Introduction to Bacteriology

GRAM POSITIVE COCCI:

 Staphylococcus
 Streptococcus
 Pneumococcus
 Enterococcus

GRAM NEGATIVE COCCI:

 Meningococci
 Gonococci

GRAM POSITIVE BACILLI:

 Bacillus
 Clostridium
 Corynebacterium
 Mycobacterium

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  Actinomyces
 Nocardia
 Listeria

GRAM NEGATIVE BACILLI:

Enterobacteriaceae family:

 Escherichia
 Klebsiella
 Salmonella
 Shigella
 Proteus
 Yersinia
 Citrobacter

Oxidase +ve: (Contain Cytochrome Oxidase)

 Vibrio
 Pseudomonas
 Campylobacter Meningococci and
 Helicobacter
 Bordetella Gonococci are also
 Brucella Oxidase Positive
 Hemophillus

Others:

 Rickettsia

Atypical pneumonia causing organisms:

 Chlamydia
 Mycoplasma
 Legionella

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 Staphylococcus

Staphylococcus aureus -> Pathogenic -> Coagulase +ve

CONS -> Commensals (good bacteria) -> Usually do not cause infection but under
some predisposing conditions become pathogenic

Staphylococcus
Virulence factors: Responsible for pathogenesis ->

1) Cell surface factors

Protein A → Anti-phagocytic

2) Enzymes

a) Coagulase:- MC associated with S.aureus

8 types of coagulase present (A,B,…..) -> MC is Type A

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Present only in S. aureus

Pro-thrombin like activity => Converts fibrinogen to fibrin (clot) → Localised

pyogenic infection (abscess with thick pus) -> Prevents spread of infection

Types:

 Bound coagulase (bound to surface of bacteria) (Clumping factor)

Bound coagulase + Plasma => Agglutination (Immediately clumps observed)
Called Slide coagulase Test

 Free Coagulase (not bound to surface of bacteria)

Free coagulase first binds to CRF (Coagulase Reacting factor) present in
plasma => Converts fibrinogen to fibrin (clot)
Called Tube coagulase Test
(Takes minimum 4 hours for reaction to occur)

b) DNase →

Breakdown of DNA => thinning of Pus

c) Hyaluronidase →

Breakdown of connective tissue/ground substance Cellulitis

=> Spread of Infection with thin pus

d) Staphylokinase →

Fibrinolytic => Breakdown of clot => Spread of Infection

3) Toxins :-

a) Hemolysins:

Complete hemolysis aka Beta hemolysis (Complete breakdown of RBCs)

α - Most important

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β – Shows Hot & cold phenomena (Produced at 37°C but hemolysis seen at 4°C)

γ + Panton Valentine toxin (Leucocidin: damage to WBCs) => Synergohymenotropic

toxin

δ – Also causes hemolysis

b) Toxic Shock Syndrome Toxin - Type-1 /Pyrogenic Exotoxin

Super Antigen- exaggerated Immune response => Damage to host tissue => Toxic
shock Syndrome

c) Enterotoxin –

Damages intestine => Food poisoning

12 types of enterotoxin [A, B,... ] => MC is Type A

Super Antigen

Preformed toxin (produced before entering host) => Incubation period less (1-6 hours)

Causes food poisoning → vomiting, diarrhea

d) Exfoliative toxin:

Causes SSSS (Staphylococcus Scalded Skin Syndrome)

Severe form:

 In Children → Ritter's disease

 In Adults → Toxic epidermal necrolysis => Nikolsky's sign (Underlying skin
visible after removal of epidermis)

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Clinical Manifestations:
Skin and Soft tissue infections:-

Abscess (MC)

Cellulitis

Botryomycosis

Systemic diseases:

Infective Endocarditis -> Damage to native valves >> Prosthetic valves

Nosocomial pneumonia (Hospital acquired) (MC caused by S.aureus)

Osteomyelitis (MC caused by S. aureus)

Diagnosis:
i) Microscopy:

Gram stain -> GPC in clusters

ii) Culture:

Selective media →

o Mannitol salt agar (MC)

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 o Salt Milk agar
o Ludlam's medium

iii) Identification (Biochemical test):

Catalase +ve

Coagulase +ve

MRSA (Methicillin Resistant Staphylococcus Aureus)

Staphylococcus Aureus- Resistant to all Beta lactam antibiotics

 Penicillin
 Cephalosporin
 Monobactam
 Carbapenens

Nowadays, Cefoxitin used as marker instead of Methicillin

Mechanism: Gene transfer

If mec A gene present on SCC (Staphylococcal chromosomal casette) => MRSA

Detection of MRSA:

i) Phenotypic method: MC -> Disk Diffusion Method

Antibiotic disks used => MC: Cefoxitin disc => If Resistant -> MRSA

MC used method -> Cefoxitin disk diffusion method

ii) Molecular method: MC -> PCR -> Detect presence of mec A gene

If mec A gene present -> MRSA

Treatment of MRSA:

DOC → Vancomycin (Glycopeptide)

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VRSA (Vancomycin Resistant Staphylococcus Aureus)

Use Daptomycin (MC)

Or Linezolid

Or Streptogramins

Or 5th gen Cephalosporins

CONS (Coagulase Negative Staphylococcus)

Commensals

Coagulase -ve

If predisposing factors present → Cause diseases

Staphylococcus epidermidis Staphylococcus saprophyticus

Novobiocin sensitivity test :- Novobiocin sensitivity test :-

Sensitive Resistant

Present on Skin Present on Female Urethra

Predisposing factors: Predisposing factors:
Any skin injury (Trauma, Surgery) Sexually active young women
 S. epidermidis enters blood  Leads to UTI
 Can lead to SSI (Surgical Site
Infection)
MCC of SSI: S.aureus
 Can lead to Infective endocarditis
within 12 months of surgery
(Prosthetic valve >> Native valve)

 Prosthetic valve Infective Endocarditis → after 12 months of surgery => MC

Cause is Streptococcus viridans (Streptococcus viridans >> Staphylococcus
epidermidis)

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 Prosthetic valve Infective Endocarditis → within 12 months of surgery => MC
Cause is Staphylococcus epidermidis (Staphylococcus epidermidis >>
Streptococcus viridans)

 MC Cause of Native Valve Endocarditis: Staphylococcus aureus

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GPC- Streptococcus, Pneumococcus & Enterococci
Streptococci
Catalase Test → -ve

(Helps to differentiate Streptococcus, Pneumococcus and Enterococcus from Staph)

Hemolysis → Beta-hemolysis (complete)

(Helps to differentiate Streptococcus from Pneumococcus and Enterococcus)

GPC in chains

Lancefield classified Streptococcus into 20 groups based on Carbohydrate Ag:-

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Virulence Factors:

1. Cell surface factors:

Protein M -> Anti-phagocytic

2. Enzymes:

a) DNase [A,B,C,D] (streptodornase)

MC -> DNase B

Breakdown of DNA present in Pus => Thinning of pus

b) Hyaluronidase

Breakdown of connective tissue => Spread of infection (Cellulitis)

c) Streptokinase

Fibrinolytic activity => Spread of infection (Cellulitis)

3. Toxins

a) Hemolysin (Streptolysin)

Streptolysin S [0₂ Stable]

 Can cause hemolysis on blood agar as can tolerate O2

 Less Immunogenic (Less Ab formed against it)

Streptolysin 0 [02 labile]

 Can't cause hemolysis on blood agar in aerobic condition as O2 labile

 Highly immunogenic ( [ASO/ASLO] [Anti-Streptolysin O] easily detectable)

b) TSST-Type 2 / Pyrogenic Exotoxin:

Super Ag → exaggerated immune response => Causes host Tissue damage => Toxic
shock Syndrome

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Clinical manifestations: Group A streptococcus
1. Throat infection (Pharyngitis)

Detect ASO Ab (Most important) or Anti-DNase Ab or Anti-hyaluronidase Ab

Complication: Acute Rheumatic Fever (ARF)

Due to Ag mimicry between: Cell wall Ag of Streptococcus and Human Ag:

 C-carb Ag mimics Cardiac valve Ag in humans

 M-protein Ag mimics Myocardium Ag in humans

DIAGNOSIS:

ASO titre <200 IU/ml => Throat infection

ASO titre >200 IU/ml => ARF

2. Skin Infection (Pyoderma): Cellulitis, Erysipelas

Anti-DNase Ab or Anti-hyaluronidase Ab >> Anti ASO Ab

Complication: PSGN (Post Streptococcal Glomerulonephritis)

Due to molecular mimicry between:

Cell membrane Ag of Strep and Glomerular vascular inner membrane in human

DIAGNOSIS:

Anti DNAse B titre <300 IU/mL => Skin Infection

Anti DNAse B titre >300 IU/mL => PSGN

Clinical manifestations: Group B streptococcus

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 1. Neonatal meningitis
(MCC of neonatal meningitis: Group B Strep)

Diagnosis of Streptococcal Infections:

1. Microscopy:

Gram stain → GPC in chain

2. Culture:

Selective culture media: Crystal violet blood agar

3. Identification:

Catalase -ve

Hemolysis on blood agar → Beta hemolysis

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Pneumococcus:
[Streptococcus pneumoniae]

 Catalase –ve
 Hemolysis on blood Agar → α hemolysis (Partial hemolysis)
 GPC in pairs [lens/lanceolate shaped, capsulated]

Virulence factors :-

1) Capsule –> Anti-phagocytic

2) IgA1 proteases -> Causes breakdown of secretory IgA (provides local immunity)

(Secretory IgA => Secreted by mucous cells of epithelial lining of respiratory tract,
GIT, Genitourinary tract)

3) Pneumolysin -> lysis of cell membrane->Host cell death

Clinical manifestations: Pneumococcus:

 Lobar Pneumonia
 Pyogenic/Bacterial Meningitis
 Sinusitis
 Otitis media

Diagnosis:
1. Microscopy:

Gram stain: GPC in pairs - Lens shaped, capsulated

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 2. Culture:

No selective media

On Blood Agar:

 α hemolysis (Partial)
 Carrom coin appearance/Draughtsman appearance (d/t presence of
AUTOLYSIN enzyme -> Lysis of pneumococcal colonies in concentric circle
manner)

3. Identification:

Catalase –ve GPC

Hemolysis on Blood agar -> α hemolysis

Optochin sensitive

or Bile soluble

or Inulin fermenter

Prevention (Vaccine):

i) Capsular Vaccine: PPSV-23 [no. of serotypes included in vaccine]

(Pneumococcal Polysaccharide Vaccine)

Contains Capsular Polysaccharide Ag

70% of serotypes in PPSV-23→ Causes meningitis in adults (therefore beneficial

more in adults)

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Contraindicated in children < 2yrs age → as less Immunogenic in them because
Capsular Ag is T cell Independent Ag => So Ab won’t be formed in good amount

ii) Conjugate Vaccine: PCV-13 (no. of serotypes included in vaccine)

(Pneumococcal Conjugate Vaccine)

70% of serotypes in PCV-13 → Causes meningitis in children

So, can be given even in < 2yrs of age

Enterococcus:
GPC Catalase → -ve

Hemolysis on Blood agar => γ hemolysis (Misnomer so no hemolysis)

GPC in pairs (spectacle shaped, non-capsulated)

Clinical manifestations: Enterococcus:

Various Clinical Manifestations:

 UTI (MC)
 Wound infection
 Intra-abdominal infection.

Diagnosis:
1. Microscopy:

Gram Stain -> GPC in pairs, spectacle shaped non-capsulated.

2. Culture:

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No selective media

Blood Agar → No hemolysis (γ hemolysis)

3.Identification:
 Catalase –ve
 No Hemolysis
 Toleration Test:
Enterococcus can tolerate 6.5% NaCl
Enterococcus can tolerate 40% bile
 PYR Test +ve
 Bile Esculin Test (MC)
Tube slant Green Culture media containing Bile and Esculin
If Enterococcus grows =>turns black from green => +ve test
As Enterococcus can tolerate 40% bile and hydrolyse Esculin to Esculitin
(black)

Enterococcus:

E. faecalis E. faecium

MC causing infection Less common causing infection

Less drug resistant More drug resistant

Sorbitol fermenter Arabinose fermenter

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 GNC - Meningococci & Gonococci
Meningococci [Neisseria meningitidis]
Pink colored GNC, in pairs, lens shaped, capsulated

(Gonococci are kidney shaped and non-capsulated)

Intracellular – Present usually within pus cells (neutrophils)

Virulence factors -

1) Capsule :- Anti-phagocytic
2) IgA1 protease :- breakdown of secretory IgA
3) Endotoxin – Lipopolysaccharide → Causes endothelial
injuries

Clinical manifestations:

 Skin rashes (MC manifestation)

 Meningitis (Severe infection)
 LPS →
 Due to endothelial injuries -> thrombus formation -> Intravascular
coagulation -> DIC (Disseminated Intravascular coagulation)
 Due to endothelial injuries => ↑ Vascular permeability → Plasma
leakage => Decrease in BP => Septecemia & shock
 Waterhouse Friderichsen Syndrome: Severe and Fatal
Includes Skin rashes, meningitis, DIC, Septecemia and shock
Bilateral adrenal hemorrhage, multi-organ failure

Diagnosis:

i. Microscopy:

Gram Stain → GNC, in pairs, lens shaped, capsulated, intracellular

ii. Culture:

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Selective Media →

Thayer Martin medium (MC used)

Modified New York medium

iii. Identification:

Maltose fermenter (Gonococci -> Maltose Non fermenter)

Prevention: (Vaccines)

1) Capsular Vaccine (Includes 4 serotypes (Quadrivalent) → A,C,Y,W)

6 serotypes cause meningitis (A,B,C,X,Y,W)
Serotype B not included as leads to Severe side effects
Serotype X not included as least to cause meningitis
Recombinant B serotype vaccine has been developed => not associated with
side effects.
Contraindicated :- <2yrs of age as less immunogenic

2) Conjugate Vaccine:
Highly Immunogenic even in < 2 yrs of age

Gonococci [Neisseria gonorrhoeae]

GNC in pairs, kidney shaped, non-capsulated

Intracellular

Virulence factors -

1. Pili / Fimbriae: Attachment/Adhesion to

urinary tract epithelium
2. IgA1 proteases: Causes breakdown of
secretory IgA

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 3. Endotoxin: Lipo-oligosaccharide => endothelial Injury -> DIC or Septicemia and
Shock

Clinical Manifestations:

1. In Men - Urethritis -> Pustular discharge from Urethra (Gonorrhea)

MCC of Urethritis -> Gonococci

Complication: Perianal abscesses (water can perineum)

2. In Women - Vulvovaginitis /Cervicitis

Complication: Peri-hepatic inflammation and Peritonitis [Fitz Hugh Curtis Syndrome]

3. In Neonates – Baby may acquire infection during delivery => Ophthalmia

Neonatorum (pustular discharge from eye d/t gonococci)

4. DGI- Disseminated Gonococcal infection (Polyarthritis and endocarditis)

DIAGNOSIS:

1. Microscopy -

Gram stain → Intracellular, kidney shaped GNC in pairs, non-capsulated

2. Culture:

Selective Media-

o Thayer Martin medium

o Modified New York medium

Transport media-
Moraxella:
o Amies Media
o Stuart Media Gram Negative

3. Identification: Cocco-bacilli

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Maltose Non-fermenter

Urethritis:
Inflammation of Urethra

MCC of urethritis: Gonococci

Non-Gonococcal Urethritis (NGU):

Bacteria: (MC)

o Chlamydia trachomatis (MC)

o Mycoplasma hominis

Viruses:

o Herpes Simplex Virus (HSV)

o Cytomegalovirus (CMV)

Fungi:

o Candida albicans

Parasite:

o Trichomonas vaginalis

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 GPB- Bacillus & Clostridium
Bacillus
 Aerobic, non-bulging spore producing GPB
 Non-bulging endospores produced after culture

PATHOGENIC SPECIES:

B. anthracis

 Causes Anthrax disease

 Capsulated
 Spores: Central

B. cereus

 Causes food poisoning

 Spores: Terminal/ Sub-terminal
 Non-capsulated

B. anthracis:
MC agent of Bioterrorism

Virulence factors -

1) Capsule: Anti-phagocytic

2) Toxin -

Anthrax toxin

1. Protective Factor (PF) → Binding fragment => Binding to host cell

2. Edema Factor (EF) -> Active fragment -> Increased cAMP

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 3. Lethal factor (LF) -> Lysis fragment -> Causes Host cell lysis

C/F:-

Anthrax - Zoonotic disease (Cattle & sheep → human)

1. Cutaneous Anthrax: [Hide Porter's disease]

Hide/Skin of Animals comes in contact with bare skin of humans (If abraded leads to
entry of organism)

Papule -> Malignant pustule → Black eschar

2. Pulmonary Anthrax: [Wool Sorter's disease]

B. anthracis spores if inhaled

Lead to Hemorrhagic Pneumonia

3. Intestinal Anthrax:

Severe Gastroenteritis

Diagnosis:

1. Culture -

 Selective Media→
PLET media [Polymyxin, Lysozyme, EDTA, Thallous acetate]
 Solid media -> (Blood Agar/ Nutrient Agar)
Medusa Head Appearance

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  Solid media + Penicillin →
String of pearl appearance

 Tube media (gelatin stab culture) ->

Inverted fir tree appearance

2. Microscopy -

 Gram stain - GPC arranged in chain

 Bamboo stick appearance – Central
spores

3. Immunological -

Ascoli thermo-precipitation test (Detects capsular Ag from the sample (putrid))

B. cereus:
Virulence factors - Toxins

Emetic toxin:

 Causes Vomiting
 Preformed toxin (IP: 1-6 hours)
 Source → Chinese fried rice

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Diarrheal toxin

 Causes Diarrhea
 Postformed toxin (IP: 16-24 hours)
 Source: Meat & Vegetables

Diagnosis:

1. Culture

Selective Media: MYPA (Mannitol, Egg Yolk, Polymyxin, Phenol red agar)

2. Microscopy

Gram stain (terminal endospore, non bulging)

Clostridium
Anaerobic, bulging spore producing – GPB

Clostridium perfringens /welchii: Gas gangrene - Club Shape (Sub-terminal

endospore), Capsulated

Clostridium tetani: Tetanus - Drumstick appearance (Round, terminal endospore)

Clostridium botulinum: Botulism – Sub-terminal

Clostridium difficile: Pseudomembranous Colitis - Sub-terminal

Clostridium perfringens:

Virulence factors:

1) Capsule – Anti-phagocytic
2) Toxin – α, β….
α toxin (Most important) as Lecithinase activity (Phospholipase)

C/F:

 Cellulitis -> Myonecrosis -> Gas gangrene

 Food poisoning => Post formed toxin

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 Meat, vegetables

Diagnosis:

1. Culture

Selective media -> RCM (As anaerobic)

Anaerobic culture methods have to be used in case of

aerobic media

2. Microscopy

Gram stain → Sub-terminal bulging spores- Club shaped

3. Toxin demonstration

α toxin

o Has Lecithinase property

o Also has Hemolysin property

a) Naegler's reaction (Lecithinase property)

Egg yolk agar taken as it contains lecithin

Half of it has Anti alpha toxin with sample -> No opalescence -> As anti-toxin
neutralizes the alpha toxin

Other half doesn’t contain Anti alpha toxin -> Opalescence seen surrounding the
colonies

b) Reverse cAMP test (hemolysin property demonstrated)

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Blood agar taken

Arrow head hemolysis => Point where α toxin hemolysin (Clostridium perfringens) and
cAMP factor hemolysin (Group B Streptococcus) meet (SYNERGISTIC ACTION) =>
Enhanced hemolysis at meeting point

c) Double zone hemolysis (Target hemolysis)

α toxin (hemolysin) -> Partial hemolysis (α)

θ toxin -> Complete hemolysis (β)

Blood agar taken

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Clostridium tetani

Virulence factors: Toxins

Tetanolysin -> Host cell lysis

Tetanospasmin -> Spastic paralysis (tetanus)

C/F:

 Tetanospasmin inhibits GABA

 Continuous Contraction of muscles (spastic paralysis) → Tetanus
 Tetanus
 Lock Jaw (due to continuous contraction of Masseter muscle)

Diagnosis:

1. Culture -

Selective media - RCM

2. Microscopy -

Gram stain - Terminal bulging spore => Drumstick Appearance

3. Toxin demonstration - (In vivo demonstration -> in mice)

Mouse (sample added) => Inject tetanus anti toxin => No paralysis

Mouse (sample added) => Spastic paralysis

Clostridium botulinum:

Virulence factors: Toxin

Botulinum toxin (8 types) -> A B C₁ C₂ D E F G

 C₂- enterotoxin => Preformed toxin (Short IP => Source: Meat and vegetables)
=> Causes food poisoning
 Remaining → Neurotoxin => Cause flaccid paralysis

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C/F:

Neurotoxins inhibits Ach (excitatory neurotransmitter)

 Continuous muscle relaxation

 Flaccid paralysis

Diagnosis:

1. Culture
Selective → RCM
2. Microscopy
Gram stain → GPB with sub terminal spores
3. Toxin demonstration → (in vivo -> mice)
Mouse (sample) → Botulinum antitoxin injected -> No paralysis
Mouse (sample) → Flaccid paralysis

Clostridium difficile:

Causes Pseudomembranous colitis

Commensal in intestine

Predisposing factors:

Long term use of antibiotics: As they kill the other important commensals =>
Decreased local immunity => C. difficile acquires Virulence factors

Ceftriaxone > Clindamycin > Ampicillin

Virulence factors -

Toxins:

o Exotoxin A
o Exotoxin B

C/F:

o Diarrhea (Clostridium difficile associated diarrhea)

o Pseudomembranous colitis

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Diagnosis:

1. Culture: (stool)
Selective media – CCFA (Cefoxitin Cycloserine Fructose Agar)
Sometimes contains egg yolk also
2. Microscopy
Gram stain → Subterminal bulging endospore
3. Toxin demonstration – In vitro methods
Molecular → PCR (best) -> Detect toxin genes
Immunological → ELISA / Rapid test -> Detect toxins
Only pathogenic C. difficile will contain the toxin

GPB - Corynebacterium & Mycobacterium

Corynebacterium diphtheriae
Causes Diphtheria

Mc Leod’s Biotypes Severity of disease PTBA

Corynebacterium Severe Daisy head colonies

diphtheriae gravis (Paralytic & hemorrhagic)

Corynebacterium Intermediate Frog’s egg colonies

diphtheriae intermedius (Hemorrhagic)

Corynebacterium Mild Poached egg colonies

diphtheriae mitis (Obstructive)

Virulence factors:

Toxin:

Diphtheria toxin (Phage coded toxin (Lysogenic conversion))

Optimal Iron Concentration required: 0.1 mg/l

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Excess iron inhibits Diphtheria toxin production

 Inhibits elongation factor EF2

 Decreased Protein synthesis

C/F:

Diphtheria → production of pseudomembrane

i) Respiratory diphtheria

o Faucial diphtheria (MC) => Bull neck appearance d/t formation of

pseudomembrane on tonsils

o Laryngeal diphtheria => Most severe form as l/t Airway obstruction

ii) Cutaneous diphtheria → Skin

iii) Systemic diphtheria :

CNS (Cranial nerves)

CVS (Cardiomyopathy)

Diagnosis:

1) Culture

Selective Media:

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a) Loeffler's serum slope (LSS)

6-8 hours -> growth (earlier detection possible)

Better demonstration of metachromatic granules is possible

Disadvantage:

If incubated for a long time, Commensals will also grow in LSS

b) PTBA (Potassium Tellurite Blood Agar)

Black Coloured colonies

Advantage: Highly selective media for C. diphtheriae

Disadvantage: takes > 48 hours for growth

c) Tinsdale media

Modified PTBA: PTBA + cysteine

Black coloured colonies with brown halo

2) Microscopy

Gram stain:

Metachromatic granules grow better after

culture

 Club shaped
 V or L shaped colonies -> Chinese letter
pattern or Cuneiform pattern

Albert’s (MC) or Niesser's or Ponder's stain -

Metachromatic granules or Volutin granules or Babes

earnst granules

 Store energy as Polymetaphosphate

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3) Toxin Demonstration

Both in-vivo and in-vitro methods possible

Molecular method → PCR

Immunological:

→ ELISA / Rapid test (MC used)

→ ELEK's gel precipitation test

ELEK's gel precipitation test

 Horse serum agar taken

 Filter paper strip soaked in diphtheria antitoxin placed
 Streak the sample perpendicular to filter paper strip
 Both diphtheria toxin and antitoxin will meet and lead to precipitation
 If +ve → Indicates diphtheria toxin is released.

Mycobacterium
- Mycobacterium tuberculosis

- Mycobacterium leprae

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- Rest all are grouped under 'Atypical Mycobacteria’ or ‘Non tuberculosis
mycobacteria (NTM)’

Mycobacterium tuberculosis

Virulence factors:

1. LAM (Lipoarabinomannan) Antiphagocytic

2. Mycolic acid -> Resp. for acid fastness

C/F:

Pulmonary TB – Involves lungs

Extra Pulmonary TB- Involves Other organs

Diagnosis:

1. Microscopy

a) Z/N staining (Modified Acid Fast stain)

Small pink rods in blue background

b) Direct Immunofluorescence test (Best)

 Detects Ag
 Specific Ab coated with fluorescent
dye

2. Culture

a) Liquid Culture - MGIT method (Mycobacterial growth Indicator tube)

 Automated method -> BACTEC method

 Growth in 3-4 weeks

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b) Solid culture –

LJ (egg based media)

Middle brook 7H10/7H11 (Agar based) (BEST)

Growth in 6-8 weeks

3. Molecular methods → PCR

1. CBNAAT-(Cartridge Based Nucleic Acid Amplification test)

aka gene Xpert

 Identify Mycobacterium tuberculosis

 Gives AST → only to Rifampicin (as Core drug for Rx of TB)
 Turnover time: Within 2 hours gives result (PREFERRED)

2. Line Probe Assay (LPA):

 Identify Mycobacterium tuberculosis

 Gives AST → to all drugs
 Takes approximately - 2 days.

4. Immunological:

a) ELISA based Method-

Quantiferon TB gold assay (IGRA) - Interferon Gamma Release Assay

Blood Sample → T cells taken (If sensitized would release interferon γ) + Add
Mycobacterium TB antigens

=> Release of Interferon γ measured

b) Rapid test

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 o Gives result within 30 minutes
o Detect MPT 64 Ag (differentiates M. tb from atypical mycobacteria)

c) Skin test (Tuberculin/ Mantoux test)

Type IV hypersensitivity test

0.1 ml PPD (Purified protein derivative) injected → Inject intra-dermally on flexor

aspect of forearm

Reading → after 48-72 hours (Never before 48 hours)

If Induration → >10mm → +ve

Atypical mycobacterium (NTM)

Runyon's Classification

1) Slow growers -> Take > 7 days for growth

a) Photochromogens: Pigment produced in presence of light

 M. kansasii
 M. marinum
 M. simiae

b) Scotochromogens: Pigment produced in dark only

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  M. scrofulaceum
 M. gordanae
 M. szulgai

c) Non- chromogens: No pigment produced

 M. avium- intracellularae complex (MAC)

 M. ulcerans
 M. xenopi

2) Rapid Growers:- takes < 7 days for growth

 M. fortuitum
 M. chelonae & abcessus
 M. smegmatis

C/F:

1. Cutaneous disease

M. marinum → (MC)

 Fish tank granuloma

 Swimming pool granuloma

M. ulcerans → (Produces Mycolactone toxin) (No other mycobacteria produces toxin)

 Buruli ulcers

2. Lymph node disease

M. scrofulaceum ->

Lymphadenitis/ Lymphadenopathy

3. Respiratory disease

Most of atypical mycobacterium cause respiratory disease

MAC - (MC to cause resp. disease) aka Battey bacillus

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Causes pneumonia

Diagnosis:

Same as M. tb

Rapid test:- MPT 64 Ag -> -nt

Mycobacterium leprae

Virulence factors:

1. LAM (Lipoarabinomannan) Antiphagocytic

2. Mycolic acid -> Resp. for acid fastness

Ridley Jopling’s Classification -> Used for Leprosy

TT --------------> Mild

BT

BB

BL

LL ---------------> Severe

TT, BT, BB -> Tuberculoid Leprosy (MILDER FORM)

BL, LL -> Lepromatous Leprosy (SEVERE FORM)

C/F:

Tuberculoid leprosy [TT, BT, BB]

 Milder
 Pauci bacillary
 Few lesions present (skin & nerves)
 CMI → good
 Lepromin test +ve

38
Lepromatous leprosy (BL, LL)

 Severe form
 Multi bacillary
 Multiple lesions (Skin and nerve)
 CMI inadequate
 Lepromin test -ve

Lepra reaction

Hypersensitivity reactions during the course of the disease

TYPE I:

Due to type IV hypersensitivity

TH1 immune response -> predominate response

Occurs Usually during BB stage (Borderline leprosy)

TT BB LL

(Reversal Reaction) (Downgrading reaction)

Rx: Glucocorticoids

TYPE II:

Due to type III hypersensitivity

TH2 immune response → predominates here

During LL stage, progresses and involves Other organs also (testes, kidney, eyes)

Skin lesions: Papules -> nodules [Erythema Nodosum Leprosum (ENL)]

Rx:

 Glucocorticoids
 Clofazimine
 Thalidomide

39
Diagnosis:

1. Microscopy

i) Z/N stain: Acid fast Globi (arranged in cigar

shaped bundle)

ii) Direct immunofluorescent test (BEST)

2. Culture

Non cultivable

Therefore, Animal inoculation method used:

 9 banded Armadillo
 Foot pad of mice

3. Immunological Method

 ELISA
 FLA – ABS (Fluorescent Leprosy Ab Absorption test) – Indirect
immunofluorescent test

4. Skin → Lepromin test

 Type IV hypersensitivity
 0.1 ml PPD → Inject intra-dermally on flexor aspect of forearm

Readings taken :

After 48 hours (Early Fernandez Reaction)

 Erythema > 10mm → +ve

21 days (Late Mistuda Reaction)

Nodule > 5mm → +ve

40
 GPB - Actinomyces, Nocardia & Listeria
Actinomyces:
MC pathogenic species of Actinomyces: Actinomyces israeli

o Anaerobic
o Filamentous bacteria
o Elongated GPB

CLINICAL FEATURES:

Actinomycosis:

i) Cervicofacial actinomycosis (MC) aka Lumpy jaw

=> Angle of Jaw is involved (MC)

ii) Extra Cervico-facial actinomycosis

 CNS -> Brain abscess

iii) Actinomycetoma (Madura foot)

Chronic subcutaneous granulomatous condition with discharging granules

DIAGNOSIS:

1) Microscopy

HPE:– Histo-pathological Examination done of crushed granules containing

Actinomyces => Sunray appearance d/t filaments

2) Culture

Selective Media - Thioglycolate broth as anaerobic bacteria => Fluffy balls at

bottom of thioglycolate broth

41
Nocardia:
MC pathogenic species of Nocardia: Nocardia asteroids

Acid fast, aerobic filamentous GPB

CLINICAL FEATURES:

1) Pulmonary Nocardiosis (MC form of Nocardiosis in humans)

 Pneumonia
2) Extra Pulmonary Nocardiosis
 CNS -> brain abscess
3) Actinomycetoma (Madura foot)
Chronic subcutaneous granulomatous condition with discharging granules
MCC of Actinomycetoma: Nocardia
MCC of Actinomycosis: Actinomyces

DIAGNOSIS:

1) Microscopy

HPE - Histo pathological Examination - crushed granules: Sunray appearance

2) Modified Acid fast staining:

Long, Pink coloured filamentous branched bacteria seen in blue background

(Mycobacteria: Short, pink coloured rods)

3) Culture:

 LJ Media
 SDA Media

Special technique used: Paraffin Bait

technique

42
Listeria:
MC pathogenic species: Listeria monocytogenes

Cold enrichment GPB (Lower temperature prefers growth)

Source :- Refrigerated food

CLINICAL MANIFESTATIONS:

1. Food poisoning => Severe vomiting & diarrhea

2. Can lead to Abortion, premature delivery

3. Neonatal meningitis

DIAGNOSIS:

1. Microscopy

Hanging drop method- to demonstrate motility

Differential motility shown by Listeria

o Non motile at body temperature 37°C

o Motile at room temperature 25°C (TUMBLING MOTILITY)

2. Culture

Selective culture media→ PALCAM Agar

43
 GNB - Enterobacteriaceae
Escherichia:
MC species: E. coli

Normally commensals, some pathogenic bacteria cause diarrhea and UTI

Diarrheagenic E. coli

a) Enteropathogenic E. coli [EPEC]

C/F: Diarrhea in children

Virulence factor:

o Non-toxigenic
o Bundle forming pili helps in attachment
o LEE - Locus of Erythrocyte Effacement factor

b) EnteroToxigenic E. coli (ETEC)

C/F: Traveller's diarrhea

Virulence factor:

o Labile Toxin (Increase in cAMP) => Similar to cholera toxin

o Stable Toxin (Increase in cGMP)

c) EnteroAggregative E. coli (EAEC)

C/F- Traveller's diarrhea

Virulence factors:

o EAST-1 toxin

d) EnteroInvasive E. coli [EIEC] (similar to shigella)

C/F: Dysentery (As invasive)

44
Virulence factors:

VMA: Virulence Marker Ag => Responsible for invasiveness

Test for invasiveness: Sereny's test (Rabbit Eye conjunctivitis)

e) EnteroHemorrhagic E. coli (EHEC) [0157: H7 -> MC]

C/F- Dysentery, HUS (Hemolytic Uremic Syndrome)

MCC of HUS: EHEC

Virulence factors:

Verocytoxin -> Acts by inhibiting ribosomes -> inhibiting protein synthesis

Identification:

Sorbitol Mac Conkey agar taken as EHEC O157:H7 is Sorbitol Non-fermenter (Pale
colonies) unlike other species of E.coli

Detection of toxins of E. coli:

ELISA (MC used), PCR, Tissue Culture test

Klebsiella tribe:
o VP +ve
 Klebsiella (LF) (Motile)
 Enterobacter (LF) (Non-Motile)
 Serratia (NLF) (Non-Motile)
 Hafnia (NLF) (Non-Motile)

45
Klebsiella:
Non-motile

C/F:

 Pneumonia
 Meningitis
 UTI
 Soft tissue infections

Salmonella:
C/F:

Typhoidal salmonella: Typhoid fever

o S. Typhi
o S. Paratyphi A
o S. Paratyphi B

Non-Typhoidal Salmonella: Gastro enteritis & other infections

o S. Typhimurium

Carrier state:- Ag persists in the body even after recovery. Shed organism in
secretions leading to transmission of infection.

Carriers based on site of shedding of bacilli:

i) Urinary carriers
ii) Stool carriers

Carriers based on time of shedding of Bacilli:

i) Convalescent → upto 3 months

ii) Temporary → upto 1 year

iii) Chronic → > 1 year

46
DIAGNOSIS:

1. Culture:

Selective media:

o XLD agar (xylose lysine deoxycholate agar) Selective media for both
o DCA agar (Deoxycholate Citrate Agar) Salmonella and Shigella
o SSA (Salmonella Shigella Agar)

o Wilson Blair Medium (Only for Salmonella) (black colour colonies seen)

2. Immunological: WIDAL TEST

O Ag H Ag Vi Ag

Cell wall Ag Flagellar Ag Capsular Ag

Present in all salmonella Present in all motile Present in all capsulated

salmonella salmonella like S. Typhi
etc)

=> Less Immunogenic => Highly Immunogenic => Less Immunogenic

Few Ab's formed and are More Ab’s formed and are Few Ab's formed and
cross reactive (React with non-cross reactive disappear early (with
O Ag of other species Highly specific recovery)
also) Persists only in carriers

Widal Agglutination Test -

Take 4 test tubes and add:

→ Sample + TO Ag => +ve in all three S.Typhi, S.Paratyphi A, S.Paratyphi B

→ Sample + TH Ag => +ve in S.Typhi

→ Sample + AH Ag => +ve in S.Paratyphi A

47
→ Sample + BH Ag => +ve in S.Paratyphi B

Diagnosis of Carriers:

a) Carriers in community: Sewer-swab test

b) Screening test → Vi Ab test

c) Confirmatory test -> Stool / urine culture => Demonstration of Ag

Shigella:
Non-motile

i. Group A → S. dysenteriae (Mannitol non-fermenter) → Most severe

ii. Group B → S flexneri

iii. Group C → S. Boydii

iv. Group D → S Sonnei (LLF (late lactose fermenter))

Two LLF:

Shigella sonnei

Citrobacter

Virulence factors:

 Enterotoxin + VMA (invasiveness)

Detected by Sereny's test: Rabbit eye conjunctivitis
Causes Dysentery
 Shiga toxin → similar to verocytotoxin of EHEC
Inhibiting ribosomes -> Inhibiting protein synthesis
C/F- Dysentery, HUS

Diagnosis:

1. Culture:

Selective Media: XLD, DCA, SSA

48
Proteus:
C/F:-

o UTI (MC) => Can precipitate phosphate stones (Struvite stones)

o Soft tissue infection

Diagnosis:

i) PPA test: Detects PAD enzyme (Phenylalanine Deaminase)

=> Converts Phenylalanine to Phenyl Pyruvic Acid (PPA)

ii) Diene's Phenomenon :

On culture -> Shows Swarming

If two strains of proteus grown on Blood agar:

 If swarming doesn't merge -> Strains of Proteus are different

 If swarming merges -> Strains are similar

Yersinia:
MC Species causing human infection: Yersinia pestis

Non-motile

C/F:

49
Plague: Zoonotic disease (Rat => Humans)

 Bubonic: lymph node

 Pneumonic: lung
 Septicemic: Blood vessels

Modes of acquiring infection:

 Rat flea
 Droplet inhalation

Diagnosis:

1. Wayson's staining (Bipolar staining -> Only poles get stained) => Methylene
Blue used
 Safety pin appearance

2. Culture:
Selective → CIN agar
Nutrient ghee broth -> Stalactite growth (hanging from
surface)

Citrobacter:
C/F:-

 UTI

Diagnosis:-

 Late lactose fermenter

50
 GNB - Oxidase Positive Organisms
Vibrio:
Classification:

a) Gardner's and Venkataraman Ramakrishnan (VR)

El Tor Classical

VP +ve VP -ve

Ogama Inaba Hikojima

(A,B) (A,C) (A,B,C)

51
b) On basis of Salt requirement:

Non halophilic Vibrios

 V. cholerae (Can tolerate salt upto 6% but does not require salt for its
growth)

Halophilic Vibrios

 V. parahemolyticus
 V. vulnificus
 V. alginolyticus

Vibrio cholerae:

Virulence factor:

Cholera toxin → Increased cAMP

C/F:

Cholera (life threatening diarrhea)

Usually no fever

Diagnosis:

1. Microscopy:
Gram stain: Comma shaped GNB arranged in
parallel rows
Fish in stream appearance
Hanging drop:
Darting motility

2. Culture
Transport media: Sach’s
Cary Blair medium Buffered
Autoclaved sea water Glycerol Saline
does not

52
 VR medium
Enrichment media: To inhibit Commensals
Alkaline peptone water
Monsur's peptone water
Selective media:
TCBS (Thiosulphate Citrate Bile Salt Sucrose Agar)
-> Yellow color colonies (Sucrose fermenter)

3. Identification
1) String test: +ve for all Vibrios
Organism suspension + Bile salt [0.5% sodium deoxycholate]
=> becomes Turbid and mucoid
=> Forms string when we try to lift it up with a loop

2) Cholera red reaction: +ve only for V. cholera

(V.cholerae suspension in culture media) Nitroso- indole compound +
concentrated H₂S04 => Red colour

53
V. cholerae 0139 (Bengal Strain)

Same as V. cholerae 01 Except:

 Capsulated
 Causes mild form disease

- 1st case → in Chennai → in 1992 (Caused Epidemics in → West Bengal)

Restricted to India unlike O1

V. cholerae 01 → Causes Pandemics (7 pandemics)

1st 6 pandemics – due to V. cholerae 01 Classical Biotype

7th pandemic – due to V. cholerae 01 ELTOR Biotype

Halophilic vibrios

i) V. parahemolyticus

C/F:

 Gastroenteritis
 Wound infection

Diagnosis:

1. Microscopy
 GNB
 Actively motile (not darting motility)
 Safety pin appearance in Bipolar staining
2. Culture
TCBS media => green color colonies (Sucrose non-fermenter)
Wagatsuma agar => Causes Hemolysis → Kanagawa phenomenon
Salt-tolerance test => Maximum upto 8% NaCl

54
ii) V. vulnificus

C/F:

 Wound infection

Diagnosis:

Max. upto 10% NaCl

iii) V. alginolyticus

C/F: Wound infection, eye and ear infections

Diagnosis: More than 10% NaCl

Pseudomonas aeruginosa
Virulence factors:

Exotoxin A → ↓ EF₂ -> Decreased Protein synthesis

Action similar to Diphtheria toxin

55
C/F:

 Swimmer's ear in children

 Malignant otitis externa in diabetics
 Ecthyma gangreosum

MC infections in:

o Burns patient
o Cystic fibrosis patient
o Ventilator associated pneumonia

MC contaminant of disinfectants (As Cetrimide is a growth factor for Pseudomonas)

Diagnosis:

1. Microscopy
GNB
Twitching motility
2. Culture
Selective media: Cetrimide Agar

Burkholderia
1. B. pseudomallei

Usually present in oil and water

C/F:

Malleidiosis

 Localised skin infection (abscesses)

 Lung infection → pneumonia

AKA Vietnam time bomb disease (due to long latency period)

Diagnosis:

1. Culture

Selective media: Ashdown medium- purple wrinkled colonies

56
 2. Identification

Oxidase +ve, Motile

ii) B. mallei (zoonotic disease)

Horse Human

(Direct contact, Droplet inhalation)

C/F: Glander's disease

 Localised skin infections

 Lung infection → pneumonia

Diagnosis: Identification

Oxidase -ve, non-motile

iii) B. cepacia:

Usually present in soil & water and I/V fluids

C/F:

 Lung Infection → especially in cystic fibrosis patient

 Nosocomial infections as contaminant in IV fluids

Campylobacter jejuni
Oxidase +ve

C/F:

Diarrhea

Diagnosis:

1. Microscopy
Gram stain – Small 'S' shaped (gull wing shape)
Hanging drop- Darting motility

57
 2. Culture
Selective media: Skirrow's medium

Helicobacter pylori [H. pylori]

Oxidase +ve

C/F:

Diarrhea

Peptic Ulcer Disease -> Gastric adenocarcinoma

DIAGNOSIS:

1. Microscopy
Warthin starry silver impregnation method
Hanging drop → Cork screw motility (Lophotricous: 5-7 sheathed flagella at
one end)

2. Culture
Selective media: Skirrow's medium

IDENTIFICATION:

1) Rapid Urease Test → +ve (Performed after Endoscopic biopsy)

2) Urea Breath Test → Non-radioactive Carbon (13C) given to patient and Mass
Spectrometer used to detect whether Urea broken down or not

58
3) Stool Antigen Test → ELISA

Haemophilus influenza b:
Oxidase +ve

MC causes infections in children

C/F:

Epiglottis and meningitis in children

Pneumonia in adults

Diagnosis:

Culture :-

Selective media -> Filde’s Digest Agar

Satellitism:- Fastidious organism (Requires factor V (Intracellular – within RBCs) &

factor X (Extracellular))

Therefore, easily grows on Chocolate agar as RBCs lysed in it

Grows better around S.aureus as it produces hemolysins => Release of Factor V

59
Bordetella pertussis
Oxidase +ve

C/F: Whooping cough

Virulence factors- Pertussis toxin (Increased cAMP)

Diagnosis:

1. Culture
Selective media -
 Bordet- gengue medium
 Bisected Pearl Colonies
 Aluminium paint appearance

Culture microscopy

Gram stain: Thumb print appearance

60
Brucella melitensis:
Oxidase +ve

C/F:

Brucellosis / Malta fever / Undulent fever -> Zoonotic disease

Triad:

 Fever & night sweats

 Hepatosplenomegaly (RES - Reticulo Endothelial system)
 Myalgia / Arthralgia (MES – Musculoskeletal system)

Cattle ------------> Human

Mode:

 Direct contact
 Droplet inhalation
 Ingestion:
Raw milk, under-cooked meat
Vegetables and water contaminated with excreta of animals.

Diagnosis:

1. Culture
 Selective media: Trypticase Soy Agar (Provides high concentration of
protein)
2. Immunological
 Standard agglutination test
 Sample + LPS Ag of B. abortus => Detection of B. melitensis Ab (CROSS-
REACTIVE)
 Diagnosis In animals →
Milk ring test
Rose Bengal card test

61
 GNB - Rickettsia & Atypical Pneumonia Organisms
Rickettsia & related organisms:
Rickettsia, Orientia, Coxiella, Ehrlichia, Bartonella

Rickettsia and Orientia:-

 Intracellular organisms
 Cause Endothelial injury
 Transmitted by vector
 Non-cultivable -> Grow only on tissue culture -> McCoy and Hela cell lines
 Weil-felix test performed to diagnose => Heterophile Agglutination test
Ab of Rickettsia and Orientia are detected using -> Ag of Proteus (OX-K, OX-
2, OX-19) as Heterophile Ag
 Characteristic Clinical manifestation: Black eschar → not seen always

Disease Organism Vector Transmission Weil-felix test

Typhus group

Epidemic typhus R.prowazekii Louse Autoinoculation OX19, OX2 ±

Endemic typhus R. typhi Flea Autoinoculation OX19, OX2 ±

Scrub typhus Orientia Trombiculid Bite OX-K

(Chiggerosis) tsutsugamushi mites(larval
form: chiggers)

Spotted fever group

Rocky mountain SF R. rickettsiae Tick Bite OX19

62
Indian tick typhus R. conorii Tick Bite OX19, OX2
(Mediterranean +ve
spotted fever)
AND
Bouttenese fever

Rickettsial pox R. akarii Mite Bite -ve for all 3

Ag
Coxiella, Ehrlichia, Bartonella:

Culture: Non-Cultivable

Tissue culture → McCoy & Hela cell lines [except Bartonella (cultivable) → on blood
agar]

Weil felix test -ve for all 3 Ag

Disease Organism Vector Transmission

Q-fever (Query) Coxiella burnetti - Inhalation of spores

HME (Human Monocytic Ehrlichia chaffeensis Tick Bite

Ehrlichiosis)

HGE (Human Granulocytic Ehrlichia ewingii Tick Bite

Ehrlichiosis)

Trench fever (5 days fever) Bartonella quintana Louse Autoinoculation

Oraya fever (Carrion's Bartonella Sandfly Bite

disease) Bacilliformis

Cat Scratch disease Bartonella henselae - Cat scratch/Cat

Bite

63
Chlamydia

Intracellular organism

Non-cultivable

 Tissue culture → McCoy, Hela cell lines

Species:

Chlamydia trachomatis TRIC

 Serotypes A,B,C → Trachoma (TR)

 Serotypes D-K → Inclusion Conjunctivitis (IC), NGU (Non-gonococcal urethritis)

Chlamydia Trachomatis LGV

L₁, L₂, L3 → Lympho Granuloma Venereum (LGV)

Chlamydia psittaci

Has many serotypes → Psittacosis (Atypical pneumonia) (zoonotic disease -> parrots)

Chlamydia pneumoniae

One serotype -> Atypical pneumonia

Life cycle:

Reticular body => Intracellular form of Chlamydia => Replication form => Active
metabolism should be going on (Unlike viruses, they contain DNA and RNA)

Elementary body => Extracellular form of Chlamydia => Infective form => No active
metabolism

Mycoplasma pneumoniae (Eaton's agent)

 Lack cell wall

64
  3 layered cell membrane (contains sterol) → gliding motility
 Resistant to all cell wall inhibitor antibiotics

C/F:

o Atypical pneumonia
o NGU

Diagnosis:

 Microscopy:
Dienes Method (Staining method) (Methylene Blue used – Poured on culture
plate)

Culture::

Selective media → PPLO Agar (Pleuro Pneumonia Like Organism) :- Fried egg colonies

Immunological tests- (Heterophile Agglutination Tests)

Cold Agglutinin test: Detect Ab's of Mycoplasma → Human O blood group RBC Ag is
used

Streptococcal MG test: Detect Ab's of Mycoplasma → Streptococcal MG Ag

Best Test: PCR

Legionella pneumophila:

C/F:

Atypical pneumonia associated with diarrhea

Pontiac fever (mild flu like illness)

Diagnosis:

Culture

Selective media – BCYE agar (Buffered Charcoal Yeast Extract)

65
 GNB - Spirochetes
Spiral in shape

Treponema
Borrelia
Leptospira

Treponema
Pathogenic Treponema

Non-cultivable

Therefore cultivated using Animal inoculation – Rabbit testes (MC)

1. Venereal Treponema (Sexual route)

T. pallidum var. pallidum -> Causes Syphilis
(Nicol's strain – Strain maintained in Rabbit Testes)
2. Non-Venereal Treponema (Non Sexual route)
T. pallidum var. endemicum → Endemic Syphilis/Bejel
T. pallidum var. pertenue → Yaws
T. carateum → Pinta

Non-Pathogenic Treponema -> Cultivable (SMITH’S NOGUCHI’S MEDIA)

T. phagedenis (Reiter’s Strain)

T. refringens (Noguchi’s Strain)

T. pallidum var. pallidum:

C/F: Syphilis (STI)

STAGES:

1° Syphilis

66
 o Hand chancre
o Regional lymphadenopathy

2° Syphilis

o Maculo papular lesions

o Generalised lymphadenopathy

Papules if appear in Intertregenous area (Skin fold area) => called Condyloma lata

Latent syphilis

o No lesions
o Asymptomatic
o Serologically +ve → Ag & Ab +ve

3° Syphilis

o Severe destructive lesions involving skin, Muscles, bones (Gumma)

o CNS (Neurosyphillis)
o CVS

Congenital Syphilis

Triad:

 Interstitial keratitis
 SNHL (sensorineural hearing loss)
 Hutchinson's teeth

Diagnosis:

1. Microscopy:
Thin, slender, spiral shaped
Dark ground microscope:–
No stain used
Live & motile bacteria
Flexion-extension / Cork- screw / Lashing
motility

67
 Bright field microscopy:-
Silver impregnation method used -> Fontana stain

2. Culture
Animal inoculation - Rabbit testes

3. Immunological methods:
Non-Treponemal / standard tests: (NTT)
Non-specific Ab of treponema (Reagin Ab's) => Cardiolipin Ag extracted from
Ox’s heart
a) Wasserman test- CFT (complement fixation test)
b) Kahn's test - tube flocculation test (Precipitation)
C) VDRL - Slide flocculation test (MC)
d) RPR - Slide flocculation test

Investigation of choice: 1st test to be performed => NTT

Screening test, not confirmatory
Prognostic test → monitor treatment response

Treponemal Tests / Specific tests:

Specific Ab detected by using specific Treponemal Ag
1. FTA-ABS → Fluoroscent Treponemal Ab Absorption test => Indirect
immunofluoroscence Assay (IFA) (gold standard)
2. TPI (Treponema Pallidum Immobilisation Test)

68
 3. TPHA (Treponema Pallidum Heme Agglutination Test)
4. TPPA (Treponema Pallidum Particulate Agglutination test)

Confirmatory test

Borrelia
MC species causing infection in humans: Borrelia burgdorferi

Causes Lyme's disease → migratory annular rashes => Erythema migrans

Transmitted by Vector: Ixodes Tick

Borrelia recurrentis:

Causes - Epidemic Relapsing fever

Transmitted by Vector: Louse

Note:- Endemic Relapsing fever (transmitted by tick)

Caused by other species of Borrelia

Borrelia vincenti:

Causes Vincent’s Angina => Necrotising gingivostomatitis

Diagnosis:

1. Microscopy - same as Treponema

2. Culture
Selective media: Modified Kelley's media

69
Leptospira:
MC species causing infection in humans: Leptospira interrogans

C/F:-

Leptospirosis or Weil’s disease →

o Involves RES (Reticuloendothelial System) -> HSM (Hepatosplenomegaly)

o Renal failure (Helps to differentiate Leptospirosis from Brucellosis)

Zoonotic disease:
Rat urine => Human (Direct contact/Indirect contact)

Diagnosis:

1. Microscopy
Same as Treponema
2. Culture
Selective media: EMJH broth
Leptospira grows densely just below the surface K/A
Dinger's ring

70