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2nd MBBS MICROBIOLOGY - MUHS EXAM ORIENTED NOTES

(Apurba Shastri Reference | All High-Priority Topics)

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TOPIC 1: MYCOBACTERIUM TUBERCULOSIS & PULMONARY TUBERCULOSIS

A. Morphology of M. tuberculosis

• Shape & Size: Slender, straight or slightly curved rods, 1-4 µm × 0.3-0.6 µm
• Gram stain: Gram-positive (weakly; practically not useful)
• Special stain: Acid-fast bacilli (AFB) - due to high mycolic acid content in cell wall
  • Ziehl-Neelsen (ZN) stain: Appears red (magenta) against blue background using carbol fuchsin
  • Auramine-Rhodamine (fluorescent stain): Appears yellow-green fluorescence - more sensitive than ZN
• Non-motile, Non-sporing, Non-capsulated
• Arrangement: Singly or in small clumps; may show "cord factor" (serpentine cords) - virulence factor = trehalose dimycolate

ZN STAIN MNEMONIC: "Red Rods on Blue Background" = AFB POSITIVE

B. Cultural Characteristics of M. tuberculosis

Media:

1. Lowenstein-Jensen (LJ) Medium - most commonly used (egg-based, inspissated)
   • Colonies appear in 3-8 weeks
   • Colony: Rough, dry, wrinkled, buff-colored, cauliflower-like = "Eugonic growth"
   • Inhibitory agent: Malachite green (inhibits other bacteria)
2. Middlebrook 7H10/7H11 agar - transparent medium (detects colonies early, ~3 weeks)
3. Middlebrook 7H9 broth - liquid medium
4. Pyruvate egg medium (Dorset) - M. bovis grows better (dysgonic)
5. Petragnani medium - for contaminated specimens

Key Cultural Tests:

• Niacin test: M. tuberculosis = POSITIVE (accumulates niacin) - differentiates from other mycobacteria
• Nitrate reduction: POSITIVE
• Catalase test: Positive at room temperature, negative at 68°C (heat-labile catalase)
• TCH (Thiophene-2-carboxylic acid hydrazide): M. tuberculosis = Resistant (grows); M. bovis = Sensitive

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C. Pathogenesis of Pulmonary Tuberculosis

Primary (Ghon's) Complex:

Inhalation of droplet nuclei (1-5 µm, contain 1-3 bacilli)
           ↓
Deposited in alveoli (middle/lower lobe)
           ↓
Phagocytosed by alveolar macrophages
           ↓
Bacilli survive INSIDE macrophages (inhibit phagolysosome fusion)
           ↓
Cell-mediated immunity develops in 2-8 weeks
           ↓
Granuloma formation (Tubercle/Granuloma):
  - Central caseous necrosis (cheese-like)
  - Surrounded by epithelioid cells, Langhans giant cells
  - Peripheral lymphocytes, fibroblasts
           ↓
GHON'S FOCUS = Lung lesion (subpleural, lower part of upper lobe)
GHON'S COMPLEX = Ghon's focus + Hilar lymph node enlargement

Fate of Primary Complex:

1. Healing (most common) - calcification - Ranke complex
2. Progressive primary TB - in immunocompromised
3. Post-primary (reactivation) TB - occurs after years of latency

Post-Primary TB:

• Occurs in apex of lung (high O2, low lymphatic drainage)
• Cavity formation - bacilli reach thousands/mL in cavity wall
• Spread: bronchogenic, lymphatic, hematogenous
• Miliary TB - hematogenous spread → multiple tiny foci in all organs

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D. Laboratory Diagnosis of Pulmonary Tuberculosis

Specimens:

• Sputum (3 specimens: spot-morning-spot), BAL, Gastric lavage (children), pleural fluid, CSF, urine, tissue biopsy

Step-by-Step Diagnosis:

• Minimum bacilli required for smear positivity: 5,000-10,000 bacilli/mL
• Grading (RNTCP/WHO):
  • Scanty: 1-9 AFB/100 fields
  • 1+: 10-99 AFB/100 fields
  • 2+: 1-10 AFB/field
  • 3+: >10 AFB/field

• Proportion method (LJ) - standard
• MGIT-based DST - rapid
• Minimum inhibitory concentration (MIC)

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RECENT ADVANCES IN TB DIAGNOSIS:

• Detects M. tuberculosis DNA AND rifampicin resistance (rpoB gene mutation) simultaneously
• Result in 2 hours
• Sensitivity ~88%, Specificity ~99%
• Recommended as first-line test by WHO/RNTCP for:
  • All presumptive TB cases
  • HIV-associated TB
  • Drug-resistant TB suspects
• Cartridge = automated PCR (hemi-nested real-time PCR)

• Detects resistance to Rifampicin (rpoB) and Isoniazid (katG, inhA) = First-line LPA
• GenoType MTBDRsl = Second-line LPA (detects fluoroquinolone, aminoglycoside resistance)
• Result in 24-48 hours

• Comprehensive resistance profiling
• Epidemiological typing (molecular epidemiology)

• Detects latent TB infection (LTBI)
• Types:
  • QuantiFERON-TB Gold In-Tube (QFT-GIT)
  • T-SPOT.TB (ELISpot)
• Antigen used: ESAT-6 and CFP-10 (encoded in RD1 region - absent in BCG strains)
• Advantages over TST (Mantoux): Not affected by BCG vaccination, more specific
• DOES NOT diagnose active TB

• Inject 5 TU PPD (Purified Protein Derivative) intradermally
• Read at 48-72 hours
• Positive: Induration ≥10 mm (≥5 mm in HIV/immunosuppressed)
• Indicates past exposure or LTBI (does NOT confirm active disease)

• Truenat MTB/MTB Plus - chip-based PCR (India-made, point of care)
• Loop-mediated isothermal amplification (LAMP)
• 16S rRNA gene sequencing - identify NTM species

• ADA (Adenosine Deaminase) in pleural/CSF fluid - suggests TB
• LAM (Lipoarabinomannan) urine test - for HIV+TB (Alere Determine TB LAM)

MNEMONIC FOR TB LAB DIAGNOSIS: "SMCG-RIND"
S = Smear (ZN / Fluorescent)
M = Molecular (GeneXpert/CBNAAT/LPA)
C = Culture (MGIT/LJ)
G = IGRA (QuantiFERON/T-SPOT)
R = Radiological (CXR - cavities, infiltrates)
I = Immunological (Mantoux)
N = NAATs (Truenat, LAMP)
D = DST (Drug Susceptibility Testing)

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TOPIC 2: SPIROCHETES - CLASSIFICATION, SYPHILIS DIAGNOSIS

A. Classification of Spirochetes

Morphology of T. pallidum:

• Thin, delicate, tightly coiled spirochete; 6-20 µm × 0.09-0.18 µm
• 8-20 regular coils (regular, rigid coils = distinguishes from Borrelia which has irregular coils)
• Cannot be stained by ordinary stains
• Seen by: Dark-field microscopy, silver impregnation (Fontana's/Levaditi), direct immunofluorescence
• Motility: Corkscrew (to-and-fro + rotation) motion

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B. Primary Stage Syphilis - Laboratory Diagnosis

Primary Chancre:

• Hard, painless, indurated ulcer at site of inoculation (incubation: 10-90 days, mean 21 days)
• Seronegative in early primary syphilis!

Laboratory Tests for Primary Syphilis:

• Scrape the base of the chancre
• Place on slide with saline, cover slip
• Examine under dark-field microscope
• T. pallidum: bright, highly motile spirochetes with corkscrew motion
• Must examine FRESH specimen within 20 min
• Limitation: cannot be used for oral lesions (commensal treponemes present)

• Uses fluorescent-labeled anti-T. pallidum antibody
• More specific than DFM (works even on oral lesions)

• Highly sensitive and specific
• Not routinely available

• VDRL/RPR may be negative
• Treponemal tests become positive slightly later

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C. Serological Diagnosis of Syphilis

Classification of Tests:

SYPHILIS SEROLOGY
│
├── NON-TREPONEMAL (Non-specific/Reagin tests)
│   ├── VDRL (Venereal Disease Research Laboratory)
│   └── RPR (Rapid Plasma Reagin)
│
└── TREPONEMAL (Specific tests)
    ├── TPHA (T. pallidum Haemagglutination Assay)
    ├── TPPA (T. pallidum Particle Agglutination)
    ├── FTA-ABS (Fluorescent Treponemal Antibody-Absorbed)
    ├── TPI (T. pallidum Immobilization test) - gold standard, rarely used
    ├── EIA/ELISA (Enzyme Immunoassay)
    └── Western Blot

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NON-TREPONEMAL TESTS:

1. VDRL (Venereal Disease Research Laboratory Test)

• Patient's inactivated serum (heated 56°C × 30 min) + VDRL antigen
• Observe for flocculation (visible clumping)
• Qualitative (reactive/non-reactive) and quantitative (titer: 1:2, 1:4, 1:8...)

• Acute BFP (<6 months): Malaria, viral infections (EBV, hepatitis, varicella), pregnancy
• Chronic BFP (>6 months): SLE, leprosy, IV drug use, old age, autoimmune diseases

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2. RPR (Rapid Plasma Reagin)

• Unheated plasma + RPR antigen card
• Rotate, observe macroscopic agglutination

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TREPONEMAL (SPECIFIC) TESTS:

3. TPHA (Treponema pallidum Haemagglutination Assay)

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4. FTA-ABS (Fluorescent Treponemal Antibody-Absorbed)

1. Patient serum absorbed with Reiter treponema (non-pathogenic) to remove cross-reactive antibodies
2. Absorbed serum placed on slide with killed T. pallidum (Nichols strain)
3. Fluorescent anti-human IgG added
4. Examine under fluorescence microscope

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5. TPPA (T. pallidum Particle Agglutination)

• Similar to TPHA but uses gelatin particles instead of RBCs
• More stable, less non-specific reactions than TPHA

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6. EIA/ELISA for Syphilis

• Detects IgG and/or IgM
• Reverse algorithm (CDC 2011): Start with EIA (treponemal) as screening → confirm positive with VDRL/RPR → if discordant → TPHA/TPPA
• High throughput, automated

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Comparison Table: VDRL vs TPHA/FTA-ABS

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Congenital Syphilis Diagnosis:

• IgM FTA-ABS - detects baby's own IgM (IgG crosses placenta from mother)
• CSF-VDRL for neurosyphilis

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TOPIC 3: CHLAMYDIAE - CLASSIFICATION, PATHOGENESIS, DIAGNOSIS

A. Classification

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B. Unique Biology (Key for Exams)

Two-stage Life Cycle:

ELEMENTARY BODY (EB)            RETICULATE BODY (RB)
- Small (0.3 µm)                - Large (1 µm)
- Dense, resistant              - Non-infectious
- INFECTIOUS form               - Metabolically ACTIVE
- Does NOT multiply             - Multiplies by binary fission
- Adapted for extracellular     - Intracellular (in vacuole)
  survival                        "metabolic parasite"
        ↓                               ↑
   Attaches to host cell        Converts back to EB
   → Endocytosis               → Cell lysis → released EBs

• Obligate intracellular parasites (lack ATP-generating enzymes - "energy parasites")
• Cannot be seen by Gram stain; stained by Giemsa or Castaneda stain
• Inclusion bodies (RBs packed in vacuole) stained by iodine (C. trachomatis has glycogen inclusions → iodine-positive)

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C. Pathogenesis of Chlamydial Infections

C. trachomatis (Genital, Serovars D-K):

Exposure to infected secretions
           ↓
EB attaches to columnar/transitional epithelium
(cervix, urethra, conjunctiva, rectum)
           ↓
Endocytosis → phagosome (chlamydial vacuole)
           ↓
EB → RB (escape phagolysosome fusion)
           ↓
RB multiplies (18-24h) → Back to EBs
           ↓
Cell lysis → spread
           ↓
Host immune response (CD4 Th1, IFN-γ, TNF-α)
→ Inflammation → Scarring (fibrosis)

• MOMP (Major Outer Membrane Protein) - attachment, immune target
• Plasmid - virulence, cytokine induction
• Heat shock proteins (HSP60) - trigger hypersensitivity reactions → scarring in PID, trachoma

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D. Complications

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E. Laboratory Diagnosis of Chlamydial Infections

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TOPIC 4: ENTEROBACTERIACEAE - CLASSIFICATION & ENTERIC FEVER

A. Classification of Enterobacteriaceae

ENTEROBACTERIACEAE
│
├── Lactose Fermenters (COLIFORM GROUP)
│   ├── Escherichia coli
│   ├── Klebsiella spp. (K. pneumoniae, K. oxytoca)
│   ├── Enterobacter spp.
│   ├── Citrobacter spp.
│   └── Serratia marcescens
│
├── Slow/Late Lactose Fermenters
│   └── Hafnia alvei, Arizona, Yersinia
│
└── Non-Lactose Fermenters (NLF)
    ├── Salmonella spp.
    ├── Shigella spp.
    ├── Proteus spp.
    ├── Yersinia spp.
    └── Edwardsiella

• Gram-negative rods
• Oxidase NEGATIVE (key!)
• Catalase positive
• Reduce nitrates to nitrites
• Ferment glucose (with or without gas)

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B. Salmonella - Diseases Caused

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C. Enteric Fever - Pathogenesis (DETAILED)

Ingestion of S. Typhi (infective dose: 10^5-10^8 organisms) in contaminated food/water
                    ↓
         Stomach (most killed by acid)
                    ↓
        Small intestine (terminal ileum)
                    ↓
   Penetrate intestinal epithelium (M cells over Peyer's patches)
                    ↓
   Taken up by subepithelial macrophages / Peyer's patches
   (Survive inside macrophages - inhibit phagolysosome fusion using Vi antigen)
                    ↓
   Mesenteric lymph nodes → Multiplication
                    ↓
          INCUBATION PERIOD: 7-21 days (average 14 days)
                    ↓
         PRIMARY BACTEREMIA (silent, transient)
         ↓ spreads via lymphatics → Thoracic duct → blood
                    ↓
         LIVER, SPLEEN, BONE MARROW (reticuloendothelial system)
         Multiplication in macrophages
                    ↓
         SECONDARY BACTEREMIA (sustained)
         = END OF INCUBATION / WEEK 1 symptoms
                    ↓
   Fever, headache, malaise, relative bradycardia (Faget's sign)
   Rose spots (bacteremic emboli in skin dermis)
                    ↓
         WEEK 2-3: Highest bacteremia
         Re-infection of Peyer's patches → Hypersensitivity reaction
         Necrosis → ULCERATION of Peyer's patches
                    ↓
         Complications: Intestinal perforation, hemorrhage
                    ↓
         WEEK 3-4: Healing (if treated) or complications

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D. Laboratory Diagnosis of Enteric Fever

Specimen by Week:

1. Blood Culture (Gold Standard - Week 1)

• 10 mL blood in 100 mL broth (1:10 ratio - dilutes bactericidal substances)
• Bile broth (ox bile) or BHI broth or automated BACTEC
• Incubate 37°C, subculture to MacConkey + Blood agar
• S. Typhi colonies: NLF on MacConkey (pale), H2S positive (black on DCA/XLD)
• Confirm: Biochemical (TSI, LIA, Urease -ve, IMViC), Serology (O, H, Vi antisera)

• Most sensitive even after antibiotics started
• Positivity ~90% throughout disease
• Used when blood cultures negative

2. Widal Test (Tube Agglutination Test)

• Serial dilutions of serum: 1:20, 1:40, 1:80, 1:160, 1:320, 1:640
• Mix with standardized Salmonella suspensions
• Incubate 37°C overnight (18-24h)
• Read: Last tube showing agglutination = titer

• Single titer: O ≥ 1:160, H ≥ 1:160 (in endemic areas) = Significant
• Non-endemic areas: O ≥ 1:80 may be significant
• Fourfold rise in paired sera = diagnostic (most reliable)

1. False positive: Previous vaccination (H rises), cross-reactions (other Salmonella), malaria, liver disease, RA
2. False negative: Early infection (antibodies not formed), antibiotic treatment
3. Cannot distinguish active from past infection
4. Not reliable in endemic areas (baseline titers high)

3. Stool/Urine Culture

• Done Week 2 onwards
• Enrichment: Selenite F broth (24h, 37°C)
• Selective agar: MacConkey, DCA (Deoxycholate Citrate Agar), XLD (Xylose Lysine Deoxycholate), Wilson-Blair (WB) agar
• WB agar: Black metallic sheen colonies (H2S + ferrous sulfate + bismuth sulfite)
• Confirmation: Biochemical + serotyping

4. Rapid/New Tests:

• Typhidot (IgM/IgG ELISA) - detects IgM (acute) and IgG (past)
• Typhidot-M - only IgM (more specific for acute)
• Tubex TF - detects anti-O9 antibody by inhibition assay (colorimetric)
• PCR - detects flagellin gene (fliC), Vi gene (viaB)
• Blood culture automation (BACTEC, BacT/ALERT) - most rapid culture method

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TOPIC 5: NON-TUBERCULOUS MYCOBACTERIA (NTM)

A. Classification (Runyon Classification)

Diseases caused by NTM:

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B. Buruli's Ulcer (M. ulcerans)

M. ulcerans enters through minor skin trauma
           ↓
Multiplies in subcutaneous tissue (cooler areas: 30-33°C)
           ↓
Produces MYCOLACTONE toxin (polyketide macrolide)
           ↓
Mycolactone effects:
  - Cytotoxic → coagulative necrosis of dermis, fat, fascia
  - Immunosuppressive → inhibits T cells, macrophage activation
  - Analgesic → PAINLESS ulcer (characteristic!)
           ↓
Massive subcutaneous necrosis with undermined edges
           ↓
Overlying skin breaks down → BURULI ULCER

• Painless nodule → ulcer with undermined edges
• Ulcer may be enormous, involving entire limb
• Necrotic base, no significant inflammation (due to mycolactone immunosuppression)
• Sites: lower limbs most common

1. ZN smear of swab/biopsy - AFB positive (clusters of bacilli in necrotic tissue)
2. PCR - IS2404 insertion sequence (most sensitive, confirmatory)
3. Culture - on LJ at 30-33°C (NOT 37°C - unlike M. tuberculosis!) - grows in 6-8 weeks
4. Histopathology - coagulative necrosis with ghost cells, minimal inflammation, AFB on ZN

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TOPIC 6: VIBRIO CHOLERAE - PATHOGENESIS & LABORATORY DIAGNOSIS

A. Morphology

• Gram-negative, comma-shaped (curved) rods - "like a comma"
• Single polar flagellum - single monotrichous - rapid darting motility
• Non-capsulate, non-sporing
• Facultative anaerobe

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B. Pathogenesis of Cholera

Ingestion of contaminated water/food
           ↓
Vibrios reach small intestine (overcome gastric acid if large dose, 
or hypochlorhydria, or with bicarbonate)
           ↓
COLONIZATION:
- TCP (Toxin Co-regulated Pili) - attachment to enterocytes
- Motility - penetrates mucus layer
- Mucinase, hemagglutinin - breach mucus barrier
           ↓
TOXIN PRODUCTION:
Cholera Toxin (CT / Choleragen):
- A-B Toxin (similar to LT of E. coli)
- B subunit (5 pentamers): BINDS to GM1 ganglioside on enterocyte
- A1 subunit: ADP-ribosylates Gs alpha protein
           ↓
Gs-α permanently ACTIVATED → Adenylyl cyclase permanently ON
           ↓
↑↑↑ cAMP (cyclic AMP)
           ↓
Activation of Protein Kinase A
           ↓
CFTR channel opens:
- ↑ Cl⁻ secretion into lumen
- ↑ Na⁺ and H₂O follow (osmosis)
- Inhibition of NaCl absorption (brush border)
           ↓
MASSIVE SECRETORY DIARRHEA
(no mucosal damage - intact epithelium)
           ↓
RICE-WATER STOOLS (watery, flecks of mucus = shed epithelial cells)
Profuse: up to 20L/day
           ↓
Dehydration, Hypokalemia, Metabolic Acidosis, Hyponatremia
           ↓
Shock, Renal failure, Death (if untreated)

• Classical (original)
• El Tor (current pandemic strain) - produces hemolysin, more resistant, Voges-Proskauer (VP) positive

• O1 (Ogawa, Inaba, Hikojima) - causes epidemic cholera
• O139 (Bengal) - non-O1, also causes epidemic cholera (1992 India/Bangladesh)
• Other non-O1 groups - only cause sporadic diarrhea

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C. Laboratory Diagnosis of Cholera

1. Direct Microscopy

• Dark-field or Phase contrast microscopy: Comma-shaped organisms with rapid shooting star or darting motility (characteristic)
• Adding specific O1 antiserum → Vibrio Immobilization Test (VIT) - motility stops if positive
• Gram stain: Gram-negative curved rods (fish in stream appearance in comma clusters)

2. Culture

3. Biochemical Tests

• Oxidase POSITIVE (unlike Enterobacteriaceae)
• Indole positive
• VP test: El Tor = POSITIVE (Classical = negative)
• String test (positive): Colony emulsified in 0.5% sodium deoxycholate → viscous string forms (indicates V. cholerae) - due to DNA release from lysed cells
• Haemagglutination: El Tor agglutinates chicken RBCs (Classical does not)

4. Serotyping

• Slide agglutination with anti-O1 polyvalent serum
• Sub-typing with Ogawa/Inaba antisera

5. Phage typing

• El Tor: sensitive to Mukerjee's phage group IV/V

6. Rapid Tests

• O1/O139 antigen detection (lateral flow assay) - rapid, point of care
• PCR - ctxA gene, tcpA gene - confirmation and epidemiology

MNEMONIC for V. cholerae lab dx: "DARK STRINGS"
D = Dark-field microscopy (darting motility)
A = APW enrichment (alkaline pH 8.6)
R = Rice-water stool specimen
K = Kill with antiserum (VIT)
S = TCBS (Selective agar) - Yellow colonies
T = Toxin (CT-ELISA, PCR ctxA)
R = Rapid agglutination (anti-O1 serum)
I = Indole positive, Oxidase positive
N = No lactose fermentation
G = GM1-ELISA (cholera toxin detection)
S = String test positive

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TOPIC 7: CORYNEBACTERIUM DIPHTHERIAE

A. Morphology

• Gram-positive, club-shaped rods (clavate ends - due to metachromatic granules at poles)
• Metachromatic (volutin/Babes-Ernst) granules - polyphosphate reserves; stain RED with methylene blue (metachromatically)
• Albert's stain: Bacilli appear green-blue, granules appear dark blue/black = diagnostic
• Neisser's stain: Granules - brown/yellow-black; body - yellowish
• Arrangement: "Chinese letter" or "cuneiform" arrangement (due to snapping division); also V, L, Y shapes
• Non-motile, Non-sporing, No true capsule
• Non-acid fast

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B. Cultural Characteristics

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C. Diphtheria Toxin - Mechanism

CORYNEPHAGE β (lysogenic bacteriophage) carries tox gene
           ↓
C. diphtheriae (lysogenized strain) produces Diphtheria Toxin (MW 62,000 Da)
           ↓
Toxin = Single polypeptide → 2 fragments:
  FRAGMENT B (Binding): Binds to host cell receptor (HB-EGF receptor)
           ↓
  FRAGMENT A (Active): Translocates into cytoplasm
           ↓
  ADP-ribosylation of EF-2 (Elongation Factor 2)
           ↓
  EF-2 inactivated → Protein synthesis ARRESTED
           ↓
  CELL DEATH (especially in heart, nerves, kidneys)

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D. Laboratory Diagnosis of Diphtheria

• Presumptive diagnosis
• Granules stain dark blue/black, bacilli green-blue
• Characteristic "Chinese letter" arrangement

• Loeffler's serum slope (rapid growth, granule demonstration)
• Blood tellurite (selective, 24-48h)
• Blood agar (hemolysis pattern)

• Filter paper strip soaked in antitoxin placed on agar
• Organisms streaked perpendicular to strip
• Toxin diffuses from colony; antitoxin diffuses from strip
• White precipitin line at 45° = toxin-antitoxin complex = TOXIGENIC
• Takes 24-48h

• Inject culture into guinea pig; protected animal gets antitoxin
• Unprotected guinea pig dies = toxigenic
• Historical reference test

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E. Four Bacteria Causing Sore Throat (Pharyngitis/Tonsillitis)

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TOPIC 8: ANAEROBES - DEFINITION & CLASSIFICATION

A. Definition

• Strict (obligate) anaerobes: Killed by even trace O₂ (e.g., Clostridium, Bacteroides)
• Aerotolerant anaerobes: Grow without O₂ but not killed by it (e.g., Lactobacillus)
• Microaerophiles: Need reduced O₂ (2-10%) (e.g., Campylobacter, Helicobacter)
• Facultative anaerobes: Grow with or without O₂ (e.g., E. coli, Staph)

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B. Classification of Clinically Important Anaerobes

GRAM-POSITIVE SPORE-FORMING RODS:

• Clostridium spp.
  • C. tetani (Tetanus)
  • C. perfringens (Gas gangrene, food poisoning)
  • C. difficile (Pseudomembranous colitis)
  • C. botulinum (Botulism)
  • C. septicum (Malignancy-associated sepsis)

GRAM-POSITIVE NON-SPORING RODS:

• Actinomyces israelii (Actinomycosis - "sulfur granules")
• Propionibacterium acnes (Acne, prosthetic device infections)
• Bifidobacterium spp.
• Eubacterium spp.
• Lactobacillus spp.

GRAM-POSITIVE COCCI:

• Peptostreptococcus (now Finegoldia, Anaerococcus) - abscesses, mixed infections
• Peptococcus niger

GRAM-NEGATIVE RODS:

• Bacteroides fragilis - most common clinical anaerobe; abdominal infections; resistant to penicillin (beta-lactamase, polysaccharide capsule)
• Prevotella melaninogenica - respiratory, oral infections
• Porphyromonas - periodontal disease
• Fusobacterium nucleatum - Vincent's angina, Lemierre's syndrome
• Fusobacterium necrophorum - Lemierre's syndrome (septic thrombophlebitis of jugular vein)

GRAM-NEGATIVE COCCI:

• Veillonella - oral flora, rarely pathogenic

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TOPIC 9: CLOSTRIDIUM & GAS GANGRENE

A. Gas Gangrene (Clostridial Myonecrosis)

1. C. perfringens type A (~80-90%)
2. C. novyi
3. C. septicum
4. C. histolyticum
5. C. bifermentans

Wound contaminated with soil/feces containing Clostridium spores
(traumatic injury, surgery, road traffic accident)
           ↓
Low redox potential in wound (devitalized tissue, foreign body,
ischemia, necrotic muscle) → favors germination
           ↓
Vegetative forms multiply → Produce TOXINS:
           ↓
ALPHA TOXIN (C. perfringens): MAJOR virulence factor
= Lecithinase C (Phospholipase C)
= Hydrolyzes lecithin (phosphatidylcholine) in cell membranes
→ Destroys RBCs, WBCs, platelets, capillary endothelium
→ Massive HEMOLYSIS + NECROSIS
           ↓
Other toxins: Beta (lethal), Theta (perfringolysin O - cholesterol-binding),
Kappa (collagenase), Mu (hyaluronidase), Nu (DNase), Lambda (protease)
           ↓
Tissue necrosis → Gas production (H2, CO2 from fermentation)
→ CREPITUS (crackling on palpation)
           ↓
Rapid spreading necrosis of muscle (myonecrosis)
           ↓
Systemic toxemia → Shock → Death (very rapid, within hours-days)

• Sudden onset of pain (out of proportion to wound)
• Wound: Swelling, bronze/dark discoloration, blisters, foul-smelling discharge
• Crepitus on palpation
• Gas in tissue seen on X-ray
• Systemic: High fever, tachycardia, shock, hemolytic jaundice
• Rapidly fatal if untreated

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B. Laboratory Diagnosis of Gas Gangrene

• Gram-positive large rods (boxcar-shaped), subterminal spores (oval)
• KEY FINDING: Absence of pus cells (neutrophils) despite severe infection (alpha toxin destroys WBCs)
• Gram stain diagnostic clue: "Fat gram-positive rods with NO PMNs"

• Egg yolk agar: contains lecithin
• One half: anti-alpha toxin serum applied
• Organism streaked across both halves
• Positive (C. perfringens): Turbid halo (opalescence) on uninhibited side ONLY (alpha toxin = lecithinase breaks lecithin)
• Protected side: No halo (antibody neutralizes)

• Stormy clot reaction in litmus milk (C. perfringens: gas + acid clotting in 4-6 hours)
• Sugar fermentation

• Muscle necrosis with ghost outlines, gram-positive rods in tissue, NO inflammatory cells

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C. Post-Operative Wound Infections - Organisms

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TOPIC 10: NEISSERIA MENINGITIDIS - MENINGITIS DIAGNOSIS

A. Morphology & Cultural Characteristics

• Gram-negative diplococci (like coffee beans facing each other)
• Kidney-bean shaped (concave facing surfaces)
• Intracellular in PMNs (diagnostic in CSF/smear)
• Capsulated (polysaccharide), pili, no flagella, non-motile

• Obligate aerobe
• Chocolate agar (heated blood agar) - optimal; lysed RBCs release X and V factors, growth factors for Neisseria
• Blood agar - also grows (unlike H. influenzae)
• Modified Thayer-Martin (MTM) medium - selective (VCN: Vancomycin + Colistin + Nystatin) - used for CSF when contamination expected
• CO₂ enriched atmosphere (5-10% CO₂ candle jar)
• Temperature: 37°C (cannot tolerate cold - CSF must be transported warm!)
• Oxidase POSITIVE
• Ferments glucose and maltose (unlike N. gonorrhoeae which ferments only glucose)

• Group A - Africa (meningitis belt), epidemic
• Group B - Europe, USA; most common in UK
• Group C - UK, USA sporadic
• Group W135 - Hajj pilgrims

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B. Laboratory Diagnosis of Bacterial Meningitis

CSF Examination:

• N. meningitidis: Gram-negative diplococci, intracellular (inside PMNs)
• Sensitivity: ~60-80% in bacterial meningitis
• Must examine IMMEDIATELY (organism dies rapidly in CSF at room temperature)

• Chocolate agar + Blood agar
• Incubate at 37°C in 5-10% CO₂ immediately
• Do NOT refrigerate - N. meningitidis is cold-sensitive
• Blood culture simultaneously
• Colonies: Small, grey, smooth, glistening, non-hemolytic

• Latex agglutination test (LAT): CSF + latex particles coated with antibodies against capsular antigens (A, B, C, H. influenzae b, S. pneumoniae, E. coli K1)
• Result in 15 min
• Useful when Gram stain negative and culture negative (e.g., after antibiotics)
• Quellung (capsule swelling) reaction with specific antiserum

• Most sensitive (can detect after antibiotics started)
• Detects N. meningitidis-specific genes (ctrA, porA)
• Serogroup determination by PCR

• N. meningitidis bacteremia (meningococcemia)
• Positive in ~50-80% before antibiotics

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TOPIC 11: NEISSERIA GONORRHOEAE

A. Morphology

• Gram-negative diplococci
• Kidney-bean/coffee-bean shaped, concave faces together
• Intracellular in PMNs (urethral smear - diagnostic in males ~95% sensitivity)
• Non-motile, Non-sporing
• Pili (fimbriae) - adhesion, antiphagocytic, antigenic variation
• Capsule: Minimal/absent
• Outer membrane proteins (OMP): Por (porin, type I/II), Opa (Opacity protein), Rmp (Reduction Modifiable Protein)

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B. Cultural Characteristics

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C. Pathogenicity / Virulence Factors

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D. Laboratory Diagnosis of Gonorrhoea

• Males: Urethral swab/discharge
• Females: Endocervical swab (NOT vaginal - commensal Neisseria present)
• Also: Rectal swab, pharyngeal swab, conjunctival swab, joint fluid (PID/DGI)

• Gram-negative diplococci, intracellular in PMNs
• Sensitivity in males (urethral smear): ~95% (highly diagnostic)
• Sensitivity in females (cervical smear): ~50-60% (less reliable - commensal Neisseria)
• Useful: Males with symptomatic urethritis (presumptive diagnosis, treat immediately)
• NOT useful: Pharyngeal/rectal specimens (commensal flora)

• Modified Thayer-Martin or chocolate agar
• 5-10% CO₂, 37°C, 24-48h
• Colonies: Oxidase positive (key - purple with Kovac's reagent)
• Oxidase test: Colonies turn pink → red → black-purple within 10-30 seconds = positive

• Sugar fermentation: Glucose ONLY (not maltose, sucrose, lactose)
• Oxidase: Positive
• Catalase: Positive
• DNase: Negative

• PCR, SDA (Strand Displacement Amplification), TMA
• Most sensitive and specific
• Can use: Urine (first-catch), vaginal swab, cervical swab, urethral swab
• Does NOT require viable organisms (no cold chain for specimen)
• Cannot test antibiotic susceptibility (limitation)
• Dual NAAT for GC + Chlamydia - recommended by UK/US guidelines

• Nitrocefin test (chromogenic cephalosporin) - turns pink/red if beta-lactamase present

• Disk diffusion or E-test on chocolate/GC agar
• Important due to rising resistance: PPNG (Penicillinase-Producing N. gonorrhoeae), TRNG (Tetracycline-Resistant), QRNG (Quinolone-Resistant), and now cephalosporin resistance (GyrA mutations)

MNEMONIC FOR N. GONORRHOEAE IDENTIFICATION:
"GONG ONLY FERMENTS GLUCOSE"
G = Gram-negative diplococcus
O = Oxidase POSITIVE  
N = Needs CO₂ (capnophile)
G = Grows on MTM (selective medium)
ONLY FERMENTS GLUCOSE (not maltose - distinguishes from N. meningitidis)

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QUICK REVISION TABLE: KEY DIFFERENTIATING FACTS

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Exam Tip (MUHS pattern): Questions marked *** are the most likely long answers. Always structure answers as: (1) Definition/Introduction, (2) Morphology/Classification, (3) Pathogenesis with diagram, (4) Lab Diagnosis step-by-step, (5) Recent Advances. This pattern scores maximum marks in MUHS written exams.