Filamentous fungi and their role in

fungalbacterial Biofilms formations

For
BACHELOR OF

SCIENCE MICROBIOLOGY

School of Basic & Applied Science

GALGOTIAS UNIVERSITY, GREATER NOIDA

UTTAR PRADESH

Student Name: Vahid

Tiya vats [19SBAS1060018]

Semester : V/ FALL 2020-21

Submitted to : Dr. Sarita Mallik

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 ACKNOWLEDGEMENT

This work was supported by the Dept. of life science, Galgotias University, Greater
Noida, Uttar Pradesh (under the summer training program) authors acknowledge
all the major and minor support got during completion of this report.

I would like to express my special thanks of gratitude to my teacher Dr. Sarita

Mallik who gave me the golden opportunity to do this wonderful project on the
topic Occurrence of Filamentous Fungi in Drinking Water: Their role on fungal-
bacterial biofilm formation, which also helped me in doing a lot of Research and I
came to know about so many new things I am really thankful who has been
supported through the completion of this project.

(Signature)
Vahid (19SBAS1060018)
B.Sc. Microbiology (Vth semester)
Division of Biochemistry
School of Basic and Applied Science
Galgotias University
Greater Noida
Uttar Pradesh, India

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 TABLE OF CONTENTS

1. ABSTRACT

2. INTRODUCTION

3. BIOFILM

4. DRINKING WATER

5. FUNGI & FILAMENTOUS FUNGI BIOFILM

6. OCCURRENCE OF FILAMENTOUS FUNGI IN DWDS

7. METHODOLOGY PROGRESSION TO STUDY FUNGI IN DRINKING

WATER

8. FUNGAL-BACTERIAL INTERACTION IN BIOFILM

9. CONCLUDING REMARKS

10. REFERENCES

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OCCURRENCE OF FILAMENTOUS FUNGI IN
DRINKING WATER: THEIR ROLE ON FUNGAL-
BACTERIAL BIOFILM FORMATION

ABSTRACT

Water is indispensable to life and safe and accessible supply must be available to
all. The presence of microorganisms is a threat to this commitment. Biofilms are
the main reservoir of microorganisms inside water distribution systems and they
are extremely ecologically diverse. Filamentous fungi and bacteria can coexist
inside these systems forming inter-kingdom biofilms. This review has the goal of
summarizing the most relevant and recent reports on the occurrence of filamentous
fungi in water distribution systems along with the current knowledge and gaps
about filamentous fungal biofilm formation. Special focus is given on fungal-
bacterial interactions in water biofilms.

KEYWORDS
o Drinking water
o Fungi
o Bacteria
o Biofilms
o Fungal-bacterial interactions

INTRODUCTION
The water that is available that can support microbial life in building materials is
commonly expressed as water activity. Different fungi have different water
requirements. Physical openings in storage facilities and lack of cover allow
microorganisms to be introduced from the air, animals, introduction of untreated
surface or groundwater. The water that is available that can support microbial life
in building materials is commonly expressed as water activity. Water activity
compares the physical properties of water in the material in question with that of
pure water to generate a scale from 0-1.0. Pure distilled water has a value of 1.0.
Although there is no sampling device that directly tests for water activity; instead
the equilibrium relative humidity is reported. The problems associated with fungi

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include blockage of water pipes, organoleptic deterioration, pathogenic fungi and
mycotoxins. Mycotoxins are secondary metabolites only produced and released
under certain conditions that can cause various health effects in humans and
animals. The secondary metabolites produced by some species can alter the taste
and smell of water. Organic acids produced by fungal metabolic processes can
increase the rate of corrosion in water pipes.
Without water, fungi are unable to grow or reproduce. A key to preventing and
controlling fungal growth in things such as buildings can be to reduce the
availability of free water by removing the water source such as repairing leaks in
pipes, running a dehumidifier in damp areas, and removing wet building materials.

BIOFILM
Biofilms are a collective of one or more types of microorganisms that can grow on
many different surfaces. Microorganisms that form biofilms
include bacteria, fungi and protists. One common example of a biofilm dental
plaque, a slimy buildup of bacteria that forms on the surfaces of teeth. Pond scum
is another example. Biofilms have been found growing on minerals and metals.
They have been found underwater, underground and above the ground. They can
grow on plant tissues and animal tissues, and on implanted medical devices such as
catheters and pacemakers.
FUNGI BACTERIA PROTISTS
Candida spp. Bacillus spp. Dexiostoma
Aspergillus spp. Listeria monocytogenes Vannella
Fusarium spp. Staphylococcus spp. Chilodonella
Pneumocystis spp. Cyanobacteria Spumella

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 Blastoschizomyces Pseudomonas Neobodo
capitatus aeruginosa

Biofilm formation begins when free-floating microorganisms such as bacteria

come in contact with an appropriate surface and begin to put down roots. This first
step of attachment occurs when the microorganisms produce a gooey substance
known as an extracellular polymeric substance (EPS). An EPS is a network of
sugars, proteins and nucleic acids (such as DNA). It enables the microorganisms in
a biofilm to stick together.
Attachment is followed by a period of growth. Further layers of microorganisms
and EPS build upon the first layers. Ultimately, they create a bulbous and complex
3D structure. Water channels crisscross biofilms and allow for the exchange of
nutrients and waste products.

Multiple environmental conditions help determine the extent to which a biofilm

grows. These factors also determine whether it is made of only a few layers of cells
or significantly more. For instance, microorganisms that produce a large amount of
EPS can grow into fairly thick biofilms even if they do not have access to a lot of
nutrients. On the other hand, for microorganisms that depend on oxygen, the
amount available can limit how much they can grow. Another environmental factor
is the concept of "shear stress". If you have a very high flow [of water] across a
biofilm, like in a creek, the biofilm is usually fairly thin. If you have a biofilm in
slow flowing water, like in a pond, it can become very thick.
Finally, the cells within a biofilm can leave the fold and establish themselves on a
new surface. Either a clump of cells breaks away, or individual cells burst out of
the biofilm and seek out a new home.This latter process is known as “seeding
dispersal”.

DRINKING WATER
Water is indispensable to life. Therefore, every effort should be done to achieve
drinking water as safe as possible. While substantial progress has been made in
increasing access to clean drinking water and sanitation, billions of people mostly
in rural areas still lack these basic services. Worldwide, one in three people do not
have access to safe drinking water, two out of five people do not have a basic
hand-washing facility with soap and water. This becomes even more dramatic in
times of SARS-Cov-2 where access to clean water and hand washing with soap are
crucial to controlling the individual and communitarian pandemic disease of
COVID-19.

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When microorganisms are present in excess as their growth may affect the
organoleptic properties of the water. These microorganisms can be found either as
in planktonic forms inhabiting bulk water or as biofilms growing on pipes surfaces.
[Douterelo et.al] Biofilms can be considered the main source of microorganisms in
drinking water distributions systems (DWDS) and are responsible for serious
effects, such as changes in the taste, turbidity, colour and odour of the water,
corrosion of metallic pipes, disinfectant demand, potential accumulation and
dispersion of pathogens and production of toxins [Flemming HC et.al]. Primary
and opportunistic pathogens are found in DWDS because they can survive water
disinfection. Special protection is provided to microorganisms embedded in
biofilms such as sharing of nutrients and metabolic products and increased
resistance to environmental stresses, such as hydrodynamic shear forces and
disinfection [Sonigo P et.al].

FUNGI AND FILAMENTOUS FUNGI BIOFILM

The most recent classification of this kingdom comprises seven recognized phyla:
Basidiomycota, Ascomycota, Glomeromycota, Microsporidia, Blastocladiomycota,
Neocallimastigomycota and Chytridiomycota [Viegas C et.al]. This latter large
group of organisms can be characterized by hyphal growth supporting macroscopic
sexual reproductive structures (e.g., truffles, mushrooms) and microscopic sexual
or asexual reproductive structures, known as moulds or filamentous fungi [Lima N
et.al]. Fungi are ubiquitous and some of them, belonging to the phyla
Chytridiomycota, are particularly adapted to aquatic environments. These fungi are
known for producing zoospores morphologically apt to propagate in running
waters. Filamentous fungi from other phyla in Eumycota are, however, mostly
adapted to the terrestrial environment, such as soil and anything in interface with
air, as they generally need a solid substrate for spore dispersal.
Many fungal species can be resistant to water treatment and disinfection. The water
treatment process may fail to remove/ inactivate all microorganisms of concern
from source water.

The four principal pathways by which people can be exposed to fungi in

drinking water are:

o Ingestion by drinking contaminated water

o Inhalation of aerosolized spores while showering or in the sauna

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 o Skin contact with contaminated water, like showering or bathing

o Introduction through mucous membranes such as the skin, eyes, and oral
cavity while showering or bathing

To identify sources of Aspergillus infection in a hospital, they found that

bathrooms had significantly higher numbers of airborne propagules than in
patient’s rooms. This increase was thought to be caused by aerosalisation
following running the tap, the shower, or flushing the toilet, which allowed for
colonization of damp micro-niches within the bathroom. It was also found that
airborne A. Fumigatus levels increased after running the shower multiple times.

Some species have the potential to cause allergic reactions and disease. There is a
risk of superficial or localized infection in healthy individuals and more severe and
invasive infection in immune-compromised patients. Water contaminated with
fungi is of importance to hospitals where immunocompromised patients undergo
treatment. Fungi present in drinking water may cause severe fungal infections in
immunosuppressed patients. In a small number of studies, drinking water supplies
have been found to be the source of infection. Aspergillus accounts for most
infections, with Aspergillus fumigatus accounting for 90% of cases.

Although there are some indications of fungal species affecting humans, little
research has been conducted on the matter and more needs to be conducted to
ensure that no harmful long-term effects are possible.

Although DWDS are not considered natural habitats for these filamentous fungi,
they can often be introduced into these environments from different pathways,
such as physical openings in storage facilities, treatment breakthroughs, leaking
joints and adapters, cracks in pipelines and/or during maintenance or mains
installation [Sammon NB et.al]. Airborne spores can be an important external
source of filamentous fungi propagules in a DWDS. Once inside these systems,
fungi can survive the oligotrophic conditions by scavenging nutrients from the
substrate which they colonize or the water in which they are inhabiting.
Consequently, their presence may then cause additional problems to the water
quality (e.g., unpleasant appearance with flocs and earthy pungent odours, the
presence of pigments, pipe blockage, a source of potentially pathogenic and
allergy-causing fungi and the presence of mycotoxins) [Bai X et.al]. To maximize
nutrient uptake, filamentous fungi will form hyphal mats. Due to their absorptive
nutrition mode, secretion of extracellular enzymes that digest complex molecules
and apical hyphal growth, filamentous fungi have a high ability to grow on

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surfaces, thus forming biofilms. Fungal survivability and proliferation in DWDS
are believed to be related to the ability to form biofilms [Sim~oes LC et.al].

Harding proposed a six-step pioneer model for filamentous fungal biofilm

formation based on models for bacteria and yeasts:
1) Propagule adsorption,
2) Active attachment to a surface,
3) Microcolony formation I,
4) Microcolony formation II (or initial maturation),
5) Maturation and,
6) Dispersal (planktonic phase) [Fernandes et.al]

This model updated by adding an initial step surface conditioning where the
surface hydrophobicity and charge of spores and the substratum play key roles in
the adhesion process. This initial physical contact can result in reversible adhesion,
followed by irreversible adhesion with the secretion of adhesive substances by
germinated spores and active gremlins [Harding MW et.al]. Spore germination will
ensue if suitable environmental conditions are met. After gremlins start to form,
they secrete hydrophobins that mediate adhesion and hyphae substratum
interaction [Zampieri F et.al]. Subsequently, hyphal differentiation produces a
complex hyphae net (mycelium) that grows in all directions enclosed within a
polymeric extracellular matrix, where quorum sensing molecules, similarly to
bacterial biofilms, are present. The maturation stage for DWDS biofilms should
mainly occur in reservoirs due to the requirement of a stable air water interface for
aerial growth and subsequent spore formation and air dispersion. Finally, the
dispersion stage occurs through the release of spores or different propagules in
response to environmental stresses or biological stimuli. In drinking water,
propagules can be dispersed by water flow, which can then establish new biofilms,
further spreading the presence of filamentous fungi in drinking water

OCCURRENCE OF FILAMENTOUS FUNGI IN

DWDS
A wide diversity of filamentous fungi has been detected from drinking water. The
most common recovered species belong to the genera Aspergillus, Cladosporium
and Penicillium. This might be related to their ability to secrete a pigment called
melanin, which confers protection to spores against a variety of stresses, providing
these microorganisms with a competitive advantage and greater resistance to water
treatment.

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Among the isolated filamentous fungi, possibly pathogenic allergenic and
toxigenic species have also been found. This is particularly concerning since
several reports on the presence of these fungi have been obtained from hospital
water systems. In some cases, the presence of pathogenic species (e.g. Aspergillus
fumigatus; Fusarium solani) in drinking water, has led to the hypothesis of hospital
water systems serving as transmission routes for fungal infections. These results
indicate that hospital water contains high fungal diversity, including potential
pathogens. Many of the fungal species found in drinking water have also allergenic
potential. Some of these health adverse effects may arise not only from the fungi
itself but also from the production of secondary metabolites and volatile organic
compounds. Several species from both Penicillium and Aspergillus genera are
known mycotoxin producers. Mycotoxins cause a variety of health problems and
are known to be carcinogenic and capable of impairing the immune system in both
humans and animals. The concentration of mycotoxins in drinking water is likely
to be very diluted and, for the time being, has not been identified as the source of
symptoms attributable to mycotoxins.

METHODOLOGY PROGRESSION TO STUDY

FUNGI IN DRINKING WATER
The most usual isolation pathways for analysing fungi in drinking water are based
on water filtration followed by:
 Conventional Culturing Methods
 Molecular Approaches
 Membrane Filtration
 Direct Plating
 Centrifugation

Since there is no standardization approach in the isolation of fungi, the culture

medium used tends to vary among researchers. Among the most commonly
reported media are Sabouraud dextrose agar (SDA), Sabouraud glucose agar
(SGA), malt extract agar (MEA), cornmeal agar (half-strength) (CMA/2), Czapek
Dox agar (CZ), Dichloran 18% glycerol agar (DG18), Dichloran Rose Bengal
Chloramphenicol agar (DRBC), Neopeptone glucose Rose Bengal aureomycin
(NGRBA) and potato dextrose agar (PDA). Some of these media have higher
nutrition content than others, thus being more selective towards some fungal
species. Hence, an extensive culturomics approach, applying different designed
media, should be used in order to increase the probability of obtaining the highest
coverage representation of the fungi present in water.

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The recommended genetic marker for basic fungal identification is the internal
transcribed spacer (ITS) region which has already been used in the majority of
studies. Biofilm sampling from DWDS has evolved with time. The most usual
method of sampling has been performed by scrapping or swabbing water related
surfaces. Swabs were then directly plated onto solid media or previously
suspended in a sterile saline solution before being plated. Other methods for
studying biofilms in situ include cutting out pipes, which could then be placed onto
medium for culturing, or the use of sampling devices inserted into the pipe.

Pipe cut-out is, however, considered as a destructive sampling method and has
other disadvantages, such as being expensive and Labor-intensive. It is also
difficult to repeat the experiment since pipe cut-outs have to be replaced after
sampling. The use of devices inserted into pipes is, therefore, the currently
preferred sampling method. These devices are usually coupons of different
materials such as glass, polyvinyl chloride (PVC), polyethylene, acetate, cast-iron
or concrete, to encompass the high variability of materials present in DWDS. The
majority of older DW networks were made of iron-based materials. More recently,
polymeric materials such as PVC have been preferred because they are easier to
handle. Different materials have different surface physicochemical properties that
influence biofilm formation, including microbial diversity. Shortly, physical
adsorption is generally a reversible process in which one monolayer is formed and
involves nonspecific bonds.

Example of a cast-iron coupon (~15 _ 15 mm) cut out from a water distribution
system pipe in direct contact with NGRBA medium, showing the growth of a filamentous
fungus. Source from the authors

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A growing number of studies have also been using metagenomic approaches for
the detection of fungi in bulk water or biofilm samples. Different next generation
sequencing (NGS) techniques have been applied. The use of these techniques has
allowed to understand and enhance not only the knowledge of fungal presence in
water but also the microbial ecology of these ecosystems. This information has
emphasized the need to understand and develop new indicators with the potential
to be used to protect and promote water quality and safety.

(a) PVC-C tube from a water distribution system and (b) Scanning electron micrograph of
a PVC-C cut out coupon after one week in direct contact with NGRBA medium

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 Pencilin under microscope

Most relevant filamentous fungi surveys in drinking water from the last two
decades
Most frequent
Location, Date Water source Isolation method References
fungal genera
Aspergillus,
United Kingdom, Surface water Membrane filtration, Cladosporium, [Kinsey G
1996 and network centrifugation, direct Epicoccum, et.al]
plating Penicillium and
Trichoderma
United States of
America Municipal water Pipe coupons Aspergillus and [G€ottlich E
(Springfield, supply system (Biofilm) Penicillium et.al]
MO), 1997
Germany (North Acremonium,
Rhine- Drinking water Pour-plating Exophiala, [Warris A
Westphalia), Penicillium and et.al]
1998-99 Phialophora
United States of Alternaria,
America (Little Water Membrane filtration, Aspergillus, [Anaissie EJ
Rock, AR), distribution swab Paecilomyces and et.al]
1997e2000 system of a applicators Penicillium
Hospital

FUNGAL-BACTERIAL INTERACTION IN BIOFILM

DWDS biofilms are complex communities with a high number of co-inhabiting
microorganisms. This diversity leads to a variety of complex relationships
involving inter- and intra-species interactions. Although intra-species interactions
may play an important role in the coexistence of some micro biomes, biotic
interactions between distantly related organisms across the kingdoms of life also
regulate the composition of these communities. In many micro biomes, bacteria
can coexist with different eukaryotic microorganisms, including fungi. These
interactions encompass, of course, biofilms. As the microorganisms are closely

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embedded in an extracellular matrix, inter-kingdom biofilms containing bacteria
and filamentous fungi can be considered a closer level of fungal-bacterial
interaction, but this aspect is still poorly understood. Different factors may affect
these inter kingdom interactions, including the presence of quorum sensing (QS)
molecules. QS is a mechanism employed by microbial species to coordinate
community behaviour. It relies on the production, release and detection of small
signaling molecules, which in turn modify gene expression. Eukaryotes, and in the
scope of this review, filamentous fungi, have the ability to interfere with bacterial
communication by producing molecular signals that interact with bacterial QS.
These compounds are called quorum sensing inhibitors (QSI). They can mimic the
structure or function of auto inducers, act as antagonists to the QS molecules as
well as interfere with the stability and function of the regulator protein or the auto
inducer synthase and hydrolysate signaling molecules. As filamentous fungi do not
have an active immune system, they must rely instead on chemical defence
mechanisms. For this reason, they have been studied regarding their QSI potential,
in particular the genus Penicillium.

Different studies have found different relationships between fungi and bacteria. In
mixed biofilms, both organisms can exist as dual complexes or fungi may offer
biotic support for the establishment of a bacterial biofilm. However, it has also
been shown that fungi often colonize pre-established bacterial biofilms, and due to
their different ecological requirements, it has been suggested that it can lead to a
positive relationship between these microorganisms [Doggett MS et.al]. Reports of
negative relationships may be observed due to culturing processes, where both
fungi and bacteria are in direct competition for resources [Gonçalves AB et.al].
This variety in findings could, in sum, be a consequence from several factors, such
as the difference in the composition of isolated species from the water systems,
differences in methodologies or different biological mechanisms at play. For this
reason, a need arises for further research to explore the different correlations
between fungi and bacteria and what are the factors influencing these interactions.

To demonstrate the heterogeneity in findings, in a single study, performed on

bacterial-fungal biofilms in flowing water photo processing tanks using a model
community, it was difficult to determine which interactions were present. Some
species showed increased growth rate in mixed cultures while others showed a
reduction, however, all species were present in a lower number than in single
cultures, which was considered to be a result of limitation and competition for the
nutrients available. A positive coexistence between the bacteria Pseudomonas and
the fungi Basidiobolus in in situ biofilms. This could be due to ability of the fungi
to produce extracellular enzymes that allow them to degrade high molecular weight
compounds, releasing secondary metabolites that can potentially be used, in this

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case, by Pseudomonas. In another study, a correlation was observed between the
relative abundance of certain bacterial taxa such as Proteobacteria and
Basidiomycota. The same study also confirmed the presence of Acremonium and
Neocomospora from early stages of biofilm formation to a more developed
biofilm, forming essential communities with bacteria. Fungal contribution, in
particular Ascomycota, is very important to the microbial ecology of real DWDS.
One of the roles of filamentous fungi in drinking water biofilms has consequently
been associated with providing building blocks and/or biotic support through their
hyphae for the establishment and colonization of surfaces by bacteria.
It was observed that intertwined fungal hyphae increased the cell number of this
bacterium, revealing a possible protective role of the fungi towards bacteria.
Despite this protective effect, it was observed that when inoculated at the same
time, bacteria could inhibit filamentous fungal spore germination and its
development into a biofilm in the first 24 h of interaction. If the possibility of
biofilm formation is provided for the fungi, or if a pre-established fungal biofilm is
present, then, an advantage may also be given to opportunistic bacteria to replicate
and proliferate in inter-kingdom biofilms inside DWDS [Flemming HC et.al].
Several new approaches are also being undertaken to help the removal of
pathogenic microorganisms from DWDS.

CONCLUDING REMARKS

Filamentous fungi are present in all environments, and water distribution systems
are no exception. Several studies have demonstrated the presence of these
microorganisms, including concerning pathogenic, toxigenic or/and allergenic
species.

The present work provides an updated overview of the occurrence of filamentous

fungi in DWDS from the past two decades. Emphasis was given to biofilm
formation along with the interaction with bacteria in fungal-bacterial biofilms.
Several gaps and drawbacks were detected from the start and some
recommendations arise from these needs. Detection and/or isolation of filamentous
fungi in DWDS should follow a uniform approach. Different methodologies,
including culturomics and culture-independent methods, should be used to
complement one another. Looking at community analysis, NGS techniques have a
fundamental role and are also of great importance to analyses interactions between
microorganisms. Even with some advances in the study of fungal-bacterial
interactions, much more work is needed to fully elucidate these interactions.

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With differences in the methodology being used and the microorganism being
studied, there is a complex variety of observable relationships. For this reason,
there is a necessity to further investigate the different interactions between fungi
and bacteria in DWDS and what factors are affecting such associations.
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