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MICROBIOLOGY - MUHS EXAM NOTES (Serotype 2 MBBS)

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1. STREPTOCOCCUS

A. Streptococcus pyogenes - Non-Suppurative Sequelae

Two Major Non-Suppurative Sequelae:

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1. Rheumatic Fever (RF)

S. pyogenes pharyngitis
        |
M protein antigen stimulates antibody production
        |
Antibodies cross-react with:
  - Heart (sarcolemmal antigens) --> Carditis
  - Joints (synovial tissue)     --> Migratory polyarthritis
  - Brain (caudate nucleus)      --> Sydenham's chorea
  - Skin (subcutaneous tissue)   --> Nodules, Erythema marginatum

• Major: Carditis, Polyarthritis, Chorea, Erythema marginatum, Subcutaneous nodules
• Minor: Fever, raised ESR/CRP, prolonged PR interval

• Primary prevention: Treat strep throat with penicillin within 9 days
• Secondary prevention: Monthly benzathine penicillin (1.2 MU IM) for 5 years or until age 21

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2. Acute Glomerulonephritis (AGN / PSGN)

• After pharyngitis: 10-14 days
• After pyoderma: 3-6 weeks

S. pyogenes infection (nephritogenic strain)
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Streptococcal antigen (NAPlr/SPE-B) deposited in glomeruli
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Antigen-antibody complex formation (Type III hypersensitivity)
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Complement activation --> Inflammatory damage
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Hematuria, proteinuria, hypertension, oliguria

• Elevated ASO titer (pharyngitis-associated)
• Elevated anti-DNase B (skin-associated - MORE sensitive)
• Low serum C3 complement
• Hematuria ("cola-colored urine")

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B. S. pneumoniae vs. S. viridans - 8 Differences

Memory tip: "OBIN" - Optochin, Bile solubility, Inulin, Neumoniae = Pneumoniae is POSITIVE for all

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2. CLOSTRIDIUM

A. Clostridium botulinum - Pathogenicity & Prevention

Toxin (Pathogenicity):

• Neurotoxin - blocks ACh release at neuromuscular junction
• Produces flaccid (descending) paralysis
• 7 types (A-G); Types A, B, E cause human disease
• Type A most potent; Type E associated with fish/seafood

Toxin ingested (preformed in food) / produced in vivo
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Toxin enters bloodstream
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Binds to presynaptic membrane of NMJ
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Cleaves SNARE proteins (synaptobrevin/SNAP-25)
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Blocks ACh vesicle fusion --> No ACh released
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FLACCID PARALYSIS (descending: diplopia --> dysphagia --> respiratory failure)

1. Food-borne: Preformed toxin in canned/preserved food (home-canned vegetables, fish)
2. Wound: Toxin produced in contaminated wound
3. Infant botulism: Most common - spores ingested (honey) --> colonize infant gut --> toxin produced in vivo ("floppy baby syndrome")
4. Iatrogenic: Cosmetic/therapeutic misuse

• Boiling food for 10 minutes before eating (destroys toxin - heat labile)
• Pressure cooking destroys spores (121°C, 15 min)
• Do NOT feed honey to infants under 1 year
• Proper canning techniques (commercial canning)
• Trivalent botulinum antitoxin (types A, B, E) for treatment/post-exposure

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B. Gas Gangrene (Clostridial Myonecrosis)

Pathogenesis:

Traumatic wound with soil/fecal contamination
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C. perfringens spores germinate in anaerobic, ischemic tissue
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Produces ALPHA TOXIN (lecithinase/phospholipase C) [MAJOR VIRULENCE]
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Also: collagenase, hyaluronidase, DNase, theta toxin (perfringolysin O)
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Alpha toxin hydrolyzes membrane phospholipids
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Destruction of RBCs (hemolysis), muscle cells (myonecrosis)
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Gas production (H2, CO2) in tissues --> Crepitus
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Spreading necrosis, toxemia, systemic shock, DEATH

• Severe pain at wound site (earliest symptom)
• Crepitus on palpation (gas in tissue)
• Brown/bronze discoloration of skin
• Foul-smelling serous discharge ("dishwater" exudate)
• Systemic toxemia: tachycardia, hypotension, jaundice

Laboratory Diagnosis of Gas Gangrene:

• Large, rectangular, "box-car" shaped Gram-positive rods
• Few or NO pus cells (toxin kills neutrophils)
• Absence of spores (in tissue specimens)

• Robertson's cooked meat medium (anaerobic)
• Blood agar (anaerobic): Double zone hemolysis (inner complete + outer partial)
• Egg yolk agar: Nagler reaction positive (opalescent zone around colonies)

• Half of egg yolk agar plate treated with anti-alpha toxin
• C. perfringens produces opalescence on untreated half only
• Inhibited on treated half = POSITIVE Nagler reaction

• "Stormy clot/fermentation" in litmus milk: acid + gas = "stormy fermentation" (classic)

• Lecithinase positive
• Lipase negative (differentiates from C. novyi)

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C. Nagler Reaction - Principle, Procedure, Use

Principle:

Procedure:

EGG YOLK AGAR PLATE
|___________________________|
| CONTROL SIDE | ANTITOXIN SIDE |
|              | (spread anti-alpha toxin) |
|              | Allow to absorb 30 min   |
|______________|___________________________|
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Inoculate C. perfringens across BOTH halves (single streak)
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Incubate anaerobically 37°C, 24-48 hrs
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RESULT:
Control side: Opalescent (pearly) zone around colonies
Antitoxin side: NO opalescence (inhibited)
= POSITIVE NAGLER REACTION

Use:

1. Presumptive identification of C. perfringens
2. Diagnosis of gas gangrene
3. Differentiating C. perfringens from other clostridia
4. Detecting lecithinase activity

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D. Immunoprophylaxis of Tetanus

Active Immunization (DPT/Td Schedule - India):

• Contains tetanus toxoid (formaldehyde-inactivated tetanospasmin)

Passive Immunization:

Wound Management (Post-exposure):

WOUND TYPE + IMMUNIZATION STATUS
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         |---> Clean, minor wound + >3 doses given --> NO prophylaxis needed
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         |---> Tetanus-prone wound (>6 hrs, crush, deep, dirty)
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                    |---> Immunized (>3 doses) --> TT booster if >5 years
                    |---> NOT immunized --> TT + HTIG (different sites)

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3. SALMONELLA

A. Salmonella Typhi - Enteric Fever - Laboratory Diagnosis

Specimen by Week:

WEEK 1: BLOOD CULTURE (most important, highest yield ~85-90%)
WEEK 2: BLOOD + URINE + WIDAL TEST
WEEK 3: STOOL + WIDAL TEST (peak titer)
WEEK 4: STOOL + URINE
Throughout: BONE MARROW (gold standard - 90% yield even after antibiotics)

Blood Culture (Week 1 - Best):

• 10-15 mL blood in 100 mL bile broth (1:10 ratio - dilutes bactericidal factors)
• Incubate 37°C, subculture on MacConkey and blood agar at 24, 48, 72 hrs
• Non-lactose fermenting (colorless/pale) colonies on MacConkey
• H2S producing (black center) colonies on SS agar
• Biochemical: TSI agar - Alkaline slant / Acid butt / H2S / Gas
• Final confirmation: Serotyping with O, H, Vi antisera

Widal Test (Agglutination Test):

• S. Typhi O (TO) antigen
• S. Typhi H (TH) antigen
• S. Paratyphi A H (AH)
• S. Paratyphi B H (BH)

• Significant titer: O ≥ 1:160, H ≥ 1:160 (in endemic area)
• Diagnostic (single sample): O ≥ 1:80 suggestive, H ≥ 1:160
• Four-fold rise in titer in paired samples (10-14 days apart) = definitive

• O antibody: Granular, compact, heavy clumps - NOT dispersed on shaking
• H antibody: Fluffy, cotton-wool clumps - DISPERSED on shaking

• False positive: Other febrile illnesses, previous typhoid vaccination, malaria
• False negative: Early disease, prior antibiotics
• Cross-reactions with other Salmonella species

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B. Laboratory Tests in FIRST WEEK of Enteric Fever:

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C. Co-Agglutination Test (CoA Test)

Protein A (on S. aureus) + Anti-Salmonella IgG (Fc binds to Protein A)
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Add patient's serum/urine/CSF (containing Salmonella antigen)
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Antigen-antibody reaction --> AGGLUTINATION = POSITIVE

• Detects S. Typhi antigen in blood, urine, CSF
• Useful in Week 1 when antibodies (Widal) not yet formed
• Can detect both Vi antigen and O antigen
• Fast (result in 3-5 minutes)
• More sensitive than Widal in early disease
• Useful when patient has received antibiotics (culture may be negative)

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4. NEISSERIA - Non-Gonococcal Urethritis (NGU/NIGU)

Causative Organisms:

Clinical Features:

• Dysuria, urethral discharge (mucoid/mucopurulent, less than gonorrhea)
• Discharge less profuse and less purulent than gonococcal urethritis
• May be asymptomatic in males (especially Chlamydia)
• In women: cervicitis, PID, infertility

Diagnosis:

• Urethral swab: >5 PMNs/HPF (high power field) = urethritis
• No gram-negative intracellular diplococci (GNID) = NGU (vs. gonorrhea)
• NAAT (Nucleic Acid Amplification Test) for C. trachomatis/N. gonorrhoeae
• Culture: negative for gonococcus

Treatment:

• Azithromycin 1g single dose OR Doxycycline 100mg BD x 7 days
• Treat partner simultaneously

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5. STAPHYLOCOCCUS

A. Staphylococcal Wound Infection - Laboratory Diagnosis

Specimen: Pus/wound swab

Steps:

• Gram-positive cocci in clusters ("grape-like bunches")
• PMNs present (pus cells)
• No spores, no capsule

• Blood agar: Golden/cream-yellow colonies, beta hemolysis (zone of clearing)
• MacConkey agar: Small pink/colorless colonies (ferments lactose weakly)
• Mannitol salt agar (selective): S. aureus ferments mannitol --> YELLOW colonies; others = red colonies
• Chapman's medium: Same principle as mannitol salt agar

• Slide coagulase: Detects bound coagulase (clumping factor) - fast screening
• Tube coagulase: Detects free coagulase - definitive (clot in tube in 1-4 hrs)

• Kirby-Bauer disc diffusion
• Check for MRSA (Methicillin-Resistant S. aureus) - use oxacillin/cefoxitin disc
• MIC testing for serious infections

• Epidemiological tracing of hospital outbreaks

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B. S. aureus - Four Important Diseases

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C. Staphylococcal Food Poisoning

• Heat stable (survive 100°C for 30 min - unlike the bacteria)
• Not destroyed by reheating food - key epidemiological point

Contaminated food left at room temperature (20-37°C) for >4 hours
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S. aureus multiplies and produces ENTEROTOXIN (preformed)
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Toxin ingested (bacteria may die, but toxin remains)
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Toxin acts on vagal afferents in gut --> stimulates vomiting center
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RAPID ONSET (1-6 hours): profuse vomiting, nausea, cramps, diarrhea
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Self-limited (12-24 hours), NO FEVER (since no invasion)

1. Short incubation (1-6 hours) - shortest of all food poisonings
2. No fever (toxin-mediated, not infection)
3. Vomiting predominates over diarrhea
4. Self-limiting (12-24 hours)

• Clinical (short incubation + no fever + vomiting)
• Culture food and patient's stool/vomit for S. aureus
• Phage typing of isolates from food and patient
• Detect enterotoxin by ELISA/RIA in food

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6. CORYNEBACTERIUM DIPHTHERIAE

A. Pathogenicity of Diphtheria

Mechanism of Toxin Action:

Tox gene (bacteriophage beta) --> Diphtheria toxin (2 fragments: A and B)
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Fragment B: BINDS to heparin-binding EGF receptor on host cell
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Toxin-receptor complex internalized
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Fragment A: ACTIVE unit - ADP-ribosylates EF-2 (Elongation Factor 2)
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Inhibits protein synthesis IRREVERSIBLY
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CELL DEATH in heart, nerves, adrenal glands, kidneys

Local Disease (Throat):

• Toxin + bacteria --> necrosis of epithelial cells
• Fibrinous exudate + necrotic cells + RBCs + WBCs form PSEUDOMEMBRANE
• Pseudomembrane: grayish-white, tough, adherent, bleeds on removal ("leathery")
• Located on tonsils/pharynx; can extend to larynx (croup, airway obstruction)

Systemic Effects (Toxin spread via bloodstream):

1. Myocarditis (most serious complication) - occurs in Week 2-3; arrhythmias, heart block
2. Neuropathy - palatal palsy first (nasal regurgitation), then ciliary paralysis, then peripheral neuritis
3. Adrenal hemorrhage - rare

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B. Metachromatic Granules (Volutin Granules/Babes-Ernst granules)

• Composed of polymetaphosphate (storage form of phosphate)
• Stain red/violet with blue dyes (metachromasia)
• Located at poles of the cell (POLAR GRANULES)

1. Albert's stain: Granules stain blue-black, rest of cell green
2. Neisser's stain: Granules stain brown-black, cell body yellow
3. Ponder's stain / Toluidine blue: Granules appear red against blue background

• Characteristic of C. diphtheriae
• Helps differentiate from C. pseudodiphtheriticum (no granules)
• Also seen in C. xerosis but less prominent

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C. Toxigenicity Tests

1. Elek's Immunodiffusion Test (In vitro - Gold Standard)

Tellurite blood agar plate:
|__________________________________|
|       ← Antitoxin paper strip →  |
|  TEST STRAIN  |  POSITIVE CONTROL |
|  streaked     |  streaked         |
|  perpendicular to strip           |
|___________________________________|
Incubate 37°C, 48 hrs

POSITIVE: White precipitin line of identity between test and control
NEGATIVE: No precipitin line

2. Guinea Pig Virulence Test (In vivo)

• Inject test culture filtrate into one guinea pig + culture filtrate + antitoxin into another
• Toxin-producing strain: first GP dies (necrosis); second GP (protected by antitoxin) survives

3. PCR

• Detects tox gene directly - most specific and sensitive
• Used in reference laboratories

4. Cell culture cytotoxicity test

• Cell lines (Vero/HeLa) inoculated with toxin; cytopathic effect (CPE) observed

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7. VIBRIO CHOLERAE

A. Gardner and Venkataraman's Classification

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B. Classical vs. El Tor Vibrios - Differences

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C. Laboratory Diagnosis of Cholera

1. Direct Examination:

• Wet film (Hanging drop preparation): "Shooting star" / darting motility of vibrios
• Gram stain: Gram-negative curved "comma-shaped" rods
• Dark field microscopy: Rapid, reveals characteristic motility

2. Culture:

• TCBS (Thiosulfate Citrate Bile Salt Sucrose) agar: Selective; V. cholerae = yellow sucrose-fermenting colonies
• Alkaline peptone water (APW) pH 8.6: Enrichment medium (vibrios prefer alkaline); vibrios grow as surface pellicle in 6-8 hrs
• GYNB (Gelatin Yeast extract NaCl Broth): Alternative enrichment

3. Serology (Confirmation):

• Slide agglutination with Poly O1 antiserum --> agglutination = V. cholerae O1
• Further type with Ogawa and Inaba monospecific antisera
• If non-agglutinating: test with O139 antiserum

4. Biochemical Tests:

• Oxidase: POSITIVE
• String test: POSITIVE (bacteria suspended in 0.5% sodium deoxycholate forms viscous string)
• TSI: Alkaline slant/Acid butt, no H2S, no gas
• Cholera Red reaction (Indole + nitroso compound): Pink-red color

5. Rapid Tests:

• Dipstick (crystal violet cholera dipstick): Field use
• Co-agglutination, immunofluorescence

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D. Halophilic Vibrios

1. Vibrio parahaemolyticus

• Requires 2-4% NaCl
• Found in coastal sea water, seafood (crabs, prawns, oysters)
• Causes: Explosive watery diarrhea (seafood gastroenteritis), occasionally dysentery
• Kanagawa Phenomenon: Virulent strains produce Thermostable Direct Hemolysin (TDH) which causes beta-hemolysis on Wagatsuma agar (human blood agar) but NOT on sheep blood agar = Kanagawa POSITIVE = virulent

2. Vibrio vulnificus

• Requires NaCl for growth
• Found in warm coastal waters, raw oysters
• Causes: Severe wound infections (exposure to seawater), primary septicemia (oyster ingestion in liver disease patients)
• High mortality (>50%) in immunocompromised/liver disease patients

Kanagawa Phenomenon:

V. parahaemolyticus strains
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        |---> Kanagawa POSITIVE (TDH producing)
                 | Beta-hemolysis on Wagatsuma agar (human RBCs)
                 | Associated with HUMAN DISEASE
                 | Virulent
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        |---> Kanagawa NEGATIVE (no TDH)
                 | No hemolysis on Wagatsuma agar
                 | Environmental strains
                 | Not virulent for humans (usually)

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8. CHLAMYDIA TRACHOMATIS

A. Four Diseases Caused by C. trachomatis

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B. Laboratory Diagnosis of Trachoma (most common/exam-asked)

• Intracytoplasmic inclusion bodies: Halberstaedter-Prowazek (HP) bodies
• Blue-staining mass in cytoplasm of conjunctival epithelial cells
• Inclusion bodies cap the nucleus

• Inclusions stain brown (glycogen-containing matrix)
• Specific for C. trachomatis (unlike C. psittaci and C. pneumoniae which do NOT contain glycogen)

• McCoy cells (irradiated) or HeLa 229 cells
• Inoculate with specimen, centrifuge to enhance infectivity
• Stain with iodine or immunofluorescence after 48-72 hours
• See intracytoplasmic inclusions

• Fluorescent-labeled monoclonal antibody against MOMP (Major Outer Membrane Protein)
• Elementary bodies (EBs) fluoresce apple-green
• Rapid, sensitive

• Detects chlamydial LPS antigen
• Less sensitive than DIF but useful for screening

• PCR, LCR (Ligase Chain Reaction), SDA
• Can use urine, cervical/urethral swabs
• Standard of care for genital chlamydia

• Micro-immunofluorescence (MIF): Detects type-specific antibodies
• Complement fixation: Genus-specific; useful for LGV (>1:64 diagnostic)

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C. Chlamydia vs. Virus - Differences

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Chlamydia Developmental Cycle (Diagram):

ELEMENTARY BODY (EB) - Infectious form
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        | Enters host cell by endocytosis
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RETICULATE BODY (RB) - Metabolically active, non-infectious
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        | Multiplies by binary fission (6-8 times)
        | inside phagosome (resists lysosomal fusion)
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INCLUSION body forms (collection of RBs, then condenses to EBs)
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        | Cell lyses
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Mature EBs released --> infect new cells
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(Cycle = 48-72 hours)

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Quick Comparison Tables for Exam

Non-Suppurative Sequelae Summary:

Classical vs. El Tor vs. O139:

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Source: Murray PR et al. Medical Microbiology 9e; Jawetz Melnick & Adelberg Medical Microbiology 28e. Apurb Shastri MUHS Pattern presentation.

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