Chapter3.pptx

CHAPTER 3
SYSTEMATIC
BACTERIOLOGY
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Part I
General Microbiology
UNIVERSITIES PRESS PVT LTD

Dr Sonal Saxena, MD
Director Professor and Head of the Department of Microbiology
Maulana Azad Medical College,
New Delhi
and
Dr Amala A Andrews, MD
Maulana Azad Medical College,
New Delhi

CLASSIFICATION, NOMENCLATURE AND TAXONOMY OF
BACTERIA
Enable easy identification of microbes
Bergey’s manual — systematic classification
Carolus Linnaeus introduced a classification system consisting of domain, kingdom, phylum, class,
order, family, genus and species
Subspecies and serotypes are based on antigens
Clone — derived from a single cell; almost all are identical
Colony — represents one clone
Strain — derived from a single source
Kingdom → Division → Class → Order → Family → Tribe → Genus → Spe

CLASSIFICATION
1) Phylogenetic
Represented as a branching tree
One characteristic—for division at each branch or level
2) Adansonian classification
Numerical taxonomy
All characteristics are taken into account
Several characteristics are compared
3) Molecular or genetic classification
Degree of genetic relatedness
Based on the genes, DNA relatedness
G:C ratio (guanine:cytosine)—Constant within the same
species
Used for classifying viruses
4) Intraspecies classification
Beyond species level
Diagnostic and epidemiological
Classified as:
 Biotypes: Biochemical properties
 Serotypes: Antigenic features
 Phage types: Bacteriophage susceptibility
 Colicin types: Production of bacteriocins

MOLECULAR
EPIDEMIOLOGY
Intraspecies typing by newer techniques has led to the evolution of molecular
epidemiology. These methods can be largely classified as follows:
Phenotypic (study of expressed characteristics):
1) Electrophoretic typing
2) Immunoblotting
Genotypic (direct analysis of genes, chromosomal and extra-chromosomal
DNA)
1) Plasmid profile analysis
2) Restriction endonuclease analysis
3) Southern blotting
4) Polymerase chain reaction
5) Nucleotide sequence analysis

TYPE
CULTURES
Contain representatives of all species
Maintained by International Reference Labs
Made available for study and comparison
ATCC (American Type Culture Collection)
NCTC (National Collection of Type Cultures [Public Health
England])

GENERAL CONCEPTS OF PATHOGENESIS AND DISEASE
SPECTRUM
Bacterial infections can be:
Acute, life-threatening—bacterial meningitis, anthrax
Chronic, indolent—tuberculosis
Mild infection—streptococcal sore throat
Primary/secondary infections
Pathogenesis
Chain of events that lead to infection
Interaction between source, host and pathogen

PATHOGENESIS
Fig. 3.1 Diagram showing the entry,
invasion and damage to host tissue by
pathogenic bacteria

PATHOGENESIS
Exposure to the pathogen (bacteria) and route of entry
Bacteria enter through an appropriate route
Table. 3.1 Routes of entry of some disease-causing bacteria
Encounter host
phagocytes
Exhibit tissue
tropism
Multiplication
Production of toxins
and enzymes
Cell destruction Tissue damage
Prevention of
host defense
Disease
manifestation
Clinical
features

PATHOGENESIS
Incubation period: Time between
exposure (entry of pathogen) and the
first manifestation of symptoms (and
clinical signs)
Fig. 3.2 Source-host-pathogen encounter

OVERVIEW OF
IMPORTANT BACTERIA
ASSOCIATED WITH
HUMAN HEALTH AND
DISEASE
Gram-positive cocci: Staphylococcus,
Streptococcus, Pneumococcus, Enterococcus
Gram-positive bacilli: Bacillus,
Corynebacterium, Actinomycetes, Clostridium
Gram-negative cocci: Neisseria, Moraxella
Gram-negative bacilli: Enterobacterales,
vibrio, non-fermenters
Miscellaneous bacteria: Mycobacteria,
spirochetes, Mycoplasma, Chlamydia, Rickettsia

GRAM-POSITIVE
COCCI
1) Staphylococci
Spherical, 1 µm diameter
Arrangement: Grape-like clusters
Grow in ordinary culture media
Produce potent toxins, enzymes
and other metabolites
Develop resistance to antibiotics
Fig. 3.3 Gram-positive cocci in clusters:
Staphylococcus aureus (Source: Dept of
Microbiology, Pondicherry Institute of
Medical Sciences [PIMS], Puducherry)

GRAM-
POSITIVE
COCCI
Common infections caused by Staphylococcus aureus
 Localised suppurative lesions: Skin and mucosa
 Disseminated: Bbloodstream infections, osteomyelitis,
meningitis, pericarditis pneumonia
Coagulase-negative staphylococci (CoNS)
 S. epidermidis, S. lugdenunsis, S. haemolyticus, S. hominis
 Commensals in the body
 S. epidermidis: causes most common CoNS infection in
humans
 Biofilm formation: Important in pathogenesis
 Causes healthcare-associated infections in
immunocompromised hosts

GRAM-
POSITIVE
COCCI
2) Micrococci
Gram-positive cocci in tetrads, pairs or irregular clusters
Rarely cause infection in healthy hosts
3) Streptococci
Gram-positive cocci in chains or pairs
Part of normal flora
Streptococcus pyogenes: Spreading pyogenic infections
and non-suppurative lesions, acute rheumatic fever and
glomerulonephritis

GRAM-
POSITIVE COCCI
Classification of streptococci
Fig. 3.4 Classification of streptococci

GRAM-POSITIVE COCCI
Classification of streptococci
1. Based on oxygen requirement
 Facultative anaerobes
 Obligate anaerobes
2. Based on hemolysis on blood agar
Alpha-hemolytic: Greenish discolouration around colonies (partial
hemolysis), e.g., viridans group of streptococci, pneumococci
Beta-hemolytic: Clear zone of complete hemolysis, e.g., Streptococcus
haemolyticus
Non-hemolytic: Fecal streptococci (Enterococcus)
3. Based on carbohydLancefield’s groups (Rebecca Lancefield): 20
groups—A-H and K-V.; S. pyogenes (under group A) is further classified into
80 M-types by Griffith typing based on M proteins on the cell surface
Fig. 3.5 Hemolysis on blood agar (Source: Public
Health Image Library, ID 10861 / CDC / Dr. Richard
Facklam)

GRAM-POSITIVE
COCCI
Infection-causing streptococci
• Streptococcus pyogenes
• Streptococcus agalactiae
These are the medically important
members of this group.
UNIVERSITIES PRESS PVT LTD Table. 3.2 Medically important streptococci and their characteristics

GRAM-
POSITIVE
COCCI
4) Pneumococci (S. pneumoniae)
Gram-positive diplococci; capsulated
Flame-shaped/lanceolate appearance
India ink stain: Capsule visualisation
Infections
Meningitis
Lobar pneumonia
Bacteremia

GRAM-POSITIVE COCCI
5) Enterococci
Enterococcus group comprises E. faecalis, E. faecium, E.
durans
Oval diplococci (cocci at an angle to each other)
Usually non-hemolytic (can be alpha- or beta-hemolytic)
Infections
Urinary tract infection
Wound infection
Endocarditis
Infections of biliary tract, septicemia, etc.
Fig. 3.6 Enterococci—electron micrograph showing oval cells
arranged in pairs (Source: PHIL, ID 266/ Janice Haney Carr /
CDC)

ANAEROBIC
GRAM-
POSITIVE COCCI
Peptostreptococci
Normal flora of vagina, intestines and mouth
Can grow well with 10% CO2 in aerobic atmosphere
Infections
Puerperal sepsis (Peptostreptococcus anaerobius)
Other genital infections
Wound infections, UTI
Osteomyelitis
Abscesses of internal organs (P. magnus)

GRAM-POSITIVE RODS
1) Corynebacteria
Gram-positive rods
Non-sporing, non-acid fast, non-
motile
Arrangement: characteristic
pairs/palisades
Chinese letter or cuneiform
arrangement
Club-shaped swellings present
Possess metachromatic granules
(Babes-Ernst granules or
polymetaphosphate granules)
Infections caused by corynebacteria
Corynebacterium diphtheriae —
Diphtheria
Other corynebacteria
Non-lipophilic corynebacteria: C.
pseudotuberculosis, C. ulcerans
Diphtheria-like lesions
Lipophilic corynebacteria:
C. jeikeium, C. urealyticum
Infective endocarditis
Affect immunocompromised hosts
Fig. 3.7 C. diphtheriae showing cuneiform
arrangement (V and L shapes) (Source: Dept. of
Microbiology, PIMS)

GRAM-POSITIVE RODS
Commensal corynebacteria
In throat, skin, conjunctiva, etc.
Similar to diphtheria bacilli
Stain more uniformly than C. diphtheriae
Palisade arrangement
Cause infection in immunocompromised patients
Diphtheroids

GRAM-POSITIVE RODS
2) Bacillus
Gram-positive bacilli with spore
Bacillus anthracis
 Major pathogen
 Human infection by contact/inhalation of spores from
animals/their products
 Aerobic, non-motile
 Bamboo stick/boxcar appearance (arranged in chains)
 Infection: Anthrax
Fig. 3.8 Bamboo stick appearance in Gram
stain (Source: Dept. of Microbiology, PIMS)

Spores of Bacillus
Central/sub-terminal and elliptical/oval
Non-bulging
Optimum temperature for sporulation: 25–30°C
Highly resistant to physical and chemical agents
Destroyed by glutaraldehyde, formaldehyde, hydrogen peroxide and beta-propiolactone at
higher concentrations
Formed in culture/in soil, but not in living body
GRAM-POSITIVE RODS
Image courtesy: Doc. RNDr. Josef Reischig, CSc., CC BY-SA 3.0 <https://creativecommons.org/licenses/by-sa/3.0>, via Wikimedia Commons

GRAM-POSITIVE RODS
Bacillus cereus
Seen in soil, vegetables, food items (milk,
cereals, meat, etc.)
Infection: food poisoning associated with
contaminated cooked food (e.g., Chinese fried
rice)
Bacillus thuringiensis
In soil
Biological pesticide and larvicide
Used to control mosquitoes and other
insects
Source of genes in making pest-resistant
food crops
Bacillus species as sterilisation indicators
(controls)
Used as biological indicators in sterilisation
controls
Spores of
B. atrophaeus (subtilis var niger)
Geobacillus stearothermophilus

GRAM-POSITIVE RODS
3) Actinomycetes
Slender, gram-positive bacteria (cell wall has muramic acid)
Non-motile, non-sporing, non-capsulated
Form branching filaments that break up into bacillary and coccoid forms
Resemblance to fungi
Mostly free-living in soil
Classification
Anaerobic/microaerophilic: Actinomyces, Bifidobacterium, Arachnia, Rothia
Aerobic: Nocardia, Actinomadura, Dermatophilus, Streptomyces

GRAM-POSITIVE RODS
Infections by Actinomycetes
Chronic, granulomatous infection with indurated swellings, multiple sinuses and sulphur
granules
Actinomycosis
 Endogenous infection
 Caused by Actinomyces israelii, A. naselundii, etc.
Nocardia
 Cause TB-like lesions of lungs in immunocompromised patients
 They are acid-fast and resist decolourisation with 1% H2SO4

GRAM-POSITIVE RODS
4) Clostridia
Gram-positive anaerobic bacteria with spores
Motile (with peritrichate flagella) except C. perfringens and C. tetani type VI
Stately motility
Non-capsulated except C. perfringens and C. butyricum
Classification
Molecular classification: based on 16SrRNA sequences
Phenotypic classification: based on morphology, culture and biochemicals

GRAM-POSITIVE RODS Table. 3.3 Morphological and biochemical classification of
clostridia

GRAM-POSITIVE RODS
Spores of clostridia: Wider than bacillary bodies
Arrangement of spores:
Central (equatorial)—C. bifermentans
 Spindle-shaped bacillus
Subterminal—C. perfringens
 Club-shaped bacillus
Oval and terminal—C. tertium
 Tennis racket-shaped bacillus
Spherical and terminal—C. tetani
 Drumstick appearance
Fig. 3.9 Sporulating clostridia (Source: Dept. of
Microbiology, PIMS)

VIABILITY OF
CLOSTRIDIAL
SPORES
Table. 3.4 Viability of spores of
clostridia

GRAM-
NEAGTIVE
COCCI
Two important organisms:
1) Neisseria
Some species are pathogenic; others are commensals
2) Moraxella
Generally colonisers; cause pneumonia occasionally
Diplococci (pairs of cocci)
Aerobic, non-sporulating, non-motile
Oxidase-positive

GRAM-NEAGTIVE
COCCI
1) Neisseria
Two important pathogens: Neisseria meningitidis, Neisseria gonorrhoeae
Others: commensals of the mouth and upper respiratory tract—N. lactamica, N. flavescens, N. subflava
Infections: Meningitis by N. meningitidis; gonorrhea by N. gonorrhoeae
Table. 3.5 Differential characteristics of neisseriae

GRAM-NEAGTIVE COCCI
2) Moraxella
Diplococci/coccobacilli
Catalase- and oxidase-positive
Moraxella catarrhalis: Opportunistic pathogen
Other species: Commensals (M. lactamica, M. bovis, etc.)
Infections: M. catarrhalis causes otitis media in children and lower respiratory tract infection in
immunocompromised and elderly individuals

ANAEROBIC
GRAM-
NEGATIVE
COCCI
Veillonella
Gram-negative cocci — pairs/chains/groups
Normal flora of mouth, intestinal and genital tracts

ANEROBIC GRAM-
NEGATIVE BACILLI—
MEDICALLY
IMPORTANT
Bacteroides
Porphyromonas
Prevotella
Fusobacterium
Leptotrichia

GRAM-NEGATIVE RODS
Enterobacteriaceae
Large group of biochemically and antigenically varying organisms
Non-sporing, non-acid fast, gram-negative rods
Mostly gut commensals
Classification systems
1) Bergey’s classification
2) Edwards–Ewing classification
3) Kauffmann and White classification (only for Salmonella)
Species further classified into biotypes, serotypes and bacteriophage types

GRAM-NEGATIVE RODS
Table. 3.6 Enterobacteriaceae—major genera associated with human infections

GRAM-
NEGATIVE
RODS
1) Escherichia coli (Chapters 24 and 27)
Morphology
Gram-negative, straight rods
No specific arrangement
Gut commensals
Infections
Urinary tract infections
Diarrhea
Bloodstream infections

GRAM-NEGATIVE RODS
2) Shigella (Chapter 16)
Short, gram-negative rods
Non-motile, non-sporing, non-capsulated
Classification (based on biochemical and
serological characters)
 S. dysentriae (subgroup A): has 12
serotypes
 S. flexneri (subgroup B): 6 serotypes
 S. boydii (subgroup C): 18 serotypes
 S. sonnei (subgroup D): single serotype
Infections
 Bacillary dysentery
 S. dysentriae associated with hemolytic
uremic syndrome

GRAM-NEGATIVE RODS
3) Salmonella (Chapters 21 and 24)
Gram-negative motile rods (except S. gallinarum and S. pullorum)
More than 2,000 serotypes
Most important: Salmonella Typhi
Infections
Typhoidal: Enteric fever (typhoid)—caused by bacilli which are exclusively human parasites
Non-typhoidal: Gastroenteritis, septicemia, localised infections—caused by bacilli that are
primarily animal parasites

GRAM-NEGATIVE RODS
Classification and nomenclature of Salmonella
By DNA hybridisation studies
1) Species enterica
 Has 6 subspecies
 Salmonella Typhi belongs to subspecies enterica (Salmonella, species enterica, subspecies
enterica, serovar Typhi)
2) Species bongori
 Has more than 2,500 serovars or serotypes

GRAM-NEGATIVE RODS
4) Yersinia enterocolitica
Two species associated with human infection
 Y. enterocolitica
 Y. pseudotuberculosis
Infections
 Y. enterocolitica: Acute bacterial enteric disease
 Y. enterocolitica and Y. pseudotuberculosis: Acute mesenteric lymphadenitis (mimics
appendicitis)

GRAM-NEGATIVE RODS
5) Klebsiella pneumoniae
Short, plump, straight, gram-negative rods
Capsulated and non-motile
Mucopolysaccharide capsule mucoid
colonies in solid media
Commensals in gut and saprophytes in soil
and water
3 species based on biochemical reactions
 K. pneumoniae
 K. ozaenae
 K. rhinoscleromatis
Infections
Community-acquired pneumonia
 Healthcare-associated infections

GRAM-NEGATIVE RODS
6) Enterobacter cloacae
Seen normally in feces, sewage, soil and water
Resistant to several classes of antibiotics
Infections
Urinary tract infections
Healthcare-associated infections

GRAM-NEGATIVE RODS
7) Serratia marcescens
Forms prodigiosin, a pink/magenta non-diffusible pigment
Only one species is medically important: S. marcescens
Multidrug resistance seen in hospital strains
Infections
Healthcare-associated infections
Meningitis, endocarditis
Septicemia, peritonitis, etc. Fig. 3.10 Non-diffusible red pigment-
producing Serratia spp. on blood agar
(Source: PHIL, ID 10544/ Dr. Negut/ CDC)

GRAM-NEGATIVE RODS
8) Proteeae
Tribe Proteeae has 3 genera:
i. Proteus
ii. Morganella
iii. Providencia
Normal intestinal commensals and opportunistic pathogens
Produce urease (except some Providencia strains)
Enzyme phenylalanine deaminase present

GRAM-
NEGATIVE
RODS
Proteus
Gram-negative, non-capsulated
Pleomorphic and motile rods
‘Fishy/seminal’ odour produced in cultures
Important species are:
 P. mirabilis: Urinary and nosocomial pathogen
 P. vulgaris: Less common
 P. hauseri: Recently identified
 P. vulgaris and P. mirabilis produce abundant hydrogen
sulphide

GRAM-NEGATIVE RODS
Swarming of Proteus
By P. vulgaris and P. mirabilis
Due to presence of flagella
Methods of inhibition of swarming
 Agar concentration 6%
 Incorporating chloral hydrate (1:500)
 MacConkey agar (due to presence of bile)
Infections: Urinary tract and hospital-acquired infections
Fig. 3.11 Swarming of Proteus on blood
agar (Source: Dept. of Microbiology,
PIMS)

GRAM-NEGATIVE RODS
9) Vibrio cholerae
Gram-negative, curved, comma-shaped bacilli
Actively motile by a polar flagellum
Vibrios: In marine environments and surface waters
Vibrio cholerae: Causes cholera
Stained films of mucous flakes from cholera cases: ‘Fish in a
stream’ appearance
Darting motility of V. cholerae: Resembles ‘swarm of gnats’
Fig. 3.12 Vibrio cholerae- comma-shaped
gram- negativerods (Source: Dept. of
Microbiology, PIMS)

GRAM-NEGATIVE RODS
Resistance of cholera vibrios
Susceptible to heat, drying and acids
Resist high alkalinity
Vibrios are susceptible to common disinfectants
Infections
V. cholerae causes cholera
 As isolated cases and epidemics

GRAM-
NEGATIVE
RODS
Halophilic vibrios
High requirement of sodium chloride
Natural habitat: seawater and marine life
Human disease caused by
 V. parahaemolyticus
 V. alginolyticus
 V. vulnificus
Infections
V. parahaemolyticus: Food poisoning and gastroenteritis

NON-
FERMENTING
GRAM-
NEGATIVE
RODS
Group of unrelated bacteria
Acquired resistance to wide spectrum of drugs
Cause healthcare-associated infections
Seen in environment, soil and water
Contaminate and survive in antiseptics and disinfectant
solutions
Pseudomonas and Burkholderia: Bacilli
Acinetobacter: Coccobacillus

NON-FERMENTING GRAM-NEGATIVE RODS
1) Pseudomonads
Saprohytic
Colonise hospital environment, equipment and moist areas
Infections
 Infections in immunocompromised patients
Pseudomonas
Slender, gram-negative bacilli
Actively motile by polar flagellum

Pathogenic factors of Pseudomonas
Pigment production (pyocyanin)
Multidrug resistance
Multi-disinfectant resistance
Survival in wide temperature range
Biofilm production
Infections:
Healthcare-associated infections like
Catheter-associated infections
Ventilator-associated pneumonia

2) Burkholderia
Burkholderia cepacia
Can grow in common disinfectants
Inherently resistant to most antibiotics
Infections
Fatal necrotising pneumonia in cystic fibrosis patients
Hospital-acquired urinary, respiratory and wound infections
Peritonitis, endocarditis and septicemia

Burkholderia mallei
Non-motile
Infections: Glanders (occupational infection)
Burkholderia pseudomallei
Resembles B. mallei, but motile
Safety pin appearance in stained smears
Infections: Melioidosis (glanders-like disease); also known as Vietnam time-bomb disease

3) Acinetobacter
Gram-negative coccobacilli
Aerobic, non-motile, oxidase-negative
Most common: Acinetobacter baumannii
Multidrug-resistant
Infections
 Biofilm based catheter-associated infections
 Bloodstream infections, ventilator-associated pneumonia

NON-FERMENTING
GRAM-NEGATIVE RODS
4) Haemophilus
Oxidase-positive, gram-negative coccobacilli
Fastidious — needs factor X (hemin) and factor V (NAD) for growth
H. influenzae: Small, pleomorphic and capsulated
Satellitism: Seen when H. influenzae is grown near S. aureus
 Shows its dependance on factor V (in high concentrations near
staphylococcal growth)
Infections: Pneumonia and meningitis in children
Fig. 3.14 H. influenzae: (a) on blood agar showing satellitism
around S. aureus streaks (Source: Dept. of Microbiology,
PIMS) and (b) demonstration of the requirement of XV
factor

Other Haemophilus species
Haemophilus ducreyi
Short, ovoid, gram-negative bacillus
In end-to-end pairs or short chains
Smear: ‘school of fish’ or ‘railroad track’ appearance
Infection:
Chancroid (soft chancre) — Sexually transmitted infection

5) Brucella
Small, gram-negative coccobacilli
Primarily infect goats, sheep, cattle, buffaloes, pigs, etc.
Infection to humans by contact with infected animals or their
products (zoonotic infection)
B. melitensis, B. abortus, B. suis
Infection
Human brucellosis or undulant fever
Fig. 3.15 Brucella—gram-negative
coccobacilli (Source: Dept of
Microbiology, PIMS)

6) Bordetella
B. pertussis, B. parapertusis, B. bronchiseptica
B. pertussis
Small, gram-negative coccobacillus
Capsulated, non-motile, non-sporing
Bipolar metachromatic granules present
Infection: Pertussis or whooping cough
B. parapertusis
Whooping cough (infrequent cause)

ANAEROBIC GRAM-NEGATIVE BACILLI
1) Bacteroides
Non-motile, strict anaerobes
Slender rods/branching forms/coccobacilli
Virulence factor: Capsular polysaccharide
Normal flora of intestinal, respiratory and female genital tracts
Important species
 B. fragilis
 B. melaninogenicus

ANAEROBIC GRAM-NEGATIVE BACILLI
2) Fusobacterium
Long, thin or spindle-shaped bacilli
F. nucleatum: Normal flora of mouth
F. necrophorum: Produces exotoxins
Infections
Deep-seated infections (liver abscess) and brain abscess, pleuropulmonary sepsis, oral
infections
Leptotrichia has a single species — L. buccalis
 Old name: Fusobacterium fusiforme
 Causes Vincent’s angina

MISCELLANEOUS
BACTERIA
1) Mycobacteria
Resist decolourisation with dilute mineral acids
 Due to mycolic acid in the cell wall
 Known as acid-fast bacilli (AFB)
Slow-growing, aerobic, non-motile, non-capsulated
Has 3 groups:
Obligate parasites
Opportunistic pathogens
 Saprophytes
Virulence factors: Cord factor, lipoarabinomannan

MISCELLANEOUS BACTERIA
Table. 3.7 Some common mycobacteria and their habitats
Mycobacteria
• Mycobacterium tuberculosis and Mycobacterium bovis cause tuberculosis
• Saprophytic mycobacteria (e.g., M. phlei) do not cause human infections

Classification of mycobacteria
Based on their ability to produce pigments when grown in the presence or absence of light
Classified into 4 groups
Group I — photochromogens
Pigment produced in light; but not in dark
Yellow-orange pigment
M. kansasii, M. marinum, M. simiae
Can cause human infections

Mycobacteria
Group II — Scotochromogens
Form pigmented colonies (yellow-orange-red)
even in the dark
Distributed in the environment
M. scrofulaceum: Causes scrofula (cervical
adenitis)
M. gordonae (tap water scotochromogen):
Rarely causes lung disease
Group III — Non-photochromogens
No pigment production
M. avium, M. intracellulare, M. xenopi, M.
ulcerans
Opportunistic infection in
immunocompromised individuals

MISCELLANEOUS
BACTERIA
Mycobacteria
Group IV — rapid growers
Heterogenous group with rapid growth
Colonies appear within 7 days of incubation
Mycobacterium tuberculosis
Long, filamentous, straight or slightly curved rod
Mycobacterium bovis
Straighter, shorter and stouter

Mycobacterium leprae
Straight/slightly curved rods
Less acid-fast than tubercle bacillus (5% sulphuric acid)
Globi: Bacilli bound together by a lipid-like substance (glia)
Cigar bundle appearance: Parallel rows of bacilli in the globi
Globi in undifferentiated histiocytes: Virchow’s lepra cells/foamy cells
Cannot be cultivated in bacterial media/tissue culture
Generation time: 12–13 days on average
Causes leprosy

2) Spirochetes
Elongated, motile, flexible bacteria, twisted spirally along the long axis
Endoflagella present (between outer cell membrane and cell wall)
Size varies
Mostly free-living saprophytes
 Some are obligate parasites
Human pathogens belongs to three genera
 Treponema
 Borrelia
 Leptospira Fig. 3.16 Schematic representation of comparative
morphology of different spirochetes

Table. 3.8 Characteristics of important human spirochetes

MISCELLANEOUS
BACTERIA
3) Mycoplasma, Ureaplasma and Chlamydia/Chlamydophila
Mycoplasma and Ureaplasma: Smallest free-living bacteria
grown in cell-free medium
Infections
Pneumonia by M. pneumoniae
Urogenital infections by Ureaplasma urealyticum
 M. hominis
 M. genitalium

Mycoplasma
No cell wall
No fixed shape/size
Can pass through filters; resistant to penicillin and cephalosporins
Are gram-negative; better stained by Giemsa stain
Classification
i. Genus Mycoplasma
ii. Genus Ureaplasma
Infections: Mycoplasma — atypical pneumonia and non-gonococcal urethritis along with
Ureaplasma

Chlamydia
Obligate intracellular
Predilection for squamous epithelial cells and macrophages of respiratory tract
Cell wall similar to that of gram-negative bacteria; but do not have peptidoglycan
Cannot be cultured in artificial media
Classification
i. Genus Chlamydia: Has single species — Chlamydia trachomatis
ii. Genus Chlamydophila: Has two species — Chlamydophila pneumoniae, Chlamydophila psittaci

Chlamydia
C. trachomatis: sensitive to sulphonamides
C. psittaci: resistant to sulphonamides
Chlamydiae have 2 forms
i. Elementary body: extracellular, infective form
ii. Reticulate body: intracellular growing and replicating form
Infections
C. trachomatis: Trachoma and non-gonococcal urethritis
C. psittaci: Pneumonia and generalised infection
C. pneumoniae: Atypical pneumonia

Table. 3.9 Mycoplasmas responsible for human infections or colonisation: diseases, species and site of colonisation
Table. 3.10 some common Rickettsial infections in humans

MISCELLANEOUS
BACTERIA
Ricketssiae
Diverse organisms with common features
Intracellular growth
Transmission by blood-sucking arthropod vectors (lice, fleas,
ticks, mites)
Small, gram-negative bacilli
Similar to viruses, but are bacteria
Cell wall has peptidoglycan
Metabolic enzymes; has both DNA and RNA
Susceptible to anti-bacterials

Classification of Rickettsiae
Rickettsiaceae: Rickettsia and Orientia
Anaplasmatacea: Ehrlichia, Wolbachia, Anaplasma and Neorickettsia
Other organisms with similar epidemiological and clinical features
Bartonella
Coxiella, Rickettsiella, Piscrickettsia
Eperythrozoon

OTHER DISEASE-CAUSING BACTERIA OF SIGNIFICANCE IN HUMANS
•Yersinia, Pasteurella, Francisella
•Alcaligens, Flavobacterium
•Campylobacter, Helicobacter
•Legionella, Gardnerella, Listeria
•Spirillum, Streptobacillus, Klebsiella granulomatis
They belong to different families
Cause various types of diseases

Yersinia pestis
Short, plump, gram-negative bacillus
Giemsa/methylene blue stain—bipolar
staining (safety pin appearance)
Non-motile, non-sporing
Pleomorphic; slime layer
Infection: Plague
Fig. 3.17 (a) Y. pestis in a blood smear exhibiting a safety pin
appearance (bipolar staining) and (b) Y. pestis grown on chocolate
agar (Source: PHIL, IDs 14555 and 12475/ CDC)

NORMAL
FLORA OF
THE HUMAN
BODY
Benefits of host-bacterial association
Host gets some essential nutrients (vit K—gut)
Aids metabolism of indigestible compounds
Provides defence against colonisation of
pathogens
Aids host immunity
Helps in prevention of inflammatory disease
during colonisation, but some act as
opportunistic pathogens

NORMAL FLORA OF THE HUMAN BODY
Human skin colonised by organisms in the environment
Natural flora depends on the area of the body
Face, neck, hands, buttocks: Streptococci, staphylococci, Candida
Nasopharynx: Colonisation within 2–3 days of birth
Mouth: Anaerobes and Candida
 At birth: Micrococci, streptococci
 In 2–5 days, replaced by bacteria from mother and attendant
 Within 12 hr of birth, alpha-streptococci found in upper respiratory tract
Gut flora: Anaerobes (Bacteroides, Clostridium, etc.) and coliform bacteria

NORMAL
FLORA OF THE
HUMAN
BODY
Vaginal flora: Depends on pH and enzyme content
 At puberty: E. coli, Doderlein’s bacilli, yeast
 Pregnancy: S. epidermidis, Doderlein’s bacilli, yeast
 Includes G. vaginalis, Bacteroides species, Candida, alpha-
streptococci
Probiotics: Live microorganisms in normal human body.
 Inhibit pathogens in gut
 Have effects on mucosal barrier function and interaction
with antigen-presenting dendritic cells
 Used in antibiotic-associated diarrhea
 Most common: Bifidobacterium, Lactobacillus

NORMAL
FLORA OF THE
HUMAN BODY
Fig. 3.18 Longitudinal
distribution, frequency of
concurrence and densities of
bacteria making up the normal
flora of the human
gastrointestinal tract

NORMAL FLORA AND THE IMMUNE SYSTEM
The normal flora provides minimum stimulation for the immune system
Implication of alteration of normal bacterial flora
Factors causing alteration of flora
 Prolonged antibiotic therapy, immunosuppression/immunodeficiency
 pH alteration
 Increased virulence of commensals
Pseudomembranous colitis
 Due to elimination of normal flora by indiscriminate antibiotic usage
 Clostridium difficile multiplies
 Results in diarrhea

THERAPEUTIC USE OF GUT
FLORA
Fecal flora for treating chronic GIT ailments
Fecal microbiota transplantation
Gut bacteria from healthy donor to patient via colonoscope
Bacteria colonise the gut
Treatment of irritable bowel syndrome
Bob Blaylock, CC BY-SA 3.0 <https://creativecommons.org/licenses/by-
sa/3.0>, via Wikimedia Commons

DIAGNOSIS OF BACTERIAL INFECTIONS
1. Clinical diagnosis
2. Laboratory investigations
i. Pre-test process (pre-analytical phase): Specimen collection and transport
ii. Testing (analytical phase)
iii. Post-test process (post-analytical phase): Reporting to clinician and archiving of results
Factors for correct identification of bacteria are
 Appropriate clinical specimen at right time from correct site
 Documentation of test result and communicating
 Application of test results during treatment

SPECIMEN COLLECTION AND TRANSPORT (PRE-TEST)
Precautions for sample collection
Aseptic collection of adequate sample
Sample collected in a sterile container
Adequate PPE to be worn by the person collecting the sample
Specimen to be transported without delay in tightly closed containers
If delay occurs, store sample at an appropriate temperature for a specified amount of time

SPECIMEN COLLECTION AND
TRANSPORT (PRE-TEST)
Table. 3.11 Sites of infection and corresponding samples
for microbiological tests

PROCESSING OF SAMPLES IN THE LABORATORY (TEST
PROCEDURE)
Rapid processing within the biosafety cabinets
Steps in isolation and identification of bacteria
 Gross examination of specimen
 Microscopy: Gram stain, acid-fast staining, fluorochrome staining
 Culture: Specific medium, overnight incubation at 37°C
Common tests used to identify gram-negative bacteria:
 Hanging drop test for motility
 Breakdown of carbohydrates

PROCESSING OF SAMPLES IN THE LABORATORY (TEST
PROCEDURE)
 Tests for the production of gas and hydrogen sulphide
 Citrate utilisation, production of enzymes like oxidase and urease
 Serotyping with specific antisera
Tests for gram-positive cocci
 Coagulase test
 Hippurate hydrolysis
 Bacitracin susceptibility

PROCESSING OF
SAMPLES IN THE
LABORATORY (TEST
PROCEDURE)
Antibiotic susceptibility test (AST): Kirby–Bauer disc diffusion method,
automated VITEK/MicroScanSystems
Rapid diagnostic method: Automated culture and identification systems
(MALDI-ToF, VITEK, MicroScan, etc.)
Immunological tests for rapid antigen detection: Latex agglutination test —
CSF in acute pyogenic meningitis
Molecular methods: PCR and its modifications
Serological tests for the detection of IgM and IgG
Detection of IgG: The presence of IgG denotes exposure to the infectoon

Standard reporting formats
Clear and easy to understand
Physician or patient is able to get an accurate and reliable picture
Effective communication between the clinician and laboratory personnel
REPORTING OF RESULTS (POST-TEST)

ETHICAL ISSUES INVOLVED IN PROCESSING AND REPORTING
TEST RESULTS
1. Collection of samples
Informed consent from the patient
Ensure the patient’s privacy and respect person’s
autonomy
2. Processing and storage of samples
Handled with care and processed as required
Additional unwarranted testing must not be performed
without the prior consent of the patient/their legally
authorised representative
Samples may be stored only up to the period specified
3. Reporting of results
Confidentiality of the test results must be maintained
Conveyed only to the treating doctor/requisitioner and
the patient
4. Use of biological material for a purpose other than what
was intended
Areas of human research and clinical trials
Material that is collected as part of the study with due
consent must be used for what it is intended and not for
any other purpose
5. Sharing of results, data and patient’s details
Ensure that the patient’s details and test results are not
disseminated to others
Prior permission is to be sought from the patient for
publication in literature
Bound by the medical code of ethics

QUALITY CONTROL IN CLINICAL MICROBIOLOGY
LABORATORY
To ensure the quality of care
Quality check (QC) procedure
 Adhere to prescribed standards for each and every medium/test/procedure
 Quality of the procedures must be regularly checked by quality control (QC) measures
QC: Procedures are tested against standard controls whose range of activity is known

QUALITY CONTROL IN CLINICAL MICROBIOLOGY LABORATORY
QC (quality control)
Performed periodically to ensure the quality of the laboratory procedure
The frequency and type of QC vary according to the test, medium or reagent
Automated systems: Calibration of the instruments is to be done to give reliable results
Needs to be mandatorily performed and documented for verification

QUALITY CONTROL IN
CLINICAL MICROBIOLOGY
LABORATORY
Laboratory accreditation
A process by which an external agency (a regulatory body)
assesses a laboratory’s practices by checking the processes and
procedures and certifying specific tests to see if they meet the
standards of the International Organization for Standardization
(ISO)
The National Accreditation Board for Testing and Calibration
Laboratories (NABL) provides certification for tests in India

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