BASIC

MICROBIOLOGY
Dr V R Yamunadevi
QUESTION 1
⦿48 y old female patient pus sample

⦿Gram stain – few gram positive cocci in clusters

and few pus cells

⦿Empiric choice of antibiotics

 OBJECTIVE
⦿Gram-stain and morphology of various bacteria
⦿Commensal flora in the body and where select
organisms are pathogenic
⦿Various types of organisms and their clinical
significance
 CLASSIFICATION OF BACTERIA
⦿Based on grams
stain(differential
stain)

⮚ Gram positive
⮚ Gram negative
DIFFERENCES
SUBUNITS FOR CELL WALL CONSTRUCTION

N-acetylmuramic acid N-acetylglucosamine

Pentapeptide in nascent

D-ala-D-ala
 Second layer of cell wall
CELL WALL ASSEMBLY cross-linked to the lower
layer

Layer of cell wall

with cross links of 5
glycines (gray)

A subunit is added to
the growing chain

Transpeptidase (PBP) forms a 5-glycine bridge between

peptides
GRAM POSITIVE ORGANISMS AND THEIR
ARRANGEMENT
⦿COCCI
◼ Staphylococcus(clusters)

◼ Micrococcus(Tetrads)

◼ Streptococcus(pairs,
short chains, long chains)

◼Enterococcus( pairs,
short chains)
GRAM POSITIVES BACILLI
⦿ Rods
◼ Corynebacterium

◼ Clostridia

◼ Bacillus

◼ Listeria (CSF)

◼ Lactobacillus
GRAM POSITIVE BRANCHING
FILAMENTOUS BACTERIA-
Actinomycetes
🞆 Actinomyces

🞆 Nocardia (Modified acid

fast stain)

🞆 Streptomyces
⦿Rods
◼Mycoplasmas (cell wall
deficient ,very small no
action with beta lactams
so atypical)
 GRAM
POSITIVE
BUDDING
YEAST CELL-
CANDIDA
(FUNGUS)
 VERY HIGH SPECIFICITY FOR SOME COMMUNITY ACQUIRED
SYNDROMES
◼Urethral discharge- Gram negative
diplococci- N. gonorrheae

◼Well collected sputum- Gram

positive capsulated diplococci-
S. pneumoniae

◼Community acquired meningitis

CSF Gram stain

◼Useful to direct therapy especially

in positive blood culture specimens
ANAEROBIC ORGANISMS
⦿ GN Bacteria: Bacteroides, Prevotella,
Porphyromonas, Fusobacterium
⦿ GP Bacteria: Actinomyces, Propionibacterium,
Bifidobacterium, Eubacterium, lactobacillus,
Clostridium
 CLINICAL SIGNIFICANCE
⦿Persistence of infection despite adequate
therapy with an appropriate therapeutic
regimen
⦿Infections of specific body sites • Brain
abscess, • Endocarditis • Prosthetic devices
or graft infections • Bacteremia
⦿Long-term therapy needed
⦿Election of an active agent is critical for
disease management
 RESIDENT FLORA/ COMMENSAL

⦿Most areas of the body in contact with the

outside environment harbor resident microbes;
large intestine has the highest numbers of
bacteria.
⦿Internal organs and tissues and fluids are
microbe-free.
⦿Bacterial flora benefit host by preventing
overgrowth of harmful microbes – microbial
antagonism.

19
BURDEN OF COMMENSALS OR
NORMAL FLORA
NORMAL FLORA OF HUMAN
BACTERIA FUNGUS

Skin Micrococci Candida

Staphylococcus aureus (nares)
Staphylococcus epidermidis),
Corynebacterium, Propionibacterium acnes
Streptococci (alpha, gamma)

URT Staphylococci, Streptococci (such as Candida

Streptococcus agalactiae), Diphtheroid bacilli
spirochetes, Neisseria, Haemophilus,

LRT sterile
 BACTERIA FUNGUS

ORAL Staphylococci, Streptococci Candida

CAVITY Lactobacillus ,Actinomyces, Neisseria,
Fusobac, Haemophilus,
Veillonella,Treponema
GIT Upper intestine Candida
Lactobacilli and Enterococci
Lower intestine and colon
anaerobes - Bacteroides ,Clostridium
E.coli, Proteus, Enterobacter

URINARY CoNS, alpha streptococci ,Lactobacillus , Candida

TRACT Corynebacterium , Bacillus sp

GENITAL Streptococci ,Lactobacillus , Candida

TRACT Corynebacterium
RESIDENT FLORA OF GIT
MOST COMMON NORMAL
COMMENSALS/ FLORA
⦿CoNS (eg: S.epidermidis)

⦿Diphtheroids (Corynebacterium)

⦿Micrococcus

⦿Streptococcus viridans

⦿Candida sp
COLONISER VS INFECTION
⦿Not the normal flora ⦿Treat only if your
⦿Presence of patient clinical
microorganisms at condition correlates
levels that provoke with the culture
neither symptoms growth
nor immune response
but when
circumstances are
favourable multiply
and cause infection
CONTAMINANT
⦿Organisms found in the environment which could
have contaminated the specimen while collection,
transportation or processing in lab
⦿Most common contaminant Bacillus sp (GPB)
STAPHYLOCOCCUS
⦿Coagulase positive
– Staphylococcus aureus
⦿Coagulase negative(less virulent)
Staphylococcus epidermidis
Staphylococcus haemolyticus
Staphylococcus lugdunensis(except)
Staphylococcus saprophyticus
⦿Organised abscess
⦿40% of normal person have SA colonization
⦿Methicillin resistance conferred by SCC mec
cassettes
⦿Health care associated or Hospital associated
MRSA(HA-MRSA)
⦿Community Acquired MRSA(CA-MRSA)
PREDISPOSING FACTORS- MRSA
⦿Prosthetic devices & implants like plates
and catheters
⦿Chronic Infections and prolonged hospital
stay ,Burns and trauma
⦿Immunocompromised and Hormonal
changes and stress
⦿Common usage of towels and razors
⦿Poor personal hygiene due to over
crowding
 CONS
⦿80% of are methicillin resistant- plasmid
mediated mostly hospital acquired
⦿S. lugdunensis, a more virulent CONS.
Infections are treated similarly to those caused
by S. Aureus
⦿Most common resident flora on skin
⦿Important aspect of treatment of most CONS
infections is their ability to form bio-films on
biomaterials(adhesin) so important in infection
of prosthetic material
 CLINICAL SIGNIFICANCE
⦿ True bacteremia range from 10% to 25% when
coagulase-negative staphylococci are isolated
from blood cultures

⦿Fever or other signs of infection (e.g.,

leukocytosis or leukopenia, hypotension)

⦿ Multiple cultures
CLINICAL SIGNIFICANCE OF
PNEUMOCOCCUS
1.Normal inhabitant of human, become
pathogenic when host compromised.
2.Single most agent prevalent in pneumonia
and otitis media.
VIRIDANS GROUP
⦿ Resident in the mouth and upper respiratory tract.
⦿Imp. Species are: Str. mitis, Str. mutant, Str.
Salivarius ,Str. Sanguis.
⦿Following dental procedures, they may cause
transient bacteremia and get implanted on damage
or prosthetic valves or in congenital diseased heart.
STREPTOCOCCUS PYOGENES
⦿Blood cultures are positive in more than 50%
of invasive infection
⦿Swabs collected for diagnosis should be
immediately to lab for better growth
 ENTEROCOCCI
⦿Enterococcus faecalis and Enterococcus
faecium are the most clinically relevant
species
⦿Cephalosporins, Clindamycin, Cotrimoxazole
Aminoglycosides are not effective clinically.
 E. Coli
Klebsiella
Enterobacter
Citrobacter
Serratia
Enterobacteriaceae Proteus
Morganella

GNB
Pseudomonas
Acinetobacter
Non- Burkholderia
enterobacteriaceae Stenotrophomon
as
Lactose Fermentation

Pathogenic
bacteria
GRAM NEGATIVE COCCI
⦿Diplococci- Neisseria meningitidis
Neisseria gonorhoea

⦿Clusters –Moraxella catarhallis

 CHARACTERISTICS OF COMMUNITY
AND NOSOCOMIAL INFECTIONS
COMMUNITY ONSET HOSPITAL ONSET
ORGANISM Escherichia coli Klebsiella Spp &
others
TYPE OF ESBL CTX-M ( CTX-M15) SHV(SHV-2& SHV 5)
and TEM (TEM-26,
TEM-51)
TYPE OF Most often UTI, also Respiratory tract, intra
INFECTION bacteremia & abdominal &
gastroenteritis bloodstream
infections
TYPE OF Most isolates not clonally Most often clonally
EPIDEMIOLOGY related, although clusters related
have been described
 General Overview
• Opportunistic Pathogens of Humans
• Clinically Important Aerobic Gram-Negative
Bacilli Include:
• Aerobic non-fermenters: 10-15% of clinical
isolates
• Clinically important
✔ Pseudomonas aeruginosa; Burkholderia
cepacia; Stenotrophomonas maltophilia;
Acinetobacter baumannii; Moraxella
catarrhalis: Account for >75% of all
clinical isolates of aerobic non-
fermenters
⮚ Oxidative gram-negative bacilli, including
Pseudomonas spp., produce acid from
glucose or other carbohydrates only in the
presence of oxygen (nonfermenters).
⮚ Pseudomonas aeruginosa oxidizes but does
not ferment glucose.
Multi-Drug Resistant (MDR) A. baumannii are
among the most “problematic pathogens”
encountered by clinicians
PA AND ATB-
MAJOR THREAT : CARBAPENEM R
⦿ß-lactamases-all classes represented
◼Cephalosporinases,
◼class A ESBLs (PER),
◼OXA ESBLs (OXA-10, -14),
◼Carbapenemases (KPC and GES), MBLs

⦿Loss of permeability (porins and efflux)

WHY SPECIMEN COLLECTION
IS IMPORTANT?
 FUNDAMENTALS OF SPECIMEN
COLLECTION
⦿Cultures before antibiotics
⦿Ask for smears wherever app(flora, pus cells)
⦿Actual site of infection with no contamination
⦿Optimal time of collection( no 24hrs sample)
⦿Sufficient quantity
⦿Appropriate device(swabs in OT) & containers
⦿Labelled culture container
⦿Do not pour formalin for any micro cultures
⦿No indwelling catheters
 SPECIMEN COLLECTION AND TRANSPORT
Sample Quantity Time
Blood 10ml in each bottle <2hrs

Pleural fluid , Ascetic fluid 25ml -50ml the yield is more sample– <15min
Blood culture bottle
CSF 5-10ml-Mycobacteria, Bacteria 3ml <15min

Tissue scrapings collect material and place in sterile tube. <15min

Saline may be added to avoid drying

Sputum 5-10ml and not saliva(Bartlett score) <1hr

AFB culture
Pus aspirated and sent in a sterile test tube <1hr

Deep ulcer with necrosis the infected material from the side wall <1hr
is aspirated with a sterile needle
Formed Feces avoid for C.difficle <1hr
Hanging drop – V.cholerae
Urine & urine routine 5-10ml <1hr
 MICROBIOLOGICAL DIAGNOSIS

Microscopy

Culture
Direct DNA
(ID+Susc)

Subunit
RNA
detection

Microbiological
investigations Antigen
detection

Antibody
detection
Indirect Serology

Biomarkers
 BLOOD CULTURES
⦿Two sets

Chlorhexidine- 30sec
Tincture iodine- 2 min
BARTLETT SCORE TO ASSESS
QUALITY OF SPUTUM
No. of neutrophils per 10 X low power field Grade
<10 0
10-25 +1
>25 +2

No. Of epithelial cells per 10 X low power field

10-25 -1
>25 -2
A final score of 0 or less indicates contamination with
saliva, so repeat specimen
COLONY FORMING
UNITS(CFU/ML)
⦿Bronchial wash/BAL/endotracheal aspirate-
to assess the severity or true infection
⦿Urine sample>10⁵ one organism is significant
⦿Growth of 2 or more different bacteria or
polymicrobial growth –contamination
⦿Pyuria -≥10 white blood cells/high-power field is
indicative of pyuria
⦿Always correlate clinically
ANTIMICROBIAL SUSCEPTIBILITY
TESTING
⦿Minimal inhibitory
concentration (MIC) :
The lowest concentration
of drug that inhibits
growth of the organism

⦿Specific for each drug-

bug combination

⦿Lower the MIC, more

susceptible is the
organism.
E-TEST
E test also known as the epsilometer
test is an ‘exponential gradient’
testing methodology
Plastic strips 5X50mm with a predefined
gradient of
◼ One antibiotic

One strip per antibiotic

Wide range of antibiotics
Easy to use
Following incubation , a
symmetrical inhibition ellipse is
produced.
The intersection of the inhibitory
zone edge and the calibrated
carrier strip indicates the MIC value.
AVAILABLE SYSTEMS
⦿MicroScan WalkAway

⦿Biomerieux Vitek 2

⦿BD Phoenix
 REFERENCES
⦿Konemann’s color Atlas and Textbook of Diagnostic
Microbiology 7th edition.
⦿Mandell , Douglas And Bennett. Principles and Practices
of Infectious Diseases. 9th Edition
⦿CLSI guidelines.Susceptibility tests for ESBL producing
Enterobacteriaceae.M100-S23.51-55.