of the commensal microbiota in tweaking physiology becomes espe-
cially clear when routinely raised explicit sans pathogen (SPF) mice are compared to
sans germ (GF, axenic) mice. The gut microbiota is personally associated with the
turn of events and guidelines of the safe framework, particularly as for nearby
86
Fig. 6.1 The microbiome and immunity

K. K. Godisela et al.
Fig. 6.2 The immune

system, microbiota, and
tumors are tightly associated
mucosal insusceptibility. Mainly focuses on GF mouse models as shown below:

(1) littler mesenteric lymph hubs (MLH) and unusual higher endothelial arteries
through poor lymphocyte restricting (Smith et al. 2007); (2) less and littler Peyer’s
patches which need germinal focuses (Lecuyer et al. 2014); and (3) absence of
lymphoid follicles inside the intestinal lamina propria (LP), however nearness of
early crypto patches which can form into useful secluded lymphoid follicles atop
microbial habitation(Bouskra et al. 2008). Systemic native immune amelioration is
additionally impacted by the symbiotic microbiota, with different paths of proof
demonstrating stimulatory consequences for myelopoiesis at the degree of
granulocyte-macrophage progenitors inside the bone marrow including in the
periphery, just as on the capacity of dendritic cells, macrophages, and neutrophils
(Gorjifard and Goldszmid 2016).
This chapter focuses on:
1. The connection between infection and malignant growth.

2. The inclination of malignancy patients to obtain infection disease(s);
3. The activity of the microbiota in malignant growth vulnerability;
4. Nitty Gritty of the microbiome in malignant growth treatment.
6.4 Infection and Malignant Growth
Human microbiome interruption is related to different kinds of cancer and for each
report of the International Agency for Cancer Research (IARC), just 10 species were
recognized as cancer-causing specialists to humans (de Martel et al. 2012). The
intensity just as marks of immune reactions might be directed by the cross-reactivity
among tumor and microbial antigens or by the incitement of example pattern
recognition receptors (PRRs) via microbe-related antigens, Moreover,
microorganisms have been appeared to impact carcinogenesis for various malignant

growth types, influencing different host factors, directing disease trademarks,
adjusting inflammation, affecting the genomic stability of host cells, and creating
metabolites that can epigenetically control host gene expression (Bultman 2014)
(Fig. 6.2). A full conversation of the job of the microbiota is as a matter of fact past
the extent of this section
As per ongoing publications, oncoviruses are responsible for almost 12% of
human malignancies and are significant factors in the actuation of oncogenesis.
These are Epstein–Barr Virus, Human Papillomavirus, Hepatitis Virus, and HIV
(Luo and Ou 2015). Microorganisms contamination of H. pylori infect over half of
the world populace, rendering it the most regular disease on the planet (Camilo
2017). It is the most focal factor of gastric disease (GC) chance; because of its
qualities, H. pylori can adjust to the outrageous acidic states of the stomach, to set up
contamination and upset mucosal homeostasis of the host, bringing about gastric
pathogenesis and at last disease. A few publications connect the nearness of fungal
diseases with a higher danger of creating malignancy. A few strains of filamentous
organisms, including Aspergillus flavus, Aspergillus parasiticus, and Aspergillus
nomius, produce aflatoxins ready to incite liver malignancy and some can advance
malignancy movement, such is the situation of expedient Candida albicans
contaminations (Martins et al. 2008; Ramirez-Garcia et al. 2011).
6.5 The Inclination of Malignancy Patients to Obtain Infection

Disease(s)
Microorganisms replicate quickly and stay alive in thickly involved biological

niches. Supplement accessibility inside these small-scale natural surroundings is
restricted; in this way, organisms go after assets, for example, amino acids sugars
(Curtis et al. 2014), zinc (Gielda and DiRita 2012), iron (Deriu et al. 2013), and
oxygen (Litvak et al. 2019) and anaerobic electron acceptors (Herp et al. 2019). A
higher microbial decent variety suggests more organisms using an increasingly
flexible pool of metabolites, representing a test for any bacterium to flourish. Any
perturbation bringing about lost microbial burden or assorted variety destabilizes the
microbial biological system, making an open door for strains with expanded well-
ness to multiply. Furthermore, it predisposes the host to diseases caused by either
indigenous or exogenous pathogens. It makes the host more susceptible to infection
from either indigenous or exogenous pathogens.
The elements of the immune system are sorted out to keep up parity amid the
microbiota and to battle against the majority of the microbial attacks. Interestingly,
in immune-compromised patients actuated by immunosuppressive medications,
including malignant growth chemotherapy, these capacities are hindered and people
are at an expanded danger of infection. However, these disease treatments change
the associations flanked by the host and microbiota.
6.6 The Activity of the Microbiota in Malignant Growth

Vulnerability
Microorganisms take an interest in host metabolic actions, and their metabolites can
instigate inflammatory processes, meddling to be decided of tissue cell multiplica-
tion and demise. The emerging of malignant growth in certain areas could be
actuated by microbial dissimilarities, for example, the more prominent powerless-
ness to disease in the large intestine because of the elevated microorganism thickness
when contrasted with the tiny intestine (Burgess et al. 2014). Subsequently, the
microbiota of all organs of the body is extraordinary, and their impact on irritation
and carcinogenesis is likewise unmistakable in every organ. There is a developing
admiration about the effect of microbiota at various anatomic locales on resistance
and for various pathologic situations, including malignancy. This has been incredi-
bly encouraged by the utilization of next-generation sequencing, which has extended
our comprehension of the expansiveness and capacity of the microbiota ahead of
conventional culturing techniques.
Dysbiosis because of malignancy prompted immunodeficiency, chemotherapy
action regimens, or antibiotic employ, could likewise elevate the threat of blood-
stream and Clostridium difficile contaminations, by upsetting the gut microbiome’s
capacity to oppose pathogen immigration or by debilitating the intestinal barricade.
Tai and colleagues depicted that the recurrence pace of Clostridium difficile diseases
in hospitalized oncologic kids was more than multiple times higher contrasted with
those without malignancy (Tai et al. 2011). Wang and the team likewise discovered
diminished diversity plus a bounty of the oral microbiome in patients by intense
lymphoblastic leukemia (Wang et al. 2014). Another examination exhibited those
pediatric oncology patients by intense lymphoblastic leukemia, beneath a few circles
of treatment, demonstrated diminished microbial variety (Rajagopala et al. 2016).
The metabolic action of bacteria might actuate, impair or increase healing toxic-
ity. An examination starting 1993 announced the limit of the microbiome to meddle
through medicines in a gathering of Japanese who developed Herpes zoster while
experiencing malignancy. Herpes zoster drug is changed by the typical stomach-
related microbiota into a section that made the drug to treat malignant mortally
poisonous. In this line of proof, Karin and collaborators expressed that a flawless
microbiome is required for the fruitful control of tumor movement (Karin et al.
2014). Microbiota utilizes pivotal systems, for example, inflammation, metabolism,
and genotoxicity to adjust cancer origination (Schwabe and Jobin 2013). Recent
research also suggests that the gut microbiome’s intention may influence the
response to cancer treatment by balancing the inflammatory response of the host cell.
6.6.1 Pancreatic Cancer
Pancreatic cancer stays one of the main sources of cancer-related demise worldwide
and has a poor forecast (Zambirinis et al. 2014). It is, indeed, a perceived inflamma-
tion determined malignancy, with enormous preclinical and clinical proof
demonstrating that microscopic organisms likely impact this procedure, since they
enact immune receptors, sustaining malignancy-related inflammation. In light of past
research in the preceding decade, the gut microbiota could prompt modified ade-
quacy of pharmacotherapeutics in malignant growth treatment (Thomas 2017).
Although the express job of the microbiota in host immunity, particularly in the
tumor-explicit tumor microenvironment (TME), stays muddled, the collaborations
between tumor control and gut microbiota have gotten more interlaced than any
other time in recent memory (Alexander et al. 2017; Yi et al. 2018). Microbial
diversity changes, including bacterial species, for example, Actinomycetes,
Fusobacterium, Bacteroides, Neisseria, Streptococcus, Porphyromonas, and
Bifidobacteria have been related with pancreatic malignancy (Mitsuhashi et al.
2015; Ren et al. 2017). These microorganisms inside tumors may animate host
immune reactions and create useful or problematic effects on anticancer treatment,
as controlled by pharmacological systems, just as the significant reaction pathways
(Cogdill et al. 2018).
6.6.2 Breast Cancer
As previously stated, danger may be linked to the unique microbial environment

found in the tissue of the birthplace. An ongoing report announced that the microbes
found in breast tissue differ with and without breast malignant growth. The
researchers distinguished more significant levels of Comamonadaceae,
Enterobacteriaceae, Bacillus, Staphylococcus, and Bacteroidetes in breast malignant
sample plus, interestingly, healthy tissue uncovered more elevated levels of
Prevotella, Lactococcus, Streptococcus, Micrococcus, and Corynebacterium spe-
cies. Higher relative bounties of microorganisms that could cause DNA to injure
in vitro were recognized in breast malignancy patients, just like a reduction in some
lactic acid microscopic organisms, known for their advantageous well-being
impacts, including anticarcinogenic properties. This examination brings up signifi-
cant issues regarding the job of the mammary microbiome in balancing the danger of
breast cancer development (Urbaniak et al. 2016).
This reality raises a significant issue: would women be able to modulate their
breast microbiome to forestall elevated levels of Escherichia coli or Staphylococcus
aureus colonization? A few examinations uncovered that drinking fermented food,
for example, kefir, is related to a lower danger of breast malignancy. Different
examinations utilizing living models indicated to orally ingested Lactobacillus
may have a defensive job in opposition to breast malignancy improvement
(Urbaniak et al. 2016). Considering this, forthcoming investigations are expected
to exhibit the job of probiotics as a protective measure in opposition to breast
malignancy. It was uncovered that a few antibiotics agents, for example, metronida-
zole, clarithromycin, and ciprofloxacin prompt a decrease within the biodiversity/
plenitude of a few bacterial networks upsetting the balance of the gut microbiome,
which comprises a higher danger of breast malignant growth (Jakobsson et al. 2010).
Another investigation created by Goedert and collaborators distinguished a
connection among fecal microbiome lesser assorted variety in postmenopausal

female and breast cancer. This examination demonstrated that 87% of the females
had estrogen receptor-positive tumors when contrasted with healthy manage females
(Goedert et al. 2015).
6.6.3 3.3 Colorectal Cancer
Colorectal cancer (CRC), along with breast, prostate, and lung cancer, is one of the
most well-known cancers. It likewise shows up more much of the time in males than
females and, in the two sexes joined, shows the fourth most noteworthy death rate,
after lung, breast, and prostate cancer (Beaulieu 2020). The human gastrointestinal
microbiome assumes a significant function in managing immune standing and, in
this way, is a further possible prescient biomarker used for CRC immunotherapy
(Xu et al. 2020). Preclinical investigations have discovered that the degrees of
explicit microbes are significantly elevated in tumors contrasted with those in
close by usual tissue (Sivan et al. 2015). Bolster the speculation that a wellspring
of between topic heterogeneity concerning programmed cell death protein1 (PD-L1)
restorative efficiency might be the symphony of gastrointestinal microorganisms
(Vetizou et al. 2015).
Additionally Vetizou et al. established that the efficacy of the cytotoxic T-
lymphocyte-associated Ag 4 (CTLA-4) obstruction is impacted by the synthesis of
Bacteroides fragilis and additionally Bacteroides thetaiotaomicron and
Burkholderiales microbiota. To utilize the gastrointestinal microorganisms as a
prescient biomarker in CRC, a superior comprehension of the useful function of
microbiota is required, just like a progression of clinical examinations making an
interpretation of preclinical outcomes to endorsed biomarkers (Vetizou et al. 2015).
6.6.4 Gastric Malignancy
Gastric malignancy is a major worldwide health issue, with more than one million
new cases each year (Bray et al. 2018). Gastric chronic illness with Helicobacter
pylori incites diminished acid discharge, which might animate the expansion of
diverse microscopic organisms in the gastric mucosa. This change within the gastric
microorganisms could prompt harm to the gastric mucosa and put into cancer. These
outcomes are as per a preceding publication, indicating lesser bacterial variety in
patients among gastric malignant contrasted and patients with non-atrophic gastritis
(Aviles-Jimenez et al. 2014).
6.7 Nitty Gritty of the Microbiome in Malignant Growth Treatment
As of late reports exhibited in translational investigations that microscopic

organisms inside the gut of malignancy patients can regulate retaliation to anticancer
immunotherapy. In particular, by looking at the gut microorganisms of melanoma
patients, huge contrasts were seen in the variety and make-up of patient gut
microbiomes in responders to non-responders (Routy et al. 2018). There is develop-
ing proof for the influence of the gut microorganisms on the reactions to different
types of malignant treatments; there are additionally a few components throughout
the gut microscopic that may impact such responses. Nevertheless, it is turning out to
be progressively certain to anticipate that there is a multifaceted bidirectional
connection between the gut microscopic organisms and antimalignancy treatments;
as well as, yet not restricted to, chemotherapy, physical radiation, molecularly
focused on treatment, checkpoint inhibitors, cytokine-based treatment, receptive
cell treatment (RCT), and operators that target inborn immunity (Table 6.1). A
Table 6.1 Multidimensional relationships between the gut microbiota, the tumor microenviron-
ment, and systemic immunity
Treatment
type Tumor Gut Systemic response
Chemotherapy Antitumor apoptosis Gut dysbiosis brought Bacterial translocation
impact by Th1 and about by changed to lymphoid organs;
Th17 effector cells proportion of gut increment of Th1 and
microbiota Th17 subset expanded
Checkpoint Develop dendritic cells Extension in CD11b Enlistment of CD4+
and agonists initiate CD4+ and DC actuation instigates Th1 and CD8+ T cells
CD8+ T cells, IL-12 ward Th1
diminished intratumoral reaction in the lamina
FOXP3 Tregs, high propria, decrease in
intrinsic effector cells incidentally
determined colonic
Tregs
Cytokine Modifies resistant Treatment causes Systemic inflammatory
therapy homeostasis in the expanded intestinal reaction prompts
tumor builds TNFα, harmfulness and gut proinflammatory
apoptosis, neutrophil shortening. Sodium cytokine reaction and an
invade. Th1 and IL-17 take-up and increments expansion in a
increment in LPS cause a supplement course
pathogenic enactment
inflammatory reaction
Adoptive cell Multiplication of Lymphodepletion Increment in LPS serum
therapy completely actuated T triggers intestinal level, cytokine sink,
cells intercede tumor injury prompting harm exhaustion of
slaughtering and an to gut epithelium and administrative T cells,
expansion in cytokines bacterial translocation MDSCs, NK cell, and B
IL-2,7, and 15 to mesenteric lymph administrative cells
hubs
concise portrayal of the cooperation of organisms with every one of these helpful
systems is incorporated later.
6.7.1 Chemotherapy
Chemotherapeutic regimens encompass to upset the intestinal boundary and advance

antitumor adequacy in mice as well as humans. Firstly in mice and humans by way
of sophisticated malignancies, secondly as in transplantable mice for CRC, cure with
cyclophosphamide exhausted circulating T regulatory cells and supported Th1 and
Th17 memory reactions (Ghiringhelli et al. 2007). Additionally, these progressions
were related to the rebuilding of natural killer cell and T cell work plus the
translocation of certain segments of the gut microorganisms to auxiliary lymphoid
organs (Viaud et al. 2013). Furthermore, chemotherapeutic factors have been
demonstrated, for example platinum-related oxaliplatin and cisplatin medicines,
which can lead to explicit microorganisms (L.johnsonii and E. hirae) being
translocalized into 20 lymphoid organs beginning with the intestinal lumen
(Alexander et al. 2017).
This understanding showed that ideal chemotherapeutic adequacy was to some
extent the consequence of undamaged microbes, which helped to make myeloid-
derived usefulness in the tumor microenvironment (TME) (Iida et al. 2013). Taken
jointly, these examinations bolster the idea that chemotherapeutics could differen-
tially impact gut microbes in a huge amount of settings.
6.7.2 Checkpoint Inhibitors
Different examinations have demonstrated the powerful capacity of the microbes to

control the antitumor reaction of the immune response framework with regard to
checkpoint blockade (Bullman et al. 2017; Geva-Zatorsky et al. 2017; Zitvogel et al.
2017). Furthermore, the ability of gut microorganisms to potentiate the gut’s reaction
to the fringe is regulated to some extent, with specific bacterial populations seen as
distinct from testee and non-testee patient cohorts. The immune reaction related with
checkpoint restraint employing hostile to programmed cell death protein 1 (PD-1)
has been additionally contemplated, whereby an improved cluster of differentiation
8+ T cell antitumor role has been appeared in malignancy, corresponding with the
nearness of a “positive” microbial signature, include Bacteroidales and
Burkholderiales (Vetizou et al. 2015). Moreover, key bits of knowledge into the
basic and particular components engaged with against cytotoxic T-lymphocyte-
associated protein 4 and hostile to PD-1 motivating interspecies populace dynamics
may provide further understanding into the general instruments driving microscopic
organisms interceded ameliorate of anticancer immunity. Take note that a recent
study found that administering live microbial cultures to testees during immune
checkpoint blockade can result in a stronger anticancer response than untreated
testees, though these perceptions could be due to a combination of factors including

diet, lifestyle, and well-being (Backhed et al. 2007).
6.7.3 Microbial Metabolites
Short-chain fatty acids (SCFA) are the microbial metabolites that secure intestinal
mucosal obstruction and epithelial cells in opposition to death and harm, diminishing
tissue penetrability and inflammation (Fukuda et al. 2011; Mathewson et al. 2016).
Besides, SCFAs assume a crucial job in producing and controlling Treg
lymphocytes restricting to inhabitant and systemic inflammation (a sign of malignant
growth) (Arpaia et al. 2013). This may theoretically clarify the great impacts of
abundant SCFA-producing microorganisms Blautia on account of hepatocarcinoma.
Recent reports recommend that microbial metabolites intercede messages among the
commensal microorganisms and the immune response, influencing the harmony
among pro- and anti-inflammatory procedures.
Besides, a high-fiber diet, normal of veggie lover propensities, brings about
increasing the SCFAs pool (Tomasello et al. 2016). The SCFAs are a subset of
saturated fats, chiefly represented by butyrates, propionates, and acetic acid
derivations, and they are the aftereffect of a multifaceted fermentation procedure
of digestible to non-digestible polysaccharides. They are for the most part created in
the proximal colon and need the presence of explicit microscopic organisms includ-
ing especially Firmicutes and Bacteroides phylum (Tan et al. 2014). There is copious
proof that an elevated level of SCFAs encourages healthy microorganisms and has
different advantages, for example, mitigating and anti-tumorigenic impacts, just as
antimicrobial activities.
6.7.4 Cytokine Therapy
Cytokine-based treatments (e.g., interleukin-2, Interferon-gamma) may likewise be

affected by the microorganisms; particularly as gut bacterial dysbiosis is connected
to distort immune activation which couples with irregular manufacture of provoca-
tive cytokines. Research connecting the individual gut microbiome to provocative
cytokine making have uncovered the impact of bacterial commensals controlling
fundamental immune action with means host–bacterial collaborations, for example,
interferon α and interferon γ that partner with explicit bacterial metabolic pathways,
for example, metabolisms of palmitoleic acid and tryptophan to tryptophol transla-
tion. Palmitoleic acid hampers the proinflammatory cytokine reaction of the host cell
(Schirmer et al. 2016; Ter Horst et al. 2016). As an important element of the Human
Functional Genomics Project (HFGP), they described the environmental, hereditary
and bacterial factors associated with cytokine production in a cohort of five hundred
healthy participants by various microbial stimuli.
6.7.5 Adoptive Cell Treatment
Adoptive cell treatments (ACT) are an additional hopeful way to deal with handling
progressed oncological ailments. As of late research work, a rodent model of cervical
malignancy, announced to adoptive T cell treatment viability was essentially
affected by the make-up of the gut microorganisms when the mouse was medicated
with versus without antibiotic agents or heterologous fecal transfer. The transloca-
tion of gut microbes to the i.p. space, because of epithelial layer damage, can incite
momentary contamination with the systemic rise of interleukins (IL-12) that
influences the viability of adaptive cell treatment (Uribe-Herranz et al. 2018).
Altogether, these findings feature the indispensable pretended by the gut
microorganisms in regulating foundational plus anticancer adequacy of adoptive
cell treatment.
6.8 Concluding Remarks
Gut microbiota, the “overlooked organ,” influences different human physiological

procedures prevalently resistance and it is still ineffectively investigated until this
time. As the investigation of the microbiome is growing, various endeavors are being
prepared to incline the range on the “ideal” microbiome region. Focusing on the gut
and intracanceral microbiome might give already unexplored intend to develop a
systemic treatment for various kinds of tumors. These possibly will go from
straightforward nutritional changes to novel adoptive cell-based treatments. Fecal
microbiota transplantation (FMT) is a clinical methodology that restores solid
microorganisms in the colon by introducing stool by colonoscopy or bowel purge
from a healthy person contributor. The relative simplicity with which the microbiota
can be concentrated makes it particularly interesting, given that bacterial modulators
are a useful extra for a surviving anti-tumor regime, especially as they have a
significant impact on the effectors of the immune system. Anyway complications
exist with so much methodology, as we do not yet have the foggiest idea about the
synthesis of “ideal” gut microorganisms in the milieu of malignant growth cure, nor
do we identify whether this would concern across medicines just as from corner to
corner malignant types.
Furthermore, the decision of a specific methodology will be optional for thorough
and cautious identifying in a clinical preliminary, as unquestionably there is an
absence of accord in regards to which populations to target, how to focus on these,
and what end-focuses to quantify. Evidently, a lot of research needs to be done to
additionally explain the effect plus targetability of the gut microbiota on malignant
growth and to completely fathom its whole potential. In any case, current endeavors
will without a doubt end up being significant.
Commensal microbes in the gut lumen affect the immune system within the
mucosa of the gut, exhausting mesenteric lymph nodes, and so on. The resistant
framework in like manner can modify the gut microbiota. Goblet cells form a thick
mucous defensive layer that protects the mucosa. Without germ creatures, this
mucosal layer is severely deficient. Plasma cells in the lamina propria transmit IgA
into the lumen of the gut. Paneth cells transmit different number of hostile to
microbial peptides; their development is upgraded because of motioning from
nearby insusceptible cells in light of the microbiota.
The microorganisms in the digestive tract can regulate tumor advancement
through different various systems that can be predominantly separated into immedi-
ate and roundabout. The immediate components (blue line) are those in which
microbial items legitimately advance tumor development. Of note, it has been
indicated that a few inadequacies in the immune system can permit the development
of certain procarcinogenic microbes. The backhanded instruments (red line) are
those in which the microbes fundamentally cannot advance tumor commencement
and development except if they cooperate with the invulnerable framework, which at
last advances disease. At long last, it is additionally conceivable that inadequacies in
explicit components of the immune response permit the extension of specific
microorganisms, which, thus, initiate a protumorigenic immune reaction (blue +
red line).
Acknowledgements The authors are grateful to their respective academic institutions for the
support extended.
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Dynamics of Respiratory Microbiome
Profiles Contributes to Imbalance and Lung 7
Dysbiosis in the Respiratory Tract
Harish Babu Kolla, Prakash Narayana Reddy, and

Abstract
Lungs and the other regions of respiratory tract are not sterile. Respiratory system
consists of separate and balanced microbial communities called respiratory
microbiome. Respiratory microbiome is the set of diverse microbial communities
residing on the mucosal surface of respiratory tract. The presence of microbiota
in respiratory tract is identified by 16S rRNA amplicon sequencing and the
diversity of microbes in the respiratory tract was clearly understood. Microbiome
equilibrium is maintained in healthy state of an individual. The composition of
microbiome gets altered during disease. During health conditions the shape of
microbiome is maintained by three detrimental factors: microbial immigration,
emigration, and relative reproduction of microbes. Sometimes balance among
these three factors is disturbed and the constancy of microbiota is affected. This
condition is called as respiratory dysbiosis. There are certain factors such as
smoking, antibiotics, chronic respiratory disorders (CRDs), and lung transplanta-
tion that contribute to imbalance and dynamic change of microbiome in respira-
tory tract. In this chapter, we discussed briefly the changes in microbial
communities in response to these factors.
Keywords
Microbiome · Respiratory tract · Mucosal epithelium · Dysbiosis · Microbial

diversity
H. B. Kolla · P. N. Reddy (*)

Department of Biotechnology, Vignan’s Foundation for Science Technology and Research
(Deemed to be University), Vadlamudi, Guntur, Andhra Pradesh, India
P. V. Bramhachari
Ltd. 2021
https://doi.org/10.1007/978-981-16-3156-6_7
102 H. B. Kolla et al.
7.1 Overview of Respiratory Microbiome
Respiratory system is not sterile and free from microbes. Lungs and other
components of respiratory system have a separate microbial composition called
respiratory microbiome. The microbiome helps in immune system regulation and
protecting against several pathogenic microbes during a healthy state. This
normal microbiome composition gets altered during disease conditions. When an
individual is diseased, the normal microbiome of the respiratory systems gets
slightly altered towards composition of pathogenic microbial communities probably
Gammaproteobacteria, a class of bacteria that causes several respiratory system
associated infections (RSAI) (Huffnagle et al. 2016). Because the respiratory tract
is warm and moist, this environment is markedly convenient for microbes to grow.
Till now there is no scientific evidence to show about sterile respiratory environ-
ment. Respiratory microbiome is different from the normal gut microbiota in its
nature and dimension. The nature of microbiota in lungs is more dynamic than to GI
tract. The movement of microbes from mouth to anus is unidirectional, whereas in
the case of respiratory tract it is bidirectional. This is because of the variant
environmental conditions in gastrointestinal and respiratory tracts contributing to
distinguished microbial communities.
Presence and dynamics of respiratory microbiome is better explained with life in
Antarctica as a model example. There might be a presence of small and constant
microbial communities even in health conditions. If the environmental conditions in
lung alter from optimal parameters, long-term microbial colonization takes place
(Dickson et al. 2014a, 2015). Microbes in microbiome are characterized by
16SrRNA profiling. Microorganisms are identified, classified and the phylogenetic
relationship among them was studied in human microbiome project (HMP).
16SrRNA amplicon sequencing from 300 adults in human microbiome project
decoded that the nasal microbiota comprise species of Staphylococcus,
Corynibacterium, Propionibacterium, and Moraxella. Further investigations of the
project have revealed that the major organisms are P. acnes, C. accolens,
C. kroppenstedtii, S. aureus, and S. epidermidis from the metagenomic studies
(Human Microbiome Project Consortium 2012). Microbial communities of these
bacteria were significantly different from the smokers and non-smokers (Charlson
2010). And from the reports of Brisgaard et al, it was known that the oral cavity also
contains several hundreds of microbial communities in teeth, palates, tongue, and
tonsils. Culture-independent studies have revealed that the occupants of oral cavity
are the species of Prevotella, Veillonella, Streptococcus, Haemophilus,
Fusobacterium, Neisseria, and Corynebacteria (Brisgaard et al. 2008). The respira-
tory microbiome is divided into upper and lower respiratory tract microbial
communities. The upper airway tract initiates from buccal cavity, nose and
terminates finally at the lungs. Mucus covers the upper respiratory tract. Lower
airway tract continues to alveoli which are lined by a phospholipoprotein pulmonary
surfactant that has bactericidal activity towards certain alien pathogenic bacteria.
This property has made alveolar space distinct from the GI tract and upper respira-
tory tract. The surfactant layer of the alveoli contains free fatty acids along with
7 Dynamics of Respiratory Microbiome Profiles Contributes to Imbalance and. . . 103
sphingomyelins and primacy dipalmitoyl phosphatidylcholine, which are

phosphatidylcholine-containing lipids. These are mainly responsible for fighting
against the pathogens when entered into respiratory tract. Respiratory microbiome
is a community of many microbes. Many research works with the involvement of
metagenomics, bioinformatics, and metatranscriptomics approaches generated the
data and information focusing on the bacterial communities in the respiratory tract.
But there are also communities of several microbes such as virus (virome), fungi
(mycobiome) that play an important role during healthy and diseased states. Even
though the fungal communities were not characterized by culture methods, they are
characterized at a molecular level. Microbiome of respiratory tract has an impact on
the upshot of chronic respiratory disorders such as asthma, COPD, cystic fibrosis,
bronchiectasis, etc. (Linh Nguyen et al. 2015).
7.2 Human Microbiome Project and Molecular

Characterization of Respiratory Microbiome
Most of the microbiological works have focused on disease-causing pathogens and

their mechanisms of pathogenesis. But the endogenous flora was poorly studied and
understood. After the completion of the mega Human Genome Project, in 2001
Relman et al. called for the second project on human microbiota (Relman and
Falkow 2001). This is a 5-year project made with a budget of nearly 150 million
dollars by NIH. The main objective of this project is to understand the microbiome of
human body and analyze the genomic data of the microbiota in human body. In this
project, samples were collected from 250 volunteers and the genome of the microbial
communities from the different regions of human body was sequenced (NIH HMP
working group, The NIH Human microbiome project 2009). Microbial communities
and their molecular characters were studied from oral, nasal, gut, airways in men and
along with vagina in females. Before HMP, it was assumed that the human lung and
the respiratory tract are sterile. Previously the source for microbiological research is
culture-based studies. But for respiratory microbiome studies, sampling is the main
challenge due to its low biomass and unavailable non-invasive sampling
technologies. Bronchoscopy is one of the techniques used to study and examine
the respiratory tract. But due to the high risk of pharyngeal contamination of samples
associated with bronchoscopy, it is not being used for examining microbial
communities in the respiratory tract (Dickson et al. 2014b, 2015). In HMP, microbes
are characterized by culture-independent methods by sequencing the gene/genome
of the microbes. The sequence data was assembled and the microbial interactions
along with their evolutionary route and phylogenetic relationships are studied based
on 16S rRNA analysis. HMP has two phases: the jumpstart phase and second phase.
The jumpstart phase is the initial phase of HMP started in 2007. In this phase, four
sequencing centers, the Board Institute, Baylor college of Medicine, Washington
University School of medicine, and J. Craig Venter Institute, were supported by
NIH. Nearly 500 bacterial reference genomes were sequenced up to July 2009 in the
jumpstart phase. 16S rRNA sequencing of microbial communities was performed
Fig. 7.1 Schematic

representation of molecular
characterization of normal
human microbiome
from the four regions in males and five sites in females. Microbial communities of
human microbiome are clustered into 5 phyla based on 16S rRNA sequence profile
(Pace 1997; Woese and Fox 1977; Stahl et al. 1984) (Fig. 7.1). These include
Firmicutes, Bacteroides, Proteobacteria, Actinobacteria, and Fusobacteria. The com-
position of these phyla varies in different regions in human body. For example, the
resident microbes of oral cavity and lung are almost the same, but there is a
significant difference in the proportion of bacteria in respiratory tract. Members of
Actinobacteria are high in nasal tract than in oral cavity.
7.3 Determinants of Respiratory Microbiome
Three factors determine the shape of respiratory microbiome. These three are
microbial immigration, elimination, and relative reproduction of microbial
communities in airways. Microbiome composition was balanced by these three
detrimental factors during health (Dickson et al. 2014c). In disease states, these
factors are disturbed resulting in alteration of respiratory microbiome composition.
Sources for immigration of microbes are microaspiration and inhalation of air
(Lighthart 2000; Gleeson et al. 1997; Huxley et al. 1978; Quinn and Meyer 1929;
Amberson 1954). Elimination of microbial communities was accomplished by
innate and adaptive immune defenses, cough, and mucociliary clearances. During
health, a balance between microbial immigration and emigration was established
(Dickson et al. 2014a; Dickson and Huffnagle 2015; Venkataraman et al. 2015)
(Fig. 7.2). Inflammation is an important factor that regulates reproduction of micro-
bial communities in the respiratory tract and shapes immunity. Inflammation affects
the growth conditions in respiratory tract such as temperature, pH, availability of
nutrients and oxygen. The epithelial lining of the respiratory tract gets damaged
during inflammation and bacteria adhere to the unshielded or exposed zones of
respiratory lining. When bacteria binds to damaged cells in the respiratory lining,
epithelia produce cytokines such as IL-25, IL-33, and thymic stromal lymphopoietin
Fig. 7.2 Determinants of respiratory microbiome
(TSLP). These factors could contribute to additional inflammation by releasing IL-5

and IL-13 resulting in Goblet cell hyperplasia, a condition of Goblet cell expansion
in allergic states with IL-13 production. This leads to an anaerobic environment in a
system with excessive mucus secretion that enhances bacterial colonization by
inhibiting phagocytosis. During health, the lung or respiratory environment does
not allow robust microbial growth. Inflammation alters the regional factors and
further contributes to the imbalanced composition in the microbiome.
7.4 Reactive Nitrogen Species (RNS) in Respiratory Tract

Inflammation
Reactive nitrogen species (RNS) are produced in response to inflammatory stimuli.

These RNS are synthesized enzymatically by inducible nitric oxide synthase (iNOS)
in leukocytes and myelocytes (Bogdan 2015). Nitric oxide (NO) in reaction with free
oxygen species O2 gives peroxynitrite (ONOO) which further breaks into nitrites
(NO2) and nitrates (NO3). Nitrates (NO3) act as a terminal electron acceptor and
support the anaerobic respiration and outgrowth of Gammaproteobacteria via deni-
trification. Gammaproteobacteria gets benefits from the inflammation in the respira-
tory tract (Rivera-Chavez et al. 2013; Winter et al. 2013; Winter and Baumler 2014;
Lopez et al. 2012, 2015; Bliska and van der Velden 2012; Vazquez-Torres and
Baumler 2015; Spees et al. 2013). RNS has antimicrobial properties that destroy the
native microbes in respiratory tract resulting in the alteration of microbiome compo-
sition towards Gammaproteobacteria.
NO + O2 ! ONOO ! NO3 + NO2.
(Nitric (Peroxynitrite) (Nitrate) (Nitrite) Oxide)
7.5 Dynamics of Respiratory Microbiome in Health

and Disease
The normal composition of microbial communities gets altered during disease and
with the treatment of antimicrobial agents resulting in disruption of native
microbiome (Alicia and Allan 2018). The state of imbalance in the microbial
communities is called as respiratory dysbiosis. Certain factors contribute to the
dysbiosis of respiratory microbiome majorly smoking, antibiotics, chronic respira-
tory disorders (CRDs), and lung transplantation. Altered respiratory microbiome and
byproducts of inflammation initiate pathogenesis in respiratory system, therefore
affecting the constancy of determinants of respiratory microbiome, i.e., balance
among the three factors, immigration, emigration, and inflammation, is lost
(Fig. 7.2).
7.5.1 Smoking
Cigarette smoke contains various harmful water-soluble compounds and toxic gases
such as Nicotine, ammonia, benzopyrene, hydro-quinone, CO, CO2, formaldehyde,
cadmium, nitrogen oxide, acetone and acrolein, etc (WHO Global Tuberculosis
control 2011). Nicotine is the main pharmacogenic factor that causes addiction to
cigarette and it is easily absorbed by mucus membrane in oral and respiratory lining
affecting the innate immunity (Bjartveit and Tverdal 2005). Smoking is one of the
major factors that affect the lung microbiome by causing inflammation in the
respiratory tract. In a study conducted by Zhang et al., 40 numbers of eight-week-
old male mice was grouped into smoking and non-smoking mice (20 each), and
exposed smoking group mice to smoke for 2 h per day for 90 days. Concentrations of
IL-6 and C-reactive protein (CRP) were determined by ELISA and microbial
communities were identified by 16S rRNA profiling. From this study, it was
concluded that weight of smoking mice increased very slowly compared to
non-smoking group. Although there is no change in IL-6 and CRP concentration
between the two groups, there is congestion and inflammation densely in the
smoking group than in non-smoking mice. Also, there is a change in microbial
composition in both the groups at the phylum and genus level. Proteobacteria and
Firmicutes are the dominant phyla in both smoking and non-smoking groups.
Trichococcus, Escherichia, Shigella, and Oxalobacteriaceae are the unique taxa
identified in the smoking mice group. And in non-smoking mice, Oceanospirillales,
Lactobacillus, and Lactobacteriaceae members are identified to be unique (Zhang
Fig. 7.3 Effect of smoking on respiratory microbiome
et al. 2018). Apart from disturbing the microbiome in respiratory tract, smoking
exasperates lung inflammation by overexpressing TNF-α (tumor necrosis factor-α),
IL-6, and monocyte chemoattractant protein-1 (MCP-1) (Gualano et al. 2008;
Vlahos et al. 2006; Guerassimov et al. 2004). And also smoking can increase the
incidence of pulmonary tuberculosis (Fig. 7.3). This was well documented by the
Department of Pulmonary Medicine in their research in the year 2014, where
patients are categorized into group I (control) and group II (n ¼ 60). From the
common radiological findings, it was identified that the incidence of pulmonary

tuberculosis increased with the duration of smoking time among the patients (Mishra
and Srivatsava 2017).
Some observations concluded that smoking can disturb only oral microbiome but
not lung microbiome. This observation was found out by Alison et al. in lung HIV
microbiome project (LHMP), 2013 promoted by National Heart, Lung and Blood
Institute in characterizing the lung microbiota. Oral washes and Bronchoscopic
Alveolar Lavages (BAL) are collected from sixty-four enrolled volunteers
(45 non-smokers and 19 smokers) (Fig. 7.3). Microbial communities were
characterized by culture-independent methods by 16S rRNA sequence. Most of
the oral native bacteria are also present in lung. Specific bacteria such as
Enterobacteriaceae, Haemophilus, Ralstonia, and Methylobacterium are in the
outer portion of lungs. Tropheryma was present only in lungs (not a resident of
oral microbiome). The lung microbiome did not witness any alteration in
communities due to smoking. Mouth microbes differed between smokers and
non-smokers especially in Gemella, Neisseria, and Porphyromonas (Morris et al.
2013).
“Vicious Circle” concept proposed by Mammen et al. explains that the harmful
compounds in cigarette smoke can impair the innate immune defense and disturb the
microbiome of respiratory tract resulting in disproportion and disturbance in taxo-
nomic composition and relations among the microbial communities (Mammen and
Sethi 2016). Direct exposure and contact of cigarette smoke through the respiratory
air tract can lead to respiratory diseases such as asthma, COPD, cystic fibrosis, and
lung cancer. The effect of smoking in causing diseases and dysbiosis is represented
in Table 7.1. The impact of smoking on the microbiome can be direct or indirect.
Smoking suppresses immunity, deprives oxygen availability, and promotes biofilm
formation (Huang and Shi 2019) (Fig. 7.3).
7.5.2 Antibiotics
Antibiotics are being widely used to fight against infections. But tremendous and
overuse of them is a menace to mankind. Antibiotics inhibit and kill the microbes.
Due to the bactericidal property of antibiotics, the beneficial microbes in the
microbiome may get altered resulting in change in composition of microbiome
(dysbiosis). Even the results from the reports regarding the effect of antimicrobial
agents on the microbiome are unclear; several research works are currently focusing
on the mechanisms underlying antibiotic-mediated dysbiosis. Many antimicrobial
agents are the derivatives of toxic and harmful elements such as lead, silver, arsenic,
and mercury. For example, the first industrially synthesized antibiotic Salvarsan is a
derivative of arsenic (Waksman and Woodruff 1940). The main problem associated
with antibiotic usage is the evolution of antibiotic-resistant microbes. Firstly, the
microbes in the community that are highly susceptible to antibiotics are eliminated
and only the resistant species remains alive (Table 7.2). These resistant microbes
now adopt some of the potential mechanisms such as drug degradation, antibiotic
Table 7.1 Influence of smoking on respiratory microbiome

Disease References Sampling Rich microbes Depleted microbes
Asthma Simpson Subgingival plaque • Fusobacterium. Not identified
et al. sample of human • Prevotella.
(2016) • Selenomonas.
Airway Charlson Nasopharyngeal and Oropharynx Oropharynx
(2010) oropharyngeal • Megasphaera. • Capnocytophaga.
swabbings of human • Veillonella spp. • Fusobacterium.
Nasopharynx- • Neisseria spp.
• Eggerthella. Nasopharynx
• Erysipelotrichacea. • Shigella spp.
• Anaerovorax.
• Eubacterium spp.
Airway Zhang Lung sample of mice • Trichococcus, • Oceanospirillales.
et al. • Escherichia. • Lactobacillus.
(2018) • Shigella, • Lactobacillaceae.
• Oxalobacteraceae. • Enterobacter.
• Acidimicrobiales.
• Caulobacteraceae.
•
Phyllobacteriaceae.
• Raoultella.
• Caulobacteraceae.
Table adapted and modified from Huang and Shi (2019)
efflux from membrane pumps, changing the membrane domains making antibiotics
impermeable into a cell, etc., to defeat the functionality of antibiotics. After a period,
these resistant strains multiply in respiratory tract. The composition of microbiota
before and after antibiotic usage may be the same, but the genomic shape of
microbial communities differs (Looft and Allen 2012; Jernberg et al. 2010;
Raymond et al. 2016). Antibiotics at higher concentrations conk outs the respiratory
system badly. Sometimes antibiotics trigger the expression of pathogenic virulence
factors and lead to biofilm formation. Biofilms are a defense property of microbes
towards antibiotics. These physiological alterations in the microbial ecosystem can
affect the health in causing surface-associated infections in the respiratory tract
(Martínez 2017) (Fig. 7.4).
7.6 Chronic Respiratory Disorders
Chronic respiratory disorders (CRDs) such as chronic obstructive pulmonary disease

(COPD), asthma, cystic fibrosis (CF), and idiopathic pulmonary fibrosis (IPF) alter
the dynamics of microbiome by affecting the immigration, emigration, and regional
conditions of respiratory ecosystem resulting in twisting out the shape of respiratory
microbial ecosystem. CRDs are commonly due to gastroesophageal reflux, elevating
the volumes of microaspiration. Mucociliary clearance and cough are the factors
responsible for the elimination of microbes from the ecosystem. Chronic lung
Table 7.2 Antibiotic-resistant bacteria in respiratory microbiome

Resistance
Antibiotic Resistant microbe mechanism References
Ampicillin Haemophilus influenza, ampicillin- Penicillinases Maddocks
susceptible Staphylococcus aureus and May
(1969),
Connell et al.
(2013)
Penicillin Streptococci Penicillinases Kundsin and
Miller (1964)
Β-lactam Haemophilus influenza, β-Lactamases Armbruster
antibiotics Streptococcus pneumonia et al. (2010),
Budhani and
Struthers
(1998)
Amoxicillin Streptococcus pneumonia β-Lactamases Weimer et al.
(2011)
Chloramphenicol Streptococcus pneumonia Chloramphenicol Sorg et al.
acetyltransferase (2016)
Ceftazidime Pseudomonas aeruginosa β-Lactamases Sherrard et al.
(2016)
Gentamicin Staphylococcus aureus Aminoglycoside- De Leon et al.
modifying (2014)
enzymes
Imipenem and Pseudomonas aeruginosa β-Lactamases Kataoka and
ceftazidime Tanaka
(2003)
Adapted and modified from Vandeplassche et al. (2019)
Fig. 7.4 Antibiotics on normal respiratory microbiome

diseases modulate the inflammatory response in the respiratory system. This was
demonstrated by Segal et al. by comparing the levels of microbiome detected in
BAL. Levels of anaerobic oral commensals like Prevotella and Veillonella rise
abundantly during the CRDs associated with inflammation and rise in lymphocyte
levels (Segal et al. 2013). Generally, CRDs are characterized by exacerbation period.
Pulmonary function declines due to exacerbations with a significant rise in mortality
and morbidity rates.
7.6.1 COPD
Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disorder that

is characterized by airflow blockage in the respiratory tract (Fig. 7.5). This is caused
due to emphysema or chronic bronchitis. Smoking is the major risk factor in the
development of COPD in humans. Factors that contribute to airflow obstruction
include alveolar wall destruction, enlargement of mucus glands in bronchi, and
hyperresponsiveness in the airway (Barnes 2000; Tashkin et al. 1992). Many
culture-independent methods have determined a diverse pulmonary microbiome in
COPD exacerbations from BAL and sputum cultured samples. These exacerbations
are due to inflammation and dynamic changes in respiratory microbiome (SZE et al.
2012; Millares et al. 2014; Huang et al. 2014; Pragman et al. 2012). Haemophilus,
Pseudomonas, and Moraxella communities are abundant during COPD. Sometimes,
the microbiome proportion can alter towards the Proteobacterium in COPD exacer-
bation. COPD exacerbations may also be triggered by viral infections (Hewitt et al.
2015). The relationship between viral infections and respiratory microbiome was
poorly understood. When patients are infected with rhinovirus experimentally, they
developed features of COPD (Papi et al. 2006; Rohde et al. 2003; Mallia et al. 2011;
Seemungal et al. 2001). It was known clearly that microbial communities shift
towards Proteobacteria phylum and Pseudomonas species increase during COPD
exacerbations (Molyneaux et al. 2013; Millares et al. 2014). GOLD stage 4 is the
severe stage in COPD. Sequence studies from COPD patients demonstrated that
Fig. 7.5 Schematic

representation of normal and
COPD airways. Airflow
obstruction in COPD air tract
is due to mucus glands
enlargement and
accumulation of mucus
narrowing the airway and
blocking the passage of air
levels of Firmicutes increased severely in GOLD stage 4. An increase in Firmicutes

contributes to an increase in Lactobacillus genus. Alveoli and airways in COPD
patients contain a distinct microbiome (Dickson et al. 2013). Introduction of a new
pathogen into the respiratory tract can lead to inflammation (Mammen and Sethi
2016). In a study conducted by Pragman et al. it was observed that Firmicutes
dominated both Proteobacteria and Actinobacteria (Pragman et al. 2012). In another
cohort study, Proteobacteria, Firmicutes, and Actinobacteria were found to be the
most prevalent phyla in both normal and exacerbated patients. Streptococcus and
Haemophilus are identified at higher levels in both groups (Millares et al. 2015).
COPD is majorly caused by smoking. Smoking alters the dynamics of host–
microbe interactions and results in respiratory abnormalities (Garmendia et al. 2012).
Cigarette smoke is a risk factor to many of the beneficial microbes in respiratory tract
(Arcavi and Benowitz 2004). Somehow antibiotic treatment also disturbs the micro-
bial diversity and distribution during COPD (Flagnan et al. 2007). Dynamic changes
in respiratory virome mediated by smoking could lead to COPD. Lung contains
some virus (virome). Virus-like phages cause opportunistic infections by mediating
the immune microbial competition (Klainer and Beisel 1969). Gregory et al. first
conducted a study and demonstrated that smoking lowers the virome community by
changing the levels of IL-8 and arachidonic acid. Both IL-8 and arachidonic acid are
the factors that are responsible for COPD (Gregory et al. 2018). Several factors that
are responsible for COPD and their effects on the microbiome and clinical outcomes
of dysbiosis need to be investigated further.
7.6.2 Asthma
Microbiome composition slightly alters towards the Proteobacteria. This was

identified by Hitly et al. in asthma patients. Proteobacteria, especially Haempohilus
is the dominant microbial population in the bronchial trees of asthmatic patients to
controls (Hilty et al. 2010; Marri et al. 2013; Huang et al. 2011). Majority of the
asthma exacerbation cases are due to viral infections (Nicholson et al. 1993;
Johnston et al. 1995). Some of the fungal species can also contribute to uneasiness
during asthma. From sputum culturing, Aspergillus and Penicillium were found to be
the dominant fungal species in asthmatics (Agbetile et al. 2012) (Fig. 7.6).
Several studies have strongly focused on the food habits in studying asthma
exacerbations. Some works strongly suggest that foods with high fiber content
reduce the risk of asthma. High fiber helps in the normal functioning of a healthy
microbiota. High fiber foods improved and raised the levels of short chain fatty acids
(SCFA) and ensured protection against asthma inflammation in murine models
(Trompette et al. 2014). Furthermore, many research works on ovalbumin-induced
asthma have discussed that native microbiota promotes Th 17-dependent neutrophil
inflammation (Lemaire et al. 2011). Similarly, antibiotics administration can lead to
inflammation in the airways and reduces microbial diversity (Russell et al. 2013).
Interactions among the host-microbial communities in the respiratory tract are very
much essential in understanding the mechanisms underlying the dysbiosis during
Fig. 7.6 Normal airway, inflamed airway, and asthmatic airway
asthma (Fig. 7.6). As like COPD, normal airway is obstructed in asthma. According
to “microbiota hypothesis” (framed by correlating the antibiotics, allergies, and gut
microbiota) deviation in GI microbiota due to antibiotic usage and poor food diets in
western areas leads to disruption of immunologic tolerance (Huffnagle 2010)
(Fig. 7.5). Smoking is another risk factor in shaping the microbiome of lower airway.
Based on 16SrRNA sequencing, the bacterial species that are dominant in
ex-smokers were found to be Fusobacteria, Firmicutes, and Bacteroidetes.
Proteobacteria is lower in non-smokers (Colak et al. 2015; Simpson et al. 2016).
7.6.3 Cystic Fibrosis
Cystic fibrosis (CF) is caused due to mutation in the CFTR gene (cystic fibrosis
transmembrane conductance regulator), where several organs such as lungs and GI
tract are affected. Water and ion concentration in epithelial tissues are maintained in
equilibrium by cAMP-regulated chloride and bicarbonate transport channel. This
equilibrium in water to ion concentration is disturbed during CF disease (Saint-Criq
and Gray 2017). During CF, the mucus-producing cells are mainly affected by
respiratory dysbiosis leading to mucus accumulation in the air tract (Fig. 7.7),
thereby disturbing the normal environment in the airway (Willger et al. 2012).
Pseudomonas aeruginosa, a gram-negative bacillus is relatively linked to CF clini-
cally (Harris and De 2007; Tunney et al. 2008). Apart from Bacteriome, Virome
(Virus) and mycobiome (Fungi) were also characterized from the respiratory tract of
the CF patients. Species of Candida, Aspergillus, Scedosporium, and Malassezia are
detected in CF (Delhaes et al. 2012; Willger et al. 2014; Mounier et al. 2014; Willner
et al. 2009; Kramer et al. 2015). An unknown set of viral and bacteriophage groups
may also play a major role in disease progression (Billard et al. 2017; Fancello et al.
2011; Willner et al. 2009). CF is associated with chronic infections and bronchiec-
tasis (enlargement of lung airway). Some other bacteria such as methicillin-resistant
Staphylococcus aureus (MRSA), Burkholderia cepacia, and some non-tuberculosis
Fig. 7.7 Healthy airway and

CF airway with mucus
accumulation
bacteria like Mycobacterium abscessus and Mycobacterium avium are also involved
in contributing to morbidity in pulmonary CF patients (Gilligan 2014; Dasenbrook
et al. 2010; Martiniano and Nick 2015). It is very difficult to diagnose and treat some
pathogens like Achromobacter spp (ruhlandii and xylosoxidans), Stenotrophomonas
maltophilia, etc. during CF. Anaerobic bacteria in respiratory airways play a key role
in downregulating the expression of the NOS (nitric oxide synthase) gene and
release IL-6, IL-8, CSF (colony stimulating factor), and granulocyte macrophages
in excess amounts. Germella, Actinomyces, Neisseria, Granulicatella, Rothia are the
genera that are found frequently in the airway of CF patients (Fig. 7.7) (Acosta et al.
2017; Rogers et al. 2003; Coburn et al. 2015; Surette 2014). Obligate anaerobes like
Veillonella, Prevotella, Porphyromonas, and Fusobacterium, etc. are also seen in
CF airways (Sherrard et al. 2016; Huang and LiPuma 2016; Mirkovic et al. 2015). In
more than 30% of children (<5 years) cases and 10% of adults with CF,
Haemophilus influenza was identified as the more frequent and potential colonizing
pathogen in the respiratory tract (Lipuma 2010). The proportion of lung microbiome
during CF may not be constant. It changes over time. For example, microbial
diversity is more in childhood than in adult age. A decline in microbial diversity
shifts the composition of lung microbiome towards Pseudomonadaceae (mostly
P. aeruginosa as the dominating one). This is because of over antibiotic usage and
the selection of drug-resistant microbes in the community (McGuigan and Callaghan
2015; Cox et al. 2010; Zhao et al. 2012).
7.6.4 Idiopathic Pulmonary Fibrosis
IPF is a fatal chronic respiratory disease that occurs in the lung parenchyma (Raghu
et al. 2015). Unlike COPD and asthma, several active pathogenic microbes are
responsible for exacerbation in IPF (Collard et al. 2007) (Fig. 7.5). Streptococcus
and Staphylococcus are the pathogens that are majorly involved in causing IPF (Han
et al. 2014). In a study conducted by Molyneaux et al, Haemophilus, Streptococcus,
Neisseria, and Veillonella were identified as enriching pathogens in IPF patients by
analyzing the BAL fluid (Molyneaux et al. 2014). Signaling of defective toll-like
receptor TLR3 initiates disease in IPF. rs3775291(L412F) is an SNP in TLR3 that
leads to a change in leucine to phenylalanine encoding a different protein. L412F is a
potential marker associated with defective TLR3. L412F polymorphism results in
inflammation and desensitized interferon response towards TLR3 activation. This is
due to defective pulmonary fibroblasts in IPF. This L412F polymorphism is the
major confirmation for mortality and morbidity in IPF patients (O’Dwyer et al.
2013). Apart from the alterations in the microbiome or pathogenic entry, presence
of a certain microbe in the lung microbiota can drive towards IPF progression
(Knippenberg et al. 2015). For example, pneumolysin is a Pneumococcus-associated
toxin that causes fibrosis in animal alveoli.
7.6.5 Lung Transplantation
In many of the final stages of respiratory disorders, lung transplantation is the main
and only option left (Christie et al. 2012). Infections are associated with mortality
and morbidity in patients who have undergone lung transplantation during pneumo-
nia and bronchiolitis obliterans syndrome (BOS). BOS is a chronic infection that
damages the air passages in lung (Botha et al. 2008; Husain and Singh 2002;
Khalifah et al. 2004). Lung transplantation may alter the host defense in patients
disturbing the regional microbiota (Duncan and Wilkes 2005; Kotloff and Thabut
2011). Through the culture-independent methods and metagenomic approaches,
Pseudomonas aeruginosa and fluorescens were identified as the dominant species
in patients who have undergone lung transplantation. Out of the two, P. aeruginosa
is the most predominant pathogen with a high risk of BOS. Whereas P. fluorescens
confers little acute infection only. The two species aeruginosa and fluorescens of
Pseudomonas have unique microbial and genomic characteristics and are more
divergent than the other microbes in the microbiome (Dickson et al. 2014b).
7.7 Conclusions and Future Perspectives
With advances in metagenomics and next-generation sequencing methods, it became

easy in sequencing the genome of microbes and assembling the genomic data.
Culture-independent methods have potentially identified the components of respira-
tory microbiome in different regions of respiratory tract such as alveoli, upper
respiratory tract, etc. Understanding the microbiome of respiratory tract in normal
homeostatic health conditions and during disease exacerbations helps in studying the
microbiome signatures and dynamics and also it is very useful in therapeutic
approaches during any imbalance in the microbiome. With advanced
instrumentation facility and emerging novel technologies in the fields of microbiol-

ogy, genomics, and molecular biology, it became very easy in studying the dynamics
of respiratory microbiome and its interaction with the other microbiomes in the body
such as oral microbiome, skin, vaginal, and gut microbiome.
Acknowledgments The authors gratefully acknowledge Vignan’s Foundation for Science, Tech-
nology and Research (deemed to be university) and Krishna University, Machilipatnam for the
support extended.
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Understanding the Interplay Between
the Host Immune–Microbiome Interactions: 8
A State of the Art Review
Kuraganti Gunaswetha, Edla Sujatha, and

Abstract
The microbiome and immune system are effectively impacting each other to
endure, characterizing the healthy individual’s dysfunctional equilibrium. The
human gut has the most complex microbiota of the multitude of non-sterile
cavities, with a solid effect on host homeostasis and immunostasis, making it
fundamental for looking after health. Simultaneously, mammalian immunity is
formed by resident bacteria. At the point when this immune system–microbiota
partnership is working appropriately, it takes into account the acceptance of
defensive reactions to pathogen just as the upkeep of regulatory pathways
engaged with the support of resilience to harmless antigens. The investigation
of the microbiome–immune system crosstalk has shown a solid association
between microbial communities and the advancement of hypersensitive
infections and asthma. Interruption of the microbiome affects the host’s safe
reaction and can prompt infection pathogenesis. Disease and remedial medicines,
then again, affect microbial populaces. A preview of the present status of the
microbiome–immune system in host weakness to pathogens, extreme hypersen-
sitivity responses, autoimmunity, chronic inflammation, and cancer research is
emphasized in this review. The turn of events and use of next-generation DNA
sequencing strategies have changed gut microecology, considering new
experiences into the synthesis of the intestinal microbiota and it connects to an
Kuraganti Gunaswetha, Edla Sujatha and Pallaval Veera Bramhachari contributed equally with all
other contributors.
K. Gunaswetha · E. Sujatha (*)

Department of Microbiology, Kakatiya University, Warangal, Telangana, India
P. V. Bramhachari
Ltd. 2021
https://doi.org/10.1007/978-981-16-3156-6_8
124 K. Gunaswetha et al.
assortment of diseases. We additionally examine how we have gotten familiar

with the associations between resident microbes and the immune system, just as
the outcomes of these outcomes for human health.
Keywords
Microbiota · Immune system · Epithelial barrier · Host–microbial relationship ·

Gut microbiota
8.1 Introduction
The alliance between the microbiome and immune system has proven to be the most
significant regarding human health and disease. The historical outlook of the
immune system and microbiome interaction came into understanding in 1798 by
the outbreak of smallpox and its vaccination study by Edward Jenner. According to
Edward Jenner, the exposure of viruses from cowpox scabs can eventually protect
the individual from the smallpox virus (Yoshikawa et al. 2014). Later Louis Pasteur
proved this technique of vaccination against fowl cholera and anthrax disease. He
demonstrated that the old culture of Pasteurella multocida bacterium and heated
culture of Bacillus anthracis of fowl cholera and anthrax disease have not caused
any disease, respectively (Berche 2012). Thus he showed that general principles of
vaccination could be applied to other diseases other than smallpox to protect humans
against other infections. Later in the 1890s the protection caused by vaccination as
stated by Emilvon Behring and Shibasaburo Kitasato that the presence of some
antibodies in blood are associated with protection against disease (Hajj Hussein et al.
2015). Paul Ehrlich later proved that antibodies not only protect animals against
bacteria but also against some foreign toxins too. The idea of the presence of
antibodies as protective agents in blood was proved and accepted by many scientists.
The relation between microbes and the immune system was studied and enlighten by
introducing the techniques and discoveries like passive immunization for tetanus
toxin, phagocytes, tuberculin reaction by Robert Koch, anaphylaxis by Charles
Richet, clonal selection theory by Medawar, radioimmunoassay by Rosalyn
Yalow, the principle of the production of monoclonal antibodies by Kohler and
Milstein. The study of the microbiome and immune system gained a huge spectacu-
lar growth in research which continues to the present.
Antibodies are protein in nature and their appearance in blood serum is triggered
when the animal is exposed to foreign substances that are infectious. The foreign
bacterium as toxin substances that induce the production of antibodies is called
antigens. The immune response against antigens can be categorized into antibody
and cell-mediated immune responses. The cell-mediated response functions through
detection and elimination of viral infected abnormal cells and chemically modified
cells, where on the other hand antibody-mediated response protects against bacteria
8 Understanding the Interplay Between the Host Immune–Microbiome. . . 125
and parasites that act as non-cell-associated invaders. In contrast, cell-mediated

immunity provides resistance to many bacteria and parasite infections, while
antibodies provide immunity against viruses and contribute towards graft rejection.
The study of the immune module enables us in understanding the different
physiopathological behavior of the human microbiome which is the complex mix-
ture of bacteria, fungi, protozoa, viruses, and archaea (Geva Zatorsky et al. 2017).
The genetic immunological factors and notably diet and environmental biodiversity
are the two core drivers of microbiome composition in humans. The genetic immu-
nological factors are more localized to the urinogenital tract microbiome (Gupta
et al. 2017). Hence the composition of the human microbiome has its importance
according to its site of accuracy (Mezouar et al. 2018). In mammals, the host
differential organismal lineage through time and type of diet from evolution are
the two factors that altered the microbiome composition that acts on different subsets
of bacteria at different temporal scales (Groussin et al. 2017). The human
microbiome composition is different or can be said smaller in size compared to
that of their ancestor gut lineage. This substantial change in the microbiome is due to
environmental changes and diet respective of industrial and western world environ-
ment (Clayton et al. 2016).
Mycobiome also a composite microbiome in the human lineage induces the
human immune system. Till now more than 400 heterogenous fungal species have
been isolated from the human gut and skin region (Halwachs et al. 2017; Belkaid and
Naik 2013). The host physiology is usually influenced by bioactive molecules
secreted by fungal species, especially the gut fungal microbiome through Dectine-
1 receptor which regulates the host immune system (IS) (Iliev et al. 2012). Any kind
of changes in the composition of the bacterial microbiome and mycobiome lead to
chronic diseases that may include gut inflammation, colitis exacerbation in the host.
8.2 Microbiome–Immune System Interaction
The human immune system and diverse gut flora have co-evolved to form a defense
barrier against pathogens along with developing tolerance towards beneficial
microorganisms. This mutualistic relationship of the host immune system and gut
microbiome enables the researchers to study the gut microflora as 70–80% of
immune cells are found in the gut itself (Ley et al. 2006; Kumar et al. 2017).
Host-microbiome mutualism to a great extent surpasses the sole metabolic and
nutritional aspects and includes the interaction between the microbiome and host
immune system. Co-evolution, however, has produced unavoidable interconnections
between the physiologies of microbial communities and their hosts that reach out to
past metabolic functions. In the connection between the microbiota and the immune
system, the interconnections are especially evident (Hooper et al. 2012).
8.3 Experimental Tools for Analyzing the Microbiota Immune

System Interactions
Quite a bit of our present comprehension of microbiota–immune system interaction

has been procured from “gnotobiotics.” Gnotobiotic animals also are known as
germ-free animals, especially rodents, have become basic experimental tools for
figuring out which host immune capacities are hereditarily encoded and which
requires microbial interactions (Hooper et al. 2012). Germ-free animals are removed
from the mother’s womb surgically without exposing it to bacteria, fungi, viruses,
and any kind of eukaryotic parasites and raised in sterile isolators. The germ-free
animals are observed to be having altered immune systems lymph nodes, hearts,
metabolisms, lungs, and even reproductive capacities (Heijtz et al. 2011). Germ-free
animals can fill in as living test tubes for the inception of a single microbial species or
a particular defined mixture of species; these can also be a tool for studying their
microbiologically sterile state (Luczynski et al. 2016).
The advancement of gnotobiotic experiments are of firstly, the germ-free mouse
strains can be both genetically targeted and wild-type inbred isogenic strains, where
the different constituents of the immune system contribute towards the host-
microbial mutualism would thus be able to be ascertained by looking at the impacts
of microbial colonization in genetically modified and wild-type mice (Geuking et al.
2011; Vaishnava et al. 2011). Secondly, the composition of human microbiota can
be defined from the polymorphism of the bacterial gene using the next genome
sequence by encoding the 16S ribosomal RNA sequence. These analyses enable us
to characterize the distinct effects on the host immune system by constructing
defined microbiota (Talham et al. 1999). The species contrasts can be shut utilizing
mice with a characterized adapted microbiota, with this developing technology.
Forthcoming, there is even the possibility of humanized isobiotic mice that addition-
ally have a refined immune system (Goodman et al. 2011). A third development has
been the improvement of trial systems that permit the dissecting of the mutualistic
relationship between the host immune system and microbial colonization. This
cannot be accomplished by antimicrobial treatment alone since small portions of
the bacterial colony persist. The explore of the deletion of bacterial strains enables
the study of mucosal immune induction, functional protection, and memory of the
host immune system since after deletion of bacteria the animal becomes germ-free.
The deletion of bacteria can only be grown in culture but does not persist in vivo
(Hapfelmeier et al. 2010). By comparing germ-free and colonized mice using
metabolomic and transcriptomic tools the effect of inhabitant microbiota on host
physiology can be obtained. DNA microarrays, nuclear magnetic resonance spec-
troscopy, and mass spectroscopy are some of the tools which have prompted a point
by point comprehension of how microbiota shape numerous aspects of development,
host physiology, and immunity (Peterson et al. 2007) as well as by giving significant
bits of knowledge into how microbiota impact metabolic signaling in mammalian
hosts (Holmes et al. 2011).
8.4 Interaction of Microbiota–Immune Homeostasis
The microbial community in the human gut plays a crucial part in the development
of the immune system and protection against pathogens in much diversified benefi-
cial functions. The gut microbiota of the host is ascertained by mainly host genetics
and environmental exposure. Intestinal bacteria regulate the immune homeostasis by
sending the signals for regulating anti- and pro-inflammatory host immune responses
and priming the systemic immune responses by T regulatory cells (Tregs) and T
helper 17 (Th17) cells (Korn et al. 2007). Inflammation and sepsis are common
issues that are caused by the disruption of the interplay of host intestinal tissue and
microbiota. Differentiation of T regulatory cells is generally facilitated by gut
commensal microbiota, where induction of Th17 cells is caused by segmented
filamentous bacteria (SFB) (Chaudhry and Rudensky 2013). Changes in the config-
uration of Th17 cells may lead to disease susceptibility as they are important in
maintaining a defense mechanism against invading bacterial pathogens (Sheridan
and Lefrançois 2010).
8.5 Innate Immunity with Epithelial Barrier Defense
To decrease the invasion and tissue damage caused by bacteria it is at most important
to keep the distance of intestinal microbiota with intestinal epithelial cells (IECs)
(Brown et al. 2013). Gnotobiotic mice and antibiotic treated mice show lower
expression of bacterial pattern recognition receptors due to the absence of bacteria
in IECs which leads to the increase in disease susceptibility. The innate immune
epithelial barrier includes the presence of a mucus layer also called mucin glycopro-
tein secreted by goblet cells, which is a vital component in segregating the
microbiota from the intestinal epithelium (Faderl et al. 2015). The large intestine is
covered with both outer and inner mucus layers, whereas the small intestine is
covered with an inner discontinuous mucus layer secreted along the apical surface.
Susceptibility towards pathogen and commensal bacteria-induced inflammation is
proportional to the thickness of the inner mucus layer by limiting the direct contact
of bacteria with epithelial cells, where the outer mucus layer provides glycans to the
mucosa-associated microbiota (Taherali et al. 2018). Along with the mucus layer,
innate epithelial barrier defense includes antimicrobial peptides (AMPs) secreted by
paneth cells. Extensively secreted AMPs play a crucial role in segregating the
microbiota from the epithelium; therefore, a decrease in the number of paneth cells
from epithelium results in an invasion of the epithelial barrier by symbiotic and
pathogenic microbes leading to inflammation (Peterson and Artis 2014). The innate
epithelium barrier defenses also have innate lymphoid cells (ILCs) which confine
much of the microbial community of the lumen of the intestinal tract. The role and
secretion of mucus, AMPs in innate epithelial barrier defense are explained in
Fig. 8.1.
128
Fig. 8.1 Anatomical containment of the microbiota along the intestine. The intestinal epithelium barrier comprises a single layer of enterocytes or colonocytes,
K. Gunaswetha et al.
protected by the immune system. In the small intestine, mucus laer is discontinuous, with fewer goblet cells. The enterocytes are enriched with paneth cells that
8
⁄
Fig. 8.1 (continued) secrete AMPs, which can cross-link with the mucus layer. Through this barrier, sampling of MAMPs can be mediated through antigen
uptake by M cells and gobet cells to dendritic cells (DCs), along with direct transepithelial luminal sampling from DCs. RORγt ILCs can sense microbial signals
and produce IL-22 to aid in IEC barrier function. Commensal-specific IgA is produced by plasma cells in the lamina propria, mediated by DCs in a T cell-
independent mechanism. The large intestine uses a thick, continuous mucus layer to compartmentalize the microbiota, with IgA and AMPs having a secondary
role. (Brown et al. 2013)
Understanding the Interplay Between the Host Immune–Microbiome. . .
129
8.6 Innate Immune Cell’s Barrier
The epithelial enterocyte barrier cells secreting the usual amount of mucus, AMPs,
pattern recognition receptors (PRRs), and secreted IgA molecules promote intestinal
homeostasis and tolerant response to the microbiota, where the microbiota is
segregated away from the IECs, and the intestinal immune system directs a largely
tolerant response to the resident commensal bacteria (Maynard et al. 2012). The
tolerogenic immune cell responses to the microbiota are regulated by the secretion of
cytokines TGF-β, BAFF, IL-33, TSLP, and APRIL stimulated by the microbe-
associated molecular patterns (MAMPs) by binding to the pattern recognition
receptors (PRRs) (De Souza and Fiocchi 2016). This cytokine condition enriches
CD103+ dendritic cells (DCs), which aid in the improvement of Treg cells secreting
IL-10 and TGF-β. Treg cells and CD103+ DCs invigorate the production of
commensal-specific IgA. IECs barrier wholeness is enhanced by the secretion of
IL-22 by RORγt ILCs in this specific condition. DCs and macrophages secret IL-12
and IL-23 which in turn promote TH1 and TH17 response. These T helper cells
secrete high levels of IFN-γ and IL-17A, respectively, and T-bet+ ILCs also accu-
mulate to produce IFN-γ (Brown et al. 2013). Infringement in the epithelial barrier
by the microbiota n this circumstance can likewise prompt more significant levels of
B cells secreting commensal-specific IgG (Fig. 8.2). In immune deficiency diseases
like HIV, IBD infections the innate barrier defect occurs and a potentially harmful
pro-inflammatory response stimulates by the intestinal immune system to the
microbiota to clear invading bacteria by promoting dysbiosis (Hunt et al. 2014).
CD4+ T cells are commonly divided into regulatory T (Treg) cells and conven-
tional T helper (Th) cells. Regulatory T cells (Tregs) are important for the induction
and maintenance of peripheral tolerance (Yong et al. 2007). It is observed that in the
large intestine the proportion of Treg cell proportions is significantly larger in the
CD4+ T cell population, whereas in the small intestine there are only fewer CD4+ T
cells and Th17 cells. Nevertheless, the gnotobiotic mice or antibiotic-treated mice
have depicted an excellent reciprocicity of this condition which occurred by the
absence of transcription factor Helios expression (Alexander et al. 2014). When
these gnotobiotic mice were colonized with commensal microbiota the restoration of
Tregs was observed this might due to these cells have a unique TCR repertoire that
confers specificity for commensal microbiota, and/or that commensal microbiota
products can influence the Th17:Treg balance, potentially by favoring Treg cell
differentiation or expansion (Littman and Pamer 2011).
The innate lymphoid cells (ILCs) of lymphoid lineage, developed from common
lymphoid progenitor cells, emerge as the first line of defense at mucosal barriers by
lacking rearranged Ag-receptors. Three groups of ILCs regulate cytokine production
as encounters with microbial and inflammation. cNK cells and helper ILC1 secrete
th1-type cytokine IFN-γ that are included in group 1 ILCs where IL-4, IL-5, and/or
IL-13, TH2-type cytokines production is characterized by group 2 ILCs, fetal
lymphoid tissue-inducer (LTi) cells, and ILC3s with cytotoxicity receptor (NCR).
NKp46 (NCR + ILC3s) are included in group 3. Group 3 ILCs develop and produce
8
Understanding the Interplay Between the Host Immune–Microbiome. . .
Fig. 8.2 Immune factors that are engaged with a functional barrier in the intestinal immune tract and permitting the concurrence of commensal bacteria,
131
standing out from a barrier defect prompting dysbiosis and dysregulated immune responses (Brown et al. 2013)
IL-22 a Th17-type cytokine through transcription factor RORγt and also protect the
mucosal tissues from damage and inflammation.
8.7 Influence of Microbiota on the Immune System
8.7.1 Outside-In Signals of Microbiota
The microbiota colonization influences the formation of the lymphoid tissue, and the
development of the subsequent immune system was studied by comparing gnotobi-
otic mice and colonized mice which revealed that commensal microbiota influences
the immune system through outside-in signals. As it is well known that gastrointes-
tinal tissue contains a high number of myeloid and lymphoid cells embedded in
lymphoid tissue, which are indeed influenced by microbiota in developing the
lymphoid structures which leads to the adequate function of immune system cells
(Hooper et al. 2012). The influence on lymphoid structures can be studied through
commensal microbiome since it confers resistance through direct commensal–path-
ogen interaction by consuming particular nutrients that are required for pathogen
growth and also produce a small chain of fatty acids like acetate which limit the
pathogen growth (Momose et al. 2008; Fukuda et al. 2011). The host-microbial
homeostasis is maintained when the hosting immune system responds to the com-
mensal microbiota through TLR-MyD88 (Toll-like receptors) signaling pathway.
Toll-like receptors (TLRs) perceive particular pathogen-related sub-atomic
examples and assume a basic job in innate immune responses. They take part in
the mainline of defense against attacking pathogens and assume a critical job in
survival, immune cell regulation, inflammation, and proliferation (Kawasaki and
Kawai 2014). The enactment of the TLR signaling pathway starts from the cytoplas-
mic Toll/IL-1 receptor (TIR) domain that associates with a TIR domain-containing
adaptor, MyD88 (Myeloid differentiation primary response 88). Upon incitement
with ligands, MyD88 initiates IL-1 receptor-related kinase-4 (IRAK-4) to TLRs
through the interaction of the death domains of both molecules (Reuven et al. 2014).
RegIIIγ is an epithelial antimicrobial protein that is induced by the MyD88
depended on bacterial signals, lipopolysaccharide (LPS), and flagellin proteins
also inducing this expression (Vaishnava et al. 2011). The flagellin protein
stimulates the production of IL-23 through TLR5 expressed by
CD103 + CD11b + dendritic cells of lamina propria, which in turn stimulate the
IL-22 expression by innate lymphoid cells (Kinnebrew et al. 2012). The outside-in
signals can be studied in understanding the microbiome in influencing and shaping
the host immune system by comparing germ-free gnotobiotic and specific pathogen-
free mice. Generally, the role of commensal microbiota in shaping the host immune
system through outside-in signals is studied by comparing the axenic (gnotobiotic)
mice and specific pathogen-free mice. A reserch report stated that commensal
microbiota when colonized in axenic mice, developed Peyer’s patches, mature
mesenteric lymph nodes, increased levels of antimicrobial peptides, high production
of immunoglobulin A (IgA), dendritic cells (DCS), B and T cells, and also developed
goblet cells with a thick layer of mucus, which were absent in early axenic mice and
were normalized with the colonization of commensal microbiome (Macpherson
et al. 2004; Sommer and Backhed 2013).
8.7.2 Inside-Out Signals of Microbiota
To maintain the integrity of the immune system, the host immune system should
have homeostatic relation with microbiota, which is accomplished by maintaining
the controlled interactions between microbiota and host tissue along with the limited
composition of microbial affiliation. Inside-out signal deals with the internal, physi-
cal, and chemical control of the immune system on microbiota localization and
composition of the microbiota (Hooper et al. 2012). As the immune system counters
microbiota at an enormous rate compared to any other organ, it should control as
well as avoid the unnecessary innate immune signals that emerge from variation in
microbiota consortia without causing damage to the host metabolic functions. In the
intestine, to reduce the pathological reactions caused by microbial interaction with
the epithelial surface is achieved by separating the mucosal immune response
through stratification and compartmentalization (Mezouar et al. 2018).
Stratification is reducing or minimizing the interaction between the intestinal
luminal microbiota with the epithelial cell surface, which is achieved by immune
effectors which function in stratifying the microbiota. Mucin glycoproteins form a
thick viscous layer at the intestinal epithelial cell surface which is secreted by goblet
cells that are embedded in enterocytes of the intestine. The mucus layer is
differentiated in the colon, the outer mucus layer is exposed to a high number of
bacteria where the inner mucus layer withstands the bacterial penetration due to
dense impermeable mucus layer which consists of O-glycosylated MUC2 mucin
with ZG16 and β-defensins proteins (Hansson and Johansson 2010; Bergström et al.
2016). On the contrary, small intestine contains a heterogeneous mucus layer that is
outer and inner mucus layers which are not distinct (Johansson et al. 2011). The
intestinal mucus layer is rich in paneth cells, RegIIIγ C-type lectin is antibacterial
and also minimizes the bacterial penetration which is controlled by toll-like receptors
(TLRs) and intestinal specific immunoglobulin A (IgA) produced by dendritic cells
by interacting with B and T cells in Peyer’s patches (Macpherson and Uhr 2004;
Cash et al. 2006; Salzman et al. 2007). Where, in gnotobiotes, the commensal
bacteria stratification is dependent on IL-22 and IL-23 produced by the ILC3 (Mao
et al. 2018).
Mucosal compartmentalization is the function that confines the commensal bac-
teria from penetrating the luminal epithelial barrier by less exposure to the systemic
immune system (Mezouar et al. 2018). Although some of the intestinal bacteria
inevitably penetrate the laminal intestinal epithelial barrier causing immune
responses like phagocytosis by lamina propria macrophages such as dendritic cells
(DCs) that are embedded along lamina propria of the epithelial lining and then
migrate to the mesenteric lymph node (Iliev et al. 2012). These DCs in the
mesenteric lymph node induce the secretion of protective IgA antibodies and get
distributed along the surface of the lamina by activating the B and T cells through
interacting with the Peyer’s patch (Macpherson and Uhr 2004). Later these DC’s
cells after activating B and T cells leave through the thoracic duct and reach systemic
circulation and induce the mucosal response throughout the mucosal surface of the
host (Macpherson and Uhr 2004). Defective in the compartmentalization was
observed in immune-deficient mice, i.e., in engineered mice, the priming of serum
IgG antibodies response toward commensal bacteria is due to lack of IgA antibodies,
indicating the bacteria exposed to the systemic immune system. This is because the
mice do not have TLR-mediated MyD88 signals for recognizing bacteria, which
results in the successful penetration of commensal bacteria by crossing epithelial
barrier and phagocytic cells (Slack et al. 2009).
8.8 Microbiota Protection Against Autoimmune Diseases
The interaction between the host immune system and microbiota favors the autoim-
mune disease by segmented filamentous bacteria in Type1 diabetes (T1D), where the
damage of pancreatic islets of Langerhans leads to the imbalance of insulin secre-
tion. With some genetic predispositions and diabetogenic T cell populations with
defined CD4 and CD8, non-obese diabetic (NOD) mouse acts as a prompt model of
T1D (Schmidt et al. 1999). In the isogenic NOD colony, the prevalence of T1D
depends upon the presence of both pathogenic and microbiota diversity since they
act as determining factors (Hooper et al. 2012). Where, on the other hand, congenic
NOD mice with single-locus MyD88 deficiency show the same incidence of T1D as
the parent NOD strain (Kendall et al. 2009). The over access of intestinal microbiota
penetration with an epithelial barrier, interaction of commensal microbiota with the
systemic immune system are the major effects of host-microbiome mutualistic
relation which occurs due to the deficiency of MyD88 (Kinnebrew et al. 2010;
Carvalho et al. 2012). Since commensal bacteria help in the development of the host
immune system in immune-deficient patients, a little change in the commensal
bacteria community affects and triggers the inflammation responses. For example,
lesions in salivary glands, lungs, and feet are caused due to defect in tyrosine
phosphate SHP-I signal that leads to an autoinflammatory syndrome which is
microbiota dependent (Croker et al. 2008).
8.9 Microbiota in Modulating Immune Responses in Cancer
The effect of microbial composition on carcinogenesis and inflammation is tissue-

specific. Generally, the tumor-promoting effects of microbiota on the host immune
system are spontaneous, genetically driven, and cancer-induced through carcinogen
(Yu et al. 2010; Lofgren et al. 2011; Li et al. 2012; Grivennikov et al. 2012). It was
observed that the microbiota shows both tumor-promoting and also anti-tumor
effects. For example, the bacterial extract mixture of Bacillus Calmette-Guerin
(BCG) is used in the treatment of bladder cancer (Dias et al. 2018). The anti-
tumor effects are due to the innate immunity by converting tumor tolerance to the
anti-tumor response by TLR and NOD-like receptors of bacterial components
(Fukata and Abreu 2007; Garaude et al. 2012). Pattern recognition receptors
(PRR) generally initiate regulatory responses by monitoring microbial status and
barrier integrity may also promote resistance to cell death and by triggering cancer-
promoting inflammation. Genotoxins and tumor-promoting metabolites are the
carcinogenic molecules that are released by microbes, which are recognized by the
TLRs and microbe-associated molecular patterns (MAMPs) that promote carcino-
genesis (Ochi et al. 2012; Sheflin et al. 2014).
The recent method was developed by studying the commensal microbiota of the
host in immune therapy by adverting of ICBs. ICBs are the immune checkpoint
blockers that exhibit the function of reactivating T cells in an ineffective tumor
microenvironment in response to tumor antigens. The efficacy of ICB is greatly
influenced by the gut microbiome community type and number (Schwabe and Jobin
2013). The blocked off two checkpoints Cytotoxic T lymphocyte-association protein
4 (CTLA-4) and programmed-cell-death protein 1 (PCD-1) by monoclonal
antibodies are well known. Recent studies emphasized that distinct gut bacterial
species greatly influenced the immune-stimulatory and anti-tumor effects of the
CTLA-4 antibody (Clemente et al. 2012). The composition of gut microbiota in
the host shows a high frequency of difference in responses to PD1 blocker (Sivan
et al. 2015). For example, the natural gut microbiota Lactobacillus johnsonii and
Enterococcus hirae get altered and translocated by cyclophosphamide an
immunostimulatory alkylating agent, by which the bacteria stimulate the production
of specific Th17 and Th1 cell subsets (Sharma and Allison 2015).
8.10 Role of Metabolites in Microbiota and Immune System
Microbiota and its metabolites are significant instigators of host physiology and
pathophysiology through the control of a huge range of inflammatory, metabolic,
and indeed, even behavioral procedures (Hsiao et al. 2013). Distinctive microbiome
setups produce, adjust, and corrupt a huge exhibit metabolite, thereby giving utili-
tarian complementation to the metabolic limits of the host; e.g., complex proteins
furthermore, carbohydrates that cannot be corrupted by the host can be processed by
the microbial community (Nicholson et al. 2012). Intervened by metabolite signaling
through a progression of innate immune receptors of microbial metabolites, the
microbiota relates to the intimate communication with its eukaryotic host.
Single chain fatty acids (SCFAs) are one of the most produced molecules by gut
bacteria that control multiple aspects of metabolism and immunity. PPARγ intracel-
lular receptors, the surface proteins GPR41 and GPR43, and GPR109a butyrate
receptors detect the SCFAs like propionate, acetate, and butyrate which are the
products of dietary fiber fermentation (Alex et al. 2013). Administration of SCFAs
leads to alterations in hematopoiesis, resulting in an intensified myeloid output due
to elevated numbers of myeloid precursors (Khosravi et al. 2014). These precursors
promote the clearance of systemic infection and ameliorates the allergic reactions
(Balmer et al. 2014; Trompette et al. 2014).
8.11 Advancements to Identify Mechanisms of Immune

Interaction
The recent novel mechanisms have been created by the scientist to study the
microbial host interactions. By performing integration analysis of microbiota and
host data sets complementing them with the results of visual analysis for assessing
both the microbiome and host immune system can be successfully done by high
dimensional technologies t deciphering the novel pathways (Siebert et al. 2019). The
integrated high dimensional “omic” techniques are used to measure a vast number of
analytes/metabolites which serve as biomarkers that release in different proportions
based on response to treatment of diseases (Matson et al. 2018). The study of these
analytical biomarkers leads to the study of immune responses to therapy and
hypotheses of mechanical insights into the immune system (Arneson et al. 2017).
The high dimensional omic technologies include metatranscriptomic sequencing,
shotgun metagenomics, and metabolomics, these advancements profoundly describe
the phenotypes of both host and microbiome generating speculations concerning
immune modulation of significant disease environment (Lozupone 2018). Due to
poor comprehension of what these organisms, transcripts, and genes do, translation
of results and potential ramifications of correlations between organisms, transcripts,
and qualities turn out to be a very test in utilizing these progressions.
8.12 Conclusion and Future Perspectives
Even though propels in cutting edge (next-generation) sequencing and bioinformat-

ics have been significant drivers of progress in human microbiome research, there
have likewise been other key advances that have empowered unthinking bits of
knowledge. These remember propels for our capacity to culture diverse intestinal
microbes and to genetically manipulate bacteria so that the impacts of addition or
loss of specific functions can be assessed, the development of animal models, for
example, gnotobiotic mice for building up causality, and mix of other front line
innovations, for example, metabolomics. Despite these gains, there are as yet
numerous difficulties, for example, the high number of qualities and metabolites of
obscure capacity and of bacteria with ineffectively comprehended properties that
dodge development and the absence of accessibility of tools to genetically manipu-
late the majority of those that we can culture. The most energizing and translational
work incorporates complex multi-omic and bioinformatics strategies with corrobo-
rative test work to build up an unthinking connection between microorganisms and
disease.
It is currently certain that the safe framework assumes a focal job informing the
structure of the microbiota just as its nearness to have tissues. Simultaneously,
inhabitant microorganisms give flags that encourage ordinary insusceptible frame-

work improvement and impact the resulting safe reactions. Interruption of these
complex and dynamic connections can have significant ramifications for host health.
Notwithstanding, there are as yet significant gaps in our comprehension of how the
host immune system regulates the microbiota, and of how the microbiota shape has
invulnerability. The inquiries that remain are testing and will require the advance-
ment of new tools and approaches. At last, these endeavors should prompt further
understanding to have microbial connections and give compelling new chances to
improve human health.
Acknowledgments Kuraganti Gunaswetha, Edla Sujatha, Pallaval Veera Bramhachari are grateful
to Kakatiya University, Warangal and Krishna University, Machilipatnam for the support extended.
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Part III
Microbiome for Human Health: Clinical
Applications
Intestinal Microbiome–Macromolecule
Signaling That Mediates Inflammation 9
and Immune System Interaction
S. Anju, Y. Aparna, K. Anuradha, Pallaval Veera Bramhachari, and

J. Sarada
Abstract
An established microbiome maintains the homeostasis of the host systems and

entails specified tasks in stages of development of the host immune system.
Synchrony between the host metabolism and the microbiota is critical for a
competent immune system. Clostridium strains have been initiating the produc-
tion of Tregs, which considerably are recognized to curb induced colitis in the
experimental conditions. Faecalibacterium prausnitzii is a clostridial organism
protecting patients from the onset of inflammatory bowel disease (IBD). The
signal transduction pathway of Bacteroides fragilis is known to regulate the
production of factors responsible for the differentiation of IL-10 secreting
Tregs. Polysaccharide A secreted by Bacteroides fragilis induces Treg cell
development utilizing TLR2 signaling pathway. It can also potentiate and trigger
the signal transducer of transcription factor 3 –STAT3 and recruit the Th17 cells.
The gut biota also is responsible for sustaining the recruitment of Th1/Th17 and
protect against pathogens. Studies reveal CD+8 cells delegating cell-mediated
response get triggered as part of the skin flora Staphylococcus epidermidis.
An alteration in the normal flora results in an imbalance of the regulatory
network. The loss of equilibrium in the commensal community is termed as
‘dysbiosis’. The role of dysbiotic microbiota is indispensable in the creation of
an inflammatory environment in the gut. Dysbiosis in the microbial flora can also
lead to chronic inflammation as part of colonic carcinogenesis. These microbes
secrete the pro-inflammatory MAMPs or metabolites that may exert damage on
S. Anju (*) · Y. Aparna · K. Anuradha · J. Sarada

Bhavan’s Vivekananda College of Science, Humanities, and Commerce, Sainikpuri, Secunderabad,
India
P. V. Bramhachari
Ltd. 2021
https://doi.org/10.1007/978-981-16-3156-6_9
146 S. Anju et al.
the host organs. Fusobacterium nucleatum triggers the inflammatory signaling

pathways and functions to shield the cells from the immune system. It manages to
abolish the NK cell-mediated destruction of the cancerous cells. It also causes
enriched myeloid cell infiltration. Even macrophages and dendritic cells that
primarily mediate inflammation are induced by the microbiota and their meta-
bolic products.
Keywords
Microbiota · Intestinal microbiome · Inflammation · Immune system
9.1 Introduction
There are about a thousand different types of bacteria in the gut microbiota part of
the colon of humans among which the majority of them remain uncultured. The
microbiome part of the animal system in the gut has a very specific metabolic profile
that is likely to complement that of the host. This results in the unique biochemical
profile of any individual and therefore results in the health status as well as the
immunity. The human gut microbes have a collection of metabolic enzymes that can
complement the host metabolism. Certain enzymes are unique to these
microorganisms which help the host in the biosynthesis of vitamins, and breakdown
of polysaccharides, polyphenols, etc. Several studies involving the fecal microbiota
have established the role of the microbiome in the development of gastrointestinal
diseases. There is also evidence indicating the establishment of diabetes and obesity
as an influence of gut microbes (Rowland 2018). Above all this, there is known to be
a bidirectional communication through the gut–brain axis (Yarandi 2016; Rowland
et al. 2018) (Table 9.1).
Several signaling pathways trigger the immune system as an interaction based on
the metabolic products and the microbe’s part of the gut. In certain cases of obesity
and metabolic syndrome-related disorders, there is evidence that the gut microbes
interact with the innate immune receptors and lead to disorders like acute inflamma-
tion. Contrarily, the right interactions of the probiotic microbes can overcome the
negative outcomes that lead to obesity and are known to release the anti-obesity
factors. There is an inevitable role of the microbes in the development of the
gut-associated lymphoid tissue (GALT) and immune tolerance (Cavalcante et al.
2015). The inflammatory and oxidative stress responses that are supposed to regulate
cardiovascular function are modulated by adenosine monophosphate-activated pro-
tein kinase (AMPK)/nicotinamide adenine dinucleotide phosphate (NADPH). The
concentration of AMPK/NADPH is identified to have been a result of the metabo-
lism of short-chain fatty acids by the gut microbiota. The mucosal immune system is
the primary site for many allergens and the development of immunological response.
To tackle the allergic responses on this basis is to contemplate the probiotic diet as
one of the solutions (Rautava et al. 2005).
9 Intestinal Microbiome–Macromolecule Signaling That Mediates Inflammation. . . 147
Table 9.1 Pathways of carbohydrate metabolism and end products of various gut microbes
Pathway of carbohydrate Metabolic end product
Gut microbiota metabolism released
Methanobrevibacter Methanogenesis Methane
smithii
Roseburia sps Butyryl pathway Butyrate
Blautia Wood Ljungdahl pathway Acetate, formate
hydrogenotrophica
Desulphovibrio Sulphate reduction Hydrogen sulphide
Ruminococcus bromii Glycolysis Ethanol
Eubacteriumrectale Butyryl pathway Butyrate
Eubacterium halli Glycolysis Lactate
Anaerostipes sps Butyryl pathway Butyrate
Coprococcus cactus Butyryl pathway Butyrate
Coprococcus eutactus Butyryl pathway Butyrate
Faecalibacterium Butyryl pathway Butyrate
frausnitzii
Coprococcus comes Butyryl pathway Butyrate
Megasphaera elsdenii Acrylate pathway Prpionate
Bacteroidetes Succinate pathway Propionate
Veillonella sps Succinate pathway Propionate
There exists a host–microbe cross-talk, an important factor for the reciprocal

advantage and undisturbed coexistence. It is also assumed that a greater understand-
ing of probiotic interactions can lead to the elucidation of gut-related or many other
disease aberrancies. They are a means to induce the anti-inflammatory effects which
are essential for immune regulation. There are diverse immune-regulatory responses
that are known to modulate the TH1 and TH2 regulator T-cell subclass. These cells are
regulated and suppressed concerning the specificity of the antigen and based on the
clonal anergy mechanism. There are gut-derived regulatory cells TH3 and TR1 that
show the effects by the production of the cytokines (transforming growth factor)
TGF-β and IL-10, respectively. A great deal of control is established in protecting
the host from the atopic immune response and autoimmune disorders by generating
an adequate immune response (Rescigno 2014). Microbiome and metabolome
evaluation has revealed that microbial metabolites like short-chain fatty acids,
acetate, and butyrate are important in the immunity, inflammation, regulation of
epigenetic mechanisms, and overall intestinal integrity (Neu and Pammi 2018).
9.2 Interactions of Microbiota and Immune System
The intestinal microbiome has an indispensable role in the metabolism of complex

macromolecules and simultaneously channelizing them to various physiological
functions. The microorganisms in the gut establish in the surface epithelial cells
and interact with the mucosal lymphoid tissue, to result in the maturation of the
148 S. Anju et al.
immune system. In the early developmental phases, the immune system educates
itself to distinguish between beneficial and harmful microbes. One of the studies has
established the role of the zebrafish gut microbiota in maintaining the alkaline
phosphatase activity and maintaining the expression of the glycan (Bates et al. 2006).
The microbiota in various parts of the human body tends to establish the homeo-
stasis along with the immunostasis (Veronica Lazar 2018). The gut of human beings
is heavily populated with bacterial species which are of numbers nearing
100–400 1012. The mucosal layer of the GIT (gastrointestinal tract) is a double
layer that is formed by the O-glycosylated mucin protein which is encoded by the
MUC2 gene of the mucin protein family. The bacterial species mostly adhere to the
outer mucus layer; however, the inner mucus layer forms the physical barrier that
limits the entry and interaction of bacteria with the epithelium. The majority of the
microbial species that get established in the intestinal tract are acquired through the
mother’s milk in early life. They are generally predominated by Bifidobacterium and
Lactobacillus species. With the advancement of the formative years from childhood
to adult life, augmentation of the food sources increases the complexity and diversity
of the bacterial population. These bacterial species are now included with
Bacteroides, Parabacteroides, and Clostridium (Firmicutes). Bacterial numbers
increase progressively toward the large intestine from the stomach or duodenum.
The largest diversity of taxa and the numbers of bacterial species are identified in the
colon that is represented as 109–1012 c.f.u. (colony forming units) per ml that is
about 99% of the total GI population (Arnold et al. 2019).
The development and the mediation of the immune system that is part of the
gut-associated lymphoid tissues (GALT), Peyer’s patches, isolated lymphoid
follicles, and mesenteric lymph nodes have been known to be influenced greatly
by the gut microbiome. During the immune system development of an individual,
the distinction between the self and nonself by the innate cells is by and large due to
the microbes and their products released in the host system. They are responsible for
the activation and maintenance of the innate hematolymphoid cells, natural killer
cells (NK cells), cytotoxic, non-cytotoxic, and helper lymphoid cells. Also, the NK
cells and ILC1 produce huge amounts of IFN-γ, antimicrobial peptides (AMPs),
granulysin, defensins, and RegIIIγ that regulate the ecological balance of the popu-
lation of the microbiota and also bring in the component of the immune surveillance.
The microbial products like tryptophan, polysaccharide A, and α-galactosylceramide
are known to stimulate the production of IL-22, IL-17, and IgA that play a key role as
immune mediators. Thus, the microbes need to be part of the host as they involve as
a closed framework along with the immune system to either suppress or stimulate the
immune response (Belizário et al. 2018).
A chronic inflammatory condition of the gastrointestinal tract, clinically the
condition referred to as inflammatory bowel disease, is a perfect example of the
immune response showing exaggeration. Many studies have reflected that this
condition is influenced largely by dietary factors. Some of the crucial components
in the diet such as the fiber content and vitamin D are known to have importance in
the development of the disease. The imbalance in the gut microbiome also greatly
alters the nutritional status of the host and that has an impact on the innate and
Fig. 9.1 Host–microbiota interactions mediating inflammation
adaptive immune responses. Microbial metabolites derived from their metabolism

supplement the host nutrition, and these macromolecules mediate the regulation of
the immune responses that can lead to a specific measure of the inflammation
expressed (Celiberto et al. 2018).
Filamentous bacteria, Bacteroides, Proteobacteria, and Acinetobacter that are
associated with closer to zones of intestinal epithelium raise the strongest immune
response. The capsular polysaccharide of Bacillus fragilis stimulates anti-
inflammatory cytokines like IL-10, leading to colonization and promoting necessary
immunosuppression in the intestine. Simultaneously, the outer membrane vesicles of
these bacteria activate the autophagy that induces the T regulatory cells resulting in
suppression of mucosal inflammation. Enteric Citrobacter, Clostridium ramosum,
and Mucispirillum are some of the commensal bacteria that are relevant, and regulate
the immune responses that are critical in maintaining the intestinal homeostasis
(Blander et al. 2017). (Fig. 9.1).
9.3 Mediators of Inflammation
The gut microbiome and immune system are essential parts of gut–brain communi-
cation that involves neuroendocrine and autonomic nervous systems. Enteric
neurons communicate the intestinal conditions to intestinal muscularis macrophages
via β2-adrenergic receptors and to the vagus nerve. Numerous members of
microbiota produce neurotransmitters and neuropeptides like dopamine and acetyl-
choline. These molecules in a study have been found to induce intestinal epithelial
cells of the mouse to release molecules that modulate signaling within the enteric
nervous system. Also, it has been concluded that microbes, like Enterococcus and
Bacteroidetes, noticeably increased in numbers in the presence of GABA-
supplemented groups. KEGG enrichment analysis revealed that the nitrogen metab-
olism, sphingolipid signaling pathway, sphingolipid metabolism, and microbial
metabolism in different environments were enriched in the GABA1 group (Rees
et al. 2018).
150 S. Anju et al.
Fig. 9.2 Mediation of neuroinflammation by microbiota
Alterations in microbial metabolism were very much concurrent with changes in

the abundances of Enterococcus and Bacteroidetes. In conclusion, GABA supple-
mentation can improve intestinal mucosal immunity by promoting jejunal SIgA
secretion, which might be related to the T-cell-dependent pathway and transformed
gut microbiota structure and metabolism (Zhao et al. 2020). Intestinal inflammatory
disorders are identified with neurophysiological and behavioral symptoms. Some
disorders of the central nervous system (CNS) are found to be accompanied by
intestinal complications. Certain observations indicate that intestinal and nervous
system physiologies are functionally linked. Multiple pathways that are having
bidirectional communication between the intestine and the CNS are collectively
referred to as the gut–brain axis. Some of the microbes naturally colonizing the
mammalian gastrointestinal (GI) tract play a causative role in regulating CNS
function, development, and host behavior. However, members of the gut microbiota
are potent modulators of intestinal, systemic, and CNS-resident immune cell func-
tion, implying the role of gut–brain interactions that may involve the host immune
systemic disorders associated with the gut microbiota including neuroinflammatory,
neuropsychiatric, and neurodegenerative disorders that also have significant inflam-
matory manifestations (Fung 2020) (Fig. 9.2).
Current advances in technology have enabled deep sequencing and analysis of
members and signals of microbial communities. In a healthy state, the microbiome is
composed of commensals and their genes and phenotypes that may be selected by
the immune system to endure in symbiosis. These extremely synchronized signals
are modulated by a network of microbial and host metabolites (Kleinstein et al.
2020; Rosen and Palm 2017).
Shreds of evidence in a study indicate that modulation of central nervous system
by the microbiome occurs mostly through a neuroimmune and neuroendocrine
mechanism that involves the vagus nerve communication which is mediated by
several microbially derived molecules like short-chain fatty acid, secondary bile
acid, and tryptophan metabolism. These molecules propagate signals by the interac-
tion of endocrine cells and the mucosal immune system. But some cross the intesti-
nal barrier and thereby enter the systemic circulation. Some may cross the blood–
brain barrier in addition to generating these metabolites that activate endogenous
central nervous signaling mechanisms. The microbiota can independently produce
several neuroactive molecules such as gamma-aminobutyric acid, norepinephrine,
and dopamine that bind to relevant receptors eliciting the host immune response
(Gilbert et al. 2018; Zinöcker and Lindseth 2018).
Very important signaling pathways are carried out in the gut by microbes and
their metabolites. These macromolecules communicate with central nervous system
that is involved in the communication with the endocrine system. There are at least
12 different types of cells with several subtypes and subgroups which along with the
cells of the intestine that get triggered with combinations of molecules are crucial in
these signaling pathways. Intestinal cells that are interspersed between epithelial
cells throughout containing more than 20 different types of signaling molecules that
are released in the response to chemical or mechanical stimuli. These molecules
enter the systemic circulation and reach the central nervous system. These molecules
regulate the receptors involved in satiety and hunger, and thereby have been
identified on the cells which are activated by microbial metabolites including bile
acids. Endogenous molecule synthesis like bile acids is heavily influenced by the
downstream metabolism by the gut microbiota. Expression of farnesoid X receptor
(FXR) that is activated by bile acid leads to the production of fibroblast growth factor
19 (FGF19). Furthermore, this leads to action on peptide or neuropeptide that is
supposed to work for improved central regulation of energy and glucose metabolism.
Some of the intestinal G protein-coupled bile acid receptors, like TGR5 expressed on
the intestinal surface as L cells, are also activated by bile conjugates that result in the
glucose homeostasis. Commensal bacteria with the gut-associated immune system
have a substantial role in the gut microbial regulation of autoimmunity and inflam-
mation. Microbiota influences the development and function of the central nervous
system of resident immune cells like microglia influencing its maturation (Martin
et al. 2018).
Products of bacterial metabolism like short-chain fatty acids (SCFAs) and hydro-
gen sulfide act as messengers to colon epithelial and immune cells. SCFAs are
known to be very crucial in maintaining colon homeostasis. Acetate, propionate,
and butyrate are the three categories of SCFAs produced in the colon by bacterial
fermentation of carbohydrates. These are identified as an important source of energy
for epithelial cells in the colon. SCFAs interact with the ligands in the intestine and
modulate glucose metabolism. SCFAs regulate (peroxisome proliferator gamma coo
activator) PGC1α which is a master regulator in mitochondrial biogenesis. These
molecules together coordinate in glucose uptake, oxidative phosphorylation, and
fatty acid oxidation. An inflammasome NLRP3 (nucleotide binding oligomerization
domain) is activated by the SCFAs that induce the release of IL-18. This event
regulates the microbiome consortium and takes over the inflammatory responses.
Bacteria like E.coli and Salmonella in the intestine are known to produce
152 S. Anju et al.
Table 9.2 Cytokine modulation: gut mucosal innate mechanisms

Lactobacillus sps. IL-22
Bifidobacterium IL-10
E. coli IL-6, IL-8
Microbial DNA INF -β
Epithelial cells and IL-10,IL-22,IL-4, IL-6, IL-33,IL-18, IL-13,IL-21,IL-17,IL-1β,
submucosa cells TNF, IFN, TGF-β
anti-inflammatory hydrogen sulphide as a by-product of the degradation of sulfur-

containing amino acids (Jackson and Theiss 2019; Blander et al. 2017) (Table 9.2).
More intake of calories leads to the accumulation of fat, leading to lipotoxicity
that results in the production of effector molecules (cytokines) and cells that are
primarily involved in innate immunity. This causes a low-profile inflammatory
condition due to the accumulation and activation of mast cells, dendritic cells, and
macrophages of several tissues. Several inflammatory pathways are activated during
the attenuation of insulin signaling. This in turn leads to several metabolic
abnormalities. The incidence of chronic inflammation in obesity-related cases is
mainly due to the immune mediators triggered in impaired insulin action (Boulangé
et al. 2016.
Nutrients influence the way the immune cells are triggered for any immune
response. Most importantly, the macrophages that are known to function differently
in various pathological conditions are accordingly stimulated by diversified
macromolecules. Macrophages exist in two major phenotypes (M1 & M2) that are
stimulated differentially based on the consumption of glucose, oxygen, and gluta-
mine. The signal molecules involved in the activation are bacterial
lipopolysaccharide (LPS), pro-inflammatory components TNF, interferon-γ, IL-1,
interleukin-4, and interleukin-13. Anti-inflammatory cytokines, TGF, IL-10, and
glucocorticoids are crucial in the regulation of the inflammation. The effector
activities of the macrophage M1 phenotype are supported by the chemical mediators
ROS (reactive oxygen species), ATP, NADPH, nitric oxide, and utilization of the
glucose (Belizário et al. 2018).
9.4 Mitochondria and Gut Microbiome Signaling
Epithelial barrier function is essential in intestinal homeostasis. Histone deacetylase

(HDAC) inhibitors when investigated have been found to regulate immune
responses. In human and murine colonic epithelial cell lines, the presence of the
HDAC inhibitors givinostat and vorinostat improved transepithelial electrical resis-
tance in inflammatory conditions. They also silenced the passage of macromolecules
across the epithelial barrier. Mechanistically, these regenerative effects could be
linked to increased secretion of transforming growth factor beta1 and interleukin-8,
paralleled by differential expression of the tight junction proteins claudin-1, claudin-
2, and occludin (Friedrich et al. 2019).
The gut microbes are known to be a dynamic key role-player in regulating the
mitochondrial function of the intestinal and in turn influence the immune cells as
well as the epithelial cells of the gut. The immune system is greatly influenced by the
gut microbiome and the mitochondrial reactive oxygen species from the mitochon-
drial system. Epithelial barrier integrity and intestinal epithelial mitochondria both
serve as a site of action as well as facilitators of inflammatory pathways. Studies on
colonic epithelial cells targeting the mitochondrial activity showed stimulation of gut
microbiome immune pathways related to inflammation by an AMPK-mediated
mechanism. Decreased oxidative phosphorylation is identified to trigger intestinal
inflammation. A pro-inflammatory cytokine TNF-α, which plays a central role in
IBD inflammation, inhibited mitochondrial function, promoted mtROS accumula-
tion, and thereby resulted in impaired barrier function. These mtROS produced in the
immune cells play an important role in the eradication of several pathogens by a
bactericidal effect. NLRP3 inflammasomes produce pro-inflammatory cytokines that
invade bacteria. Fermentation products of the gut microbiome such as short-chain
fatty acid-induced mitochondrial reactive oxygen species production and the
immune cells and produced during mitochondrial respiration enhance the oxidative
phosphorylation activation and induce Toll-like receptors (TLRs) 1, 2 or 4 at the
plasma membrane or endoplasmic reticulum membrane. This controls the rate of
bacterial or viral infection and subsequently induces the release of accumulation
macrophages as well as neutrophils. However bacterial-induced mitochondrial
responses are detrimental to gut epithelial tissues. Epithelial cells of the intestine
when showed mitochondrial dysfunction the tissues had lost the ability to tolerate the
commensal microbiome and were dependent on the IFNα and TNFα. Mitochondria
have an inevitable role in maintaining the homeostasis of the intestine. It can initiate
the mediation of the innate and adaptive immune responses, in turn promoting
inflammation and immune pathways. Functions of mitochondria are crucial to
propagate the role of epithelial barrier integrity during inflammation and play an
important role in tumorigenesis. The intestinal microbiome modulates mitochondrial
signaling in mucosal cells. Studies to understand mechanisms connecting the gut
microbiota and mitochondria of the host during the pathogenesis of IBD and CRC
will be the turning point in the targeted therapy. Bacterial and mitochondrial
signaling is known to inhibit the apoptotic pathway in the intestinal epithelium. A
better understanding of whether direct bacterial toxins or bacterial metabolites are
involved in the mechanism whereby bacterial-to mitochondrial signaling enhances
colon tumorigenesis is a future gap in the field that can be explored and build new
avenues in the treatment strategies (Jackson and Theiss 2019).
9.5 Cytokines Influenced by Gut Microbiota
There are a variety of cytokines produced as a result of the interactions generated due
to the number of metabolites released by the gut microbiota. Cytokines play a very
important role in generating inflammatory responses in various infectious diseases
and thereby develop intestinal and extra intestinal inflammations. This results in
154 S. Anju et al.
generating host inflammatory responses which are otherwise a consequence of

microbiota cytokine interactions. Cytokine production may be directly influenced
by the microbiota, or sometimes, there are indirect means by which they become
responsible for it. These interactions lead to cytokine-mediated molecular pathways
throughout the body. These innate inflammatory cytokines are responsible for the
intestinal homeostasis (Knight et al. 2018).
Pro-inflammatory interleukin IL-1b initiates a series of cascades of other
cytokines and substances that promote inflammation. Stimulation of inflammation
and protective role of IL-1b is regulated by the influence of the microbiota and
therefore maintains the intestinal homeostasis. Colonic bacteria are known to raise
the levels of pro-inflammatory cytokines in the gut and promote the formation of the
inflammasome complex. IL-18 promotes the inflammatory responses by inducing
the INF-α and also downregulates the IL-22. It is observed that during IBD,
alterations in gut microbiota and raise in the macrophages and T-helper cells
occur. High levels of IL-6 are found during the E.coli infection that triggers the
pro-inflammatory responses as a protection to the host. A large range of gut
microbiota in the epithelial cells as well as submucosal region produce cytokines
IL-18, Il-6, TNF, INF, IL-33, IL-17, IL-21, Il-13, IL-1β, and Il-10, IL-22, IL-4,
TGF-β, respectively. The gut has a complex interplay of the cytokines, and therefore,
there are critical conditions established whenever conditions that disturb homeosta-
sis are established. Interactions among cytokines based on the concentration levels
and the types of receptors or cells there may be anti-inflammatory and
pro-inflammatory mediators in the gut. Certain pro-inflammatory mediators are
known to promote epithelial proliferation essential for wound closure or healing.
On the contrary, it may also promote carcinogenesis if the actual function of healing
is not taking place and there is a deviation in the mechanism. It is interesting to note
that cytokine profile of the gut can encourage the survival and functioning of the
microbiota synergistically to the host mechanisms. At the same time, there is also
suppression of the microbiota as adverse effects wherein the immune system is
activated to eliminate the microbes in the gut and hence may lead to depletion or
reduction of certain species of the normal flora in the intestine (Mendes et al. 2019).
9.6 Conclusion
Microbiota host interactions along with many macromolecules generated and

introduced through various pathways modulate inflammatory responses establishing
critical conditions. Intestinal inflammation may be due to the activity of microbiota,
in correlation with the state of host health or disease or maybe certain other
components that are yet to be elucidated. Controlled immune responses that generate
appropriate amounts of mediators to either promote or suppress inflammation are
very crucial in maintaining the host homeostasis. Host inflammatory response and
microbial interactions are dynamic and complex that is important in the regulation of
the pro-inflammatory and anti-inflammatory pathways. There is a fact about an
indispensable link between the human microbiome and diseases. Analysis of the
microbiome components by methods like DNA sequencing of the genomes from the
human samples and further introspection using transcriptomes, proteomes, etc., can
be instrumental in understanding the dynamics of this invisible community.
Acknowledgments We acknowledge principal and management of Bhavan’s Vivekananda Col-

lege of Science, Humanities and Commerce, Sainikpuri, for their support and encouragement in
writing this book chapter.
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Microbiome Diagnostics and Interventions
in Health and Disease 10
Nandini Dasgupta, Alka Srivastava, Amrita Rao, Vrishali Murugkar,
Ravi Shroff, and Gautam Das
Abstract
The growing evidence of literature, correlating the importance of balancing

noninfectious microbes inhabiting our bodies in disease and health, has given
birth to a new field of medicine—microbiome therapeutics. Composition of
microbiome evolves with us right from birth, impacted by several factors like
an individual’s genetic makeup, quantity and quality of the different foods that we
consume, and the environment that we interact with. A change in the composition
of our microbiome (gut, skin, lung, gastric, vaginal, oral) may trigger or predis-
pose us to a disease condition before clinical manifestation of symptoms. The
current trend to better understand these correlations in health and disease is by
leveraging metagenomics, metabolomics, data mining, artificial intelligence, and
machine learning tools for human microbiome diagnostics. Encouraging results
have been obtained with therapeutic strategies using prebiotics, probiotics, sig-
naling molecules, antimicrobial peptides, and microbiome transplant in
alleviating disease symptoms and promoting well-being. This is generating
increased interest in the medical and scientific community and awareness in the
public. The emerging concepts of ‘smart sampling’ using 3D printed devices,
engineering diagnostic and therapeutic bacteria using synthetic biology, and
using microbiome engineering to restore niche-specific balance are some addi-
tional paths that scientists are pursuing to arrive at a viable solution. In this article,
we will address the challenges and potential solutions of microbiome diagnostics
and therapeutics.
N. Dasgupta · A. Srivastava · A. Rao · V. Murugkar · R. Shroff · G. Das (*)

miBiome Therapeutics LLP, Mumbai, Maharashtra, India
Ltd. 2021
https://doi.org/10.1007/978-981-16-3156-6_10
158 N. Dasgupta et al.
10.1 Introduction
Hundred trillion invisible living beings (grouped together as the microbiome) live
inside humans. A delicate balance between the quality and quantity of the microbes
that live inside our body is vital for our well-being. Growing scientific evidence
suggests that the microbiome is intricately connected to human health, wellness, and
treatment of certain disorders. As the cells of our bodywork tirelessly to perform the
necessary functions for us to live, so do the microbial communities within our body
right from our birth. They keep performing jobs that benefit us like breaking down
foods, aiding nutrient absorption, protecting against pathogens, and aiding in immu-
nity. According to scientific reports, around 1000 different species of bacteria reside
in our gut; therefore, any imbalance (‘dysbiosis’) can result in a variety of problems
to human health. An estimate suggests that 85% ‘good bacteria’ and 15% ‘bad
bacteria’ are tolerated by the human gut; deviations from this ratio cause
perturbations in the digestive system and various illnesses, also affecting our own
immunity. Many of us would have experienced that when doctors prescribe
antibiotics to kill harmful bacteria that invade us occasionally, they also recommend
consuming probiotics as supplements. This is for the simple reason that while
antibiotics do an excellent job killing ‘bad bacteria,’ they also eliminate some of
the ‘good bacteria’ from our system, thus creating an imbalance. This results in
diarrhea and many other gastrointestinal problems. Supplementing with probotics
helps in restoring the loss of the ‘good bacteria’ and gradual reversal of symptoms
associated with gut dysbiosis.
10.2 Need for Human Microbiome-Based Diagnostics
According to the World Health Organization, a state of good health is not actually
the absence of a disease; rather, it is a state of complete mental, physical, and social
well-being (WHO 1946). Based on patient examination, clinical history, associated
symptoms, and diagnostic tests, health professionals diagnose the nature of the
illness. They prescribe appropriate therapeutics, life style changes, or a combination
of both, to treat the illness and improve the health of the patient. Diagnostic is a
symptom or characteristic of value in diagnosis. Microbiome diagnostics, pertaining
to the identification of the imbalance (if any) in the human microbiome, has been
more of a research endeavor in the past. With the growing evidence in literature, of
changing microbiome profiles (both abundance and diversity) correlations in differ-
ent disease conditions, its cause and consequences on effectiveness of
pharmaceuticals (Vieira-Silva et al. 2020); the importance of microbiome
diagnostics as a companion in the clinical medical diagnostics tool box is emerging
rapidly (Raes 2016). Diagnostic applications of the microbiome in the past were
focused on the pathogenic microbes, but we now know the relevance of monitoring
nonpathogenic microbial components of commensals associated with many
noncommunicable chronic diseases. For example, Hollister et al. (2019), were
capable to clearly differentiate children with irritable bowel syndrome, also called
10 Microbiome Diagnostics and Interventions in Health and Disease 159
as I.B.S., a chronic condition, from the children who were healthy, by profiling the
intestinal microbes, their genes/pathways, and metabolites. With the whole world
reeling under the COVID-19 pandemic, a contagious disease whose pathophysiol-
ogy is not yet completely understood, microbiome diagnostics has the power to
query the changes happening at the molecular level in niche-specific microbiomes in
both cross-sectional and longitudinal studies in patients (He et al. 2020). The
knowledge gathered will empower research scientists and health professionals to
build an appropriate repertoire to counter the pathology of SARS-CoV2.
Human orifices, and organs like the gut, lung, and skin, are abundant with niche-
specific microbial species including bacteria, archaea, fungi, virus, and protozoa
(mainly gut). Each one of us acquires a largely distinctive microbiome early in life.
The same may persist with us for years or may undergo changes in compositional
diversity/abundance. Such changes may correlate with a change in the environment,
health status, or lifestyle. The niche-specific microbiome components are known to
differ between environments and populations (Integrative HMP (iHMP) Research
Network Consortium 2019), but certain indicator species are conserved across
human populations studied around the world. This suggests that a symbiotic rela-
tionship between these indigenous organisms and human physiology cannot be
ignored. However, the ‘cause-consequence’ problem of perturbations in the
microbiome that are associated with a health condition remains to be understood
in detail in several diseases. Some questions still remain: Are the molecular
components of an individual’s microbiome responsible for health outcomes? How
do they combine with and maintain critical physiological processes like the immune
system and metabolism?
Recently, gathered evidence clearly suggests that the composition of the gut
microbiome has a correlative effect via modulating at least the brain, lung, and
liver. Many reviews have described these microbiomes with respect to their modu-
lation and interplay with host factors at a greater depth.
Classically, culturing microorganisms and their identification from various clini-
cal samples were the basis of ‘germ theory’ for any etiological agent and the earliest
tool for microbiome diagnosis. But we now know the unculturable plethora of
microbes that inhabit our body, and efforts are ongoing in ‘culturomics’ to narrow
this gap by combining extensive laboratory culturing conditions followed by identi-
fication using mass spectrometry (Lagier et al. 2012; Lagier et al. 2018). Attempts at
using real-time PCR as a diagnostic for microbiome composition (Ott 2004) at a time
when Next-Gen sequencing (NGS) was just launched could not give the true picture
of the microbiome since it estimated the abundance of only the dominant 20 patho-
genic and commensal species in the intestinal bacterial flora. The advent of NGS has
revolutionized the approach that researchers have at their disposal today and together
with MALDI-TOF mass spectrometry, it is poised to take microbiome diagnosis to
the next level with precise study designing, controls, unbiased analysis of data and
reporting. A desirable outcome that would immensely help clinicians to serve their
patient better would be the availability of NGS backed biomarker diagnostic assays
for a disease condition or its prediction. How to identify these biomarkers? Novel
noninvasive diagnostic biomarkers for colorectal cancer diagnosis have been
successfully identified (Liang et al. 2017) based on metagenome sequencing analysis

of fecal bacterial marker candidates and adapted for a qPCR assay.
10.3 Challenges in Design, Analysis, and Interpretation
For niche-specific microbiome diagnostics, it would be ideal to have reference

ranges of microbial species or their metabolites (in a healthy or faulty microbiome)
that a doctor could use for differential diagnosis. Among human microbiomes
studied worldwide, the gut microbiome has probably been researched the most due
to the ease of sample collection, abundance of microbes, and apriori knowledge.
Unfortunately, due to many unresolved factors, the availability of a dependable
diagnostic test, based on microbiome analysis, is still in the developmental stage.
Though there is an abundance of microbiome data, the taxonomic changes identified
in a disease are not consistent across different studies. The underlying reasons for the
incoherence may have arisen due to variations in sample population (including diet,
lifestyle) and the different technological approaches used in the diverse studies. In
addition, a key problem in the field is to define the ‘healthy’ microbiome, owing to
the large degree of variation in the microbiome composition among healthy
individuals. Hence to stay relevant, all efforts toward identifying microbial markers
for disease diagnostics must be based on comparisons with parallel control groups of
healthy individuals (Versalovic et al. 2017).
An ambitious project launched in 2007 in the USA (Turnbaugh et al. 2007),
termed the HMP or the National Institutes of Health’s Human Microbiome Project,
was a one of its kind, large-scale initiatives to resolve the burning issues mentioned
above (Gevers et al. 2012a, b). The first phase of the program involved generating
massive amount of data and putting together the different analysis platforms to
determine the composition of the ‘healthy’ microbiome (absence of evident disease).
A baseline adult population (Huttenhower et al. 2012; Lloyd-Price et al. 2017) and
‘demonstration’ populations with specific disease states were studied to determine
characteristic ranges (for some populations) of various microbiome-host parameters.
The parameters that were included are as follows: (1) combinations of metabolic
functions that are either ubiquitous or specific to the strain; (2) enzymatic repertoires;
(3) some host factor, such as race or ethnicity. Information generated comprised of
nucleotide sequences of microorganisms and human population (http://hmpdacc.
org), protocols for body-wide microbiome sampling and data generation (Aagaard
et al. 2013), and computational methods for microbiome analysis and epidemiology
(Gevers et al. 2012a, b; Markowitz et al. 2012; Faust et al. 2012). This is a rich
community resource for the scientific community. A striking revelation from the
HMP1 was that the taxonomic classification of the microbiome was unable to
explain host health or disease phenotype; molecular functional analysis of the
microbial population or understanding personalized strain-specific makeup was a
better correlate (Human Microbiome Project Consortium 2012).
Studies of the gut microbiome from healthy cohorts of other countries such as
Denmark (Qin et al. 2010), China (Zhang et al. 2019), and India (Dhakan et al. 2019)
have corroborated the same based on observations of some degree of functional
redundancy in microbiomes in spite of compositional differences. Diet was a
significant player in modulating the composition in these studies.
10.4 An Informed Approach to Next-Gen Sequencing-Based

Microbiome Diagnostic Design and Evaluation
Metabolite-related profiling studies are beyond the chapter scope; hence, we are
limiting ourselves to nucleic acid-based diagnostics here. Given the diversity of the
human microbiome, challenge of limited clinical specimen size, and the large
number of samples in cohort studies, Next-Gen sequencing-based approaches
(Levy and Myers 2016) are the current method of choice for nucleic acid-based
microbiome diagnostics (Fig. 10.1). In order to establish baseline ranges of taxo-
nomic diversity in the HMP1 study, encompassing within and between body sites
analyses, to decipher functional commonalities and signature strains across various
subjects, NGS-based approaches were adopted. Sequencing profiles based on 16S
rRNA gene sequences of 5577 samples and 681 shotgun metagenomes spanning up
to 18 body sites and three time points each from 242 healthy adults were analyzed
(Human Microbiome Project Consortium 2012). The study was extended (HMP1-II)
to 2355 total shotgun metagenomes from 265 healthy adults to identify niche-
specific and host-associated microbial community functions and to quantify strain
personalization and retention dynamics over time (Lloyd-Price et al. 2017). Subse-
quently, three iHMP clinical studies served as models of microbiome-associated
conditions, wherein the biological properties of both the microbiome and host were
studied longitudinally. Microbial community compositions, transcriptomes and
proteomes of the microbiomes, global metabolome, and immune and clinical
markers from the host were analyzed to generate datasets. Among the conditions,
vaginal microbiome of the mother associated with preterm birth, gut microbiome of
subjects with inflammatory bowel disease, and gut/nasal microbiomes of type
2 diabetics were chosen. (NIH Human Microbiome Portfolio Analysis Team 2019;
Integrative HMP (iHMP) Research Network Consortium 2019).
Country-specific microbiome databases are required to construct meaningful
correlations in disease and for the comparison of healthy and diseased individuals.
The following critical parameters need attention for any microbiome diagnostic:
1. Design: The role of the clinician, statistician, epidemiologist, and research scien-
tist is critical in identifying:
(a) The sample population size, age group, and location consisting of healthy
and affected subjects (clinical diagnosis based, patient consent, diet, clinical
history of self and family, medications, supplements, lifestyle, ability to
comprehend instructions).
162
Fig. 10.1 A schematic illustration of few popular methods employed in the microbiome analysis. Sequence-based profiling (top and middle-left panels) has
more widespread reach due to the speed, higher throughput, sensitivity, specificity, and the ability to detect smallest signals and even dead bacteria. Culture-
based profiling (bottom-left panel) is complementary to the sequence-based profiling; a prototype graph of MALDI-Biotyper is shown, different bacteria have
different spectrum and scores. Real-time PCR (top-left panel) is very useful for diagnosis and quantification if the sequence of the organism or a biomarker for a
particular disease is known. Next-generation sequencing (middle-left panel) has led to data explosion, due to continuous advancement in the field. It is broadly
N. Dasgupta et al.
10
⁄
Fig. 10.1 (continued) categorized into two types—(a) Targeted amplicon sequencing (top-right panel), where 16S/18S ribosomal RNA gene is used as
phylogenetic markers for identification of microbes. General flow of the process is shown; (b) Holistic approach (bottom-right panel), where the entire genome/
metabolites are used for the identification of microbes. It can be further divided into three categories: (1) shotgun based metagenomics, where DNA or genome is
the target. General flow of the process is shown, where taxonomic identification is done either by mapping the sequences to the reference genome or assembling
the sequences to get gene profile and hence the probable functional pathways; (2) metatranscriptomics, where the transcribed RNA is the target (expression
profile) which gives an insight into active pathways/gene; (3) metabolomics, where the metabolites produced by the microbes are the target (not shown here)
(Allaband et al. 2019; Schlaberg 2020)
Microbiome Diagnostics and Interventions in Health and Disease
163
(b) Which niche to study? (Skin/oral/nasal/gastric/gut/vaginal) Will simulta-

neous blood tests be needed to record any other parameters?
(c) Timeline of study: Longitudinal study or cross-sectional study, sampling
frequency.
(d) Appropriate sampling, storage, and transportation: home/clinic collection,
preferred time of collection, clear instruction on what and how to collect
(stool/urine/sputum); addition of appropriate stabilizer/inhibitor for
stabilizing nucleic acids; proper storage at desired temperature (4 C
or 20 C).
(e) Controls: Inclusion of a mock community of several microbes (bacteria/
fungi/virus) at varying abundances as a positive control for the process and
LoD (limit of detection) determination; a reagent control with sterile water or
saline in place of clinical sample to serve as a negative control.
2. Data generation and analysis:
(a) Isolation of nucleic acid (DNA/RNA/both): Commercially available kits or
laboratory-developed protocol best suited for the clinical sample type can be
evaluated for nucleic acid extraction yield, quality, and removal of inhibitors
from mock community to give a statistically sound representation of the
richness and abundance of microbial species. Once the isolation protocol
meets the quality requirements, the same can be applied for the clinical
samples in the study. Efficient conversion of the labile RNA to a more stable
cDNA is critical for RNA genomes (RNA viruses) or for transcriptomic
analysis of the sample. Methods that are easily adaptable for upscaling and
automation are highly desirable to make the process efficiency user agnostic
and predictive with turn-around times.
(b) Sequencing methodology and platform (targeted amplicon-based/shotgun
metagenomics/metatranscriptomics): Generally, sequencing library prepara-
tion for targeted amplicon-based sequencing includes a polymerase chain
reaction step using DNA/cDNA, to generate amplicons of the targeted
genetic marker with adapters. The shotgun metagenomics/
metatranscriptomics library preparation approach on the other hand is not
targeted, but includes a size selection of the double-stranded DNA/cDNA
prior to adapter ligation using a series of enzymatic and mechanical
manipulations as directed by the manufacturer. Details on the choice of
sequencing methodology and platform are out of scope of this chapter.
Briefly, NGS platforms can be categorized into two major categories:
short-read (e.g., Illumina, Ion Torrent) or long-read (e.g., Pacific Biosciences
(PacBio), Oxford Nanopore’s MinION) sequencing (Fig. 10.1). For taxo-
nomic profiling, DNA-based targeted amplification of 16S/18S rRNA gene
variable region, panel of gene targets, and shotgun metagenomics are com-
monly used. For molecular function-based querying of the microbiome and
to distinguish active from dormant metabolic state, targeted transcript-based
or metatranscriptomic methodology is followed. The read depth coverage
and sequencing of single reads or paired reads are some other criteria that are
taken into consideration.
(c) Data processing tools and analysis of data: Upon data acquisition from the
sequencers, performing several quality control checks is critical to prepare
the data for downstream analytics. Examples are data trimming and the
removal of poor-quality reads. Two primary approaches to taxonomic
profiling of analysis can be employed. These include de novo assembly-
based and ‘read alignment to reference-based’ methods. Many assembly
software such as metaSPADES (Nurk et al. 2017) and MEGAHIT (Li et al.
2015, 2016) can be used to reconstruct genomes from metagenomics
sequence data. Once draft genomes are assembled, software such as CON-
COCT (Alneberg et al. 2014), or MetaBat (Kang et al. 2015) can perform
contig binning and taxonomic profiling. Examples of read-based taxonomic
profiling software include Kraken (Wood and Salzberg 2014) and
MetaPhlAn2 (Truong et al. 2015). Computational software such as QIIME
(Caporaso et al. 2010) and MOTHUR (Schloss et al. 2009) are most com-
monly employed for targeted amplicon sequence data analysis using opera-
tional taxonomic unit (OTU)-based analyses. Aligned read pairs form
contigs, followed by clustering of contigs into OTUs based on similarity to
reference sequence in a database such as Greengenes (DeSantis et al. 2006)
or SILVA (Pruesse et al. 2007) for taxonomic classification. Following
classification, community structure as measured via alpha and beta diversity
can be examined.
3. Interpretation of data for diagnostics:
(a) Correlation and association based: Comparison of the microbial diversity
and relative abundance in the healthy group vs. the disease group to derive
statistically significant correlations and associations using bioinformatic and
statistical tools that take metadata into consideration is critical for unbiased
interpretation. This topic will not be covered in detail here.
(b) Database richness and accounting for microbiota interactive network:
Ensuring database richness and updating for taxonomy and disease
associations for microbiome profiles from various studies are needed to
translate the benefit in diagnostic reporting.
(c) Predictive medicine: Since precise microbiome diagnostic needs to be
population-specific, every nation may need to determine the microbiome
biomarkers for disease diagnosis/prognosis/treatment that is relevant to
their population for best patient outcomes. Using Big Data analytics and
machine intelligence to discover those correlations and validate them is
paving the way for predictive medicine (Fig. 10.2). The following
possibilities are very encouraging for the future of medicine:
• Identify unforeseen mechanistic insights of treatment.
• Identify associations not yet detected by humans.
• Identify biomarkers defining a patient’s response to treatment.
• Predict synergism/antagonisms of combination therapies and dosage
effects.
• Potentially minimize side effects and maximize efficacy of treatment.
• Predictive modeling for the diagnosis and treatment of diseases.
166
Fig. 10.2 A schematic illustration showing the future of artificial intelligence (AI)/machine learning (ML) in microbiome diagnostics and therapeutics.
N. Dasgupta et al.
Artificial intelligence has the potential of removing the ‘one-size-fit-all’ stigma in the medical field. The progress made in sequencing technology has led to
10
⁄
Fig. 10.2 (continued) an increased interest in microbiome data and can lead to DNA-based specific diagnostics. The integration of high-resolution sequencing
data and the patient metadata or clinical data can serve as model for machine learning, which can help in detecting/identifying patterns or biomarkers or insights
missed by the human eye. AI/ML is still in nascent phase, and it has huge potential to revolutionize the medical field. However, caution is needed, in terms of
training dataset as well as ethics and privacy (Espinoza 2018; Leber et al. 2017; Topçuoğlu et al. 2020)
167
• Developing noninvasive microbiome-based diagnostics with the help

of AI.
• Create virtual preemptive and predictive in silico testing of safer, more
effective therapeutics.
Innovations in the field from designing sampling devices, sequencing platforms

with better and cheaper technologies, and algorithms for data analysis are forward-
facing. Most studies of the gut microbiome study fecal sample, which may not be the
best representation of the whole gut microbiota. It is interesting to note that an
ingestible, biocompatible, 3D-printed microengineered battery-less pill has shown
promise in vitro and in animal models including primates (Rezaei Nejad et al.
2019) to aid in this sampling process.
10.5 The Healthy Gut Microbiome
Among the inhabitant microbes of our gut, bacteria are the most predominant.
Though there are 1000 different bacterial species colonizing our gut, only about
330 of them have been characterized and classified so far. The top inhabiting phyla
consists of strict anaerobes (with their relative abundance in parentheses): Firmicutes
(64%), Bacteroidetes (23%), Proteobacteria (8%), and Actinobacteria (3%) (Gill
et al. 2006; Bäckhed et al. 2012). The constantly changing microbiome responds to
our lifestyle modifications, such as diet and exercise, and displays perturbations
accordingly (Qin et al. 2010; David et al. 2014; Wu et al. 2011).
Among the benefits we derive from our gut bacteria, commensal species such as
Lactobacillus plantarum helps in regulating the integrity of intestinal epithelium,
which acts as the first physical barrier for enteric pathogens. Short-chain fatty acids
(SCFAs) such as acetate, propionate, and butyrate play vital roles in the gut
microbiome homeostasis and host immunity. The SCFAs are produced from the
breakdown of polysaccharides (mainly dietary fiber) by specific bacteria (El Kaoutari
et al. 2013; Canfora et al. 2015; Koh et al. 2016; Miyamoto et al. 2016). It is the
tuning of the biochemical pathways in the specific bacteria that result in different
by-products though starting with the same dietary fiber source. The major acetate
producers belong to the genus Streptococcus, Prevotella, Bifidobacterium, and
Clostridium to name a few (Rey et al. 2010). Propionate is produced by Bacteroides
spp., Salmonella spp., Dialister spp., Veillonella spp., Roseburia inulinivorans,
Coprococcus catus, Blautia obeum, etc., (Louis and Flint 2017). Bacteria belonging
to Lachnospiraceae, Ruminococcaceae, and Acidaminococcaceae families are the
major butyrate producers in the gut (Duncan et al. 2002). It is reported that the
SCFAs along with G-protein coupled receptor 41 (GPR41) and GPR43 present in
intestinal epithelial cells can modulate satiety and food cravings (Kim et al. 2013a, b;
Ang and Ding 2016). Higher uptake of nutrients is facilitated by suppressing
intestinal mobility transit by the secretion of peptide YY (PYY) and glucagon-like
peptide-1 (GLP-1) stimulated by SCFAs (Chambers et al. 2018; Ang and Ding
2016).
The major representatives of archaea in the gut microbiome are different species
of methanogens and halophiles. Some examples and their relative abundance among
gut methanogenic archaea include Methanobrevibacter smithii (94%),
M. stadtmanae (23%), Candidatus Methanomethylophilus alvus, and Candidatus
Methanomassiliicoccus intestinalis (Dridi et al. 2009). Gaseous by-products such
as methane and hydrogen are generated by the anaerobes inhabiting the bowel.
The signaling among the gut microbiota, the gut, and the brain by metabolites
occurs via neuronal pathways which involves both the central and enteric nervous
systems, along with the circulatory system (Cryan and Dinan 2012; Mohajeri et al.
2018). Thus, the significance of the gut microbiome in health and disease is being
appreciated like never before by scientists, clinicians, nutritionists, and the informed
public.
The healthy adult gut microbiota is highly tolerant in accommodating minor
perturbations with respect to its diversity and abundance, due to a temporary change,
such as in eating habits, life style, or environment. In a study conducted over a course
of 5 years, the individual gut microbiome displayed 60% strain level conservation,
where the major contributors were the members of the phyla Bacteroidetes and
Actinobacteria (Faith et al. 2013). This conservation or ‘longitudinal stability’ along
with the diversity of the microbiome at individual levels or ‘interpersonal diversity’
is capable of assigning an unique ‘microbial fingerprint’ to every individual, based
on the identification of >80% of the individuals microbiome composition (Franzosa
et al. 2015). Despite its resilience potential, the recent studies indicate ‘dysbiosis’ of
the microbiome to be associated with a major change(s), such as onset of disease,
surgery, or antibiotic treatment (Morgan et al. 2012). Some examples of
noncommunicable diseases, where correlations between the gut microbiome profile
and disease status have been elucidated, will be discussed in subsequent sections of
this chapter.
Developing robust microbiome-based therapeutics to restore microbiome bal-
ance, maintain the same over a period of time, and prevent relapses of dysbiosis
poses a few challenges that need interdisciplinary approaches to offer viable and
effective solutions.
10.6 Microbiome Therapeutics
We all consume curd/yogurt in our regular food habits, but it was the curiosity and
observation of Elie Metchnikoff, who wondered how a rural Bulgarian community
with limited resources for living were able to live longer. He later found out that by
manipulating the microbiome, one can increase the life and health spans of humans.
He is the father of probiotics.
Traditionally, people in Europe and Japan have relied on fermented food products
and the active ingredients that give the health benefits in fermented food products,
are the microbes that constitute the food products.
In today’s world with high stress, reduced sleep, unbalanced diet, and lack of
exercise, it is increasingly important to balance the gut microbiome through
supplementation of probiotics. Probiotics not only help in balancing the ‘good

bacteria’ but also keep the ‘bad bacteria’ away. This in turn helps the digestive
system and overall health of an individual.
We also know that imbalance in the microbiome is the underlying cause of many
disease conditions. The progressing field of microbiome diagnostics will be best
matched, when ‘Microbiome Therapeutics’ can be customized based on the need of
every patient with the right combination of prebiotic, probiotic, and supplements
(Fig. 10.3).
10.6.1 Prebiotics Support Probotics
According to the International Scientific Association of Probiotics and Prebiotics,

prebiotics are defined as ‘a substrate that is selectively utilized by host
microorganisms conferring a health benefit’ (Gibson et al. 2017). In our microbiome,
they promote the absorption of ion and trace element such as that of calcium, iron,
and magnesium and modulate cytokine and secretory immunoglobulin A produc-
tion, via mechanisms involving microbial metabolic products (Holscher 2017).
Inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), and human
milk oligosaccharides (HMOS) are a few prebiotic ingredients in our diet that have a
strong correlation in keeping body weight under check (Kim et al. 2019). They are
found to stimulate the growth of Bifidobacteria and Lactobacillus species, thus
enhancing the availability of SCFAs within the microbiome. Higher SCFA level
positively influences satiety and food consumption via improved GLP-1, PYY, and
ghrelin production (Cerdó et al. 2019).
10.6.2 Probiotics in Food and as Supplements
Natural probiotics can be obtained from food sources such as curd/yoghurt and
fermented foods. Food products that contain probiotics are yoghurt, kefir, cheese,
tempeh, kimchi, miso, sauerkraut, and some soy beverages. Freeze-dried bacteria in
the form of tablets, capsules, powders and sachets, and ampoules containing bacte-
rial spores, are available commercially from cultured organisms at a defined compo-
sition and abundance (Table 10.1, Alfano et al. 2020). In addition, probiotic-fortified
foods are also available like juices, chocolates, flour, and cereal. Food and Drug
Administration, USA (FDA), regulations allow probiotics to be sold as supplements
and not like drugs, for healthy people. For people with illnesses such as irritable
bowel syndrome, inflammatory bowel disease, diarrhea (both infectious and
antibiotic-induced), urinary tract infections, and eczema, doctor-prescribed
probiotics may be given. Pregnant women, infants, young children, and immuno-
compromised patients should be given probiotics with caution.
10
Fig. 10.3 Functional foods have health benefits above and beyond their nutritional value, e.g., providing prebiotics and probiotics which help in balancing the
microbiome. Probiotics (right panel) are the live microbes which are beneficial for health and can be obtained from various foods which act as live culture for
171
these beneficial microbes. Prebiotics (left panel) are the food for these live microorganism (probiotics) present in different food and are associated with the
growth of beneficial bacteria. (Green et al. 2020; Markowiak and Ślizewska 2017; McBurney et al. 2019; Pandey et al. 2015; Rezac et al. 2018; Terpou et al.
2019)
Table 10.1 List of a few commercially available probiotic preparations

Brand name Strain Producer
Dicoflor Lactobacillus rhamnosus GG AGPHARMA
Enterogermina Bacillus clausii SANOFI
Enterolactis Lactobacillus casei SOFAR
Nutriflor Lactobacillus acidophilus DDS-1, Lactobacillus NUTRIGEA
bulgaricus DDS-14 Bifidobacterium bifidum,
Lactobacillus rhamnosus
Probactiol duo Lactobacillus acidophilus NCFM, Lactobacillus METAGENETICS
paracasei Lpc-37 Bifidobacterium lactis Bi-07,
Bifidobacterium lactis Bi-04
VSL#3 Streptococcus thermophilus, Bifidobacterium breve, FERRING
Bifidobacterium longum, Bifidobacterium infantis FARMACEUTICI
lactobacillus acidophilus, Lactobacillus plantarum,
Lactobacillus paracasei, Lactobacillus delbrueckii
subsp. bulgaricus
Yakult Lactobacillus casei Shirota YAKULT (Tokyo)
10.6.3 Postbiotics
Postbiotics is a therapeutically attractive emerging field that deals with the use of
nonviable by-products of microbial growth (metabolites, cell lysis components,
enzymes) that have an added health benefit when consumed. Unlike probiotics,
which are based on the administration of live organisms, postbiotics are administered
in alignment with pharmacokinetic and pharmacodynamic properties. They are
being explored since they are abundant at most body sites, are suitable for different
routes of administration, have low toxicity potential, are stable in the systemic
circulation, and scale up friendly. Bacterial exopolysaccharides (EPS) from
Bifidobacterium and Lactobacilli and extracellular vesicles (EVs) from Akkermansia
muciniphila and commensal Escherichia coli are some such postbiotic examples
(Wegh et al. 2019). There are several downsides such as pleiotropic effects, shorter
half lives, and high cell-type specificity, which need further exploration and well-
designed studies to evaluate them for therapeutics.
10.6.4 Fecal Microbiota Transplantation (FMT)
Fecal microbiota transplantation (FMT) is a procedure of administering donor fecal

suspension into the colon of a diseased recipient, thus aiming to restore the disturbed
gut microbiota and the associated disease. The first application of FMT in modern
medicine was reported in 1965 for Clostridioides difficile colitis. Though FMT has
benefited many to cure chronic conditions, it is not advisable for all conditions of gut
dysbiosis and for all categories of patients. Selection of patients for whom FMT is
effective is an important concern since long-term safety of the procedure and
outcome for the patient needs careful consideration. Nevertheless, more data from
well-designed studies will help in determining the efficacy and safety of the
procedure.
10.6.5 Research-Driven Probiotics: The Future
The rationale that the naturally occurring human-associated microorganisms offer

myriad of health benefits, is the basis of probiotic therapies. A systematic and well-
designed research on specific probiotics designed for an individual is needed (Mimee
et al. 2016). This would be possible through the studies on microbiome and
microbiome diagnostics. A precise quantization of the microbiome components,
the dosage, and regimens all require advanced science. The need of the hour is to
develop microbiome-based recommendations for probiotics and probiotics pre-
scribed for specific diseases classified as ‘prescription probiotics. Preparing them
will involve large-scale culturing and identification of various bacterial species, their
long-term storage, and potency testing of the probiotic cocktails tailored to an
individual’s need. A futuristic probiotic application wherein the probiotic strain
can be used for directly delivering anti-inflammatory and intestinal epithelial repair
factors to the intestinal tract will allow correction of multiple aberrations in a unified
manner.
Genetically engineered probiotics: The use of genetically modified organisms
(GMO) in human microbiome therapeutics is still farfetched due to the associated
regulatory clearances needed for their safety. Nevertheless, researchers have tested
the concept in animal models and have met with success. The probiotic E. coli Nissle
1917 was altered to be used as a prophylactic, in order to inhibit virulence of Vibrio
cholerae. (Hamady et al. 2010). A genetically modified derivative of a vaginal
commensal Lactobacillus jensenii able to prevent transmission of chimeric simian/
human immunodeficiency virus (SHIV) in a rhesus macaque model, when
administered is another encouraging study (Motta et al. 2012) demonstrating the
utility of engineered probiotic strains. A common side effect of chemotherapy ‘oral
mucositis,’ a condition involving ulcerative lesions, is shown to be benefited from
the topical application of an altered L. lactis engineered to secrete trefoil factor-1.
Data from an early clinical trial for the treatment of the condition displayed good
tolerance among patients and could be effective at reducing prevalence (Limaye
et al. 2013).
Engineered ‘designer’ consortia: This concept is based on building a collection
of well-characterized probiotic strains that can be custom combined in the laboratory
based on the attributes desired for the therapeutic consortia. One such example is
elaborated here. Bacteria in the gut generate urease which convert the urea produced
by the liver to ammonia and carbon dioxide. Patients with liver deficiency, neuro-
toxicity and encephalopathy are found to be associated with accumulation of sys-
tematic ammonia. In a study, mouse models were treated with antibiotic and
polyethylene glycol, resulting in reduction of endogenous microbiota. This was
followed by transplantation with a defined microbial community with low urease
activity. The microbiota reconstitution was successful in altering community-wide
metabolic activity of urea that remained stable for months (Shen et al. 2015). Such
designer consortia for human use may be seen in trials soon in the near future.
10.7 The Role of Microbiome Diagnostics and Therapeutics

in a Few Disease Scenarios
In this section, select disease conditions primarily influenced by the gut microbiome
dysbiosis are presented. A brief pathophysiology of the condition, it’s associated
microbiome dysbiosis profile and suggested therapeutics for correcting the dysbiosis
is summarized in the following sections (Fig. 10.4). Oral, vaginal, skin, and respira-
tory microbiome modulations are out of scope of this chapter.
10.7.1 Atherosclerosis
Atherosclerosis, a chronic inflammatory disease, and major contributor in CVDs

(cardiovascular diseases), is associated with plaque formation consisting of
accumulated modified lipids, calcified regions, neurotic cores, inflamed smooth
muscle cells, endothelial cells, leukocytes, foam cells and impaired lipid metabolism
and endothelial functions (Frostegård 2013). CVD is one of the leading causes of
disease and death globally (Benjamin et al. 2018).
Atherosclerosis has been linked to intestinal microbes due to a substance called
trimethylamine oxide in recent studies (Chen et al. 2016). In 2015, Cleveland Clinic
researchers observed that lecithin and L-carnitine which is present in red meat, egg
yolk, etc., can be converted to TMAO wherein intestinal microbes play an important
part thus promoting atherosclerosis and speeding up the pathological process of
cerebrovascular diseases (Wang et al. 2015a, b).
What’s the connect? Intestinal microbes can absorb foods rich in lecithin, choline,
and carnitine to produce trimethylamine (TMA, a colorless gas of a foul odor), which
is oxidized by flavin monooxygenase (FMO, FMO3 with highest activity) to TMAO
in the liver. Atherosclerosis is linked with increased TMAO in the blood (Koeth et al.
2013), which in turn is linked to the diet, intestinal microbes, FMO3 activity, gender,
and heredity of host (Seldin et al. 2016). Some mechanisms which TMAO uses to
develop atherosclerosis are hampering cholesterol reverse transportation (Koeth
et al. 2013), up-regulating the expression of macrophages CD36 and scavenger
receptor A1 (SR-A1), encouraging foam cell formation, down-regulating the expres-
sion of cholesterol absorption targets ABCG5/8 and NPC1L1 affecting cholesterol
metabolism, reducing the expression of cytochrome P450 (CYP) 7A1 and 27A1 in
the liver, which in turn reduces the transport of bile acid and clearance of cholesterol
and activates monocytes via mitogen-activated kinase and nucleic acid factor-κB
signaling pathway by developing vascular inflammation (Seldin et al. 2016). Thus, if
the density, richness, and diversity of gut microbiota are improved, it can help in
prevention and treatment of atherosclerosis. People having lower species richness
and diversity in the gut are prone to develop atherosclerosis (Menni et al. 2018).
10
Fig. 10.4 Depiction of various diseases associated with different microbiome (gut, oral, skin, vaginal) and the major upregulated and downregulated microbes
involved in said diseases (Álvarez-Mercado et al. 2019; Green et al. 2020; Kho and Lal 2018; Kosti et al. 2020; Liu et al. 2019)
175
Atherosclerotic patients are found to have a gut microbiota that is less fermenta-
tive and more inflammatory (Jie et al. 2017). Firmicutes and Bacteroides are the
major taxa present which seem to be remarkably constant (Huttenhower et al. 2012;
Faith et al. 2013). However, in patients with atherosclerosis, Escherichia coli,
Klebsiella spp., Enterobacter aerogenes, Streptococcus spp., Lactobacillus
salivarius, Solobacterium moorei, and Atopobium parvulum were found to be
increased, whereas Bacteroides spp., Prevotella copri, and Alistipes shahii were
found to be depleted (Jie et al. 2017).
Therapeutics that help: The three ‘p’ known for the well-being of gut are
probiotics, prebiotics, and polyphenols, as these help regulate the gut microbiota
composition (Marchesi et al. 2016). Since small intestine cannot absorb polyphenols
directly, the bioavailability of polyphenols depends on the gut microbiota and their
ability to convert it into components which can be absorbed by the small intestine
(Duda-Chodak et al. 2015). Few of the polyphenols which have shown some
potential mechanism in atherosclerosis are protocatechuic acid (PCA), quercetin-3-
glucuronide, 2,4,5-trimethoxycinnamic acid, gallic acid, and equol (Pieczynska
et al. 2020). Resveratrol, a natural phenolic phytochemical, works by reducing
TMAO levels by promoting the growth of commensal bacteria such as Bacteroides,
Lactobacillus, and Bifidobacterium (Jung et al. 2009; Qiao et al. 2014). The decrease
in TMAO levels by resveratrol associated with inhibited development of atheroscle-
rosis has also been proven in vivo (Chen et al. 2016).
As with prebiotics, probiotics like Lactobacillus plantarum and E. aerogenes
could lower the production of TMAO and attenuate the formation of atherosclerotic
abrasion in ApoE/mice (Qiu et al. 2017, 2018). Another prebiotic, mannan oligosac-
charide (MOS) supplement, was found to regulate gut microbiota by lowering
plasma cholesterol levels and improving atherosclerotic plaques in high cholesterol
diet-fed mice (Hoving et al. 2018). Stimulation of Akkermansia in ApoE / mice
was linked with berberine, which is found to be effective against atherosclerosis
(Zhu et al. 2018). The endothelial function in ApoE / mice seems to be improved
by administering ITFs, a prebiotic (Inulin-type fructans), as supplement. These
inulin-type fructans enhance the formation of butyrate and protect against athero-
sclerosis formation (Watzl et al. 2005; Catry et al. 2018) as per the recent studies.
Both fish oil and flaxseed oil are found to reduce TMAO by enhancing SCFAs
production and lowering LPS generation by microbes, and fish oil seems to be more
productive (He et al. 2019). Research suggests that calorie-controlled diet integrated
with supervised exercise lowers TMAO levels considerably (Erickson et al. 2019).
FMT is another therapy; however, it comes with risk as well. For example, while
beneficial flora is getting transferred, so could the endotoxins or infectious agents
present in the donor, and this could start new gastrointestinal complications. This is
the reason it has found limited use as treatment for CVD patients (De Leon et al.
2013; Brandt 2013). Further research is needed to take a look at whether or not FMT
probably prolongs different aspects of cardiometabolic disorders. Instead of fecal
contents, the transplantation of particular group of microbes can be a rational
opportunity to FMT. To better define the optimal fecal microbial preparation,
dosing, and method of delivery, further research needs to be conducted (Sanchez-

Rodriguez et al. 2020).
How the gut dysbiosis and TMAO derived from the microbiota participate in
atherosclerosis is yet to be cleared (Zhu et al. 2020a, b). New strategies to prevent or
treat the disease can be developed by better understanding of gut microbiota
composition, to the development of atherosclerosis (Pieczynska et al. 2020).
10.7.2 Hypertension
Hypertension is among the chief causes of cardiovascular disease and is responsible

for global deaths (Go et al. 2014). Although the procedure of how gut microbiota is
involved with hypertension is not quite clear, SCFAs and oxidized low-density
lipoprotein (ox-LDL) are believed to take some part in it (Ma et al. 2018).
What’s the connect? Obese pregnant women with lower blood pressure have
shown increase in butyrate-producing bacteria (Gomez-Arango et al. 2016). Gut
dysbiosis was improved by fiber and acetate supplementation in a study on hyper-
tensive mice. It led to a surge in Bacteroides acidifaciens, which seems to have a
defensive role in hypertension/heart failure (Marques et al. 2017). GPR41, GPR43,
and GPR109AA are the three G-protein-coupled receptors (GPCRs), regulated by
SCFAs. Another type expressed in the kidney is olfactory receptor 78 (Olfr78)
regulated by acetate and propionate (Tan et al. 2017). The GPCRs regulated
pathways of host can be stimulated by SCFAs which effects the secretion of renin
and in turn effects the blood pressure (Furusawa et al. 2013; Pluznick et al. 2013).
Oxidation of LDL causes vasoconstriction leading to hypertension through gut
dysbiosis (Packer et al. 2014), by boosting the expression of pro-inflammatory
cytokines which induces oxidative stress triggering the Ox-LDL stimulation
(Chawla et al. 2011; Peluso et al. 2012). Oxidation of L-arginine by nitric oxide
synthase produces nitric oxide (NO) (Ma et al. 2006). Production of NO and
endothelin-1 which maintains basic vascular tension and cardiovascular system
homeostasis (Boulanger and Lüscher 1990) is hampered due to higher levels of
ox-LDL which causes hypertension (Subah Packer 2007). Another cause of hyper-
tension is chronic low-grade inflammation (Schiffrin 2014), which occurs due to
depletion in microbial gene richness (Cotillard et al. 2013). Chronic probiotic intake
decreases preeclampsia associated with hypertension (Brantsaeter et al. 2011).
Therapeutics that help: The composition of gut microbiota is altered by consum-
ing β-glucan in such a way that it reduces the risk markers associated with CVD as
per the single-blind randomized trial (Hoving et al. 2018). When Lactobacilli
fermented milk was consumed by hypertensive humans, it lowered their blood
pressure (Seppo et al. 2003). It is observed in human trials that consumption of at
least 1011 colony-forming units along with multiple species of probiotics for 8 weeks
decreases both systolic and diastolic blood pressures (Khalesi et al. 2014). Long-
term administration of probiotics of various Lactobacillus bacteria such as Lactoba-
cillus fermentum CECT5716 (LC40), Lactobacillus coryniformis CECT5711 (K8),
and Lactobacillus gasseri CECT5714 (LC9) could decrease systolic blood pressure
in hypertensive rats (Gómez-Guzmán et al. 2015). Phenylacetyl glutamine, a gut
microbiota-derived metabolite, is negatively linked with pulse wave velocity and
systolic blood pressure (Menni et al. 2015).
10.7.3 Obesity
Obesity is the manifestation of accumulated fat and is correlated with the progression
of many diseases of metabolic origin like cardiovascular disease, type 2 diabetes
mellitus, cancer, and nonalcoholic fatty liver disease (Kim et al. 2019). The fact that
obese people live 7 years shorter than nonobese people is quite alarming (Van Hul
et al. 2018).
What’s the connect? Studies from humans and animals have clearly shown a
correlation between obesity and gut microbiome. Some examples are as follows:
(1) decreased gut diversity (Baothman et al. 2016); (2) increased Firmicutes and
decreased Bacteroidetes (Koliada et al. 2017; Mariat et al. 2009; Greenhill 2015).
This increased Firmicutes/Bacteroidetes (F/B) ratio facilitates the energy extraction,
and this in turn effects the energy storage in the adipose tissue (Mariat et al. 2009;
Bell 2015). A significant increase in Enterobacteriaceae was observed in obesity
(Balamurugan et al. 2010).
There exist a few other mechanisms for the functioning of gut microbiome in
influencing obesity. Examples include bile acids that actively help in resolving fat
uptake from diet in the small intestine, but they also hamper the growth of different
commensal bacteria such as Lactobacilli and Bifidobacteria by disorganizing their
membrane permeability (Ridlon et al. 2006; Kurdi et al. 2006). Acetate, propionate,
and butyrate (SCFAs) which are eventually consumed by various organisms are
estimated to have a production rate of 80–200 kcal/day (Riley et al. 2013). Decrease
in butyrate-producing bacteria along with reduced intake of dietary carbohydrates
such as polysaccharides, vegetable oligosaccharides, and resistant starch was
observed in obese patients (Canfora et al. 2019). Another study gave similar results
where the levels of fecal butyrate, SCFAs, and Bifidobacterium were found to be
reduced considerably in obese patients who consumed less fiber (Brinkworth et al.
2009). Metabolic endotoxemia is defined as a chronically high plasma LPS disorder
at 10–50 times less than the septic conditions of LPS and is a high-fat dietary
elevation of plasma lipopolysaccharide (LPS) as termed by Cani et al. (2007).
Dietary increases of endotoxin were linked to enhanced fat deposit, systemic and
tissue-specific inflammation, and resistance to insulin (Cani et al. 2007; Amar et al.
2008).
Therapeutics that help: Prebiotics regulate the gut microbiome composition by
improving lipid metabolism which is also seen in short-chain FOS treatment in diet-
induced obese mice (Cluny et al. 2015) and are known to have antiobesity effects
(Barengolts 2016; Nicolucci and Reimer 2017; Delzenne et al. 2011). Animals
treated with oligofructose displayed reduction in both triglyceride level and adipose
tissue mass (Cluny et al. 2015). It is also reported that α-cyclodextrins supplementa-
tion in obese mice (diet-induced) resulted in inflection of gut microbiota and SCFA
production (Nihei et al. 2018). Human breast milk is enriched with milk
oligosaccharides and serves as wonderful prebiotics candidate. As a prebiotic, it
promotes the growth of beneficial bacteria such as Bacteroides and Bifidobacterium
and hinders the pathogens such as Campylobacter jejuni, Helicobacter pylori, and
E. coli (Newburg 2000).
Recent studies have shown that when compared with placebo-treated control
animals, supplementation of Bifidobacterium species such as B. breve B3,
B. infantis, and B. longum and Lactobacillus species such as L. rhamnosus,
L. casei strain Shirota [LAB13], L. gasseri, and L. plantarum has shown obliteration
of weight gain, fat deposits, and white adipose tissue (Barengolts 2016; Kim et al.
2019).
In another study, treatment of obese adults with L. gasseri (SBT2055 and
BNR17) exhibited reduction in visceral adipose tissue as well as waist size (Kadooka
et al. 2010; Kim et al. 2018). Similar study was reported by Pedret et al, where
intervention with Bifidobacterium animalis subspecies. Lactis CECT 8145 reduced
waist size, waist circumference/height ratio, and BMI considerably (Pedret et al.
2019). L. rhamnosus CGMCC1.3724 therapy displayed weight loss in obese women
but nothing significant in obese men (Sanchez-Rodriguez et al. 2020).
Diet is a major player in obesity and has associations with gut microbiota (Brahe
et al. 2016). It has been shown that the various diet styles such as western, vegetar-
ian, gluten-free, and the Mediterranean diet disturb gut diversity (Lazar et al. 2019).
The Western diet which comprises of high amount of sugar, salt, saturated fats,
refined grains, and high fructose corn syrup with lesser amount of fiber is responsible
for decrease in total gut microbiota amount along with reduction in beneficial
bacteria such as Lactobacillus sp. and Bifidobacterium sp., thereby promoting
inflammation and changing gut microbiota to obese pattern (Bell 2015; Statovci et al.
2017). One of the mechanisms can be improving energy harvesting by increase in
Firmicutes for promoting better caloric absorption leading to weight gain (King et al.
2012).
Plant-based diet such as vegetarian and vegan diets is rich in dietary fiber and
entails plant-derived products which is known to trigger an increase in the abun-
dance of protective microbiota. This diet promotes an increase in (1) Bifidobacteria
and Lactobacillus; known intestinal barrier protectors, (2) Faecalibacterium
prausnitzii and Roseburia; butyrate producers, and a decrease in Escherichia coli
and Enterobacter cloacae; inflammation-inducing lipopolysaccharide-producing
bacteria, thus ultimately preventing obesity (Tomova et al. 2019; Glick-Bauer and
Yeh 2014). Mediterranean diet is majorly comprised of vegetables, olive oil, fruits, a
modest amount of poultry, with limited consumption of red meat and dairy products.
This dietary habit correlates with higher abundance of Lactobacillus,
Bifidobacterium, and Prevotella in the gut, which helps in preventing obesity by
improving lipid and cholesterol profiles (Garcia-Mantrana et al. 2018; Coelho and
Cândido 2019). Korean traditional diet consists of high amounts of vegetables,
fermented foods with modest consumption of legumes and fish. Such a diet helps to
prevent obesity by increasing abundance of Bacteroides (Bacteroidaceae) and
Bifidobacterium (Bifidobacteriaceae-Actinobacteria) while decreasing Prevotella
(Prevotellaceae) (Baik 2018; Lim et al. 2015). With the help of high-fiber diet,
obesity can be managed through intestinal SCFA dependent modulation of down-
stream pathways (Barathikannan et al. 2019). In high-fat diet (HFD) fed mice, when
HFD is replaced by treatment with L. rhamnosus GG, it reduces adiposity via the
heightened production of adiponectin, thereby protecting the animal from insulin
resistance as well as helping in diminishing liver adiposity (Kim et al. 2013a, b).
Pasteurized nonviable Akkermansia muciniphila as a prebiotic treatment showed an
increased ability to reduce the development of fat mass along with insulin resistance
and dyslipidemia in mice (Plovier et al. 2017; Depommier et al. 2019).
10.7.4 Nonalcoholic Fatty Liver Disease
Nonalcoholic fatty liver disease (NAFLD) is marked by hepatic steatosis and may
advance to an inflammatory condition called nonalcoholic steatohepatitis (NASH),
liver cirrhosis, and hepatocellular carcinoma. Gut microbiome and certain host
factors have been linked to this condition (Grabherr et al. 2019).
What’s the connect: A large variation in terms of phylum, family, and genus was
observed between healthy controls and NASH patients in several studies. NAFLD
patients display upregulation and downregulation of a large array of organisms; the
organisms which are enriched are Bacteroides, Ruminococcus, Lactobacillus
(Genus), E. coli (Species), Lactobacillaceae (Family), and Proteobacteria (Phy-
lum), while the downregulated organisms are Oscillibacter, Prevotella,
Ruminococcus, Coprococcus (Genus), Faecalibacterium prausnitzii (Species),
Actinobacteria, Bacteroidetes, and Firmicutes (Phylum) (Loomba et al. 2017; Del
Chierico et al. 2017; Boursier et al. 2016; Da Silva et al. 2018).
NAFLD may be associated with low abundance of Faecalibacterium prausnitzii,
a butyrate-producing bacterium from Firmicutes phylum, which was associated with
>5% fat hepatic content and increased adipose tissue inflammation (Munukka et al.
2014, 2017). A gram-negative Proteobacterium Bilophilia wadsworthia, has been
shown to aggravate high fat diet induced metabolic dysfunctions in mice. The
mechanism followed is that it lowers the butyrate metabolism, which leads to
disruption of the gut barrier (interrupted tight junctions), thus allowing the LPS
circulation from the gut lumen into the incoming portal vein of the liver. Once there,
it releases a pro-inflammatory cytokine by acting on the hepatic macrophages and
promotes a reduction of bile acids production. All of these leads to a disrupted
microbiota and hence the heightened release of LPSs (Feng et al. 2017; Natividad
et al. 2018).
Helicobacter pylori is a gram-negative Proteobacterium which is responsible for
immune resistance contributing to NAFLD. H. pylori infection can increase the
chances of NAFLD development. However, to comprehend the association between

H. pylori and NAFLD progression, more clinical studies are needed (Wijarnpreecha
et al. 2018; Ning et al. 2019).
In a healthy state, ethyl alcohol is continuously being produced in the gut. In the
healthy individual, it gets metabolized in the liver by alcohol dehydrogenase (ADH)/
other hepatic enzymes. However, when alcohol-producing bacteria like Klebsiella
pneumoniae increase in the gut, they produce reactive oxygen species (ROS)
constantly due to exceeding of liver detoxification capacity which promotes hepatic
inflammation, often ending in steatohepatitis (Yuan et al. 2019).
Obese and NAFLD animals have shown lesser abundance of Akkermansia
muciniphila, a gram-negative bacterium from the phylum Verrucomicrobia with
mucin degrading capacity than in their healthy counterparts (Everard et al. 2013;
Zhao et al. 2017).
Therapeutics that help: The consumption of processed foods and beverages
containing fructose was seen higher in NAFLD patients (Chen et al. 2017).
NAFLD progression is also associated with lower fiber, polyphenols, vitamins
(Vitamin D), and mineral nutrients (calcium) intake (Van Herck et al. 2017;
Wehmeyer et al. 2016). Polyphenols like quercetin, epigallocatechin gallate,
anthocyanins, and resveratrol have also been found to be protective (Wrzosek
et al. 2013).
It has been reported that administration of a cocktail of Lactobacillus acidophilus
ATCC B3208, Bifidobacterium lactis DSMZ 32,269, Bifidobacterium bifidum
ATCC SD6576 and Lactobacillus rhamnosus DSMZ 21,690 to adolescents for
12 weeks in the form of probiotic capsules, resulted in substantial decrease in
ALT (Alanine aminotransferase), lipid profile and intrahepatic fat content compared
to placebo group. In another study, efficacy of ‘Symbiter,’ containing 14 alive
probiotic strains of Lactobacillus + Lactococcus, Bifidobacterium,
Propionibacterium, and Acetobacter, is assessed in NAFLD patients and has
shown to improve hepatic steatosis, aminotransferase activity, TNF-α, and IL6 levels
(Kobyliak et al. 2018). Another multistrain probiotic VSL#3 has been found to
protect the integrity of intestinal barrier and diminish endotoxemia and oxidative/
nitrosative stress, thus improving liver pathology in patients suffering from various
chronic liver diseases (Loguercio et al. 2005). VSL#3 contains Bifidobacterium
longum, and it adjusts gut microbiota in such a way that increases the production
of conjugated linoleic acid (CLA); this further impacts fatty acid composition in the
liver and in a way plays a significant role in therapeutic interventions (Meroni et al.
2019). When Bifidobacterium longum is administered in combination with prebiotic
fructo-oligosaccharides (FOS), it considerably improves the metabolic and inflam-
matory markers and fibrosis scores in NASH patients (Malaguarnera et al. 2012).
10.7.5 Type 2 Diabetes
Diabetes mellitus (DM) is marked by chronic hyperglycaemia, as a result of deficits

either in insulin secretion, or in insulin action, or both. 90–95% of diabetes cases are
of type 2 (T2D) (Woldeamlak et al. 2019).
What’s the connect? Bifidobacterium, Bacteroides, Faecalibacterium,
Akkermansia, and Roseburia were found to be negatively related with T2D, while
Ruminococcus, Fusobacterium, and Blautia were connected positively with T2D
(Gurung et al. 2020). Patients treated with metformin or after undergoing gastric
bypass surgery have found to be negatively associated with B. adolescentis,
B. bifidum, B. pseudocatenulatum, B. longum, and B. dentium (Wu et al. 2017;
Murphy et al. 2017). Bacteroides intestinalis, Bacteroides 203, and Bacteroides
vulgatus were decreased in T2D patients and Bacteroides stercoris were enriched
after sleeve gastrectomy (SG) surgery in T2D patients with diabetes remission
(Wu et al. 2011; Murphy et al. 2017; Zhang et al. 2013; Karlsson et al. 2013).
Investigations have reported a negative association of Roseburia inulinivorans,
Roseburia_272, and one unclassified OTU from this genus, with disease (Murphy
et al. 2017; Zhang et al. 2013; Karlsson et al. 2013). Lower frequencies of
Faecalibacterium were reported in patients in two case–control studies (Gao et al.
2018; Salamon et al. 2018). Also, after different types of antidiabetic treatments
ranging from metformin and herbal medicine to bariatric surgery, decreased
their abundance (Tong et al. 2018; Murphy et al. 2017). Half of the T2D studies
showed that out of these five genera, Bacteroides, Bifidobacterium, Roseburia,
Faecalibacterium, and Akkermansia, at least one is reduced, suggesting that they
have a role which goes beyond serving as a biomarker (Gurung et al. 2020). There is
an increase in organisms like L. acidophilus, L. gasseri, and L. salivarius, and
reduction in L. amylovorus in T2D patients which signify species specificity
(Karlsson et al. 2013; Graessler et al. 2013; Forslund et al. 2015). L. acidophilus,
L. plantarum, and L. reuteri were found to have lower frequencies when compared
with controls in this disease (Suceveanu et al. 2018).
Patients with T2D show raised levels of pro-inflammatory cytokines,
chemokines, and inflammatory proteins. Also, increased gut permeability allows
for passage of gut microbe-derived products into the blood. This causes metabolic
endotoxemia, effects glucose homeostasis and insulin resistance in liver, muscle, and
fat, and in addition affects the digestion of sugars and production of gut hormones
that regulate glucose metabolism (Gurung et al. 2020). The microbes, like
Fusobacterium nucleatum and Ruminococcus gnavus which are potentially harmful
in T2D, induces various inflammatory cytokines (Yang et al. 2017a, b; Hall et al.
2017), in other inflammatory diseases.
The Firmicutes to Bacteroidetes ratio along with proportions of phylum
Firmicutes and class Clostridia was reduced, while class Betaproteobacteria was
increased in T2DM patients as per one of the case–control study (Larsen et al. 2010).
Also, Faecalibacterium prausnitzii and genus Blautia were diminished in T2DM
patients (Navab-Moghadam et al. 2017; Inoue et al. 2017). Increase in serum
fructosamine is associated with the decrease in Prevotellaceae and increase in

Enterobacteriaceae (Li et al. 2019a, b).
Therapeutics that help: There have been many animal studies conducted for
diabetes. An improved glucose tolerance is seen in several Bifidobacterium spps,
i.e., B. bifidum, B. longum, B. infantis, B. animalis, B. pseudocatenulatum, B. Breve
(Le et al. 2015; Moya-Pérez et al. 2015; Kikuchi et al. 2018; Aoki et al. 2017; Wang
et al. 2015a, b). In another study, an improvement in glucose tolerance and insulin
resistance was seen in diabetic mice by providing Bacteroides acidifaciens and
Bacteroides uniformis (Yang et al. 2017a, b; GauffinCano 2012). In human studies,
progress in type 2 diabetes-related symptoms is seen by administration of
L. sporogenes, L. casei Shirota, and L. reuteri used as monoprobiotics (Hulston
et al. 2015; Simon et al. 2015; Asemi et al. 2014, 2016). Lactobacilli works more
effectively as a part of probiotic cocktail than given individually in majority of cases
(Ejtahed et al. 2012; Asemi et al. 2013; Tajabadi-Ebrahimi et al. 2016;
Mohamadshahi et al. 2014). Among the several Lactobacillus species that have
been tested as probiotics, L. plantarum, L. reuteri, L. casei, L. curvatus, L. gasseri,
L. paracasei, L. rhamnosus, and L. Sakei have shown favorable effects on T2D in
mice models (Gurung et al. 2020).
There have been different mechanisms found to reduce gut permeability like
(1) Administering Bacteroides vulgatus and B. Dorei species (Yoshida et al. 2018);
(2) Butyrate, produced by Faecalibacterium, via serotonin transporters and PPAR-g
pathways (Kinoshita et al. 2002); (3) Akkermansia muciniphila, probiotic bacterium
using extracellular vesicles which improve intestinal tight junctions via AMPK
activation in epithelium (Chelakkot et al. 2018).
There has been an emerging focus on interactions between microbiota and
antidiabetic drugs in microbiome research (Gurung et al. 2020). Different
combinations have been used and been effective than when administered alone in
reducing/improving T2D parameters, like reduced hyperglycemia, adiposity,
improved fasting blood glucose, glucose tolerance, and insulin resistance in different
studies. For example, (1) probiotic Bifidobacterium animalis ssp. lactis 420, prebiotic
polydextrose in amalgamation with sitagliptin in diabetic mice (Salamon et al.
2018); (2) prebiotic polysaccharide in combination with metformin and sitagliptin
in Zucker diabetic rats’ study (Reimer et al. 2014); (3) combination of a prebiotic and
metformin in streptozotocin-induced diabetic mice (Zheng et al. 2018).
There has been inconsistency in identification of T2D-associated microbiota in
humans because of several elements such as geographic location, race, culture,
health status, and drug use. Stool samples are the preferred choice for microbiota
analysis in most of the studies due to difficulties in sampling from human intestine,
but, however, the stool sample does not represent entire gut microbiome profile
(Gurung et al. 2020).
It has been observed that vegetables, fruits, dietary fibers, and medicinal plants
reduce or prevent T2DM by raising the level of SCFAs and fine-tuning gut
microbiota. Capsaicin can increase the Firmicutes to Bacteroidetes ratio, Roseburia
number, and at the genus level reduce the quantities of Bacteroides and
Parabacteroides which could reduce pro-inflammatory cytokines, such as TNF-α
and IL-6 in obese diabetic mice (Song et al. 2017). Pumpkin polysaccharide can
exert its antidiabetic effect by increase of selective bacteria, like Bacteroidetes,
Prevotella, and Deltaproteobacteria in mice (Liu et al. 2018a, b). The Lactobacillus
rhamnosus GG fermented carrot juice can improve T2DM in rats by increasing
Oscillibacter and Akkermansia (Hu et al. 2019). The antidiabetic activity of
Momordica charantia (bitter melon) is improved by Lactobacillus fermentation,
which in turn can increase Bacteroides caecigallinarwn, Bacteroides
thetaiotaomicron, Prevotella loescheii, Prevotella oralis, and Prevotella
melaninogenica (Gao et al. 2018). The phlorizin in many fruits could regulate the
blood glucose level by reducing serum LPS and insulin resistance, increasing butyric
acid as well as increasing Akkermansia muciniphila and Prevotella (Mei et al. 2016).
It has been reported that the extracts from cinnamon bark can improve glucose
tolerance and insulin resistance by reducing Peptococcus, and the extracts from
grape pomace in diabetic mice could decrease Desulfovibrio and Lactococcus, and
increase Allobaculum and Roseburia (Van Hul et al. 2018). The inulin reduces the
effects of T2DM in diabetic mice by increasing Cyanobacteria and Bacteroides and
decreasing Deferribacteres and Tenericutes (Li et al. 2019a, b).
10.7.6 Inflammatory Bowel Disease (IBD)
IBD is a long-lasting heterogeneous, GI tract associated disorder, characterized by

an inflammatory process and materializing in the form of Crohn’s disease (CD) or
ulcerative colitis (UC) in different patients and differentially diagnosed by clinicians.
Factors that could influence host–microbiome homeostasis such as host genetics,
host immune system, shifts in diet, exposure to antimicrobials, urbanization, west-
ernization (Statovci et al. 2017) of life style, and many more could predispose and
trigger IBD.
What’s the connect? IBD was one of the three conditions that was explored in a
longitudinal study of a cohort of 132 individuals over a period of 1 year in the
Human Microbiome Program (iHMP) and is the most comprehensive study till date
(Lloyd-Price et al. 2017). Interestingly, the dysbiosis consisted of changes in tran-
scription profile of several microbial species and associated changes in host bio-
chemical parameters such as considerable transition in acylcarnitine pools and bile
acids and heightened levels of serum antibody. IBD patients go through a cycle of
dysbiosis and non-dysbiosis phases. Taxonomic perturbations during dysbiosis
included reduction in obligate anaerobes such as Faecalibacterium prausnitzii and
Roseburia hominis and the increase in facultative anaerobes such as E. coli in
Crohn’s disease.
Therapeutics that help: Probiotics, especially Bifidobacteria, are linked to sup-
pression of mucosal inflammation in animal models of IBD. In humans, however,
the evidence for probiotic efficacy is less positive for maintenance of remission of
CD, based on meta-analysis of eight clinical trials, mainly due to variance in study
design. Inclusion of probiotics with conventional treatment for UC did not improve
the overall remission rates in mild-to-moderate UC, but it was possible to generate a
slight decrease in disease activity. The mixture VSL#3 (composed of four lactoba-
cillus strains; three from bifidobacteria and S. thermophilus) has shown positive
effects in UC treatment, whereas indications for probiotic efficacy in CD are low. At
the current stage, a conclusion has been reached that use of probiotics in IBD cannot
be recommended. Studies have been conducted using a diversity of bacterial strains
in different clinical situations, but only a few patients have been enrolled in these
studies. Hence, more randomized trials with statistically sound study design may
give us better clarity (Jadhav et al. 2020).
Among microbially derived products that have been shown to be protective in
colitis, polysaccharide A from Bacteroides fragilis and butyrate from Clostridial spp.
induce peripheral regulatory T cells. Favorable effects of FMT on UC (compared to
control treatment) have been published. However, there are many parameters such as
donor selection, administration routes, frequency of FMT and the development
of easy-to-administer formulation, that need to be optimized and validated before
FMT is ready to be offered as a standard therapy for UC.
10.7.7 Irritable Bowel Syndrome (IBS)
IBS is a disorder associated with the gastrointestinal tract and is marked by a long-
lasting, persistent discomfort and pain in abdomen and, with altered bowel
behaviors. Mainly based on the stool pattern, IBS patients can be categorized into
(1) IBS with constipation (IBS-C), (2) IBS with diarrhea (IBS-D), (3) IBS with
mixed bowel habits (IBS-M), and (4) unclassified IBS. The other associated
comorbidities linked with IBS are profoundly psychiatric in nature such as depres-
sion, anxiety, and somatoform disorders (Chong et al. 2019).
What’s the connect? In a healthy gut, the mucus epithelium and homeostatic
immune responses limit microbes both symbiotic and commensal from breaching
the epithelial barrier surface. Nevertheless, when the influx of any agent breaches the
barrier, provoking intense immune reactions, it leads to severe inflammation. This in
turn impacts the intestinal environment including the alteration in the composition of
gut microbiota (Pédron et al. 2016). Such underlying conditions with wider
consequences on the gut neuromuscular junction and gut–brain axis could contribute
to IBS pathogenesis. Gut dysbiosis accompanying the changes from beneficial
bacteria to pathogens in the human gut has been reported (Carroll et al. 2011,
2012). It is not surprising that IBS patients are highly deficient in the beneficial
activities of Lactobacilli and Bifidobacteria (Bellini et al. 2014). Interestingly, there
seems to be a direct correlation between the severity of IBS with low levels of
exhaled methane, decreased microbial richness, absence of Methanobacteriales, and
enrichment with Bacteroides enterotypes compared to that in controls (Tap et al.
2017).
Disease conditions with altered gut mycobiome profile are attracting the attention
of researchers, especially in immunocompromised patients. IBS is another condition
where the connection of microbiome alterations and the occurrence of visceral

hypersensitivity indicate the role of gut microbiome in the disease pathogenesis
(Botschuijver et al. 2017).
Therapeutics that help: Probiotics specially Lactobacillus plantarum is found to
be most beneficial in IBS by improving visceral sensitivity, intestinal permeability,
and inflammation (Ohman and Simrén 2013). Maintaining a food and symptom
diary can help patients determine which foods trigger symptoms. Specialized diets
such as where fermentable oligo-, di- and monosaccharide, and polyol (FODMAPs)
are less may improve symptoms in individual IBS patients, though the safety of this
diet such as the possibility of malnutrition needs to be monitored in the long term
(Ferreira et al. 2020).
10.7.8 Fecal Microbial Transplantation in Clostridium difficile

Infections
The commensal microbes of the gut have the capacity to defend against pathogenic
invasion by either competing for resources or waging microbial warfare. Consump-
tion of prescribed broad-spectrum antibiotics while controlling the ‘bad infection’
eliminates these beneficial commensals from the gut. Some individuals are then
predisposed to opportunistic pathogens such as Clostridium difficile resulting in
diarrhea and associated symptoms. Van Nood in 2014 reviewed the efficacy of
FMT in treating a variety of dysbiotic states (van Nood et al. 2014). It is encouraging
that FMT treatment showed around 90% success rate in treating C. difficile-induced
severe GI dysbiosis. In a patient with persistent C. difficile, associated with diarrhea,
the fecal microbiota both 2 weeks and 1-month post-FMT consisted mostly of the
bacteria from the healthy donor and normalized the patient’s bowel function, thus
demonstrating the success of the treatment. Until systematic trials evaluate both
safety and effectiveness of FMT, its efficacy is currently on a case-by-case basis. In
order to enhance treatment reliability and mitigate safety concerns, it is important to
identify the minimal subset of microbes needed to achieve therapeutic efficacy and
formulate the treatment using appropriate guidelines (Petrof et al. 2013).
10.7.9 Chronic Kidney Disease
Chronic kidney disease (CKD) is emerging health problem, and its occurrence is due
to important risk factors like diabetes, hypertension, and obesity affecting about 10%
of the population worldwide (Hall et al. 2014). The microbiome has recently gained
lot of importance and known to impart an important contribution in health and
disease (Hobby et al. 2019).
What’s the connect: Renal failure may occur due to high levels of urea in blood,
and in the presence of intestinal bacteria, it is converted to ammonia by urease
produced by intestinal bacteria leading to overgrowth of bacterial families. Patients
with end-stage renal disease (ESRD) have shown to have an expansion of uricase
and indole-forming and p-cresyl-forming enzyme-producing bacteria compared to

healthy controls (Wong et al. 2014). The most extensively studied gut-derived
uremic toxins, i.e., p-cresyl sulfate and indoxyl sulfate, arise from the colon with
increased concentration and decline in renal function (Aronov et al. 2011; Ramezani
and Raj 2014). In one study, the patients with ESRD have shown largest increase in
Actinobacteria, Firmicutes, and Proteobacteria compared to healthy controls
(Vaziri et al. 2013). Bifidobacterium catenulatum, Bifidobacterium longum,
Bifidobacterium bifidum, Lactobacillus plantarum, Lactobacillus paracasei, and
Klebsiella pneumoniae (Wang et al. 2012) are hardly seen in patients on peritoneal
dialysis.
Research from different groups have shown that patients undergoing hemodialy-
sis have higher number of Enterobacteria and Enterococci, facultative anaerobic
bacteria, and lower abundance particularly in the genera Bifidobacteria with a related
higher abundance of Clostridium perfringens than healthy controls (Kieffer et al.
2016).
In CKD and ESRD, patients who have reduced intake of fiber and decreased
colonic time have encountered comorbidities such as diabetes (Yasuda et al. 2002).
The disintegration of the epithelial barrier of the gut is mainly due to ammonia and
ammonium hydroxide derived from urea which allows absorption of the toxins
produced by microbes, thus leading to systemic inflammation which lead to anemia,
protein wasting, and cardiovascular disease (Wong et al. 2014).
Therapeutics that help: In rats with reduced renal function, AST-120, an oral
adsorbent, helps in restoring the epithelial tight junction proteins by absorbing
uremic toxins produced in gut and thus reducing the levels of endotoxin and markers
of oxidative stress and inflammation (Redman et al. 2014). There may be increase in
SCFA-producing bacteria due to increased intake of resistant starches reducing loss
of renal function, interstitial fibrosis, renal tubular damage, and activation of
pro-inflammatory molecules (Vaziri et al. 2014).
The serum levels of indoxyl sulfate have been reduced due to administration of
B. Longum orally (Takayama et al. 2003). In hemodialysis patients, plasma levels of
indoxyl sulfate have been reduced due to increase in dietary fiber (Sirich et al. 2014).
In vitro studies and in clinical studies, indoxyl sulfate has been reduced due to
Streptococcus thermophilus (Vitetta et al. 2019).
The metabolism of gut microbiota of CKD patients can be modulated by dietary
fiber which helps in improving CKD by reducing uremic toxins and also improves
uremic symptoms and comorbidities in dialysis patients. However, more clinical
studies are needed to be carried out in order to evaluate the multidimensional benefits
of a fiber-rich diet in CKD and to determine the effect of different kinds of fiber in
terms of quality as well as quantity (Camerotto et al. 2019).
10.7.10 Cancer
What’s the connect? Gut microbiome has played a significant role in tumor devel-
opment and effective anticancer therapies (Heshiki et al. 2020). Case in point is the
gut flora metabolizing bile acids which then redistribute and regulate the employ-
ment of natural killer T cells to tumorous liver cells (Ma et al. 2018). Another
example is those gut microbes which metabolize estrogen and hence potentially
altering the risk of postmenopausal estrogen receptor-positive breast cancer (Kwa
et al. 2016). Another case would be the microbes which take glucocorticoids, present
in the gut and urinary tract of men (leading to prostate cancer), and metabolize it to
produce 11-oxyandrogens (Devendran et al. 2017, 2018; Zimmermann et al. 2019).
Gut microbiota can act as a tumor promoter (Vivarelli et al. 2019). Helicobacter
pylori accounts for 90% of gastric cancers and is also considered to be a component
of stomach microbiome (Xavier et al. 2020). CagA protein (cytotoxin associated
gene A) from Helicobacter pylori was the first bacteria-derived protein to have a role
in human cancer (Hatakeyama 2017). It assists in increase of gastric cancer by
inducing the breakdown of p53 in gastric epithelial cells, by interfering with the
host’s AKT pathway (Buti et al. 2011). Beside this CagA protein, there are others as
well such as Fusobacterium nucleatum-derived effector adhesin A (FadA) and
Bacteroides fragilis-derived metalloproteinase toxin (MP toxin) which promote
cell proliferation and cancerogenic transformation of affected host’s cells by
interacting with epithelial E-cadherin of host, directly or indirectly hampering the
intercellular junctions and triggering β-catenin signaling (Murata-Kamiya et al.
2007; Rubinstein et al. 2013; Wu et al. 2007).
Similarly, Salmonella enterica effector avirulence protein A (AvrA) with its
intrinsic deubiquitinase activity activates β-catenin thereby enhancing cell prolifera-
tion and promoting colonic tumorigenesis (Lu et al. 2014). Escherichia coli-derived
colibactin and cytolethal distending toxin (CDT), when released near gastrointestinal
epithelium, release toxins. This creates break in double-stranded DNA within the
host’s epithelial cells, thus inducing a temporary cell cycle arrest, contributing to
genomic instability which ultimately leads to tumor initiation and progression in
those predisposed cells (Lara-Tejero 2000). This is generally encountered during the
dysbiosis of gut microbiome led by pathogenic infections (Halazonetis 2004; Frisan
2016).
H. pylori colonization may harm human health by causing gastroesophageal
reflux disease and its sequelae, Barrett’s esophagus, and adenocarcinoma of the
esophagus (Blaser and Atherton 2004) and benefit human health (by protecting
against asthma, multiple sclerosis, and IBD (Chen et al. 2017; Kira and Isobe
2019; Piovani et al. 2019; Gravina et al. 2018; Hosseininasab Nodoushan and
Nabavi 2019).
There are bacteria which block tumorigenesis inhibiting immune effectors and
thus induce cancer formation. Fusobacterium nucleatum indirectly helps in onset of
cancer by inhibiting its own hosts natural killer (NK) cells (Gur et al. 2015). Few
microbiota may obstruct the host hormones metabolism. Clostridium leptum and
Clostridium coccoides secrete β-glucuronidase and increased level of this enzyme
when coupled with gut dysbiosis results in deconjugating liver-catabolized and
plant-based estrogens. This in turn allows to bind and trigger the estrogen receptors
leading to cell proliferation in estrogen related tissues, i.e., as breast and
endometrium. There is a heightened risk of breast cancer due to intake of estrogen

hormones (Fernández et al. 2018).
Some gut microbiota act as a tumor suppressor. Microbial-derived SCFAs like
butyrate and propionate are able to exhibit an anticancer effect by inhibiting host’s
tumor cells histone deacetylases (Vivarelli et al. 2019). Case in point is the exten-
sively studied LPS (bacterial lipopolysaccharide), which triggers the surface toll-like
receptor 4 (TLR4) on the host’s cell. This starts a immune response (T-cell mediated)
against cancer cells (Paulos et al. 2007). Similarly, the Salmonella enterica-derived
monophosphoryl lipid A (MPL) is presently used in the vaccine as adjuvant, against
anticervical carcinoma (Paavonen et al. 2009). Furthermore, pyridoxine, a group B
vitamin from bacteria, is capable of stimulating antitumoral immunosurveillance in
host (Aranda et al. 2015). The chapter dedicated to the role of the microbiome in
immunotherapy will shed more light on that aspect.
Western diet can cause increase in intestinal tumors due to high animal fat and
protein which increases bile secretion into the GI tract (Goncalves et al. 2019).
Bilophila wadsworthia metabolizes taurine into acetate and ammonia, along with
release of hydrogen sulfide, a carcinogenic gas (Ridlon et al. 2006). In colorectal
cancer, Fusobacterium nucleatum is one of the most predominant bacteria (Bullman
et al. 2017).
Therapeutics that help: The use of probiotics synergistically with pain killers
such as opioids is being studied (Rousseaux et al. 2007). Many probiotics have
shown a potential antineoplastic activity. For example, ferrichrome, a Lactobacillus
casei-derived metabolite, is able to trigger apoptosis in tumor cells via direct
activation of JNK pathway (Konishi et al. 2016). It has been appeared in several
studies that Lactobacilli may eliminate cancerous or precancerous cells by stimula-
tion of immune cells like NK cells or dendritic cells (DC) or TH1 response in the
host (Lenoir et al. 2016; Lee et al. 2004; Baldwin et al. 2010; Takagi et al. 2008).
Lactobacillus casei when orally administered reduces the reappearance of superficial
bladder cancer (Aso and Akazan 1992). Lactobacillus rhamnosus GG (LGG) is a
gut-resident bacterium which modulates several proliferation pathways of host, for
example, mTOR or WNT (Taherian-Esfahani et al. 2016), and thus wield either
antiproliferative effects or antimetastatic effects (Orlando et al. 2016; Nouri et al.
2016; Zhao et al. 2017; Behzadi et al. 2017; Chen et al. 2017). This can influence
immune system of host, consequently helping in eliminating newly developing
cancer cells (Gamallat et al. 2016). LGG can exert an anti-inflammatory profile by
triggering an immune response within the normal untransformed gut epithelium
(Suzuki et al. 2017) as well as changing gene expression in intestinal porcine
epithelial cells and intestine myofibroblasts (Taranu et al. 2018; Uribe et al. 2018).
LGG can serve as an apt candidate as a possible adjuvant in integrated anticancer
therapies (Vivarelli et al. 2019).
The main purpose of giving probiotics especially Lactobacilli, to cancer patients,
is to reestablish the levels and function of the beneficial bacteria which gets
exhausted after the treatments, thus restoring the microbiota balance in the patients’
gut (Zitvogel et al. 2018). At the same time, probiotics may possess a likely risk of
development of opportunistic bacteria/infection and exhibit antibiotic resistance

when administered to immunocompromised cancer patients (Vanderhoof and
Young 2008; Redman et al. 2014).
In 2014, it was observed in a double-blind control trial that Bifilact (Lactobacillus
acidophilus LAC361 and Bifidobacterium longum BB536) administration could
considerably reduce both moderate and severe diarrhea induced by the pelvic
radiation treatment (Demers et al. 2014). On similar front, in 2015, a probiotic
formulation Colon Dophilus (a cocktail of ten different strains) was evaluated in a
clinical trial in metastatic CRC patients treated with irinotecan-based chemotherapy.
Prevention of diarrhea in such patients suggested that the administration of such
probiotics facilitates in the reduction of the occurrence as well as severity of diarrhea
and gastrointestinal toxicity induced by the chemotherapy and is considered to be
safe (Mego et al. 2015). In 2016, it was observed that probiotic Saccharomyces
boulardii has the ability to downregulate pro-inflammatory cytokines in treated
patients, with no beneficial effects on the postoperative infection rates (Consoli
et al. 2016). In 2017, it was observed that the epigenetic patterns of tumor tissue
from its baseline can be altered, with potential therapeutic benefits by the adminis-
tration of Bifidobacterium lactis and Lactobacillus acidophilus to CRC patients
(Hibberd et al. 2017).
10.7.11 Mental Disorders
Mood disorders, such as depression and bipolar disorder (BD), have known to cause
significant individual and socioeconomic burdens affecting around 10% of the
world’s population (Wittchen 2012). When compared to the normal population,
people with mood disorder incline toward higher mortality rates and reduced life
span (Angst et al. 1999; Kessing et al. 2015).
What’s the connect? The human gut has trillions of bacterial which are known to
play a critical role in communication between gut and brain via influencing neural,
immune, and endocrine pathways (Dupont et al. 2020). Patients with a variety of
psychiatric disordered such as BD, schizophrenia, depression, and autism spectrum
disorder display a significant difference in the composition of the gut microbiome.
Thus, there is renewed interest in treating the gut microbiome as a potential thera-
peutic target in psychiatry. Psychobiotics, a recently coined term, refers to favorable
microbes that may provide value in diagnosed mental disorders cases. The term
refers to ‘a live microorganism that, when taken in adequate amounts, produces a
beneficial health benefit in patients suffering from psychiatric illness’ (Dinan et al.
2013). The research on gut microbiome related to mood disorders is still in its
nascent stage. Although growing evidence suggest that alteration in the gut
microbiome plays a significant role in patients with mood disorders, the cause–effect
relationship is yet to be determined. In major depressive disorder (MDD) and bioplar
disorder, a consistent surge in the abundance of Actinobacteria and
Enterobacteriaceae and a decline in Faecalibacterium were observed in different
studies (Sowa-Kućma et al. 2018). Raised levels of Clostridium sp. have constantly
been observed in patients with autism spectrum disorder. These discoveries imply
that in patients with mood disorders, a decrease in bacterial genera which produces
short-chain fatty acids and an increase in pro-inflammatory genera can be linked to
chronic, low-grade systemic inflammation. On the other hand, till now, a clear
relation between gut microbiota composition and anxiety disorders has been not
been reported (Groen et al. 2018).
Exploring microbiome therapeutics: Changing the diet and incorporating
probiotics/prebiotics in order to enhance the beneficial bacteria in the gut in both
healthy and patient groups are likely to improve mood and decrease anxiety. Diet
and food habit/pattern lead to the formation of gut microbiota which in turn regulate
the host inflammation and thrombosis, causing brain disorders (Zhu et al. 2020a, b).
For depression and other mental disorders, omega-3 fatty acids, ions such as zinc and
magnesium, and plant phytochemicals are found to be more relevant nutrients
(Kaplan et al. 2015; Trebatická and Ďuračková 2015; Haider et al. 2015; Bouayed
et al. 2007; Solanki et al. 2015). Epidemiological studies have stated that traditional
diet styles lead toward better mental health and hence reduce the risk of depression
(Jacka et al. 2011; Jacka et al. 2010; Sánchez-Villegas et al. 2009; Skarupski et al.
2013; Rienks et al. 2013).
Food rich in polyphenol such as green tea, coffee, cocoa, curcumin, and other
polyphenol-rich foods is connected with better mood, less fatigue, and hence
reduced risk of depression in humans (Pham et al. 2014; Sathyapalan et al. 2010;
Pase et al. 2013; Yu et al. 2015). These foods in preclinical settings prevent dysbiosis
by inducing the growth of beneficial bacteria (Seo et al. 2015; Mills et al. 2015;
Massot-Cladera et al. 2012; McFadden et al. 2015). It emerges that microbially
transformed phytochemicals (e.g., quercetin after it has been subjected to fermenta-
tion) can alter the gut microbiota in healthy ways (i.e., growth of bifidobacteria and
decrease in the ratio of Firmicutes to Bacteroidetes) (Parkar et al. 2013). Honey is an
important diet constituent, high in phytochemicals such as phenolic acid and that of
flavonoid families (Alvarez-Suarez et al. 2013), and has antidepressant and anxio-
lytic properties too (Azman et al. 2015; Mijanur Rahman et al. 2014). Honey
flavonoids when stimulated by LPS hinder the release of pro-inflammatory
cytokines, e.g., tumor necrosis factor alpha and interleukin-1 beta from microglia
in a significant manner (Candiracci et al. 2012).
Free radicals or reactive oxygen species (ROS) lead to oxidative stress and trigger
the pro-inflammatory avalanche, which involved IL-6 and CRP, both linked with
depression. Several dietary antioxidants such as curcumin, ascorbic acid,
carotenoids, and flavonoids are found to diminish depressive symptoms, either
directly or linked to a lower occurrence of depression (Christy Harrison 2020).
Fermentation leads to transformation of bioavailable phytochemical structures
and peptides which could lead to novel therapeutics (Selhub et al. 2014). It has been
reported that consumption of fermented food results in reduced social anxiety,
particularly in people with high neuroticism scores (Hilimire et al. 2015). Fermented
food has heat-inactivated microorganisms, and their structural parts may also have
significant effects on intestinal ecosystems (Yang et al. 2014).
10.7.12 Rheumatoid Arthritis
Rheumatoid arthritis (RA), an autoimmune disorder, is dependent on/controlled

by/related to several factors, both genetic and environmental. Since it targets the
self-antigens present in the synovium, cartilage, and bone, it leads to destruction of
the joints and hence functional disability in most patient (Halpern et al. 2009; Tran
et al. 2005). Majority of the drugs available for the treatment of RA today (including
DMRDs or disease-modifying antirheumatic drugs) target cytokines, nonspecific
immune suppression, or T-cell and B-cell activation (Isaacs 2008; Kumar and Banik
2013; Benjamin et al. 2018). Besides the genetic factors, diet, smoking, and stress
are some of the environmental factors which influence the microbiota diversity/
composition and hence the onset/outcome of RA.
What’s the connect? The onset of autoimmunity may be associated with gastro-
intestinal tract. It is found that microbial composition in subjects with early rheuma-
toid arthritis differed from controls, with a reduction of bacteria from the family
Bifidobacterium and Bacteroides (Vaahtovuo et al. 2008), and a noticeable increase
of species from the genus Prevotella (Bernard 2014). Most of the studies indicated
that treatment with MTX and HCQ leads to partial restoration of dysbiotic
microbiomes to normal or alteration to increase abundance of beneficial microbial
members (Zhang et al. 2015). Methotrexate reduced the abundance of
Enterobacteriaceae and partially restored the gut microbiota in patients.
Hydroxychloroquine increased the gut microbial species’ richness and diversity.
Hydroxychloroquine plus doxycycline treatment led to the reduction in abundance
of phylum Bacteroidetes and Firmicutes (Bodkhe et al. 2019).
Recent studies in humans and mouse models of arthritis indicate that periodontal
disease is linked with a heightened risk of RA (Scher et al. 2012; Arvikar et al. 2013;
de Aquino et al. 2014). The presence of periodontitis is associated with anti-CCP
antibody levels in RA patients (Dissick et al. 2010). Treatment of periodontitis
enhances the disease activity of RA (Ortiz et al. 2009; Al-Katma et al. 2007). It is
found that periodontal bacteria such as P. Gingivalis and A. actinomycetemcomitans
induce the production of anti-CCP antibodies, which in turn leads to the arthritis thus
suggesting the link of periodontal bacteria with RA pathogenesis. Another evidence
comes from the finding that DNA of oral cavity bacteria such as Fusobacterium
nucleatum and Porphyromonas gingivalis has been detected in the synovial fluid of
RA patients (Reichert et al. 2013; Stephanie et al. 2012). In order to establish the
mechanistic link of the oral cavity bacteria with that of development of RA in
humans, more studies are needed. Oral microbiome and periodontal disease are
described in another chapter in further detail.
Exploring microbiome therapeutics: Randomized controlled trials with probiotic
bacteria like Lactobacillus rhamnosus, (Pineda et al. 2011; Hatakka et al. 2003),
Lactobacillus casei (Vaghef-Mehrabany et al. 2014), Bacillus coagulans (Mandel
et al. 2010), Lactobacillus reuteri, Lactobacillus acidophilus, and Bifidobacterium
bifidum have been carried out to determine their ability to treat RA. Randomized
controlled trials with probiotic bacteria have been studied for their ability to treat
RA, like Lactobacillus rhamnosus, (Pineda et al. 2011; Hatakka et al. 2003)
Lactobacillus casei (Vaghef-Mehrabany et al. 2014), Bacillus coagulans (Mandel

et al. 2010) Lactobacillus reuteri, Lactobacillus acidophilus, and Bifidobacterium
bifidum. These probiotics have appeared to be effective for RA patients and are
considered safe (Zamani et al. 2016). However, since there is limited data on the
interaction between drug and microbiomes and probiotics for RA, further studies are
needed for a probiotic to be used for immune homeostasis. This could be helpful in
personalized medicine where a tailor-made probiotic (targeted organism/metabolite
which is in low abundance) can be supplemented to create immune homeostasis in
patients.
10.8 Conclusion
Acknowledging the impact that the microbiome has on human health and well-being
by scientific and nonscientific communities has allowed us to get curious and explore
this area in our own way. The existing approaches of microbiome diagnostics and
therapeutics need to evolve keeping in pace with current technologies. New
microbiome diagnostic marker panels developed based on Next-Gen sequencing
can be used to diagnose (and potentially prognos) disease by clinicians. Predictive
medicine using AI/ML algorithm-based analysis of patient’s microbiome could
predict the outcome of treatment options. To revolutionize microbiome therapeutics,
the treatment options should move from general to personalized ones. Such
personalized treatment strategy can be created based on the gut microbiome of an
individual (patient-specific). The treatment options can range from ‘precision
probiotics’ where a specific microbial cocktail can be administered to alter the
species richness or ‘precision prebiotics’ where targeted microbial nutrition is
given to promote growth of certain microbes or ‘personalized dietary interventions’
or ‘targeted subtractive theory’ (Fig. 10.5). Microbiome time series analysis to
follow-up treatment success and establishment of normobiosis will allow early
intervention and tweaking of treatment plans for better patient outcomes. Neverthe-
less, though the challenges are many, it is achievable. It will be thrilling to witness
this microbiome-based therapeutic model being adopted in routine clinical practice
and becoming a true translational discipline in the near future!
194
Fig. 10.5 A schematic illustration showing various aspects associated with microbiome. Microbiome balance is affected by many factors (left panel) and leads
to disease condition (top-right panel). The use of broad range antimicrobial may lead to further disbalance the microbiome by killing the good bacteria along
with the targeted bad ones. Microbiome diagnostic (bottom-left panel) can reveal the upregulated/downregulated microbes in disease condition. The advent in
N. Dasgupta et al.
sequencing leads to better diagnostic, and the better the diagnostic, the better will be the therapeutics (bottom-left panel). Therapeutics involves the microbes
either directly (live microbes) or indirectly (metabolites from bacteria or products to support the growth of good bacteria). (Allaband et al. 2019; Espinoza 2018;
FitzGerald and Spek 2020; Raes 2016)
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Microbiome Therapeutics: Emerging
Concepts and Challenges 11
J. Sarada, S. Anju, Y. Aparna, and K. Anuradha
Abstract
The human microbiome has become a thrust area for researchers and industrial
scientists in understanding microbial roles in health and disease. With the advent
of NGS methods, good progress is made in analyzing the composition of the
microbiome and their key metabolites produced, so many researchers are now
trying to explore the potential of the microbiome as an important therapeutic tool.
Microbes in the human gut are identified as key contributors in host metabolism
and could be explored as novel therapeutics. A large number of research papers
reviewed different aspects of the microbiome and its potential role in
cardiometabolic disorders, inflammatory bowel diseases, neuropsychiatric
diseases, and cancer. Research studies made in the last 5 years proved that the
microbiome disruptions play a vital role in malnutrition, obesity and also modu-
late associations between diet and disease. Beyond the gut, the human
microbiome likely affects all organs through the immune, circulatory, and ner-
vous systems, affecting human behavior and cognitive function. Researchers
have made efforts to clarify the role of the microbiome in autoimmune disease
development and find new therapeutic approaches to treat immune-mediated
diseases. For example, bacterium A. muciniphila is viewed as a next-generation
beneficial microbe as supplementation of it protects against several
cardiometabolic features and decreases several pathological conditions, such as
obesity, type 2 diabetes, hypertension, hypercholesterolemia, and liver disease.
Hence microbiome-based therapeutics are being identified as an integral part of
the precision medicine approach because of the contribution to inter-individual
variability in diseases condition and also as a modifiable factor leading to the
J. Sarada (*) · S. Anju · Y. Aparna · K. Anuradha

Department of Microbiology, Bhavan’s Vivekananda College of Science Humanities and
Commerce, Sainikpuri, Secunderabad, Telangana, India
Ltd. 2021
https://doi.org/10.1007/978-981-16-3156-6_11
218 J. Sarada et al.
development of future therapeutics. They have the potential to influence the

therapeutic strategies in the treatment of several diseases, generate new economic
opportunities, and benefit humans around the globe.
Keywords
Microbiome · Therapeutics · Human gut · Health and disease
11.1 Introduction
The microbiome has become the central concept among academic researchers and
biotechnology-based companies to explore the hidden microbial roles in health,
disease and to understand microbiome-based interventions. Different microbial
communities are found throughout the mucosal surfaces and cavities present in the
human body and contribute to diverse physiological processes like immunity,
metabolism, nutritional homeostasis, and neuronal activity in a healthy individual
(Fig. 11.1). The human body in return provides a suitable environment for stable
colonization of this commensal microbiome.
With the advent of Metagenomic and Gnotobiotic techniques, microbiome con-
tribution to human health is being established. Variations in gut microbiome com-
position and function are connected to the etiology of several diseases. Loss or
changes in the microbiome and its homeostatic function could be due to infection,
inflammation, improper diet, and or antibiotics. The microbiota of each organ in the
human body is unique and the microbial population in different organs gives
important information about the occurrence of disease symptoms. Therefore, the
microbiome is found responsible for the clinical symptoms and drug response of
individuals. The population of beneficial microbes in healthy individuals is more
Fig. 11.1 Role of Gut Microbiome in Human Health

11 Microbiome Therapeutics: Emerging Concepts and Challenges 219
diverse and can withstand physiological changes than the disease-associated

microbiota which causes inflammation.
Several studies were made to prove that interventions to modulate the
microbiome help in improving the health of a person. Current microbiome-based
therapeutics (Table 11.1) aim at altering the gut microbiome by using live microbes
as probiotics. Instead of probiotics, metabolite-based approach or prebiotics are also
used to affect microbiome composition and/or function in a beneficial way.
The Human Microbiome Project (HMP) was launched by the National Institute of
Health in 2007, to provide expertise and knowledge to characterize the human
microbiome and understand its role in the health of a person. HMP provides
guidance and direction to unravel the role microbes play in human health, disease,
nutrition, and immunity.
Metagenomic projects assisted in the detection of the pathobionts in Colorectal
Cancer (CRC), Inflammatory Bowel Diseases (IBD), Irritable Bowel Syndrome
(IBS), Clostridium difficile infection (CDI), Cardiovascular Diseases (CVD), auto-
immune diseases, and several Non-Communicable Diseases (NCDs) like Obesity,
Asthma, Type 1 Diabetes, Eczema, etc. (Fig. 11.2). These studies help to prevent and
even cure by modulating the human microbiota. As a biomarker, the human
microbiome can be used to detect diseased conditions and has the potential for the
diagnostic and therapeutic role. This approach supports diagnosis and aids in novel
therapeutic strategies.
Microbiome-based personalized medicine has become a new therapeutic
approach especially for the treatment of cancer. Since microbiota is identified to
modulate carcinogenesis through mechanisms like inflammation and immunity, the
microbiome can be used to develop anticancer therapies.
11.1.1 Colon Rectal Cancer (CRC) Therapy
Any changes of the normal microbiome that could interrupt host–microbial

interactions lead to dysbiosis which in turn result in diseases. Dysbiotic condition
is commonly observed in several human diseases especially cancer. Colon harbors
70% of the human microbiome and thus more prone to cancer development than the
small intestine in our body. Origin and development of Colorectal Cancer (CRC)
involve alteration in the gut microbiota. Hausen reported that 20% of the cancers are
attributed to intestinal microbiota changes from normal flora to the infectious state
(Hausen 2009). Mutations in proto-oncogene, tumor suppressor genes, and genes
involved in DNA repair lead to induction of CRC. Environmental factors like
smoking, alcohol, processed foods, animal fat, and low intake of fiber and fruits
are known to trigger the development of CRC.
Gut microbiota help to maintain mucosal homeostasis and epithelial barriers. Wu
et al. and Yoshioka et al. reported that microbial metabolite butyrate initiates
dysfunction in the gut epithelia by activating pro-inflammatory mediators like
cytokines, interleukin-6, and tumor necrosis factor-α (Yoshioka 2009; Wu 2006).
Goodwin et al. stated that Bacteriodes fragilis and Enterococcus faecalis stimulate
Table 11.1 Microbiome therapeutics in various diseases
220
Types of microbiota present in

disease condition or dysbiotic Biomarker strains
Disease microbiota Depletion of microbiota for diagnosis Therapeutic microflora
Colorectal Streptococcus gallolyticus, Roseburi, Clostridium, F. Nucleatum Bifidobacteria spp.; Akkermansia
cancer F. nucleatum, E. coli, E. fragilis, and Faecalibacterium, and muciniphila, Enterococcus hirae,
E. faecalis Bifidobacterium Bacteroides spp.
Irritable bowel No information Lactobacillus, Bifidobacterium, No information Bifidobacterium, Lactobacillus, and
syndrome (IBS) and Faecalibacterium acidophilus, L helveticus, and
prausnitzii Bifidobacterium infantis Pediococcus
acidilactici and two L plantarum
strains
Inflammatory Enterobacteriaceae Firmicutes: Clostridium clade, Enterobacteriaceae Bacteroides fragilis Segmented
bowel diseases and Bacteroidetes Filamentous Bacteria (SFB) and
(IBDs) Mucispirillum, engineered
Lactococcus lactis
Clostridium C. difficile No information C. difficile Lactobacillus casei, Lactobacillus
difficile infection bulgaricus, and Streptococcus
(CDI) thermophilus, purified Firmicutes
spores
Food allergy Bacteroides, Propionibacterium, No information No information Lactobacillus rhamnosus GG (LGG)
and Klebsiella
Type 1 diabetes Bacteroidetes, Bacteroides dorei, No information No information Lactobacillaceae-
(T1D) Bacteroides vulgatus
J. Sarada et al.
11
Multiple Methanobrevibacter, ermansia, Butyricimonas No information Lactobacillus spp., Pediococcus

sclerosis Pseudomonas, Mycoplasma, acidilactici, Bifidobacterium bifidum,
Haemophilus, Blautia and Dorei Bifidobacterium animalis, and
Bacteroides fragilis
Systemic lupus Bacteroidetes members, and Firmicutes No information Supplementation with Treg-inducing
erythematosus Rhodococcus, Eggerthella, bacteria
Klebsiella, Prevotella, Eubacterium,
Flavonifractor, and Incertae sedis
Microbiome Therapeutics: Emerging Concepts and Challenges
221
222
Fig. 11.2 Dysbiosis related diseases

J. Sarada et al.
initiating preliminary responses for inflammation and transformation of epithelia and

make cancer indigenous bacteria participate in cancer induction (Goodwin et al.
2011). Dysbiosis gut microbiota causes functional imbalance by initiating
pro-inflammatory responses and epithelial cell transformation and make driver
bacteria (cancer indigenous intestinal bacteria) participate in cancer initiation. Dur-
ing the tumor developing stage, microenvironmental alterations favor the growth of
bacterial passengers or opportunistic bacteria. Comparison of fecal microbiota from
CRC patients with healthy individuals shows enriched and depleted microorganisms
differentiating CRC patients from control populations. The occurrence of Strepto-
coccus gallolyticus, F. nucleatum, Escherichia coli, B. fragilis, and E. faecalis was
more in CRC patients than in healthy people. Interestingly bacteria like Roseburia,
Clostridium, Faecalibacterium, and Bifidobacterium are found to be depleted in
CRC patients.
Diagnosis at the initial stages can prevent CRC either by surgeries or by therapy
which has a higher success rate. Generally, FOBT (Fecal occult blood test) and FIT
(fecal immunochemical test) are used along with colonoscopy for diagnosis.
Microbiome signatures in stool samples were used as microbial markers in the
early detection of CRC. Yu et al. reported that adenoma and CRC patient’s stool
samples had F. nucleatum (Yu 2017). In a meta-analysis research study, Zhang et al.
also reported that F. nucleatum becomes a biomarker for a non-invasive screening in
CRC and colorectal adenoma (Zhang 2019). Another strategy is to screen for fecal
metabolome metabolite markers like SCFA, fructose, linoleic acid, and nicotinic
acid in CRC patients.
Microbiome modulation stands as an alternative approach to prevent or treat
CRC. Strategies so far in use are fecal microbiota-transplantation (FMT), pre /
probiotics, and diet. Among all, FMT has become a hopeful strategy for CRC
patients. Since the gut microbiome stimulates the immune system, they possess
anticancer effects and become key contributors to tumor immunotherapy.
Bifidobacteria spp.; Akkermansia muciniphila, Enterococcus hirae, Bacteroides
spp exhibit anti-tumoral response by activating T-cells. The role for
A. muciniphila is identified to be prominent in anticancer immunotherapy, especially
with anti-PD-1 treatment.
Singh and his group observed that diet influences the human gut microbiome
(Singh 2017). In a recent report, it was observed that the mouse model when induced
by deoxycholic acid and fed with a western-type diet initiated intestinal carcinogen-
esis. However, with supplementation of a diet rich in fiber content and butyrate-
producers colon tumor growth could be drastically reduced (Cao 2017; Pandey
2015). Probiotics were known to inhibit CRC by producing detoxifying agents,
anti-inflammatory factors, anticancer compounds, and short-chain fatty acids
(SCFAs) which improve the intestinal barrier function.
A combination of chemotherapy and/or immunotherapy with supplementation
with diet, probiotics, and prebiotics targeting the gut microbiota is a promising
strategy. A study made by Ding et al. has shown that oral administration of
Bifidobacteria along with PD-L1 inhibitors had a synergistic effect inhibiting
tumor growth, against the individual on chemotherapy alone (Ding 2018).
Pandey et al. reported the use of synbiotics like OAT fiber/L. plantarum and
FOS/L. sporogens for therapy (Pandey 2015). Bozkurt et al. genetically modified
Bifidobacterium animalis species for butyrate and mycosporin-like amino acids
production for prebiotics effects (Bozkurt 2019). This genetically altered strain
could modulate host immunity by regulating cytokine production, macrophages
and lymphocyte proliferation. Ding et al. stated that pro /synbiotics therapies
were useful for CRC patients. Bifidobacteria spp., Akkermansia muciniphila,
Enterococcus hirae, Bacteroides spp were reported to cause an anticancer immune
response by T-cell activation.
11.1.2 Gut Microbiome and Chimeric Antigen Receptor (CAR) T-Cell

Therapy.
The targeted immunotherapeutic approach is found to be more effective and less

toxic in cancer treatment than conventional chemotherapy. Immunotherapy includes
targeting programmed cell death 1 (PD1) receptor, programmed cell death ligand
1 (PD-L1) or cytotoxic T-lymphocyte antigen-4 (CTLA-4) inhibitors, and chimeric
antigen receptor (CAR) T-cells which could control the natural solid and hemato-
logical malignancies (HM). Chimeric Antigen Receptor (CAR) T-cells are autolo-
gous T-cells targeted to a tumor-specific antigen and seem to be more promising for
patients suffering from unmanageable hematological malignancies.
CD19-directed CAR T-cells therapy was approved by FDA for cancer patients.
However, variations in clinical responses to immuno and CAR T-cells therapies call
for deeper investigations on factors influencing the therapy. Bifidobacteria longum is
known to boost response in Immune Checkpoint Inhibition (ICIs) while
Bifidobacterium bifidum is immunosuppressive. Usage of antibiotics during immu-
notherapy and CAR T-cells therapy lead to dysbiosis.
Gut microbiota negotiates with both systemic immunity and immune checkpoint
inhibition (ICI) responses and demarcates the native T-cells either into
pro-inflammatory (Th17) or anti-inflammatory (Tregs) effector cells. Then these
cells migrate to systemic circulation. Several strategies like interference in
pre-CAR conditioning, usage of antibiotics with the narrow spectrum, suppression
of CAR T-cells by Treg cells could be explored by using specific gut microbes.
In recent research investigations, it was observed that gut microbiome diversity
and composition influence response to immunotherapy. Muhammad Bilal Abid et al.
hypothesized that the modulation of the gut microbiome could enhance the CAR
T-cell response (Muhammad Bilal Abid 2019).
Bacteria like Faecalibacterium prausnitzii, Ruminococcaceae, Clostridia when
used in therapy gave superior response and survival to patients while
Enterobacteriaceae members yielded low response and decreased survival.
Akkermansia muciniphila is known to boost the immune response and increase
survival. Many clinical studies had proved that manipulation of gut microbiota
results in enhancement of immunotherapy responses.
11.1.3 Irritable Bowel Syndrome (IBS) Therapy
IBD is also known as a functional gastrointestinal disease (FGID), has a global

prevalence of 10%, and is known to affect the quality of life among the patients
suffering from it. Pathogenic symptoms like dysmotility, visceral hypersensitivity,
abnormal responses to stress play a role in the development of IBS. Diagnosis is
based on symptoms like constant abdominal pain during defecation and change in
stool frequency and form. Microbiota mediated changes in bile acid deconjugation
affect stool volume and consistency and changes in microbial fermentation affect gas
volume in these patients. IBS diagnosis poses a challenge as it lacks a universally
reliable biomarker, and heterogeneity in its progression, and nonspecific symptoms.
Enteric Dysbiosis and Small Intestine Bacterial Overgrowth (SIBO) are identified
as the main factors associated with the IBS altering mucosal permeability. Enteric
bacteria like Lactobacillus, Bifidobacterium, and Faecalibacterium prausnitzii were
fewer in these patients than in healthy individuals. For therapy presently prebiotics,
probiotics, antibiotics, diet, and FMT are in use, all targeting gut microbiota.
11.1.3.1 Prebiotics
Prebiotics like fructooligosaccharides (FOS) or galactooligosaccharides (GOS) are
identified to support the growth of Bifidobacteria and Lactobacilli and give health
benefits for the host. Supplementation of trans-GOS at 3.5 g/day improved stool
consistency and flatulence, while supplementation at 7 g/day only improved subjec-
tive universal estimation. Interestingly at two doses Bifidobacteria and Lactobacilli
abundance significantly increased. Vulevic, et al. reported that supplementation of
β-GOS improved flatulence, stool consistency, bloating, and growth of
Bifidobacteria at low (3.5 g/day) and high (7 g/day) dose groups (Vulevic et al.
2018).
11.1.3.2 Probiotics
Probiotics treatment includes supplementation of Bifidobacterium, Lactobacillus, or
Saccharomyces alone could cure the IBS-based symptoms. Meta-analysis of probi-
otic treatment identified that a treatment period of fewer than 8 weeks could improve
the overall symptoms and Quality of life (QoL). Multiple Probiotic mixtures—VSL
#3 has Bifidobacterium, Lactobacillus, and Streptococcus strains and when
supplemented decreased abdominal bloating in patients with IBS-Diarrhea. Supple-
mentation in IBS patients alone reduced colonic transit time and flatulence. Another
probiotic mixture SCM-III, containing L. acidophilus, L. helveticus and
Bifidobacterium species, was effective and reduced bloating, abdominal pain, and
improved bowel habit. Pediococcus acidilactici and two L. plantarum strains as the
probiotic mixture could significantly improve gut-specific anxiety. However, con-
tinuous use of probiotics in susceptible patients may cause infection, sepsis, and
endocarditis.
11.1.3.3 Dietary Therapy

Dietary therapy recommends the low consumption of Fermentable
Oligosaccharides, Disaccharides, Monosaccharides, and Polyols also known as
FODMAPs. As the gut microbiome quickly ferments FODMAPs and starts water
secretion and gas production, a diet low in FODMAP (LFD) helps to control IBS
symptoms. LFD with probiotic supplementation also prevents the reduction in
Bifidobacteria population. Diets rich in FODMAPs and excessive prebiotics usage
lead to dysbiosis of enteric bacteria resulting in the formation of excessive gas.
Several research groups are working to treat IBS by restoring homeostatic balance in
enteric bacteria with a suitable strategy.
11.1.3.4 FMT
FMT tries to restore the microflora in the intestine by injecting fecal suspension from
a healthy donor into the gastrointestine of the patient. A review of FMT studies
revealed that it was beneficial up to 58% of patients treated and success depended on
host–microbiome characteristics. Donors rich with Bifidobacteria species induce
symbiosis in IBS patients more efficiently. In a study made by Halkjaer et al. with
oral FMT and placebo capsules, they observed enteric biodiversity in both groups
(Halkjaer et al. 2017). However, an investigation made by Johnsen and group
revealed that prominent improvement was obtained with freshly prepared and frozen
FMT than the placebo (Johnsen et al. 2018). These observations indicate that the
efficacy of FMT depends upon the factors like bowel preparation, route of delivery.
Even though the above-mentioned strategies were found to be promising, several
limitations were also observed in these treatments. Proof of concept for a direct
relationship between bacterial overgrowth in the small intestine with IBS symptoms
exists, the linkage between the microbiome and IBS symptoms is yet to be evaluated
and needs more validated experimental designs. Prebiotic usage is often linked with
unwanted abdominal symptoms and their efficacy yet needs to be proved. Probiotics
have heterogeneous methodologies but to a greater extent have benefits for IBS
symptoms. The efficiency of probiotics requires further research on the type of
probiotic to be used, measured quantity, treatment duration, etc. FMT needs impro-
visation in course of therapy and method of administration.
11.1.4 Inflammatory Bowel Diseases (IBDs) Therapy
IBD is an intestinal disorder resulting in extended inflammation of the gastrointesti-

nal region and caused because of unusual immune reactions to intestinal bacteria.
IBD has two diseases, namely Crohn’s disease (CD) and ulcerative colitis (UC).
Inflammation of the large intestine is seen in UC wherein surface mucosal layers are
affected. Patchy transmural inflammation in any part of the digestive tract is found in
Crohn’s disease. Many countries across the world are found suffering from IBD.
Dysbiosis of the gut microbiome resulting in a decrease in anti-inflammatory
microbes like Firmicutes: Clostridium clade, and Bacteroidetes supports patient
susceptibility to colonization by colitogenic strains, like Escherichia species. Four
effective therapeutic strategies for treatment are (1) manipulation gut flora, (2) gut
immunity, (3) maintain gut homeostasis, and (4) decrease inflammation.
11.1.4.1 Gut Microbiota Mediated Immunomodulation Therapy

Hosts immunological tolerance to commensal microbiota comes from immunologi-
cal responses triggered by gut microbes. Polysaccharide A capsule of B. fragilis
induces Treg cells and aids in developing tissue homeostasis. B. fragilis stimulates
dendritic cells by secreting cytokine and interleukin-10 to curb inflammation of
mucosa and activate CD4+ T-cells to release transforming growth factor-β. Protec-
tive immunomodulatory signals are elicited to immune cells by B. fragilis through
outer membrane vesicles. Commensal Segmented Filamentous Bacteria (SFB) and
Mucispirillum elicit T-dependent IgA production. These investigations provide a
deeper understanding of the role of gut microbes in triggering or inhibiting host
immune response, which could be used later to modulate inflammatory responses in
clinical IBD (Chu et al. 2016; Lécuyer 2014; Bunker et al. 2015).
11.1.4.2 Precision Edited Gut Microbiota Mediated Therapy

This method was investigated by research Zhu et al. with a colitis mouse model (Zhu
et al. 2018). Gut inflammation caused by Enterobacteriaceae was specifically
inhibited by targeting molybdenum-dependent pathways by Tungsten
(W) treatment because tungsten can replace molybdenum, and prevents its consump-
tion in pathways during inflammation. This treatment reverses the dysbiotic state of
the gut to normal composition in colitic mice by diminishing the pro-inflammatory
markers and pathological changes. Colonization of Clostridium scindens which
makes 7α-dehydroxylation of bile acid was observed to give resistance to
C. difficile infection during precision microbiome editing experiments carried out
by Buffie et al. (2014). Multiprotein oligomers responsible for activating inflamma-
tory responses are known as inflammasomes.
Inflammasomes work on the composition of gut microbiota with pre-designed
microbiota induced T-reg cells which increase the anti-inflammatory activity in the
intestine and thus control IBD. The potential of remodeled microbiota was also
investigated in experimental colitis, by using mutant strains of Candida glabrata
lacking chitin synthase-3 had a less inflammatory response when compared with
control yeast strains. Thus in the future remodeled microbiota could be explored as
an effective treatment for colitis.
11.1.4.3 Probiotics Mediated Therapy

Two major bacterial genera, Lactobacillus and Bifidobacterium, were identified to
maintain the equilibrium between modulations in gut microbiota and repress the
overgrowth of pathogens. In the mouse model of Ulcerative Colitis, probiotics
induced T-reg cell production by regulating the growth of TH2 cells and the ratio
of Treg/TH1/TH2 cell populations. Overall protection against mucosal inflammation
in the intestine is achieved by changing TLRs and signaling pathways in a probiotic-
dependent manner. Depending on the disease context probiotic strains differ in
mechanism of action and have diverse pathways in regulation. Further studies are
required in dose, treatment duration, and strains used, to recommend probiotics in

clinical therapy of IBD.
11.1.4.4 Engineered Probiotics Mediated Therapy

Employing genetically engineered probiotic strain—Lactococcus lactis an IL10
(anti-inflammatory molecule) producer, 50% of colitis was attenuated in the mouse
model. Protective role engineered strain of L. lactis was understood when given
orally to trinitrobenzene sulfonic acid-treated mice (Steidler et al. 2000). In a DSS
colitis model, administration of modified L. lactis to produce an IL-7 like cytokine
could attenuate the inflammation by lowering the secretions of DCs. This approach
of manipulating the probiotic strain to produce anti-inflammatory cytokines seems to
be promising to treat inflammatory diseases effectively.
Enhanced levels of reactive oxygen species would develop inflammation and IBD
leading to tissue damage. Citrobacter rodentium growth results in better
oxygenation to mucosal surface. The effects of ROS are neutralized by superoxide
dismutase (SODs) secreted by intestinal mucosa making diffusible hydrogen perox-
ide from superoxide anions. Liu et al. demonstrated that engineered Bifidobacterium
longum containing recombinant human manganese SOD fusion protein successfully
expressed in mice model and successfully stopped colitis by decreasing
pro-inflammatory cytokines (Liu et al. 2018). In another study, B. longum expressing
α melanocyte-stimulating hormone an anti-inflammatory peptide is known to differ-
entially control the anti-inflammatory cytokines in experimental colitis.
Attempts were also made to utilize H47 producing EcN, i.e., bacteria’s narrow
spectrum antibiotics to kill pathogens responsible for inflammatory bowel diseases.
Spisni and colleagues demonstrated that RNAi technology targeting COX2, a
pro-inflammatory enzyme in a pathogenic E.coli strain could successfully decrease
the activity of pro-inflammatory cytokines when administered in a mouse model
(Spisni et al. 2015). Treatment of IBD was also carried out by using systemic
antibodies against cytokines. Various other strategies like engineering Lactobacilli
to recognize pro-inflammatory cytokines, cloning natural protease inhibitor, and
lipoteichoic acid-mediated secretion of IL10 by mutant L. plantarum to decrease
pro-inflammatory cytokines were proved to be effective in mouse models; however,
such methods need evaluation in humans for application.
11.1.4.5 Microbial Metabolites Mediated Therapy

In IBD patients, butyrate-producing bacteria are found to be washed out supporting
the growth of pathobionts and thus paving the way to inflammation. SCFAs pro-
duced by gut bacteria are important mediators of “protective” signals. The host
system identifies and responds to SCFA which are known to stimulate various
physiological functions, thus reducing inflammation. SCFAs control excessive
intestinal inflammation by promoting T-cell responses through T-reg cells
expressing SCFA-GPCR (G protein-coupled receptors) mechanisms or by histone
deacetylase (HDACs) inhibition. Therefore, SCFAs produced by gut microbiota are
proved to be beneficial as they promote mediators to resist inflammation and inhibit
cytokines using different mechanisms and establish homeostasis in the gut.
Administration of butyrate to colitis-induced mice showed resistance towards

migration of enteropathogens. Butyrate could induce segregation of homeostatic
macrophages and increase antimicrobial activity in macrophages. This was possible
by making shifts in macrophages metabolism, e.g., reduced glycolysis, mTOR
kinase activity, and increased bacterial autophagy protein. SCFAs, for example,
acetate was produced by fermentation of dietary fiber in the gut and triggered IL-18
production and restored gut homeostasis in the animal colitis model (Macia et al.
2015). Propionate—a less well-studied SCFA could reduce the expression of
pro-inflammatory cytokines. Non-SCFAs microbial-derived products like vitamin
B6 when supplemented along with Bacteroides spp. could clear pathogens. These
studies highlight the mechanisms of microbial metabolites in modulating the host
responses. These strategies look promising for clinical application in the future.
11.1.4.6 Intestinal Stem Cells-Gut Microbiota Mediated Therapy

Epithelial integrity and tissue homeostasis are maintained by pluripotent Lgr5+ stem
cells. Intestinal stem cells (ISCs) interact with gut microflora and change epithelial
regeneration during inflammation. When these cells are exposed to microbial
muramyl dipeptide (MDP), cytosolic NOD2 expression is increased causing
cytoprotection of stem cells against oxidative stress. In the intestine, microbes
indirectly raise stem cell numbers and influence host barrier function. Even
probiotics and diet also influence the gut microbiome and present alternate
mechanisms for enhancing the stem cell population. There is a scope for further
investigation on the interplay between ISC, probiotic, and dietary as therapeutics for
IBD treatment. Mesenchymal stem cells which are heterogeneous
non-hematopoietic, can differentiate into various cell types and participate signifi-
cant roles in gut immunity and inflammation.
Since IBD is an immune-mediated disease, therapies-based MSCs were tried in
pre-clinical models for tissue regeneration, restoring immune balance, and
microbiome. The potential role of ISCs as therapeutics was discussed in detail in
these studies (Shamoon 2019).
11.1.5 Microbiome-Based CDI Therapy
Clostridium difficile infection (CDI) has caught the attention of clinicians and
researchers alike because of its prominence and prevalence. CDI is identified with
symptoms like mild to moderate non-bloody diarrhea, abdominal cramping, and
tenderness. In severe cases, watery diarrhea with abdominal pain, fever, nausea,
anorexia, and malaise are noted. Besides, leukocytosis, elevated C-reactive protein,
and low albumin levels are present. CDIs are frequently attributed to antibiotics
altering the normal gut flora thus allowing C. difficile to nourish (Katz 2006). The
majority of the therapeutic agents designed for CDI control are antimicrobial agents,
toxin binding agents, immune modifying agents, probiotics, and FMT.
11.1.5.1 Probiotics
Probiotics are considered an effective method for restoring homeostasis in the
gastrointestinal tract and to prevent or treat CDIs. Commonly used probiotics for
CDI therapy are Lactobacillus and Bifidobacterium and the yeast Saccharomyces
(Boyle et al. 2006; Katz 2006).
In a clinical study conducted by Hickson et al., the effectiveness of Lactobacillus
casei, Lactobacillus bulgaricus, and Streptococcus thermophilus as prophylactic
agents with 115 patients of age 50 years who were on antibiotics were investigated
(Hickson et al. 2007). Within 2 days of antibiotic therapy, probiotic administration
was initiated and continued for 7 days. They observed a 17% reduction in the
prevention of CDI. However, probiotics usage has limitations because they do not
undergo rigorous testing, unlike pharmaceutical agents (Boyle et al. 2006).
Probiotics while eliminating the infectious bacteria may show certain side effects
for e.g. bloating and flatulence caused by Lactobacillus and Bifidobacterium.
11.1.5.2 FMT
FMT comes under live biotherapeutics drugs for CDI treatment and is regulated by
the FDA just like any other drug. It is an option for patients with recurrent CDI as
80% of clinical response rates were recorded. Since FMT includes the administration
of live feces-derived mixtures of microorganisms, potential safety issues are
involved. The impact of long-term manipulation of gut microbiota by FMT is
unknown and needs consent from both the receivers and donors. Limitations in
present delivery routes encourage researchers to design alternative drugs with
reduced risk and ease of delivery without compromising on the efficacy of FMT.
Another approach is the use of freeze-thawed and encapsulated material instead
of fresh feces for FMT. When compared with controls, freeze-thawed versus fresh
feces delivered through enema was good without recurrence up to 13 weeks. How-
ever, in each treatment group about one-third of the patients required two FMTs to
prevent recurrences. RBX2660 is a standardized product of live microbiota suspen-
sion developed for the treatment of recurrent CDI and proved to be effective as FMT
(Orenstein et al. 2016). SER-109 or Seres is another biotherapeutics prepared by
encapsulation of Firmicutes spores mixture obtained from human feces. Ethanol
treatment was used to reduce the spread of infectious agents in the product. Seres
have completed two clinical phase trials. In the first noncomparative clinical trial
26 out of 30 patients (86.7%) could be recovered and 96.7% of patients had a good
clinical resolution. SER-109 could turn around the loss of gut microbiota after the
treatment. In the second clinical study, SER-109 could not reduce CDI recurrence
but was found effective in aged patients. Keeping because of these limitations,
alternative approaches are under clinical trials (Khanna et al. 2016; Wilcoxb 2016).
11.1.6 Microbiome Therapeutics for Food Allergy (FA)
Food allergies are estimated to affect around 4%–6% of children and 4% of adults in
a survey made by the Center for Disease Control and Prevention. It occurs because
of the hyper-reactivity of the immune response to any component in food. FA is most
frequent in young children but can appear at any age. Food allergy correlates
dysbiosis during childhood and immune conditions later part of life. During epide-
miological surveys, it was identified that environmental factors like caesarian births,
no breastfeeding, drugs, and diet with low-fiber content and high-fat are important
contributors to dysbiosis.
In a research investigation, the fecal microbiota of infants suffering from allergy
was compared with healthy infants revealed alterations in gut microflora with the
development of allergy (Nakayama and Kobayashi 2011). Allergic group infants had
more Bacteroides, Propionibacterium, and Klebsiella in first & second months,
respectively, while Acinetobacter and Clostridium were found in the non-allergic
group of similar age. Non-allergic infants have SCFA producing Clostridium species
which prevent the growth and proliferation of Bacteroides which are most abundant
in FA. SCFAs are vital metabolites of gut flora that protect from a food allergy and
regulate the immune system in epigenetic mode. Depletion of butyrate-producers
could lead to dysbiosis and initiate FA. Therefore SCFA supplementation could be
explored further to cure food allergy.
Microbiota-directed therapy has the potential in decreasing the occurrence of
FA. One research study had demonstrated that dietary patterns have an impact on the
progress of FA and influence the gut microbiota population. Breast milk provides a
primary source of commensal microbiota in the infant’s gut and later helps in the
maturation of the microbiota. An abundance of oligosaccharides and TGF-β2 in
breast milk promotes the growth of SCFA producing Bifidobacterium and
Streptococcaceae and Ruminococcaceae members, respectively.
Modulation of gut flora is more effective through diet as well. An infant diet
enriched with fruits, vegetables, and homemade foods could lessen the FA. A high-
fiber rich diet promotes the growth of Bifidobacterium and Lactobacillus which
ferment the fiber foods and thereby increase serum SCFA levels. Studies on neonatal
prebiotic supplementation could control eczema.
Supplementation of Lactobacillus rhamnosus GG (LGG) to breastfeeding
mothers and infants could control the risk of eczema. L. rhamnosus supplementation
along with oral immunotherapy led to tolerance to peanuts in allergic children.
Similarly, administration of LGG containing extensively hydrolyzed casein formula
provided tolerance to an allergy of cow milk. Comparison of the fecal microbiota of
infants receiving EHFC + LGG with the infants receiving EHCF alone revealed
enrichment of butyrate-producing flora and high fecal butyrate levels. Butyrate is
reported as an epigenetic regulator and the use of EHCF+LGG induces methylation
of the promoter of cytokines genes. Such epigenetic regulation was observed in
children supplemented with EHCF+LGG rather than children treated with other
methods. Long-term protection was possible by targeting gut flora in children
suffering from FA. Many more trials are necessary to assess the roles of probiotic
strains in manipulating gut flora. Integration of clinical trials with epigenetics and
metabolomics data may lead to novel therapeutics (Rosita Aitoro 2017).
11.1.7 Microbiome Therapy for Autoimmune Diseases
Autoimmune diseases are caused due to intestinal dysbiosis leading to impaired gut
barrier function, increased inflammation, and immunity. Dysbiotic flora induces
neo-epitope formation by imperfect post-translational modification of luminal
proteins. This in turn aggravates systemic autoimmunity and provokes autoimmune
diseases (Lerner et al. 2016).
11.1.7.1 Type 1 Diabetes (T1D)

Type 1 diabetes (T1D) is caused due to the destruction of insulin-secreting cells,
therefore cells start depending on exogenous insulin. Several research studies tried to
establish the relationship between dysbiosis and T1D. The role of the gut flora in
humans was first investigated in four T1D children in Finland by examining their
stool samples and reported a low ratio of the Firmicutes: Bacteroidetes than controls
(Giongo et al. 2011; De Goffau et al. 2013). Later, De Goffau also reported that
children with autoantibodies against b-cells had more Bacteroidetes and less lactate
and butyrate producers in their feces samples (Li et al. 2015). In a study with
seroconverted T1D patients, Davis-Richardson et al. found plenty of Bacteroides
dorei and Bacteroides vulgatus. From these research studies, it could be possible to
consider early dysbiosis as an indication to predict T1D in genetically predisposed
individuals (Davis-Richardson 2014).
Administration of Lactobacillaceae-enriched probiotic orally protected NOD
mice from T1D by suppressing autoimmune response in the gut (Dolpady et al.
2016). In humans, a TEDDY group evaluated probiotic supplementation for 7473
children from 4 to 10 years in the age group with genetic risk for T1D during their
first year of life. Early probiotic administration was correlated with a decreased risk
of islet autoimmunity when compared with the group that received probiotics after
27 days of life or no supplementation (Uusitalo et al. 2016).
11.1.7.2 Multiple Sclerosis

Multiple sclerosis (MS) is a seriously disabling disease of the Central Nervous
System in which the immune system attacks the protective myelin of nerve fibers
and causes communication problems between the brain and the rest of the body. This
disease can cause permanent deterioration of the nerves and thus disability in young
adults. Environmental factors like a viral infection, a hypocaloric diet, vitamin D
deficiency, and dysbiosis trigger MS along with susceptible HLA alleles (Dendrou
et al. 2015). Research studies indicate that intestinal dysbiosis is one of the factors of
extraintestinal disease development.
A recent research study made on 60 Relapsing-Remitting MS (RRMS) patients,
observed an increased number of Methanobrevibacter which is involved in
recruiting macrophages and activating dendritic cells, Akkermansia playing a role
in degrading the mucus layer and promoting inflammation and decrease in

Butyricimonas—butyrate-producing bacteria which has immunomodulatory
properties in the gut of untreated patients (Cantarel et al. 2015). However, in patients
treated with IFN-b and glatiramer acetate, there was an increase in the number of
Prevotella which is associated with high-fiber ingestion and has regulatory roles via
butyrate generation. Dysbiosis in RRMS patients was also reported by Chen et al., in
their study while comparing stool samples from patients with healthy controls. They
observed a plethora of bacterial strains like Pseudomonas, Mycoplasma,
Haemophilus, Blautia, and Dorei in patients when compared with healthy
counterparts who had only Prevotella and Parabacteroides in abundance (Chen
et al. 2016a, b). Current research findings point to the gut–brain axis connection
during dysbiosis in animal models and MS patients. The relationship between
immunity in the gastrointestinal mucosa and commensal bacteria seems to promote
important physiological homeostasis for the host (Colpitts et al. 2017).
Several studies established the immunoregulatory functions of probiotic admin-
istration in MS. Treatment with Lactobacillus spp., Pediococcus acidolactici,
Bifidobacterium bifidum, Bifidobacterium animalis, and Bacteroides fragilis
improved CNS inflammation by inducing Treg cells in the gut mucosa thus promot-
ing the secretion of IL-10, transforming growth factor-b and inducing decreased
Th1/Th17 inflammatory subsets in mice model (Lavasani et al. 2010). Kouchaki
et al. reported a decrease in inflammatory markers in MS patients treated with
probiotic supplementation containing Lactobacillus acidophilus, Lactobacillus
casei, Lactobacillus fermentum, and Bifidobacterium bifidum (Kouchaki et al.
2016). This randomized double-blind placebo-controlled clinical trial analyzed
probiotic intake for 12 weeks in 60 MS patients. Although these studies give hope
for the treatment of MS, future studies are still required to determine the real role of
the gut microbiota in CNS demyelinating diseases.
11.1.7.3 Rheumatoid Arthritis

Another systemic autoimmune disorder is Rheumatoid Arthritis (RA) which is
characterized by persistent inflammation of joints. Autoantibodies like anti-cyclic
citrullinated peptide and/or rheumatoid factor are detected before RA occurrence
(Van de Wiele et al. 2017) and initiated with factors like smoking, infections, and
dysbiosis (Klareskog et al. 2006). Chen et al. identified decreased species richness
(alpha-diversity) in gut microbiota in RA patients and a positive connection among
rheumatoid factor levels and disease progression. In RA patients along with the
pro-inflammatory cytokine Eggerthella, Actinomyces, Turicibacter, Streptococcus,
and Collinsella species were more in gut microbiota (Chen et al. 2016a, b). Beta-
diversity found in the gut microbiota of RA patients was observed to have a link with
the levels of rheumatoid factor, C-reactive protein, suggesting that these parameters
play role in gut microbiota modulation (Yatsunenko et al. 2012). The establishment
of intestinal dysbiosis signature will thus help to develop therapeutic tools (Ciccia
et al. 2016; Di Paola et al. 2016).
11.1.7.4 Systemic Lupus Erythematosus (SLE)

SLE is a heterogeneous disease wherein autoantibodies mainly bind with nuclear
and cytoplasm antigens damaging the vital parts of the body (Apostolidis et al.
2011). Genetic and external factors like viral infections, and exposure to UV-B rays
are involved in pathogenesis. Several research studies provide the role of intestinal
dysbiosis in SLE development (Van de Wiele et al. 2017). Hevia et al. found
decreased Firmicutes: Bacteroidetes ratios in stool samples of SLE when analyzed
with the healthy counterparts (Hevia et al. 2016) Another study made with stool
sample analysis of SLE patients with control subjects reported low numbers of
Firmicutes and an enhanced population of Bacteroidetes members. The gut
microbiota profile of SLE patients has a predominance of Rhodococcus, Eggerthella,
Klebsiella, Prevotella, Eubacterium, Flavonifractor, and Incertae sedis genera.
Based on these reports, investigation on in vitro differentiation of T-reg, Th1, and
Th17 cells in SLE patients was carried out (Lopez et al. 2016). Treg-inducing
bacterial supplementation decreased the balance of Th17/Th1 in SLE patients,
indicating that these strains could be used as therapeutic probiotics.
11.1.8 Cardiovascular Disease (CVD)
Modulations in gut microbiome composition are known to be associated with CVD

including atherosclerosis, dyslipidemia, hypertension, and heart failure. Gut
microbiota persuades the host signaling and immunomodulatory effects through
microbial metabolites. For example, SCFA, bile acids, and trimethylamine-N-
oxide (TMAO) are contributing factors of CVD risk.
Patients with coronary artery disease contain DNA from bacterial species in the
atherosclerotic plaques (Kubinak et al. 2016). Modulation of SCFA production and
inflammatory signaling played a prominent role in inducing atherosclerosis when
experimented within mice with gut microbial transplants. Similar experiments also
demonstrated that TMAO is proatherogenic, prothrombotic, and an important under-
lying contributor to CAD risk (Hamada et al. 2002). In an investigation with animal
models, TMAO is observed to decrease the mechanism of removal of cholesterol
transport by macrophages. Intestinal microbiota could also regulate host lipid
metabolism.
Gut bacteria with their microbial enzymes make different bile acid metabolites
which activate host receptors and interfere with lipid metabolism. Short-chain fatty
acids and sodium-dependent inflammation are microbiome-mediated contributors to
hypertension (Fagarasan et al. 2010). Though the evidence suggests a fundamental
role for the microbiota in CVD, many questions remain in the mechanism they
cause. Drug pharmacokinetics and pharmacodynamics are observed to be influenced
by gut microbiota (Fukata et al. 2013). Microbiome-based precision medicine could
be designed or developed by gaining knowledge in molecular mechanisms
contributing CVD risk and drug response.
11.1.9 Microbiome-Based Therapy for Non-Communicable Diseases

(NCD)
NCDs occur in different organs of the body and become systemic diseases. Unlike
infectious diseases which are transient, latent, or lethal, NCDs are seen over a longer
duration. For example—allergy, asthma, and some autoimmune diseases come
under early-onset NCD, while CVD, metabolic disease, and neurodegenerative
disorders fall under later-onset NCDs. Generally, NCDs were thought of as diseases
of more developed countries alone, but now they are global problems affecting entire
human health. NCDs combined with microbial resistance to antibiotics had posed a
major threat to the integrity of healthcare systems and to the global economy.
Interestingly all NCDs have common environmental risk factors with altered gut
colonization patterns. A decrease in the microbiota, increase in immunologic and
metabolic deregulation are very common attributes seen in all NCDs. Fluctuations in
the gut microbiome occur because of complex changes in nutritional patterns and
environment. Early environmental changes are involved in the increase in early-
onset inflammatory NCDs like an allergic disease. The gut microbial deprivation
hypothesis demonstrates a link between reduced gut microbiota diversity and early-
onset NCDs, including atopy, eczema, and asthma. Microbiome modulation with
strategies like microbiota seeding, feeding, and rebiosis appear as promising
methods for sustainable healthcare. These methods try to treat emerging NCDs,
reduce later-life health risks, and reduce recurrent infections with personalized
medicine approaches (Fukata et al. 2013).
Recently, researchers and clinicians are also making efforts to adopt systems
biology-approach to manage NCDs. New therapeutics could be designed by
integrating metagenomic and metabolomic information which would extend effec-
tive healthcare and preventative strategies to the patients. This approach majorly
focused on the early phases of conception to birth where epigenetic programming
influenced over the life course and helped researchers to integrate human gut
microbiota information with epigenetic. These combined approaches are ultimately
direct to better strategies to control the diseases.
11.2 Conclusion
Although dysbiosis and decrease in microbiota diversity are common in almost all
diseases, yet whether a microbial deviation is the reason or result needs to be
investigated. To address the challenges faced in microbiome modulation, researchers
need to focus on microbiota signatures as biomarker development for diagnosis.
Instead of looking at microbiome composition, it is most important to study the role
of the microbiome in a disease condition. Deeper insight into the role of a healthy gut
microbiome is possible through Human Microbiome Project and MetaHit projects to
design or plan dietary modulation. Gut colonization patterns need to be developed
with prebiotics, nonconventional indigenous gut bacteria, and FMT because they
might have continuous influences on health. Therefore, research on microbiome
modulation should enhance the assessment of risk in diseases, discovery, and

efficacy of drugs.
Microbiome-based therapeutics are being identified as precision medicine
because of the contribution to inter-individual variability in disease conditions and
also as a variable factor for the improvement of potential therapeutics. They have the
potential to influence the therapeutic strategies in the treatment of several diseases,
generate new economic opportunities, and benefit humans around the globe.
Acknowledgement Authors thank the Principal and Management of Bhavan’s Vivekananda

College, for the encouragement and support.
Conflict of Interest The authors declare that there is no conflict of interest.
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Recent Advancements in Microbiome–
Immune Homeostasis and their 12
Involvement in Cancer Immunotherapy
Anusha Konatala, Fain Parackel, and Pola Sudhakar
Abstract
The microbial genome of bacteria, archaea, protists, fungi, and viruses which
colonize in humans is known as the microbiome. The population of microbes in
the human body is known as microbiota, its composition may differ with
concerning host factors like sex, age, obesity, physical inactivity, alcohol con-
sumption, smoking, diet, and polymorphisms in dominant human oncogenes. The
current chapter is ascertaining the connection between microbiota and cancer, the
role of the microbiota in cancer immunotherapy, which leads to significant
advances and scope in the etiology of cancer. Different processes are studied
and have been promising to conclude the role of microbiota in tumorigenesis and
progression, processes like genotoxicity, induction of chronic inflammation,
bacteria-mediated cell proliferation, and activation of procarcinogens show the
interference of microbiota with the tumors. More research studies must focus on
microbiota interaction with the host to define its contribution to the growth and
development of cancers and identify microbiome as a potential cancer marker and
develop personalized medicine to treat malignancies. This chapter outlines vari-
ous researches, explaining how the microbiota itself enclose a novel paradigm in
the prevention of cancer and its management. Paramount to develop microbiota-
based immunotherapy for treating cancer, few challenges in microbiome research
are to identify individual microbial species such as viruses, protozoans, archaea,
protists, and fungi that causally affect cancer phenotypes and unravel the under-
lying mechanisms. Here, we discuss a few relevant technologies and few
A. Konatala · F. Parackel
University of Windsor, Windsor, ON, Canada
P. Sudhakar (*)
Department of Biotechnology, AUCST, Andhra University, Visakhapatnam, Andhra Pradesh, India
Ltd. 2021
https://doi.org/10.1007/978-981-16-3156-6_12
240 A. Konatala et al.
challenges in studying the microbiome and their involvement in cancer

immunotherapy.
Keywords
Microbiome · Immunotherapy · Carcinogenesis · Epidemiology · CTLA-4

Cytotoxic T-lymphocyte Antigen-4
12.1 Introduction
The human microbiota, collective of primary bacteria and other microbes, like
archaea, fungi, protozoa, protozoan viruses, helminthic worms that reside in the
human body, the genetic material of microbiota is known as the microbiome. It is an
inhabitant in the human body, within the surface of the epithelial barrier, 99%
occupied in the gut and 1% in skin, vagina, nasal, and mouth. The fluctuation of
the equilibrium, which is detrimental due to the loss of beneficial microorganisms, is
known as dysbiosis. Physiology factors, lifestyle changes in the diet all affect the
health of microbiomes in the host. This dysbiosis causes inflammation in epithelial
cells and is known to cause tumor development. There are corrective treatments for
dysbiosis mentioned in this chapter, which showed promising results in the
microbiome caused by diseases. Moreover, multiple techniques and models used
in the characterization of microbes are discussed briefly in this chapter.
The gut microbiome has shown significant importance in immune cell develop-
ment and maintains equilibrium with commensal microbes. These microbes have
regulatory roles in the development of mucosal immune systems. Intestinal microbes
produce short-chain fatty acids, which play a crucial role in tumor prevention and
activation for apoptosis. Besides, they have an impact on efficacy immunotherapy in
cancer patients by blocking cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4)
and programmed cell death ligand 1 (PD-L1), which is explained below in detail
(Temraz et al. 2019).
12.2 Microbiome
The Human Microbiome Research observed 11,174 primary biological specimens in

a study conducted in 242 healthy adults (Methé et al. 2012). The microbiota has a
considerable impact on the host’s immunity, physiological functions, particularly
metabolism, cognitive and neurological function, inflammation, hematopoiesis (Roy
and Trinchieri 2017). The microbiome is first acquired by vertical transmission from
mothers during delivery and lactation period. Newborns birth by cesarean has
abundant skin microbiota of mothers compared to birth by vaginal delivery, later
possess more maternal vaginal microbiota (Dominguez-Bello et al. 2016). Total
microbial counts about ten times more than the human cells, with more genes,
concerning the human genome (Shahanavaj et al. 2015). Primarily dominant bacteria
12 Recent Advancements in Microbiome–Immune Homeostasis and their. . . 241
found in the healthy human gut are Bacteroidetes and Firmicutes; their percentage
range varies from 10% to 90% (Allaband et al. 2019). Some research studies
detailed, secretions by the bacteria make it dominant in the interbacterial competition
with a high population of B. fragilis strain (Garud and Pollard 2020). Microbiota of
the gut is known as the “second genome” as it shows a significant impact on the
internal activities of the host; thus, it is also called “forgotten organ.” The gastroin-
testinal microbiome has been observed to have a crucial effect on overall health and
serves as the best model to understand microbiota and host interactions (Schwabe
and Jobin 2013).
Gut bacteria is categorized into three types based on their function in the host:
• Symbionts: (90% in the gut) Microbes in mutualistic benefit to the host.

• Conditioned Pathobionts: Usually harmless, causes disease in unfavorable
condition.
• Pathobionts: Disease-causing microorganisms.
The pathobionts can be harmful when there is a disturbance in equilibrium

between the gut microbiota and the host due to altered dietary habits, exposure of
pathogens, the action of antibiotics, and other environmental factors like change of
weather or disturbance in the circadian clock. Alterations in the homeostasis in the
microbial communities are known as “dysbiosis” (Helmink et al. 2019a). It is
affected by the physiological and pathological changes that take place in the host
(Shui et al. 2020). Some recent finding suggests colorectal cancer (CRC), inflamma-
tory bowel disease (IBD), celiac disease, obesity may have been caused by
pathobionts. Dysbiosis leads to a leaky gut by exposure to pathogens, increasing
the intestinal permeability, promoting translocation of gut bacteria, and dysplasia of
the immune system affecting the homeostasis of the gut (Zhou et al. 2020). To treat
the above condition caused by dysbiosis through fecal microbiota transplantation
(FMT) and other novel therapies can be introduced. Gut microbiome study is in a
preliminary stage of the investigation to know the functional properties of commen-
sal bacteria, and its mechanism involved to interact with the host is not completely
understood. Moreover, multiple promising types of research have suggested gut
microbiota showcase great potential towards medical treatments of cancer and other
diseases (Kho and Lal 2018).
12.2.1 Esophageal Microbiota
The esophageal microbiome has been studied in healthy and diseased conditions
with the help of recent gene sequencing tools. It is observed that a healthy human
esophagus contains abundant Streptococcus (gram +ve), compared to the infected
esophagus, with a high amount of gram-negative bacteria (Rajagopala et al. 2017).
Microorganisms inhabiting the esophagus are Bacteroidetes, fusobacteria,
proteobacteria, and spirochetes. These microbes produce lipopolysaccharides
(LPS), which acts as an immune-activating agent in stimulating innate immune
responses that can directly treat malignancies. LPS interact with the innate immune
system by binding to toll-like receptor-4 (TLR4), resulting in the activation of
nuclear factor kappa B (NF-κB). High levels of NF-κB are observed in esophageal
adenocarcinoma patients cascading levels of inflammatory cytokines like IL-1b, Il-6,
IL-8, and TNF-a. In some research studies in mice, LPS prolongs the time of gastric
emptying, which helps in increased gastric reflux to the esophagus. Therefore, using
NF-κB host cell pathway inhibitors, probiotics, antibiotics, and microbiome in the
esophagus can prevent cancer development (Shahanavaj et al. 2015). Some detailed
studies are required to find the target in different diseases in the esophagus,
diagnosing, therapeutics, and prevention (Lv et al. 2019).
12.3 Healthy and the Unhealthy Microbiome
12.3.1 Healthy Microbiome
Characterization of microbes as healthy and unhealthy is necessary to understand

their functions and their roles in healthy and diseased conditions are critical. The gut
microbiota is diverse compared to other host sites; a healthy microbiome considered
in one host may not be healthy for others. Although there have been some patterns
found in a study conducted in patients from different zones. Healthy hosts have rich
microbiota, which harbors 1000 species of bacteria belonging to Firmicutes and
Bacteroidetes. Different proposals from researchers say that a host with favorable
gut microbiota has elicited an immune response against cancers due to antigen
presentation and enhanced T cell function. Research studies in mice explain T cell
response is defined for B. fragilis or B. thetaiotaomicron in microbiota promoting
CTL-4 blockade seen in patients. Gopalakrishnan found responses to anti-PD-1 in
skin cancer affected by gut microbiota by changing CD4+ IL-17+ cells and CD4+
FoxP3 + T cells. Restoring the efficacy of anti-PD-1 through T cells recruitment by
Akkermansia in the gut microbiota sensitizing the cancer cells was studied (Chen
et al. 2020). Bacillus polyfermenticus, a probiotic bacterium observed to affect the
development of colon cancer cells by obstructing receptors like ErbB2 and ErbB3 by
immune suppression, chronic inflammation, immune evasion (Shahanavaj et al.
2015). Moreover, probiotics destroying hepatocellular carcinoma is through SCFA
production (Zhou et al. 2020).
Symbionts inhabiting the gut of the host play role of cancer transforming agents
in distal and local carcinogenesis and involve indirectly causing induction of inflam-
mation and immune suppression. Due to disturbed equilibrium, some microbes tend
to act as a part of an unhealthy microbiome being involved in altering host physiol-
ogy and metabolism (Li et al. 2019).
12.3.2 Unhealthy Microbiome
The gut microbiota of the host affects its immune system indirectly by suppressing
and inducing inflammation leading to cancer development (Li et al. 2019). Few
observed that some bacteria promote chronic inflammation to activate macrophages,
increase reactive oxygen species (ROS) generation leading to DNA breakage and
mutations (Zhou et al. 2020). Obesity leads to dysbiosis with a high volume of
Clostridia, which produces secondary bile acid, deoxycholic acid (DCA) and
promotes hepatic cellular carcinoma (HCC) (Schwabe and Jobin 2013).
Human tissues tightly regulate growth and death promoting signals to maintain
homeostatic cell densities, tissue function, and architecture of the tissue or organ.
Disruption in these signals results in uncontrolled cellular proliferation. E-cadherin
and intercellular adhesion molecule have been a target for the intestinal bacteria to
promote epithelial cell proliferation by activation of Wnt/u-catenin pathway
(Fulbright et al. 2017).
Cyclophosphamide was found effective in translocation of the Enterococcus
hirae small intestine bacteria to spleen and colonization of Barnesiella
intestinihominis in the colon of the host; these microbes together contribute to the
antitumor immunity (Li et al. 2019). Some examples of an unhealthy and healthy
microbiota, affecting the physiological and metabolic activities of the host are listed
in Fig. 12.1.
12.4 Techniques and Tools for Microbiome Analysis
Gut microbiota is well understood in recent years with the help of advanced gene
sequencing tools and humanized gnotobiotic models (Kho and Lal 2018). These
advanced sequencing tools have helped researchers to generate millions of
sequences to study different microbial communities. Conventional techniques used
to unravel the gut microbiome are 16S ribosomal RNA, metabolic characterization
of the microbiome, gene amplicon sequencing, shotgun, single-cell RNA sequencing
by CRISPR–Cas technology, metagenomic sequencing (Elinav et al. 2019), and
next-generation sequencing tools. Of all, the composition of host–microbiota can be
defined by 16S RNA ribosome amplicon sequencing and whole-genome shotgun
(WGS) sequencing. Through WGS appropriate detection of the species, strains with
diversity within the samples can be determined, which are concluded in 16S rRNA
amplicon sequencing. The primary disadvantage of 16SrRNA sequencing is it lacks
taxonomic resolution. In either case, microorganism DNA sequence samples are
studied by next-generation sequencing technologies in comparison with known
database sequences to analyze the presence and abundance of taxa (Saus et al.
2019). Microbial community analysis can be achieved with genomic databases and
tools such as the quantitative insights into microbial ecology (QIIME), ribosomal
database project (RDP) pyrosequencing, procrustes analysis, taxonomy, and ecology
of ribosomal sequences (W.A.T.E.R.S) (Ursell et al. 2012).
Healthy Microbiota Unhealthy Microbiota
Co-medication
Dysbiosis
Fecal microbiota transplant
Environmental factors
Dietary Intervention
Internal factors Prebiotics
Examples
Examples
Bifidobacterium longum Bacteriodales

Faecalibacterium Roseburia intestinalls
Colinesella aerofaciens Parabacteroides distasonis
Parabacteroides distasonis Clostridiales bacterium
Ruminococcus spp. Porphyromonas gingivalis
Helicobacter pyroli
Effects Effects
Low Intratumor CD8+,

High CD4+ & CD8+, Low
Melanoma, NSCLC,
Treg production.
RCC.
Fig. 12.1 Healthy and unhealthy microbiota inhabiting host: Different factors like dysbiosis,
internal and external factors of host result in loss of healthy microbiota leading to unhealthy or
harmful microbiota promoting uncontrolled cell proliferation and other clinical disorders
Human-compatible preclinical models, humanizing mice, organ-chips, and

human-derived organoids are used in the study of gut microbiota and their reactions
to other tissues. Fecal transplant trials have been conducted in patients, to unwrap the
composition of essential microorganisms to help in immunotherapy for cancers
(Elinav et al. 2019). Advanced tools and genomic database consortium are most
popularly used to identify bacteria and study their effects on the host.
Standardization in this research study helps to compare the various preclinical and
clinical studies and understand how microbiota with different genomes have been
involved in the development of malignancies.
12.5 Microbiome Therapies
The microbiota damaged due to antibiotics, drugs, changes in diet, and dysbiosis can
be restored with the addition of new microbes in the gut that would mimic healthy
gut composition. New therapeutic strategies are involved in altering gut microbe to
mimic gut microbiota found in healthy humans to restore the resistance capacity of
gut microbe towards the disease (Cerdó et al. 2019). Different microbiome-targeted
therapies such as prebiotic-resistant starches (fiber), probiotics, and fecal microbiota

transplantation are in play to replenish the microbiota composition in aid to treat
diseases (Zhou et al. 2020). The role of the prebiotic and its influence depends on the
existing bacteria in the host. The combined approach of prebiotics and growing
specific bacteria is known as synbiotics, may be promising in treating diseases
(Li et al. 2019).
• Prebiotics (Food Components): Edible substance that helps to promote the

growth of defined microbes to enhance the host health by restoring the stability
of microbiota and decrease proinflammatory pathways.
• Probiotics (live Microorganism): Beneficial and active microbes are composed
mainly of yeast, Lactobacillus, Actinomycetes, Bifidobacterium, Clostridium
which assist in inhibiting harmful bacteria growth by colonizing in human
reproductive systems and intestines through maintaining microecology of
the host.
• Fecal Microbiota Transplantation: It is the transplantation of healthy human
feces to the gastrointestinal tract (GIT) of the patient to develop healthy functional
microbiota to treat extraintestinal and intestinal diseases.
In late 300 AD, in Eastern Jinn Dynasty, China Ge Hong’s “Elbow Reserve
Emergency” has a record in the treatment of patients with human waste for treating
conditions like diarrhea, food poisoning, fever, and death (Zhou et al. 2020).
Although prebiotic and probiotics have shown promising effects in several
treatments, their molecular mechanism is still unknown (Vieira et al. 2013). In an
FMT study conducted inpatient with C. difficile infection, followed by transplanta-
tion, there is increased Bacteroidetes in the gut. The microbiota composition after
FMT was like the donor and differences were observed in the metagenomic profile in
recipients. Further studies are needed to identify a specific colony that can modulate
the immunity of the host and prevent tumor development and also restore the balance
between gut microbiota and host (Seekatz et al. 2014).
12.6 Microbiome in Mice and Humans
In gut microbiome study, researchers mostly use mice, as they share similar digestive
tract. However, animal models like Zebrafish, drosophila, fruit fly, and the Hawaiian
bobtail squid have also widely been used in the study of host–microbiota
interactions. Mice genes share 99% of similarities with human genes and also
have a close resemblance with microbiome phylum as in humans (Kostic et al.
2013a).
In mice, the gut is different from the human due to low pH and oxygen tension in
the intestine affecting the fidelity of human microbiota. The changes in the glycan
profile of the mucus and around 4% of microbial gene sequences were found to be
shared between humans and mice (Fessler et al. 2019a), leaving less scope of
research using mice to mimic the human environment. Sequencing technologies
revealed that microbiota exhibit varied genetic sequences between hosts and within a
host over time. This variation of microbiota can be like one nucleotide variant, short
insertions and deletions, and more substantial structural variations like deletions,
insertions, duplications, inversions, and gene copy-number variants (Garud and
Pollard 2020). Microbiome composition in humans evolves in the first 3 years
after birth and then stabilizes and tends to remain constant until being affected by
external and internal factors. Among microbiota, the gut has most of it, with
approximately 3 10 13 bacterial cells count ten times to the number of human
cells (Roy and Trinchieri 2017).
12.7 Role of the Microbiome in Healthy Individuals
Microbiota is potential enough in transforming a variety of metabolites like some

proteins, impacting the immune response in protecting the host from cancer genera-
tion and progression (Prosperi 2020). Gut microbiota and its products have been
observed to be influencing the anticancer effect by modulating the immune system of
the host through the immunological cell death pathway (Chen et al. 2020). Eubiosis,
a rich and diverse microbiota regulating micro-ecological balance within the host,
helps to maintain immunity by activating TLR signaling pathway acting as an
adjuvant enhancing the immune response (Li et al. 2019). The gut microbiome
regulates the homeostasis of the host intestine by processing the dietary fiber
ingredients consumed by the host into digestible byproducts and plays an important
role in eliciting an immune response against invading microbes and resisting them
(Shui et al. 2020). It is demonstrated that the microbiome has been involved in the
maturation of immune cells like CD4+ T cells, CD8+ T cells, and dendritic cells
(Zhou et al. 2020). SCFA, such as butyrate, is generated by the dietary fibers through
microbial fermentation, the primary energy source for the colonocytes (Fulbright
et al. 2017). Butyrate is sensed by the dendritic cells, T cells expressing G protein-
coupled receptors, GPR41 or 43 (Zitvogel et al. 2018). Glucose obtained from
glycolysis becomes the primary carbon source for the cancer cells; this is known
as the “Warburg Effect.” In a diet with high fiber, content butyrate is produced and
due to its impaired metabolism, there is a high percentage of the butyrate making the
cancer cells starve.
Also, butyrate promotes apoptosis, inhibits histone deacetylase, regulates immu-
nogenic cell death (ICD), cellular proliferation through epigenetic modifications
(Fulbright et al. 2017). Bifidobacterium infantis involve in the differentiation of
immune cells Tregs and dendritic cells and promote Foxp3+ regulatory T cells.
Clostridium butyricum maintains intestinal immune homeostasis by regulating pan-
creatic T cells (Chen et al. 2020). Table 12.1 shows the systemic effects of gut
microbiota on the host: The following functions in the human body are affected due
to the gut microbiome impacting homeostasis leading to clinical disorders (Roy and
Trinchieri 2017).
A good understanding of the bacteria gut microbiome is established. More
research and details about the action of the virus and fungi composition and their
Table 12.1 Systemic effects of gut microbiota on the host physiology

Physiological functions Non-neoplastic pathology
• Cardiovascular and musculoskeletal.Functions • Insulin resistance.
• Metabolism. • Obesity.
• Neurological and cognitive functions. • Autoimmune.
• Hematopoiesis and myeloid cell functions. • Non-alcoholic steatohepatitis.
• Inflammation and immunity.
• Aging.
interaction and their effect on the host would give complete handling of the human
microbiota and utilize them efficiently as anticancer agents (Saus et al. 2019).
12.8 Microbiota Effects on Immune System Development
Initial colonization of intestinal bacteria depends upon gut-associated with lymphoid

tissues (GALT); similarly, Bacillus subtilis and Bacteroides fragilis gut microbes
have shown to promote GALT development (Rhee et al. 2004). GALT is a compo-
nent of mucosa-associated lymphoid and they are divided into three sections (Cebra
1999):
(a) Payer patches (containing B cell and T cell),

(b) Lamina propria (consisting of immunoglobulins, dendritic cells, mast cells),
(c) Intraepithelial leukocyte spaces (NK cells, T cells).
Gut microbiota is involved in the maintenance of the mucosal immune system,

during myelopoiesis, and the function of dendritic cells, macrophages, and
neutrophils (Gorjifard and Goldszmid 2016; Fessler et al. 2019b). The mucosal
immune system undergoes significant changes once bacterial colonization
establishes in the intestinal tract. The gut microbiome has been shown to play a
significant role in promoting NK cell differentiation, dendritic cells (Wu and Wu
2012). B. fragilis and Clostridia shape the polarity of macrophages and are observed
to be coordinated mutualistic relationships between macrophages and microbes
(Mezouar et al. 2018). Moreover, microbe-derived luminal ATP molecule activates
CD70high CD11c low cells, which promotes TH17 cell differentiation (Atarashi et al.
2008).
Multiple diseases and chronic disorders had common intestinal dysbiosis that
may have contributed to the pathogenicity of these diseases. Symbiotic bacteria are
essential for lymphoid tissue development. Germ-free mice have shown
gut-associated lymphoid tissues (GALT), developmental issues, and impaired lym-
phoid follicles compared to a pathogen-free mouse (Kim et al. 2017).
12.9 Microbiome Role in Epithelial Barrier
The epithelial barriers contain goblet cells, Paneth cells, on damage to these cells
microbes infiltrate into the blood, few acting as the procarcinogenic agents to spread
carcinogenesis and inflammation (Rajagopala et al. 2017). The gut epithelium and
its tight junctions act as a barrier for a wide variety of bacteria and internal gut
milieu, “at density up to 1012 organisms/ml intestinal content” were observed from a
unicellular layer of epithelium (Sonnenburg et al. 2004). The luminal surface of gut
epithelium cells prevents the entry of large particles and bacteria, preventing exces-
sive immunological response, which affects gut health. IgA, IgM antibodies regulate
the entry of antigen penetration across the epithelium layer. During an immune
response, bacteria generate short-chain fatty acids, and these enhance the production
of IgA (Li et al. 2019). IgA is crucial to maintain homeostasis of gut microbiota; its
deficiencies cause the growth of anaerobic organisms in the gut cells (Suzuki et al.
2004). Short-chain fatty acids like acetate inhibit the growth of other pathogens
and viruses, SCFA serves as an energy source for gut microbes (Mezouar et al.
2018).
12.10 Microbiome as a Marker
Most of the research studies explain that a specific microbe colony seems to be either
dominant or causal of cancer development and progression. Alteration in the
microbiome colonization due to antibiotics, vaccines, host genetics leads to cancers
(Shahanavaj et al. 2015). Microbiome, highly populated microorganisms reside
within the proximity of epithelium, soon be a way for the personalized medicine
development targeting the pathobionts for the cancer progression (Fulbright et al.
2017). The altered microbiome can be a useful marker for diagnosing neoplasm
primarily colorectal cancer (CRC), gastric cancer, cervical cancers. F. nucleatum
found to be highly associated with the CRC in tumoral tissue and feces of the
patients in comparison to the control individuals. SCFA, like butyrate, fructose,
linoleic acid, acts as a robust diagnostic marker for CRC with low levels found in
patients in comparison to control individuals (Saus et al. 2019).
Research studies explain that B. longum, B. adolescentis, Parabacteroides
merdae, Collinsella aerofaciens are more populated in the feces of responder
patients of melanoma and non-responders have dominated with Ruminococcus
obeum and Roseburia intestinalis colonization (Elkrief et al. 2019).
Higher bacteroidales and low B. fragilis composition masking the effect of anti-
CTLA-4 in melanoma patients was observed. Butyrate-producing bacterium and
Firmicutes like Faecalibacterium genus involved with producing a higher response
rate with more prolonged progression-free survival (Li et al. 2019).
Pancreatic cancer has been the most prominent and fourth leading cause of death,
has some difficulty in early detection due to a lack of specific biomarkers. Recent
research studies have been promising to overcome this situation in pancreatic cancer.
Porphyromonas gingivalis, an oral bacterium is found to be increased in pancreatic
cancer. Patients tend to have antibodies against the bacteria P. gingivalis ATTC
53978. Also, saliva bacterial biomarkers are specific for the detection of pancreatic
cancer (Shahanavaj et al. 2015).
12.11 Microbiome Affecting Cancer
Ongoing cancer research is focused on the human microbiota due to promising

results shown in their interaction. The unknown proliferation of the cells is due to
external and internal factors of the host affecting the development and progression of
cancer. These factors are influenced by the microbiome activities within the host,
indirectly affecting cancer. Microbiome in the host affects the remodeling of the
tissue-like angiogenesis, a part of the tissue remodeling where adequate blood flow is
developed, which is prior necessary for a tumor to get initiated. More investigation is
required to understand the mechanism involved between microbiome and angiogen-
esis interaction (Fulbright et al. 2017). Therefore, the microbiota is observed to be
important for the development of the vasculature in the intestines of the host.
Coley, in the nineteenth century, cures malignancies in humans using live
cultures. There were few initial failures in the treatment but resulted in a mixture
known as Coley’s toxin composed of attenuated Streptococcal and Serratia
marcescens. The success rate is 80% with 5 years survival rate treated around
1000 sarcoma patients in the period where the knowledge on cancer is still in its
infancy. The mechanism behind the cure was toxins secreted by the composed
mixture-induced immune response to fight against the malignant cells.
In 1863, Virchow explained the interrelation between inflammation and cancer
onset, based on the studies detailed that carcinogenesis is initiated at the site of
chronic inflammation. This concludes the direct microbiome effects on host cell
physiology and changes in the equilibrium of the tissues. Modifications in the
microbiome may result in undefined local and systemic inflammation and conditions
within the host (Shahanavaj et al. 2015).
When cells stop to divide a condition known as cellular senescence, cells in the
senescence state secrete growth factors, enable tumor growth, and the intestinal
bacteria to induce malignancy. E. coli regulates senescence-associated phenotype
(SASP) by secreting growth factors inducing tumor development and epithelial
proliferation. Therefore, this bystander proliferation and microbial induced cellular
senescence mechanisms caused due to microbial and host interactions develop
malignancies (Fulbright et al. 2017). Some bacteria within the microbiome can
induce chronic inflammation with or without an increase in the ROS, indicating
their carcinogenic potential in the host. When the epithelial barriers are damaged by
alterations, bacteria that get in direct contact with the host cell secrete toxins, leading
to host DNA damage. Bacterial genotoxins like cytolethal distending toxin (CDT)
and colibactin cause direct dsDNA damage and instability of the host genome,
including phosphorylation of histone proteins and activation ataxia-telangiectasia
mutated (ATM)–CHK2 signaling pathway. These genetic changes lead to cell
swelling and cell cycle arrest at G2/M phase. Other toxins like B. fragilis toxin
and cytotoxic necrotizing factors affect the cellular responses and thus indirectly
play a role in tumorigenesis (Schwabe and Jobin 2013).
Studies explain there is an increase in the interferon a/b signals in lung stromal
cells, which aid in resist Influenza virus infection due to the gut microbiome.
Researchers observed FMT enhances the immune system by altering the tumor
microbiota. These promising observations allow defining cancer treatment by
modifying the tumor immune micro-environment using the gut microbiota (Shui
et al. 2020).
The gut microbiome has a significant impact on treating cancer and related
toxicities in cancer-related therapies (Helmink et al. 2019). This explains that the
gut microbiome has potential in overall cancer therapy. Gut microbiome alters the
gut-associated lymphoid tissue and mucosa immune function through the interaction
with PAMPs and antigen-presenting cells and TLRs, triggering an innate response in
the host. These immune activities result in accelerated antitumor immune function
with the low number of myeloid-derived suppressor cells (MDSCs) and high levels
of tumor infiltration lymphocytes (TILs) (Helmink et al. 2019). Scientists revealed
that patients have a higher diversity of the bacteria in their gut, who responded to the
anti-PD-1 ICIs therapy compared to the non-responders. The diversity of the
microbiota mainly includes an abundance of Ruminococcus, Faecalibacterium,
Clostridiales (Gopalakrishnan et al. 2018). Further studies on the microbiota diver-
sity generate more customized and increase the efficiency of cancer immunotherapy.
Immunotherapy as a cancer treatment is an efficient way of utilizing the patient
immune system to generate an antitumor effect with less adverse effects. Different
approaches like sensitizing tumor cells as non-self to the immune system, immune
checkpoint inhibitors (ICIs), a novel therapeutic agent with promising clinical results
in malignancies. Monoclonal antibodies blocking PD-1/ PD-L-1, CTLA-4 blockade
sensitize cancer cells to the patient immune system. Recent research studies explain
that the gut microbiome affects the therapeutic efficacy of ICIs against cancer
(Li et al. 2019). B. fragilis colonized in the mouse gut flora increased TH1 responses
in the lymph nodes near to the tumor to enhance the efficacy of the CTLA-4 immune
checkpoint inhibitor blockade (Elkrief et al. 2019).
The microbiome has proven to be a double-sided sword in cancer studies; wild
type mice can combat carcinogenesis compared to germ-free mice, on the other end
it can promote carcinogenesis by inducing inflammation to intestinal cells (Li et al.
2019) when there are alterations in the microbiome due to environmental or intrinsic
factors affecting microbial structure (Zechner 2017). The chronic inflammation
caused due to microbial dysbiosis has been known to promote cancer in the site of
inflammation and also enhance the accumulation of E. Coli.
H. pylori is carcinogenic bacteria interacting with cell growth signaling pathways.
Certain bacteria and viruses are known to cause fatal disease or chronic inflamma-
tion, as primary and secondary effects would be carcinogenic nature (Li et al. 2019).
H. pylori have cytotoxin associated gene A (CagA) which produce virulence protein
VacA, ureas, NapA2; the Vac A modulates β-catenin, resulting in inflammation and
carcinogenesis (Rajagopala et al. 2017).
Fusobacterium nucleatum, an enterotoxigenic bacteria, when fed to ApcMin/+

mice, showed characteristics like human colorectal cancer with an abundance of the
same bacteria in the tissues; however, few other mouse models did not exhibit any
tumorigenesis (Kostic et al. 2013b). Other studies showed F. nucleatum virulence
protein FadA activates the β-catenin pathway; alteration with NF-kB leads to
inflammation and promotes a favorable tumor environment. Moreover, other viru-
lence proteins like RadD induce the formation of biofilm from different bacteria,
FaP2 binds to Gal-GalNAc, which promotes colonization of F. nucleatum; besides,
it inhibits NK cells (Rajagopala et al. 2017). These studies suggest more details are
required in signaling pathways between bacterial cells and host immune cells.
Certain bacterial species induce proinflammatory toxins, alteration in signaling
pathways, also the production of genotoxic substances (Helmink et al. 2019). Some
microbes are known to cause cancer other than inducing inflammation. Microbes
produce toxic substances and some microbes themselves, when mixed with blood,
get carried to distant locations in the body and can cause cancer (Rajagopala et al.
2017). Human papillomavirus, hepatitis B and C viruses, human cell leukemia virus,
Epstein–Barr virus (EBV), Kaposi sarcoma-associated virus (KSHV) human, T
lymphotropic virus one and all known to cause cancer in humans. EBV is associated
with gastric cancer (Rajagopala et al. 2017). Virus composition in the human
virome has been unexplored. Fungi and protozoa research studies are to be
focused on knowing different microbiome genome interaction with host (Elinav
et al. 2019).
12.12 Microbiome in Cancer Immunotherapy
12.12.1 CD47 Blockade with Bifidobacteria
The effect of immunotherapy in patients is influenced by the host gut’s ability to

resist invading pathogens and response to treatment. In the malignant mouse model,
scientists found that anaerobic bacteria travel to tumor sites and boost effectiveness
against immunotherapy. In tumor-bearing mice, the absence of gut bacteria did not
respond to anti-CD47 antibiotics (Shi et al. 2020). Bifidobacteria present in the
human gut travels and accumulates at the tumor site and blocks CD47 to increase the
response against immunotherapy via stimulators of interferon genes (STING). In a
similar study, mice with inactive STING pathways showed no benefit from bacteria-
immunotherapy combined approach. STING is a transmembrane protein present on
macrophages, T cells, dendritic cells. STING stimulates innate immune genes with
respect to invading viruses, bacteria into the host. STING is activated by certain
cyclic dinucleotides (CDNs) produced by certain bacteria, followed by subsequent
reaction process type I interferons (IFNs) are secreted outside the cytoplasm of the
cell (Barber 2015). Type I interferons are antiviral cytokines and regulate adaptive
immune systems (Haller et al. 2006).
CD47 (cluster of domains) is a transmembrane protein; it is present in different
cell types (Zhang et al. 2019) (Zhang et al. 2020). CD47 is an immunoglobulin
known as integrin associated protein (IAP). It is overexpressed in cancerous cells to

avoid immune responses by acting as self-cells. High levels of CD47 in cancerous
cells mask immunotherapy and its prognosis. One of its ligand, known as signal
regulatory protein α (SIRPα) is a transmembrane protein present on myeloid cells
such as monocytes, macrophages, granulocytes, and myeloid dendritic cells. Forma-
tion of CD47 and SIRPα signaling complex inhibits the build-up of myosin IIA in
phagocytic synapses, which acts as a “do not eat me” signal. Blocking this CD47 has
potential in cancer treatment and has been used in various immunotherapies. Mono-
clonal antibodies against this complex have proven to be an effective therapy for
solid tumor and hematologic malignancies (Folkes et al. 2018).
12.12.2 PD-L1 Blockade Assisted with Bifidobacterium
Bifidobacterium (gram +ve) found in the healthy gastrointestinal tract, which helps
in digestion and produces vitamin K and B, codes for carbohydrate digestive
enzymes, also used in probiotics (O’Callaghan and van Sinderen 2016). This
organism has shown antitumor activity when subjected to mice with melanoma.
Bifidobacterium, along with programmed cell death protein1 ligand (PD-L1),
abrogated cancer with enhanced CD8 + T cells (Sivan et al., 2015).
12.12.3 CTLA-4 Blockade Assisted with Bacteroidales
CTLA-4 binding achieved with monoclonal antibody studied in patients with III/IV
stage melanoma faced effects on gastrointestinal immunity (Berman et al. 2010). In
fecal microbial transplantation (FMT) study conducted in mice proved the microbial
influence of blocking of CTLA-4. B. fragilis, B. thetaiotaomicron, and
Burkholderiales played a significant role in antitumor activity with the help of
interleukin 12(IL-12) dependent T cells (Vétizou et al. 2015). Contradicting the
above statement, Bacteroides fragilis is an enterotoxigenic bacteria; its abundance
was co-related to colorectal cancer by a study conducted in 150 humans (Purcell
et al. 2017).
12.12.4 Short-Chain Fatty Acids in Treatment for Cancer
The most prominently studied SCFAs are acetate, butyrate, and propionate com-
pared to valerate and caproate. The abundant SCFA like acetate, butyrate, and
propionate is produced in the ratio of 60:20:20 (Chambers et al. 2018). Acetate,
butyrate, propionate, valerate, and caproate were used in a study to understand the
effects of SCFA in apoptosis and cancer. This study concluded that the butyrate was
more potent to compare to propionate and valerate to induce cell growth arrest and
differentiation in colon cancer cell lines. A related study showed that this ability of
SCFA depends on histone hyperacetylation effects, alteration in cell cycle regulators
p21 and CB1. Butyrate enhanced histone acetylation compared to other SCFA and
increased the rate of programmed cell death. The exact mechanisms of action is not
well known; it has been proposed that butyrate modifies chromatin structure by
inhibiting histone deacetylase resulting in hyperacetylation of core proteins. During
histone acetylation, the DNA becomes loosely packed to histone protein and is
available for transcription of specific genes like cell regulators, chemokines
(Hinnebusch et al. 2002).
The chemokines expressed by the epithelial cells like IL-8 and MCP-1 are found
to attract neutrophils and monocytes (Fusunyan et al. 1999). Butyrate, with the help
of p21 protein downregulated Cyclin B1(CB1), is found in a study conducted on
HT-29 cells. CB1 is a crucial component for health development; its increased levels
are found in colon cancer (Hinnebusch et al. 2002). It can control p53 mitotic cell
division through regulating CB1 levels and preventing neoplastic transformation
(Innocente et al. 1999). Cyclin B1 plays a critical role in cell cycle progression from
G2 to M phase, with the involvement of NF-κB. Studies are explaining that CB1 has
induced tumor malignancy in esophageal cancer (Zhan et al. 2012).
It is studied that chronic intestinal inflammation causes cancer in the intestine;
also, it leads to pattern alteration in epithelial differentiation leading to an undiffer-
entiated state. Interleukin-8, a proinflammatory cytokine induces differentiation in
epithelial cells, butyrate has shown inhibition of IL-8 also can induce differentiation
of cells in vivo (Huang et al. 1997). SCFA are known to directly activate G-coupled
protein receptors like GPR43, GPR109A, and GPR41, which activate anti-
inflammatory cascades (Venegas et al. 2019) (Lazar et al. 2018). The detailed
illustration is in Fig. 12.2.
12.13 Future Perspectives
We are in the era of the microbiome, which has more positive preclinical and clinical
research in treating cancer. Furthermore, few challenges are upfront to know how to
regulate gut microbiota and the interaction of other genomes in the microbiome to
improve the efficacy of cancer immunotherapy. Targeting cancer immunotherapy
through the microbiome can be more successful and improve immune surveillance
when the favorable components of the microbiome are completely defined. FMT in
anticancer therapy acts as a promising way to treat cancers if the donor composition
is well known. The favorable bacteria composed of Akkermansia muciniphila,
Bifidobacteria spp., E. hirae, and Bacteroides spp. are found to impact malignant
cells effectively. Finalizing the set of microbes for treating cancer can be done by
filling the research gap, knowing the interaction of other microbial genomes with
hosts like viruses, archaea, protists, and fungi would be more promising in treating
cancers and building personalized medicine.
Fig. 12.2 SCFA production and its impact in different ways on the immune system of the host.
When the host consumes a high fiber diet, interbacterial fermentation by Faecalibacterium
prausnitzii, Eubacterium rectale produces high SCFA like butyrate and activates some immune
proteins to kill cancer cells by apoptosis, ell cycle arrest at G1 phase, by downregulation of
inflammation (Canani et al. 2011) (Segain et al. 2000)
12.14 Conclusion
Microbiomes within the body can be a good source in treating cancer growth without
any adverse effect on the host body. The host–microbiome plays a crucial role in
maintaining homeostasis of the immune system and its study can be an efficient and
economical way of developing a treatment for cancer and other microbial diseases.
The interaction between the host immunity, microbiome, and cancer progression is
explained to an extent, but more studies are to be performed. Microbiomes have been
affected by many factors, these alterations modifying the favorable microbiome to
unhealthy ones. Different microbes act as a marker specific to cancer and have been
used as an early diagnostic route to detect them. Immunotherapy is an existing way
of treating cancers, microbiome playing a considerable role in the effect of immuno-
therapy enhances the antitumor effects in the patients. To conclude, we are in a state
of a holistic vision of using the microbiome as a strategy in cancer immunotherapy.
In the coming years, more studies on other genomes of the microbiomes and their
interaction would strengthen the knowledge on the microbiome and make it a
promising way to treat cancer.
Acknowledgments The corresponding author Dr. Pola Sudhakar would like to express his deepest
thanks to authorities of Andhra University, Visakhapatnam, India for providing necessary facilities
to carry out the research work and for extending constant support.
Conflict of Interest The authors declare that there is no potential conflict of interest.
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Insight into the Animal Models
for Microbiome Studies 13
Chanda Vikrant Berde, P. Salvi Sagar, V. Kajarekar Kunal,
A. Joshi Suyoj, and B. Berde Vikrant
Abstract
The microbiomes, including bacteria, fungi, and viruses, exist within and on all the
organisms, which is the current field of research. Particularly of interest are
microbiome of human and its direct impact on human health. The health and fitness
of animals, including humans, are influenced by the existence and composition of
microbial communities of the host. To date, maximum microbiome research has been
focused on the mouse as a model organism for studying the mechanisms of different
processes occurring in the microbial communities. Mouse microbiome models have
also been the primary choice for performing preclinical tests for studying
relationships between the microbiomes and host physiological, metabolic, immune,
and neurologic phenotypes. These were also used for developing methodologies to
correct functional abnormalities in these communities that lead to disease. The
mouse, however, is not a perfect model for studying different aspects of the
microbiome and for studying the host stimuli and environmental responses. Hence,
researchers have been conducting microbiome studies using other animals as well, for
example, zebrafish, pigs, and Drosophila. This chapter summarizes the microbiome
studies conducted using different models and an insight into its advantages.
Keywords
Microbiome · Animals · Models · Health · Microflora · Host–microbe interaction
C. Vikrant Berde (*)

Marine Microbiology, School of Earth, Ocean and Atmospheric Sciences (SEOAS), Goa
University, Taleigao Plateau, Goa, India
P. Salvi Sagar · V. Kajarekar Kunal · A. Joshi Suyoj
Gogate Jogalekar College, Ratnagiri, Maharashtra, India
B. Berde Vikrant
Department of Zoology, Arts, Commerce and Science College, Lanja, Maharashtra, India
Ltd. 2021
https://doi.org/10.1007/978-981-16-3156-6_13
260 C. Vikrant Berde et al.
13.1 Introduction
Microbiomes including bacteria, fungi, and viruses live inside and on all organisms
and are a growing area of research. Particularly of interest is the human microbiome
because of its direct impact on the human health. Humans carry trillions of microbes
inhabiting our bodies, resulting in creating complex, body-habitat specific, and
adaptive ecosystems. These systems are finely adapted to continuously changing
host physiology. The presence or absence of residential microbial communities has a
direct impact on the health and fitness of the animal. Dysbiosis or dysbacteriosis is
the microbial imbalance or maladaptation of the body, for example, the microbiota
composition during a number of diseases such as inflammatory bowel disease,
multiple sclerosis, types 1 and 2 diabetes, etc. (Berg 1996; Qin et al. 2010;
Turnbaugh and Gordon 2009; Delzenne et al. 2011; Kau et al. 2011). The microbiota
of humans plays an important role in possibly causing, spreading, and prevention of
human illness (Lai et al. 2014; Norman et al. 2014; Palm et al. 2015). Gut microflora
is now known as an important factor in etiology of a number of human diseases such
as obesity (Turnbaugh and Gordon 2009; Delzenne et al. 2011), inflammation (Kau
et al. 2011; Garrett et al. 2010), metabolic syndrome (Kau et al. 2011; Cani et al.
2012), and colorectal cancer (Arthur and Jobin 2013; Macdonald and Wagner 2012).
Use of humanized mouse has brought great advancement in the field of gut microbi-
ology and associated health outcomes (Turnbaugh et al. 2009; Goodman et al. 2011;
Gootenberg and Turnbaugh 2011). Normally, these models are made by seeding
germ-free mice with the bacteria derived from human. Therefore, they provide a
solid system for studying different interactions between human microbiome and
chronic diseases where use of humans as subject is not possible.
A number of potential features of healthy microbiome have been proposed. These
potential features include prevalent organisms or molecular pathways (Cani et al.
2012) and usual ecological properties, such as diversity or stability (Garrett et al.
2010; Arthur and Jobin 2013). Normally, microbiomes show a great degree of
diversity irrespective of the presence or absence of the diseased condition (Garrett
et al. 2010; Macdonald and Wagner 2012). This characteristic of the microbiomes
creates complication in identification of simple microbial agents causing disease or
present in diseased state.
Most of the studies carried out regarding microbiome to date have focused on
mouse as a model for studying how different mechanisms occur in the microbial
communities. Mouse microbiome models have been first choice for carrying out not
only preclinical tests for studying relationships between community and hosts
metabolic, physiological, immune, and neurologic phenotypes but also for develop-
ing different methodologies to correct functional abnormalities in microbial
communities, which results in causation of disease. Many reports have stated that
mouse is not a perfect model for studying various aspects of microbiome and also for
studying host stimuli and environmental response. Researchers are carrying out
microbiome studies in other animals as well as in zebrafish, pigs, and Drosophila.
13 Insight into the Animal Models for Microbiome Studies 261
Development of different animal models for microbiota studies allows studying

of microbiota subsets, i.e., causative vs. correlative factors in diseased states, and
also offers a system to reveal putative therapeutics.
13.2 Mouse as the Animal Model for Human Microbiota Studies
The advantages of mouse models are numerous, and also, the expanse of research on
the gastroenterology, genetics, and immunology of mice is much more than any
other model. The genotypes and phenotypes range offered by mouse models out-do
all other model organisms. Thus, mice models have played a very vital role in the
research concerning human gut microbiota.
The physiology and anatomical structures of humans and mouse are quite similar,
and thus, this is one of the reasons behind more use of mouse as a model organism in
biomedical research. In the case of mice and humans, gastrointestinal tract is made of
anatomically similar organs. The prominent differences in anatomy of both human
and mice intestinal tract are because of their diets, patterns of feeding, metabolic
requirements, and size of the body.
Mice and humans share the average ratio of intestinal surface area: body surface
area (Casteleyn et al. 2010), but this ratio varies between different sections of gut
between these two organisms. The average small intestine:colon length ratio is 2.5:7
in mice and humans, respectively (Treuting and Dintzis 2012), and the surface ratio
of small intestine:colon is only 18 in mice as compared to 400 in humans (Casteleyn
et al. 2010).
The cecum in mouse is relatively large in comparison to its total gastrointestinal
tract. Cecum is recognized as the chief site for the fermentative decomposition of
plant material and the synthesis of vitamin K and B, which is reabsorbed via
coprophagy. The human cecum is small, having anatomically similar structure to
colon and doesn’t hold any clear function (Treuting and Dintzis 2012).
At microscopic level, there are numerous differences in the structure of intestinal
tract of humans and mice (Treuting and Dintzis 2012). The colon of mouse consists
of thin muscularis mucosae, whereas the human colon is covered with thicker
mucosal wall. There are a number of transverse folds present along colonic mucosa
in humans. In the case of mice, transverse folds are found only in cecum and
proximal colon. These differences in the compartmentalization and structure of
colon might contribute to creating different ecological microniches holding a variety
of microbial communities.
The surface of mice intestinal crypts in mucin-producing goblet cells of proximal
colon are abundantly present, whereas their number decreases at the base of crypt, in
distal colon and rectum. In the case of humans goblet cells, they are profusely
present from cecum to rectum.
There is a difference in the presence of another type of intestinal epithelial cell,
i.e., the Paneth cell in mouse and human. The role of Paneth cells is to secrete
antimicrobial components in the lumen of small intestine. In the case of humans,
Paneth cells are present in the cecum and proximal colon. Paneth cells are uniquely
present in the cecum in mouse but are not present in entire colonic mucosa.
There is a difference in location and amount of defensins produced by Paneth
cells, and their secretion and storage have been found to be different in human and
mice (Cunliffe et al. 2001; Ghosh et al. 2002; Ouellette and Selsted 1996). The
dissimilarities in location and amount of Paneth cells and goblet cells indicate
difference in local immune responses, which might contribute to composition of
intestinal microbiota.
Laboratory mice have been instrumental for understanding role of normal flora in
many aspects of human physiology, which includes studies like angiogenesis by
Stappenbeck et al. (2002) and Reinhardt et al. (2012), bone mineral density studies
reported by Cho et al. (2012) and Sjogren et al. (2012a, b), and studies related to
innate and adaptive immune function (Garrett et al. 2010; Littman and Pamer 2011;
Hooper et al. 2012).
Mice are an important model system for studying host-microbiota interactions
that are applicable to human biology (Spor et al. 2011) because of
1. Mice share around 99% genes with humans.

2. These genes have key similarities with human gut microbiome at phylum through
family level.
There are a number of characters of mice genetics that mark the mice as a
powerful model system for studying genetics of humans in the interactions between
host and microbiota. The availability of both inbred and outbred strains, numerous
collections of knockout, knock-in, and transgenic mutants (International Knockout
Mouse Consortium [IKMC, http:// www.knockoutmouse.org]), available data and
work done by Knockout Mouse Project [KOMP, https://www.komp.org], and data
available at Mutant Mouse Regions Resource Center [MMRRC, http://www.mmrrc.
org] also make mice a preferred study model.
13.3 Germ-Free and Antibiotics Treatment Models
Germ-free (GF) animals are devoid of any microorganism in its lifetime (Wostmann
1996; Yi and Li 2012). For studying the interactions between a host and its
microbiota, germ-free animals are important experimental aids. They are colonized
with specific microorganisms and are then referred to as gnotobiotic (Fritz et al.
2013; Smith et al. 2007). Germ-free mice are bred in isolators that prevent entry of
any microorganisms. Hence, together with these special facilities, monitoring for
contamination using different methods, along with the cost, labor, and skills required
to maintain them, makes GF mice quite expensive (Fontaine et al. 2015; Nicklas
et al. 2015).
To remove the microbiota from the model animals, specific antibiotics having
different mechanisms of action are utilized, such as antibiotic polymyxin B to target
the Gram-negative bacteria and Vancomycin for Gram-positive bacteria (Atarashi
et al. 2011; Schubert et al. 2015). Thus, the composition of the animal gut may be
manipulated by using the combination of antibiotics as per the requirements
(Schubert et al. 2015; Zackular et al. 2016).
13.3.1 GF Mice as Experimental Models
GF animal models have been used to study and understand host-microbiota

interactions in various fields of study, which include lipid metabolism (Nicholson
et al. 2012), cardiology (Stepankova et al. 2010), neurogastroenterology (Diaz Heijtz
et al. 2011; Al-Asmakh et al. 2012; Neufeld et al. 2011; McVey Neufeld et al. 2015),
reproductive biology (Al-Asmakh et al. 2014; Shimizu et al. 1998), bone homeosta-
sis (Sjogren et al. 2012a, b), and so on. Another interesting observation in the
humanized rats was the maintenance of some metabolic activities in the gut micro-
flora transferred from humans to rats. The activities such as production of equol
(Bowey et al. 2003) and reduction of cholesterol (Gérard et al. 2004) have been
reported to be maintained in the microflora.
Mice models are among the best preferred tools for studying microbiota-
associated human diseases, by understanding the host-microbe interactions as
monocolonization of single bacteria is possible. The mice are made germ-free and
then inoculated with human gut microflora. These are termed the humanized gnoto-
biotic models (Goodman et al. 2011). This model thus helps in recapitalization of
microbiota composition of the human gut.
The research findings on GF, however, cannot be directly utilized for treatment
purposes. The reasons being that the bacterial species in mice gut are not found in
humans, and this microflora is influenced by numerous factors involving anatomy,
behavior, etc. (Gordon and Pesti 1971; Sommer and Backhed 2013; Kostic et al.
2013; Gootenberg and Turnbaugh 2011). In spite of these drawbacks, the GF mice is
the most preferred model system for studying host-microbe interactions.
13.4 Other Models
There are numerous invertebrate model species, which are often used in the studies
related to certain interactions between the host and its microbiota. The selection of
these invertebrate species is dependent on two factors, namely, the innate immune
system (Chu and Mazmanian 2013) and a highly restricted gut microbiota (Chaston
and Goodrich-Blair 2010). ‘Humanized’ animals, i.e., models with human micro-
flora, have been established to understand the human microbiome under controlled
conditions, utilizing highly researched and genetically manipulable mice and rats, in
addition to pigs, dogs, etc. (Hazenberg et al. 1981; Hirayama 1999; Bowey et al.
2003; Gérard et al. 2004; Kibe et al. 2005; Pang et al. 2007).
Some of the invertebrate models used are as follows:
Numerous systems have been studied, which include Heterorhabditis
bacteriophora and Steinernema carpocapsae and their respective symbionts,
Photorhabdus luminescens and Xenorhabdus nematophila (Clarke 2008;

Wollenberg et al. 2016; Singh et al. 2015; Sicard et al. 2004), Hirudo verbana and
Aeromonas veronii (Rio et al. 2009; Graf et al. 2006), and Euprymna scolopes and
Vibrio fischeri (McFall-Ngai 2014; Schleicher and Nyholm 2011). The most com-
monly employed model organisms are, however, Drosophila melanogaster and
Caenorhabditis elegans. The ease of rendering these models germ-free (Kietz
et al. 2018; Berg et al. 2016), their small size, and freedom from regulatory concerns
as in vertebrate models make these models advantageous over the other. The
drawbacks are the gastrointestinal anatomical differences with the host, the
differences in the microbiota of the gut, and inability to carry out certain studies in
these systems such as adaptive immunity in humans.
The gut microflora of invertebrates has fewer microbial species, and the compo-
sition is dependent on the environment (McFall-Ngai 2007). In the case of
vertebrates, adaptive immune response plays a role in the establishment and devel-
opment of microbiota of the gut (McFall-Ngai 2007; Maynard et al. 2012). This
leads to zebrafish Danio rerio, the simplest vertebrate system and having a diverse
microbiota, being preferred as model system for host-symbiont relationship studies.
13.4.1 Zebrafish (Danio rerio)
Over the last few decades, Zebrafish use in research has increased progressively.
Zebrafish is advantageous owing to the limited requirements of less space, availabil-
ity in large numbers, cost effectiveness, and high prolificacy. The preference for
zebrafish is also due to many similarities to its mammalian hosts (Trede et al. 2004;
Norton et al. 2008; Alsop and Vijayan 2009; Wong et al. 2013). Homology is seen in
the adaptive immune system and in the digestive system. Organs similar to mammals
Zebrafish are pancreas, gall bladder, liver, and intestine. Also, the intestinal epithe-
lial cells consist of absorptive enterocytes, goblet cells, and enteroendocrine cells
and share similarity to mammals.
Rawls et al. (2007) focused on the transparent nature of the zebrafish, which
allows real-time visualization of fluorescently labeled microbes lining the gut,
throughout. The external fertilization of Zebrafish is followed by development of
the embryo. Transparency of the embryo and larvae permits for the visualization of
developing cells and the successive development of microflora using time-lapse
microscopy. Studies concerning the host genes or signaling pathways that are
regulated by the gut microbiota can be performed (Patton and Zon 2001). Genetic
screening methods include mutagenesis, retrovirus-based insertional mutagenesis,
zinc finger nucleases, morpholino-based gene knockdown, role of RNAi in function
loss (Amacher 2008; Nasevicius and Ekker 2000; De Rienzo et al. 2012), and
genome editing using TALEN system (Bedell et al. 2012). Thus, the zebrafish has
a numerous features that make it an attractive experimental system.
Pham et al. (2008) have reported studies on early postembryonic development of
Zebrafish using relatively simple methods for the development of green fluorescent
(GF) and gnotobiotic zebrafish. GF zebrafish larvae are obtained by surface
sterilization of embryos with various antibiotics (Bates et al. 2006; Davis et al.
2016a, b). These GF larvae may be labeled, or the microflora of the larvae may be
fluorescently labeled in the larva and visualized, through the transparent body (Bates
et al. 2007; Russo et al. 2015; Singer et al. 2010). By using these techniques, the role
of microbiota can be determined in various disease conditions such as inflammatory
bowel disease (IBD) (Yang et al. 2014; Geiger et al. 2013; Brugman et al. 2009),
effect of probiotic bacteria on stress- and anxiety-related behavior (Davis et al.
2016a, b), metabolism and reproduction (Qin et al. 2014; Giorgini et al. 2010),
and immunity and pathogen resistance (Wang et al. 2016; Qin et al. 2017).
Limitations in the use of zebrafish as study model for microbiota-related research
are the differences in environmental conditions and exposures.
13.4.2 Fruit Fly (Drosophila)
Numerous studies have been carried out on the gut microflora of Drosophila in order
to understand the host-microbe interaction. The composition of gut microflora has
been studied, and many studies have shown that the microbial community is less
complex as compared to those found in mammalian gut (Corby-Harris et al. 2007;
Cox and Gilmore 2007; Ren et al. 2007; Ryu et al. 2008; Chandler et al. 2011; Wong
et al. 2011). Drosophila is therefore being looked upon as a model for host microbial
interaction studies.
It was observed in a couple of studies that the gut microflora composition was
highly subjective to diet and bacteria belonging to families Acetobacteraceae,
Lactobacillales, and Enterobacteriaceae were most dominant (Corby-Harris et al.
2007; Chandler et al. 2011). The microflora of the gut is aerobic in nature and is
easily cultured in the laboratory, which has made possible to have microbial stocks
of the microbiota available for studies on host-microbial interaction (Chandler et al.
2011; Charroux and Royet 2012; Shin et al. 2011). Thus, these factors have made
Drosophila, a model for studies on symbiosis, with huge potential to disclose new
insights into host–symbiont interactions.
13.4.3 Dogs (Canis familiaris)
There are a number of factors that make dogs the preferred models over other
models. Gastrointestinal tract of the dogs with respect to size and structure is fairly
identical to humans, in that dogs are monogastric like humans, cecum more devel-
oped than human cecum (Song et al. 2013; Misic et al. 2015), and both suffer from
diet-induced periodontal disease (Gorrel 1998; Harvey 1998). Common oral flora
includes Streptococcus, Staphylococcus, Pseudomonas, Actinomyces, Pasteurella,
Neisseria, and Porphyromonas spp. (Dewhirst et al. 2010; Sakamoto et al. 2005),
while common lung microflora includes Pseudomonas, Streptococcus, Prevotella,
and Fusobacterium (Ericsson et al. 2016; Erb-Downward et al. 2011). Thus, dogs
are an ideal model species for finding the microbiota present in other internal organ
systems such as the respiratory tract, intestinal tract, skin, etc. Also, studies on
beneficial effects of the gut microflora and probiotic benefits are being carried out
using canine models. There are numerous limitations to the usage of dogs as study
models. Like rats, dogs are costly and require housing facilities.
13.4.4 Rabbits (Oryctolagus cuniculus)
Rabbits are used not so commonly as animal models, more so due to the cost factor.
Rats or mice are preferred to rabbits. The same is true for research concerning GF
models. Rabbits have been used for research on infectious diseases caused by GI
tract pathogens (De and Chatterje 1953). The rabbits have well-developed system,
and hand-rearing cesarean-born rabbits are maintained as GF models to be used in
the studies (Lanning et al. 2000; Schousboe et al. 2001). A part of the small intestine
is reported being used in experiments on studying the effects of pathogens like
Vibrio cholerae, etc. (Taylor et al. 1958; Duncan et al. 1968; Arm et al. 1965; Sanyal
et al. 1995; Melling et al. 1976). The technique involves ligation of small intestine
and inoculation of pathogen under study in the portion of intestine and placing it
back in the abdomen (De and Chatterje 1953). There are, of course, limitations to the
use of rabbits as model species in host-microbiota interactive research mainly
including their cost, relative to rodents.
13.4.5 Pigs (Sus scrofa domesticus)
Pigs are omnivorous and have anatomical and physiological similarities with human
gut. Their gut microflora has been well-characterized (Zhao et al. 2015), and the
composition is similar to human gut microflora (Panasevich et al. 2018; Pedersen
et al. 2013; Ji et al. 2018). Microbial community is similar to the human donor
developed in germfree piglets. Hence, these GF animals are excellent models for
studies dealing with the effect of dietary changes on the establishment of the gut
microbiome. Apart from these, other features that make pigs a model of choice for
studies are size, physiology, developmental stages relative to humans, and ability to
manipulate their genome (Perleberg et al. 2018; Ryu et al. 2018). Hence, they have
been used in a number of studies such as gastrointestinal immuno-ontogeny (Sinkora
and Butler 2016), diet-induced obesity (Turnbaugh et al. 2006), xenotransplantation
(Vodicka et al. 2005), gastrointestinal physiology (Roura et al. 2016), and cardio-
vascular physiology (Hughes 1986; Gallo et al. 2017). Effects of resistant starch
(Haenen et al. 2013), high- and low-fat diets (Heinritz et al. 2016), antibiotics (Looft
et al. 2012; Allen et al. 2011), prebiotics (Berding et al. 2016), probiotics (Barszcz
et al. 2016; Riboulet-Bisson et al. 2012; Shen et al. 2010; Zhang et al. 2014; Wen
et al. 2014), and myriad other compounds (Liu et al. 2012) on the GM of pigs. Pigs
have been successfully colonized with human microflora (Wang and Donovan
2015). The housing and feeding costs as well as the size of the pig models make
them undesirable as study models.
13.5 Conclusions
The microbiome field has undergone a big change in recent years. The studies have
mainly focused our attention on the role played by the host microbiota in the
maintenance of host health. The role of the microflora of the host in the initiation
and propagation of disease has also been highlighted by this study. These
interactions are studied using model systems. Fundamental discoveries in
microbiome research can be made using the most controllable animal systems,
including nonmammal vertebrates such as the zebrafish and invertebrates such as
Drosophila and Caenorhabditis elegans. The early studies of humanized animal
models have permitted assessment of the human microbiome that would be chal-
lenging to achieve using customary human cohort studies or in vitro model systems.
Each one of the animal models described in this chapter displays some resem-
blance to the physiology of the human digestive system, thus providing valuable
knowledge from diverse angles about the gut microbiota in health and disease. The
information obtained from these studies has diversified our understanding of the
human gut microbiota in general. Although much of the research to date has focused
on the human microbiome, similar metagenomic studies can be applied to under-
stand better animals of agricultural importance as well as pets.
It can be concluded that depending on the study, the animal model may be
selected. Dogs may be used for studies related to host-associated microbiota and
interactions between host and microbes, while for research concerning nutrition,
omnivorous animals such as pigs may be used. The fruit fly offers the advantage of
microbial manipulability in the perspective of a genetically manipulable host, while
the zebrafish is good for experiments requiring a greater degree of genetic tractability
than that of complex vertebrates. Thus, depending on the line of research, the most
appropriate model for the study may be selected.
Acknowledgement The authors are grateful to their respective institutions for encouragement and
support.
Conflict of Interest The authors declare no conflict of interests.
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Bioinformatics Algorithms and Software
for Predicting Microbiomes 14
Shrikant Pawar, Yong Chiang Tan, and Chandrajit Lahiri
Abstract
Over the last decade, bioinformatics approaches have been used extensively for
analyzing microbiomes from various sources. Microbiomes can vary from exter-
nal skin to internal gut, with each of the tissue demanding different analytical
techniques. Of these, the next-generation sequencing strategy is the current trend
instead of amplicon sequencing. It is a principal method for phenotypic trait
characterization of strains, as exemplified by the shotgun metagenomics of
probiotics. Moreover, quantifying abundance is crucial and can be portrayed
using different computational steps enabling gene predictions and annotations.
This profiling or metagenome assembly is achievable by utilizing varying
algorithms. In fact, bioinformatics implications are tremendous in microbiome
predictions and this review focuses on some of the salient strategies. Essentially,
we present comprehensive coverage of the current next-generation sequencing
platforms. To this end, we add up the open-source databases and bioinformatics
algorithms and software tools for microbiome prediction and analysis. Finally, we
delineate the bioinformatics challenges for discovering microbiomes.
Shrikant Pawar and Yong Chiang Tan contributed equally with all other contributors.
S. Pawar
Yale Center for Genome Analysis (YCGA), Yale University, New Haven, CT, USA
Y. C. Tan · C. Lahiri (*)
Department of Biological Sciences, Sunway University, Petaling Jaya, Selangor, Malaysia
Ltd. 2021
https://doi.org/10.1007/978-981-16-3156-6_14
276 S. Pawar et al.
Keywords
Bioinformatics · Microbiomes · Next-generation sequencing · Metagenomics ·

Metaproteomics
14.1 Introduction
Microbes are ubiquitous on the Earth, residing even in extreme environments and
within a multicellular organism. The presence of normal human microbiota is crucial
in maintaining human health, and the alteration in microflora composition, or
dysbiosis, can cause several health complications. These can range from mental
issues such as depression to physiological changes such as hypertension, carcino-
genesis, and obesity (Yang et al. 2015; Sobhani et al. 2011; Aron-Wisnewsky et al.
2019). Recent researches have been hugely focusing on the impact of microbiota
composition on disease progression due to the discovery of various host-microbiota
links, such as the gut-lung axis and brain-gut axis (Budden et al. 2017; Mayer and
Tillisch 2011; Quigley 2017). For instance, a study by Riley et al. (2013) has
unearthed excessive exposure to low-dose antibiotics, which leads to gut microflora
dysbiosis, as a probable cause of the prevalence of obesity in the USA. Moreover,
disruptions of microflora composition can stimulate microbial pathogen introduc-
tion, thus leading to severe infections like Pseudomonas aeruginosa-associated
pneumonia (Rosa et al. 2020). In the light of the close relation between microbiota
composition and health complications, microbiome detection has become a crucial
field of medical research, especially in disease diagnosis and discovery of new
intervention strategies (Malla et al. 2019; Shi et al. 2015).
In microbiome detection, cultivation approaches have been traditionally hugely
utilized (Boase et al. 2013; Sibley et al. 2011). For example, one of the important
research areas of utilizing such approaches is determining the efficacy of probiotics.
In this process characterization of different strains is carried out for determining
overall composition (de Dios Miranda et al. 2019). Research has shown that
probiotics usually benefit humans. The identification of novel probiotics, however,
from different bacterial strains, is a necessity but a laborious task (LaPierre et al.
2019) with testing different cultures and libraries. From the prepared culture
libraries, bioinformatics analysis, if performed on different strains for characteriza-
tion, can lead to several potential candidates which then have to pass through
different regulations and clinical trials to be approved in the market (FEEDAP
et al. 2018). Therein, whole-metagenome sequencing can provide strain characteri-
zation as well as a screening of the traits based on gene expression levels (Berendsen
et al. 2016). This itself results as an extension of bioinformatics applications to
analyze and predict microbiome utilizing data mining and machine learning (Zeevi
et al. 2015).
14 Bioinformatics Algorithms and Software for Predicting Microbiomes 277
The presence of unculturable or hard to culture microbes, however, has impeded

the utilization of cultivation approaches in in-depth microbiome analyses hoping to
detect every microbe involved in the sample. Therefore, culture-independent
approaches, especially with the help of bioinformatics, have gained huge attention
and became the main trend of microbiome study due to indiscriminative coverage of
detection. Therein, metagenomic and metaproteomic studies have been developed,
of which microbiome detection can be done at the level of nucleic acid (DNA or
RNA) and proteins, respectively, via next-generation sequencing (NGS) and mass
spectrometry (MS) (Lepage et al. 2013; Zhang and Figeys 2019; Zhang et al. 2016).
For downstream analysis of the metagenomic and metaproteomic data, bioinformat-
ics approaches have been utilized via many tools and algorithms which have been
developed in the hope of increasing efficacy and accuracy. Therefore, herein, we
mainly review the various bioinformatics tools and algorithms for microbiome
detection and analysis. Meanwhile, we also briefly mention the recently used NGS
and MS platforms in microbiome detections.
14.2 Microbiomes
Microbiome composition has been an important area of study not only in life science
researches, but also in geology, archaeology, environmental studies, and other fields
of research (Ramsøe et al. 2020; Vick et al. 2018). This is due to the huge coverage
of microbial population and their indispensable roles in maintaining the normal
functioning of their residing environment while making sure of their survival. The
ubiquitous property of microorganisms indicates that they possess specific
functionalities for survival in extreme environments which can be further
manipulated for the benefits of humanity.
Environmental microbiomes. The symbiotic presence of microbial communities
has been crucial for the natural environment in carrying out recycling functions.
Examples of these are the mediation of global carbon and nitrogen cycles by the land
and ocean microbes (Canfield et al. 2010; Gougoulias et al. 2014; Sulman et al.
2014; Worden et al. 2015). Symbiotic microbiota residing in the soil has been aiding
the growth of plants, especially in terms of nutrient uptake and immune system,
thereby contributing to the smooth recycling of chemicals in the global food cycle
(Finkel et al. 2019; Hacquard et al. 2017; Müller et al. 2016). Therefore, understand-
ing crucial environmental microbiomes is important for the manipulation of natural
resources in researches. To cater to the need, plant microbial fuel cell (PMFC) was
being proposed and developed for a potent new and clean source of electricity
(Lu et al. 2015; Timmers et al. 2012; Wetser et al. 2015) through the utilization of
plant microbiota comprising the bacterial and archaeal community.
Human or animal microbiomes. The interactions between animal and symbiotic
microflora are crucial in maintaining the health and normal function of the host.
Different organs are comprised of different microflora compositions, thereby
supporting the normal activity of the respective organs in different ways. For
instance, gut microbiome has been crucial in helping food digestion, conferring
278 S. Pawar et al.
immunity against various types of diseases, and altering normal behavior of the host
(Cryan and Dinan 2012; El Kaoutari et al. 2013; Kosiewicz et al. 2011; Million et al.
2018). Moreover, vaginal microbiota, especially comprising lactobacilli, has been
crucial in preventing infections such as vulvovaginal candidiasis (VVC) (Oerlemans
et al. 2020; Tortelli et al. 2020). The involvement of gut microbiome in drug
metabolism has led to the establishment of a framework by Javdan et al. (2020) to
consider gut microbiome composition upon drug discovery. Therefore, it is crucial to
explore the microbiome for better diagnosis and cope with health complications due
to dysbiosis.
14.3 Microbiome Analysis by Sequencing
New techniques for microbiome studies have evolved with long-read sequencing
including Pacific Biosciences (PacBio) and Oxford Nanopore Technology (ONT)
platforms which are at the forefront of sequencing technology. PacBio has its unique
advantages wherein high-quality full-length sequences can be sequenced with mul-
tiple reads which are currently unavailable with short-read sequencing (Saulnier
et al. 2011). Sequencing costs have reduced significantly in the last couple of years
allowing researchers to generate replicates and improve the depth of sequencing for
microbiome samples thereby leading to improvised downstream analyses. Machine
learning algorithms like support vector machines, random forests, neural networks
on next-generation sequencing data have improvised disease state classifications and
predictions of different pathologies (van Dijk et al. 2014).
Next-generation sequencing (NGS) has made microbiome research more com-
prehensive. Illumina and Ion Torrent technologies, which came before Pacific
Biosciences and Oxford Nanopore Technologies, were only capable of generating
millions of short reads while newer technologies generate hundreds of long reads per
run. Thus, time and cost efficiency have been greatly increased thereafter. For
instance, in Illumina sequencers, DNA fragments attached to the glass slides are
amplified with fluorescently labelled nucleotides binding to the complementary
DNA sequence producing short reads (300 bp) (van Dijk et al. 2014). Pacific
Biosciences on the other hand works through engineered DNA polymerase working
on concentrated genomic DNA producing long reads (10–15 kb) but, unfortunately,
with a high error rate (Koren et al. 2012). Oxford Nanopore technologies work on
single molecule sequencing, where a single DNA strand goes through a protein
nanopore, wherein an enzyme usually helps in unwinding the double strand (Jain
et al. 2015).
Due to the presence of non-culturable microbial contaminants, NGS stood out to
be a better approach in microbiome analysis due to its indiscriminative coverage of
detection. A study by Lewis et al. (2020) has proposed the utilization of Illumina
ScriptSeq in detecting pathogens in fresh produce. The proposed NGS detection has
manifested detection not only in bacterial pathogens (Salmonella) but also viral
pathogens (phage MS2), of which such simultaneous detection of the interkingdom
microbiome could not be achieved in a single culture environment through
cultivation approach. Moreover, body fluid microbiome detection has been arising as
detection for infectious diseases. Utilizing Illumina MiSeq 16S rRNA sequencing,
NGS is a promising tool in the detection of the pathogenesis of continuous ambula-
tory peritoneal dialysis (CAPD) (Kim et al. 2020). Besides, a new pathogenesis
detection and classification through a search-based approach, involving NGS, were
established by Su et al. (2020). In this case NGS was firstly conducted, and a
microbiome novelty score (MNS) was utilized in differentiating diseased from
healthy genomic samples. Then, the diseased genomic samples were further classi-
fied based on references.
Furthermore, NGS technology has been utilized in probiotics and gut microbiome
detection. As different composition of gut microbiota affects the health of host
differently, and probiotics have been proven to improve human health, tracking of
gut microbiota composition and probiotic efficacy became an important task in
monitoring a person’s health (Kumar et al. 2020; Rawi et al. 2020). Utilizing 16S
rRNA metagenomic sequencing, Suez et al. (2018) monitored the effects of
probiotics introduction and autologous fecal-microbiome transplantation on gut
microbiome recovery after antibiotic introduction. Moreover, another research
using a similar method has proven the importance of personalized probiotic admin-
istration due to person-specific colonization resistance (Zmora et al. 2018). Besides,
through gut metagenomic analyses, bacterial species, involved in pregnancy-related
iron-deficiency anemia, were discovered (Celis and Relman 2020).
14.4 Microbiome Analysis by Mass Spectrometry
Besides NGS, which processes the sample DNA and RNA molecules for
metagenomic analysis, mass spectrometry (MS) plays a huge role in microbiome
prediction by processing protein molecules on samples for metaproteomic analyses.
Proteins are the main functional component of the central dogma of molecular
biology, and different DNA sequence codes for different amino acid sequences.
Therefore, by analyzing the protein or peptide composition in a sample, information
on taxonomy until functions and metabolic activities of the sampled microbiome can
be predicted, through amino acid sequences, and protein identification and quantifi-
cation, respectively. The increased importance of metaproteomic analysis can be
seen by the recent development of microbiome prediction tools (detailed in section
“Metaproteomic Analysis”) with MS spectra as direct input.
In metaproteomic analysis, liquid chromatography-mass spectrometry (LCMS) is
usually carried out. For MS, a triple-quadrupole mass spectrometer like SCIEX
Triple Quad™ 7500 is usually utilized for quantifications. The shortcoming of
triple-quadrupole MS is that it utilizes selected reaction monitoring (SRM) which
specifically detects only the selected compounds (Picó 2020). Fortunately, high-
resolution mass spectrometry (HRMS) is recently being introduced as an alternative
to the traditional MS due to its untargeted screening capabilities. LC-HRMS has
been prevalently utilized in the metaproteomic analysis of biological samples as
described by Pezzatti et al. (2020). Currently marketed HRMS instruments that are
280 S. Pawar et al.
frequently utilized in metaproteomic studies are the Orbitrap and Q-ToF mass
analyzers. Although Orbitrap confers higher resolution analysis compared to
Q-ToF mass analyzers, both instruments have high accuracies in mass
quantification.
14.5 Metagenomic Analyses in Microbiome Prediction
The metagenomic analysis is a crucial procedure in microbiome prediction mainly

for taxonomical identification and diversity quantification. By analyzing DNA or
RNA information extracted from a sampled microbiome, sequential information can
lead to the identification of important microbial taxa present in the sample. Fig-
ure 14.1 summarizes the main workflow of metagenomic analyses in microbiome
prediction. It comprises the usage of QUAST, a software package that handles the
metagenomic analysis of detected genomic data, and consists of QUAST (genomic
data quality assessment), MetaQUAST (match metagenomic data to references),
QUAST-LG (QUAST but specifically for large data), and Icarus (visualizing tool for
QUAST results) (Gurevich et al. 2013). Alignment of the original reads to the
assembled data for the detection of structural variants in microbiomes is performed
by MetaQUAST, where assembly refers to the alignment of genomes to the refer-
ence. This software utilizes de Bruijn graph data structure for assembly, which can
Fig. 14.1 Schematic diagram showing an overview of metagenomic analyses workflow

generate multiple genome tables and plots for understanding diversity in species, can
auto-download reference sequences for unknown species and can detect chimeric
regions reporting interspecies misassemblies (Zerbino and Birney 2008).
MetaVelvet is another tool that extends MetaQUAST application utilizing single
de novo genome assembler Velvet (Zerbino and Birney 2008). MetaVelvet
decomposes de Bruijn graph into sub-graphs thereby isolating every species genome
from short reads. The main advantages of MetaVelvet are that it utilizes multispecies
metagenomic assemblies and assembles read data with longer N50 sizes compared to
the single-genome assemblers and can avoid chimeric scaffolds forming longer
scaffolds with an increase in the predicted number of genes. Moreover, it can also
be widely applicable to metagenomic analysis (taxonomic content, functional com-
position, etc.).
Another application utilizing the de Bruijn graph is Ray Meta, it utilizes parallel
computing for handling large datasets, the k-mers are utilized for calculating cover-
age distributions, followed by aligning reads against reference genomes. It can also
utilize multiple cores to decrease memory requirements and computing time in a
high-performance computing environment (Boisvert et al. 2012). Ray Meta has wide
applications ranging from de novo genome, metagenome, transcriptome assembly,
quantification of the contig, microbiome consortia members, transcript expression
abundances, taxonomy, and gene ontology profiling of samples. Ray Meta is a
scalable distributed technique with faster fetching of data yielding taxonomic
profiles by graph coloring with unique colors for k-mers to identify taxons at low
taxonomic ranks. It is available as a distributed software having a message-passing
interface (MPI) implemented Open-MPI library with a division of tasks in several
workers with message aggregation strategy. Storage of k-mers is achieved with
double hashing with the utilization of pointers for compacting memory on high-
performance computing applications.
Another important tool developed on some of the existing tools is MetAMOS. It
can perform microbiome genome assembly using sequencing technology used by
the investigator (Treangen et al. 2013). MetAMOS is an automated, reproducible,
and traceable assembly method generating assembly, scaffolds, variant motifs,
annotations, and other analysis reports. A workflow in MetAMOS is a text file
with input sequences for filtering, assemblers, read mapping, classifier, annotation,
validation, and scaffolders. MetAMOS essentially has two main components,
initPipeline (initializing sequence libraries) and a runPipeline (actual run). Although
running MetAMOS is relatively easier, installation requires specific packages and
versions like Java (6+), perl (5.8.8+), python (2.7.3+), R (2.11.1+ with PNG
support), gcc (4.7+ for full functionality), curl, and wget. The most common cause
of a failed run for MetAMOS is a missing package or dependency.
There are several algorithms for microbiome assembly, the one which stands out
is binning. This clusters sequences into groups, after which assembly can be
performed on genomes. The importance of binning is that it prevents the mixing
of different genomes, so ideally each bin consists of a single genome overcoming the
problem of wrong assemblies connecting contigs. PHYSCIMM is another program
that essentially does binning and clustering to characterize microbial composition
282 S. Pawar et al.
and species classification (Kelley and Salzberg 2010). PHYSCIMM utilizes

supervised learning to improve clustering from well-characterized genera. While it
is important to not mix different genomes, some investigators may be interested in
multiple strains of a species with identical 16S rRNA sequences, to analyze these
couple of software, StrainPhlAn and PanPhlAn can be applied to shotgun
microbiome data (Scholz et al. 2016; Truong et al. 2017). Another binning program
worth mentioning is CONCOCT which uses sequence coverages throughout multi-
ple samples for clustering microbiomes (Alneberg et al. 2014). The CONCOCT
program uses Gaussian mixture models to cluster contigs into genomes with clusters
assigned with a variational Bayesian approach. CONCOCT’s precision of the
clusters is 0.988, with a recall of the same cluster at 0.998 and an adjusted Rand
index (precision and recall combined) of 0.983 (Alneberg et al. 2014). Thus,
CONCOCT has been effectively utilized for automatically clustering contigs into
genomes, linking cluster abundances to environmental variables, evaluating clusters
with single-copy core genes, and evaluating clusterings by comparing them to
known genome assignments. Once the assembly is completed, genes and regulatory
elements need to be annotated.
As mentioned above, short reads are relatively difficult to assemble as they are
usually fragmented. To overcome this problem, MetaGene Annotator, a microbiome
gene finding algorithm from short sequences could be utilized (Noguchi et al. 2008).
It utilizes a self-training model from inputs for predictions with a capacity to detect
prophage and horizontally transferred genes. It can also analyze ribosomal binding
sites with a prediction of the translation start sites with 96% and 93% sensitivity and
specificity, respectively. MetaGene Annotator works well on longer genomic
sequences for precise annotations with statistical models of prophage genes in lateral
gene transfers. The ribosomal binding sequences of the 30 tail of 16S RNA predict
translation starts of genes improving prediction accuracies of genes. Glimmer also
does a similar analysis with bacterial gene prediction via clustering data belonging to
the same organism (Kelley et al. 2012). Glimmer gene prediction identifies >99% of
the genes in prokaryotic genomes but falls short of accuracy with fragmented and
error-prone sequences. It does classification and clustering of the sequences before
gene prediction, utilizing a probabilistic model for predicting gene length and start/
stop presence with insertion, deletion, and stop codon substitution errors. Glimmer
also utilizes Markov models for understanding gene composition from available
training data and utilizes a flexible open reading framework to capture ribosomal
binding sites followed by scoring its log-likelihood ratio. Comparing other known
programs, other researchers have found that Glimmer predicts several more genes
from 454 reads of the human gut microbiome (Kelley et al. 2012).
Predicting genes can also be performed using FragGeneScan without assembly,
wherein, unlike binning, it utilizes hidden Markov models (HMMs) in overcoming
any frameshift sequencing errors (Rho et al. 2010). It specifically targets genes from
short reads without compromising increasing sequencing error rates. It combines a
sequencing error model with a hidden Markov model for prediction in short reads.
Authors state that it can outperform MetaGene with 62% accuracy on 1% sequenc-
ing errors and can predict significantly more genes than MetaGene with many genes
with no possible homologs.
A command-line-based pipeline, SHOGUN, was developed by Hillmann et al.

(2020) for metagenomic analysis of WMS data. After filtering of contamination in
raw sequencing data input, SHOGUN carries out sequence alignment with three
different algorithms which are Bowtie2, BURST, and UTree. Eventually, taxonomic
annotation and gene abundance prediction are generated using the last-common
ancestor (LCA) algorithm and Bayesian redistribution (BD), respectively. Similarly,
iMAP is another command-line-based pipeline that handles taxonomic assignments
of metagenomic data (Buza et al. 2019). With demultiplexed sequencing data as
input, iMAP utilizes QIIME2 pipeline and mothur-based taxonomic annotation
regarding SILVA and Greengenes classifiers, respectively (DeSantis et al. 2006;
Yilmaz et al. 2014). Besides OTU abundance generation through sequential binning,
iMAP also estimates microbiome diversity through principal component analysis
(PCA), principal-coordinate analysis (PCoA), and non-metric multidimensional
scaling (NMDS) analysis of Bray–Curtis dissimilarity coefficients. Thereafter, phy-
logenetic annotation is carried out through iTOL tree viewer.
BiomMiner is a command-line-based tool mainly developed for downstream
analysis of metagenomic data although it provides an optional built-in upstream
analysis besides the processing protocol recommended by the sequencer
(Shamsaddini et al. 2020). It has five analysis modules, which are Overview,
Alpha Diversity, Beta Diversity, Differential Abundance Analysis, and Machine
Learning. The Overview module summarizes the taxonomic abundance and
computes a rarefaction curve through Mothur v1.34. Alpha Diversity module
computes the diversity estimate value and statistically (Kruskal–Wallis test)
compares between two datasets for differential analysis. Differential Abundance
Analysis module utilizes Metastats, Linear discriminate analysis Effect Size
(LEfSe), and Kruskal–Wallis test algorithms to compute differences in abundance
between two datasets. Beta Diversity module computes beta diversity indices
through PCoA and NMDS and then quantifies the similarities in OTU features
between two datasets. Machine Learning module integrates the random forest
(RF) and supports vector machine (SVM) in assigning “importance” value to each
OTU, which indicates the accuracy of the search.
While most metagenomic analysis tools are either command-line based or with a
user-unfriendly interface which is challenging for beginner researchers, recently
introduced tools are aiming at improving the graphical user interface (GUI) while
utilizing or pipelining present algorithms. One such tool is the MicrobiomeAnalyst
(Chong et al. 2020; Dhariwal et al. 2017). Microbiome Analyst online server has
four modules, which are Marker Data Profiling (MDP), Projection with Public Data
(PPD), Shotgun Data Profiling (SDP), and Taxon Set Enrichment Analysis (TSEA).
All modules aim to confer functional annotation to metagenomic data. The MDP
module analyses the marker-gene abundance data, upon which species diversity
(alpha and beta diversity) and functional predictions are carried out. PPD prints the
summary of the submitted 16S rRNA data and compares it with the public database
to probably derive novel insights. SDP conducts differential functional analysis with
the aid of statistics on gene list or gene abundance data annotated through KEGG
Ortholog (KO), COG, or EC. TSEA module enriches taxa list with their associated
284 S. Pawar et al.
implications on a host like diseases. As a result, MicrobiomeAnalyst can infer host–

microbiome interactions from functional assignments of metagenomic data.
14.6 Metaproteomic Analyses
Besides metagenomics which analyzes and categorizes genomic data at the sequen-
tial and taxonomical level, the metaproteomic approach (Fig. 14.2) which utilizes
protein information has been developed along the way of microbiome detection and
analysis. EggNOG-mapper predicts and annotates functional information of protein
sequences based on the eggNOG (“evolutionary genealogy of genes:
Non-supervised Orthologous Groups”) HMM (Hidden Markov Models) and protein
databases (Huerta-Cepas et al. 2019; Huerta-Cepas et al. 2017). Other than func-
tional annotation, eggNOG-mapper can also be utilized in orthology analysis.
Instead of the traditional homology-based approaches, eggNOG utilizes either
HMMs or Double Index Alignment of NGS Data (DIAMOND) approach in ortholog
search, of which DIAMOND is recommended in large data size due to its speed
advantage over HMMs but with less sensitivity. In orthology assignments, eggNOG
outperformed homology-based approaches, which are Basic Local Alignment
Search Tool (BLAST) and InterProScan, in terms of precision and speed. EggNOG
accepts not only protein sequences as input but also coding sequences from
metagenomic and metatranscriptomic data. MetaGOmics is another traditional
metaproteomic analysis tool, which is an online pipeline utilizing the BLAST
algorithm to search through UniProtKB for Gene Ontology (GO) assignments
(Riffle et al. 2018). Therein, taxonomical and functional annotations can therefore
be achieved, and a directed acyclic graph (DAG) will be plotted for visualization and
analysis of the GO annotation. However, MetaGOmics only allows FASTA file
input.
Proteomic studies usually have mass spectrometry peptide spectra as output data,
and therefore, for convenience, MS spectra, as input for analyses, are usually
preferred. Trans-Proteomic Pipeline (TPP) is a pipeline that comes along with a
set of tools for MS data analysis (Deutsch et al. 2015). TPP can process MS spectral
data into a list of identified proteins. From MS spectral data, TPP will initially
conduct peptide identification, validation, and quantification, followed by protein
assignment such that finally an identified protein list is generated. After the protein
list is generated, further downstream analysis is usually carried out through external
tools for taxonomical and functional annotation. For instance, Rabe et al. (2019)
utilize PROPHANE for downstream analysis of TPP resulting in oral microbiome
analysis. PROPHANE confers downstream metaproteomic analysis with FASTA
input along with report files from external upstream analysis like
MetaProteomeAnalyzer (MPA), Scaffold, Proteome Discoverer, and tabular reports.
PROPHANE integrates the lowest common ancestor (LCA) algorithm into protein
identification and reports protein quantitation through normalized spectral abun-
dance factors (NSAF). In PROPHANE taxonomical and functional assignments,
database search will be conducted through three algorithms (DIAMOND,
14
Bioinformatics Algorithms and Software for Predicting Microbiomes
Fig. 14.2 Schematic diagram showing an overview of metaproteomic analyses workflow

285
286 S. Pawar et al.
eggNOG-mapper, and HMMER3) along with their associated databases such as

non-redundant (nr) proteins from NCBI, UniProtKB, SwissProt, and eggNOG.
Besides that, there are some tools capable of carrying out data processing from
MS spectral input to functional and taxonomical annotations.
MetaProteomeAnalyzer (MPA) is a traditional pipeline-based tool which utilizes
MS spectra as the initial input, followed by database search through UniProt, KEGG
Pathways, Enzyme Commission (EC) Classification, and NCBI Taxonomy
databases (Muth et al. 2015). As a result, taxonomic and pathway classification
along with protein identification can be retrieved. Moreover, PeptideShaker is a tool
for general proteomics data analysis (Vaudel et al. 2015). PeptideShaker utilizes
several search engines, then estimates error probabilities through target-decoy search
approach to finalize the peptide-spectrum match (PSM) list, resulting in more
accurate final search results. PeptideShaker results output can be exported in
Cytoscape (Proteomic network analysis and visualization) and Nonlinear (LC-MS
data analysis and visualization) formats for further analyses, besides the commonly
used text and graphic formats. Besides, UniPept is another pipeline-based tool in
metaproteomic analyses (Singh et al. 2019). In UniPept, the MS input data will be
searched for references through GO terms and EC classifications, therefore resulting
in functional assignment of the analyzed metaproteomic sample. Thereafter, taxon-
omy and protein identification will be generated. Van Den Bossche et al. (2020) have
introduced a pipeline combining MPA and PeptideShaker for MS input data
processing, with UniPept for downstream processing and visualizations due to its
user-friendly graphical output.
MetaLab also allows automated pipeline-based metaproteomic data analysis with
MS peptide spectra as input (Cheng et al. 2017). Upon raw data input, MetaLab
processes the data through two separate approaches, which are MetaPro-IQ iterative
search and spectral clustering, to narrow down the input data prior to database
search, which increases the temporal efficacy of the search (Zhang et al. 2016).
Thereafter, peptide identification and quantification are carried out through maxLFQ
algorithm (label-free XIC intensity-based quantification) or MaxQuant (isotope
labelling quantification), followed by taxonomy analysis by searching through the
built-in pep2tax database (Cox et al. 2014; Tyanova et al. 2016). An updated
version, MetaLab 2.0, allows the detection of post-translational modified (PTM)
proteomes, which is a feature that is rarely seen in other metaproteomic tools and it
allows more accurate and higher coverage of proteomic identification (Cheng et al.
2019). MetaLab results can be exported in Biological Observation Matrix (BIOM)
format, and therefore can be subjected to downstream analysis such as the MEGAN
tool as recommended by the author (Cheng et al. 2017; Huson et al. 2016).
As enzymatic digestion of proteomic samples, prior to MS, is usually carried out
in metaproteomic analyses wherein traditional tools like MPA are unable to handle
such cases with trimmed peptides. Therefore, to cope up with this problem, PepFunk
has been created by Simopoulos et al. (2020) for gut microbiome metaproteomic
analyses. It utilizes mass spectrometry (MS) data as input and is capable of handling
trypsin-digested protein samples. By automatically searching through their modified
KEGG database, the Integrated human gut microbial Gene Catalog (IGC) database,
functional assignments can be achieved with KEGG pathways as pepFunk output.

The IGC database was curated by the in silico trypsin digestion of proteins in KEGG
database. Sample treatment examples, which starts with microbiome cultivation
followed by trypsin digestion and mass spectrometry, were tested and elucidated
by Simopoulos et al. (2020). Like pepFunk, ProteoClade enables users to search
through in silico enzymatically digested databases using MS data as input
(Mooradian et al. 2020). Instead of the KEGG database, ProteoClade Database
(PCDB) originated from UniProt and NCBI databases and can be utilized as a
reference for the metaproteomic search. Therefore, the taxonomic classification of
the detected peptides is applicable herein. Upon benchmarking, ProteoClade has
manifested less RAM consumption and less duration of a process compared to
UniPept and MetaProteomeAnalyzer (MPA).
14.7 Combined Analysis
M2IA developed by Ni et al. (2020) is a pipeline-based tool that integrates

metagenomic and metabolomic analysis in microbiome detection. M2IA is mainly
used for differential analysis of metagenomic and metabolomic information between
two datasets. M2IA requires 16S rRNA sequencing data (OTU table in .txt and
respective sequences in .fasta or .fna), metabolome data (mass spectrometry data in .
csv), and a tabulated sample information (in .csv) as described by Ni et al. (2020).
The overall similarity between two datasets in M2IA is quantified through Coinertia
analysis (CIA) and Procrustes analysis (PA) approaches, of which a correlation
coefficient (R-value) ranging from 0 to 1, and 1 being exactly similar, is computed.
Besides that, M2IA also carries out several pairwise statistical tests to predict the
correlation between two datasets, as well as between microbiome and metabolome.
Moreover, a network plot based on a relationship between microbes and metabolites
will be plotted for visualization.
14.8 Algorithms and Tools
Several softwares utilizing varied algorithms are presently available for analyzing
and detecting microbiomes. Table 14.1 summarizes the important tools explained in
this paper.
14.9 Open-Source Databases
With information on enzymatic pathways or classes, users can utilize some specific
online repositories for performing microbiome analysis. Some of the important tools
include antiSMASH, dbCAN, and Resfams (Gibson et al. 2015; Blin et al. 2017)
which focus on metabolite synthesis and resistance pathways in different
microbiomes. Another tool with the incorporation of 12 such databases for proteins
288 S. Pawar et al.
Table 14.1 A summary of microbiome analysis tools

Types Tools Usage
Metagenomics
Tool within MetaQUAST Utilizes de Bruijn graph data structure for
software assembly
package
Standalone MetaVelvet Utilizes single de novo genome assembler velvet
tool
Software Ray Parallel computing for handling large datasets
package
Toolkit MetAMOS Perform microbiome genome assembly using
sequencing technology used by the investigator
Standalone PHYSCIMM Does binning and clustering
tool
Standalone StrainPhlAn and Shotgun microbiome data
tools PanPhlAn
Standalone CONCOCT Uses sequence coverages throughout multiple
tool samples for clustering microbiomes
Online tool MetaGeneAnnotator Microbiome gene finding algorithm from short
sequences
Software Glimmer Bacterial gene prediction via clustering data
Standalone FragGeneScan Utilizes hidden Markov models (HMMs)
tool overcoming any frameshift sequencing errors
Pipeline SHOGUN Taxonomic annotation by LCA and gene
abundance prediction by Bayesian redistribution
Pipeline iMAP Taxonomic assignment, microbiome diversity, and
phylogenetic annotation
Standalone BiomMiner 16S rRNA taxonomic abundance and diversity
tool analysis
Online tool MicrobiomeAnalyst Infer host–microbiome interaction through
metagenomic data
Metaproteomics
Standalone EggNOG-mapper Utilizes HMMs and DIAMOND approach in
tool orthology and functional predictions
Online tool MetaGOmics Utilizes BLAST to search through UniProtKB
database
Pipeline with Trans-proteomic Protein identification from MS spectral input
tool package pipeline
Online tool PROPHANE Downstream analysis tool for taxonomical and
functional annotation
Standalone MetaProteomeAnalyzer Search through UniProt, KEGG pathways, EC
tool classification, and NCBI taxonomy databases
Standalone PeptideShaker Multiple search engine with target-decoy search
tool method of error estimation
Online tool UniPept Search through GO terms and EC classifications
(continued)
Table 14.1 (continued)

Types Tools Usage
Standalone MetaLab Iterative search or spectral clustering for MS
tool spectra input, supports both label-free and isotope
labelling quantification
MetaLab 2.0 allows PTM proteome identification
Online tool PepFunk Search through enzymatically digested KEGG
database
Standalone ProteoClade Search through enzymatically digested UniProt
tool and NCBI databases
Combined
Online tool M2IA Combined metagenomic and metabolomic analysis
is InterPro, which integrates information with protein function, families, publication

information, protein domain information, etc. (Mitchell et al. 2015). Querying with a
name or sequence information from its online portal produces possible matches.
Besides that Reactome is an open-source curated repository for interpreting
interactions between biomolecules (Jassal et al. 2020). KEGG or Kyoto Encyclope-
dia of Genes and Genomes combines multiple databases with genomics and proteo-
mics data and also incorporates information about genes, proteins, and their
associated pathways, diseases, and drug databases (Kanehisa et al. 2012).
MediaWiki software is another attempt in improving deficiencies from the above
portals where a user can curate or edit the pathway structure and the child product of
this software is an open-source pathway analysis platform, WikiPathways (Kelder
et al. 2012). With an improvised strategy, MetaCyc is another open-source tool with
pathways curated from all domains of life with prioritization on metabolic pathways
from published experimental datasets (Caspi et al. 2016). The general idea behind all
these tools is an online annotation that is user-friendly and fast, as prediction and
annotation files for assembly are usually huge such that querying them on personal
computers is impossible. Considering this aspect, the Meta4 web application was
developed (Richardson et al. 2013). It can search for thousands of genes and proteins
and retrieve their original raw files. Moreover, it can also be installed on a personal
server with a user-friendly interface. It can perform several features like BLAST
against proteins or nucleotides and can also set up a private account for unpublished
user data.
Furthermore, researchers have been aggregating microbiome analysis data in
databases such as MGnify to ease future taxonomic annotations (Mitchell et al.
2020). MGnify users can search microbiome datasets through their location of
sample collection. Besides, the expanded Human Oral Microbiome Database
(eHOMD) consists of 16S rRNA reference sequences of the aerodigestive tract
microbiome (Chen et al. 2010; Escapa et al. 2018). Similarly, CORE is an oral
microbiome 16S rRNA database (Griffen et al. 2011). The BLAST search algorithm
is usually employed in microbiome databases.
290 S. Pawar et al.
14.10 Challenges
Microbiomes can vary from external skin to internal gut, where each of the tissue
calls for different microbiome analysis techniques. For example, the diversity of
human skin microbiomes for exploring skin products may call for one type of
longitudinal study, while the gut microbiomes may require another, thereby discov-
ering microbiomes challenging (Zeeuwen et al. 2012). In general, the environment
introduces uncharacterized biodiversity which makes metagenomic enzyme charac-
terization challenging. Obtaining complete assemblies of microbiomes is difficult
with a different abundant distribution of genomes in the environment (Ayling et al.
2020). Thus, quantifying abundance is important and can be profiled using different
computational steps enabling gene predictions and annotations (Fig. 14.3). This
profiling or metagenome assembly can be achieved utilizing conventional
algorithms like de Bruijn graph-based assembly. Bruijn graph-based assembly is
Fig. 14.3 Flowchart of microbiome raw data processing using metagenomics, metaproteomics and
combined
the most popular technique owing to its simplicity, scalability, and computational
time efficiency (Van der Walt et al. 2017).
14.11 Conclusion
Microbiome prediction is indeed an indispensable and rapidly growing part of

medical research. With the inability of cultivation approaches to detect unculturable
microbes, culture-independent approaches have emerged as a popular alternative to
microbiome predictions and analyses. Herein, we have summarized the important
bioinformatics algorithms, software, and portals in extracting putative genes and
proteins for functional and taxonomical characterization in microbiomes. The
advancement of technology has led to the evolution from short-read to long-read
sequencing via the NGS approach, and from MS to HRMS in mass spectrometry, in
extracting microbiome information from nucleic acid and protein samples. Mean-
while, new bioinformatics tools and novel algorithms have been frequently
introduced for downstream analyses in metagenomics and metaproteomics, with
improvements in efficacy and accuracy.
Acknowledgments The author acknowledges the support of Sunway University, Selangor,

Malaysia for providing the necessary computational facilities.
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Microbiome in Human Health and Disease: Pallaval Veera Bramhachari Editor

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Pallaval Veera Bramhachari Editor

ISBN 978-981-16-3155-9 ISBN 978-981-16-3156-6 (eBook)

Krishna University K. B. Chandra Sekhar

Machilipatnam, India Pallaval Veera Bramhachari

Part I Introduction to Microbiomes

Part II Microbiome for Human Health: From Basic Science

8 Understanding the Interplay Between the Host

Part III Microbiome for Human Health: Clinical Applications

About the Editor

Pallaval Veera Bramhachari is currently a Head and

for attending XVII Lanceﬁeld International Symposium

S. Anju Bhavan’s Vivekananda College of Science, Humanities, and Commerce,

A. Joshi Suyoj Gogate Jogalekar College, Ratnagiri, Maharashtra, India

Pola Sudhakar Department of Biotechnology, AUCST, Andhra University,

ACT Adoptive cell treatments

CRC patients Colorectal cancer patients

IBD Inﬂammatory bowel disease

MyD88 protein Myeloid differentiation primary response 88 protein

SRM Selected reaction monitoring

Pallaval Veera Bramhachari

Human beings are home to a massive invisible microbial community, which

Microbial communities · Microbiome’s diversity · Microbiomes · Human health

# The Author(s), under exclusive license to Springer Nature Singapore Pte 3

1.2 The Complexity of the Microbiomes

1.3 Co-Evolution of Microbiomes with Humans

variability is associated. In several roles, the microbiome is involved viz. production

1.4 Microbiome Dysbiosis

1.5 Significance of Microbiome Research.

1.6 The Way Forward

Pavani Sanapala and Sudhakar Pola

P. Sanapala · S. Pola (*)

# The Author(s), under exclusive license to Springer Nature Singapore Pte 13

2.2 Genital Microbiome

poises tolerance against infections during reproduction. Though inﬂammatory

2.2.1 Modulation of the Genital System

Vaginal microbiota (VMB) and endometrial microbiota can modulate inﬂammation.

The vaginal microbiota plays an essential role as a mutualistic relationship with

2.3 Skin Microbiome

2.3.1 Skin Architecture

2.3.2 Association of Microorganisms and Skin Diseases

2.3.3 Topography of Skin

Studies based on cultures illustrated variation of skin microbiota due to different

2.3.4 Skin Modulation

Additionally, acting as a physiological barrier, the skin plays a vital role as an

2.4 Oral Microbiome

2.4.1 Ecology of the Oral Microbiome

Table 2.1 Microbiota in association with oral diseases

Table 2.1 (continued)

2.4.2 Association of Microbiota and Oral Diseases

2.4.2.2 Periodontal Diseases

2.4.2.3 Oral Tumor and Recurrent Aphthous Stomatitis

2.4.2.4 Association with Systemic Diseases

2.4.3 Modulation of the Oral Microbiome

homeostasis. Understanding the mechanism of oral microbiome is necessary to

In recent years, the expansion of molecular methods in identifying the composition

Sakamoto M, Huang Y, Ohnishi M, Umeda M, Ishikawa I, Benno Y (2004) Changes in oral

The microbiome includes a diverse group of microorganisms that inhabit a

Y. Aparna (*) · K. Anuradha · S. Anju · J. Sarada

# The Author(s), under exclusive license to Springer Nature Singapore Pte 35