---

2nd MBBS MICROBIOLOGY - DETAILED ANSWERS (Q9-Q16)

---

Q9. SHIGELLA

Classification of Shigella

Note: Genetically, Shigella species are biogroups within E. coli, but historical names are retained.

---

Morphology and Biology

• Gram-negative, non-motile, non-spore-forming, non-capsulated rods (exception: S. sonnei has a capsule-like antigen)
• Facultative anaerobes, oxidase negative
• Non-lactose fermenters (colorless on MacConkey agar)
• Do NOT produce H₂S (unlike Salmonella)
• Highly infectious - as few as 10-100 organisms can cause disease

---

Infections Caused by Shigella

1. Shigellosis (Bacillary Dysentery) - the primary disease
2. Hemolytic Uremic Syndrome (HUS) - caused by S. dysenteriae type 1 via Shiga toxin
3. Toxic megacolon, intestinal perforation - rare complications
4. Reactive arthritis - post-infectious, especially in HLA-B27 individuals
5. Septicemia - rare; occurs in neonates, malnourished children

---

Pathogenicity of Shigella Dysentery

Step-by-step mechanism:

INGESTION (as few as 10-100 organisms)
        ↓
Reach large intestine (colon)
        ↓
Attach to M cells (microfold cells) in Peyer's patches
        ↓
Type III Secretion System injects IpaA, IpaB, IpaC, IpaD proteins
        ↓
Membrane ruffling → engulfment of bacteria
        ↓
Lyse phagosomal vacuole → replicate in HOST CELL CYTOPLASM
(Unlike Salmonella which stays in vacuole)
        ↓
Actin rearrangement → bacteria propel to adjacent cells
(cell-to-cell spread, evading immune clearance)
        ↓
Induce APOPTOSIS of macrophages → release of IL-1β
        ↓
Attract PMNs → destabilize intestinal wall
        ↓
Bacteria reach deeper epithelial cells
        ↓
MUCOSAL DESTRUCTION → blood + mucus + pus in stool

Role of Shiga Toxin (S. dysenteriae type 1):

• Structure: 1 A subunit + 5 B subunits
• B subunits bind to host cell glycolipid Gb3
• A subunit cleaves 28S rRNA of 60S ribosomal subunit
• Prevents binding of aminoacyl-tRNA → disrupts protein synthesis
• Results in: intestinal epithelial damage + glomerular endothelial damage (HUS)

---

Laboratory Diagnosis of Shigella Dysentery

Specimen:

• Fresh stool (mucoid/bloody portion), rectal swab
• Must be processed immediately or in transport media (Cary-Blair)

Steps:

• Wet mount: sheets of pus cells (PMNs), RBCs
• Gram stain: Gram-negative rods

• MacConkey agar: colorless (NLF) colonies
• DCA (Desoxycholate Citrate Agar): pale colonies
• XLD (Xylose Lysine Desoxycholate): pale pink colonies (no H₂S blackening, unlike Salmonella)
• SS agar (Salmonella-Shigella agar): small, colorless colonies

• Oxidase: negative
• Glucose: fermented (without gas)
• Lactose: NOT fermented
• H₂S: NOT produced
• Motility: Non-motile

• Slide agglutination with polyvalent antisera (Groups A, B, C, D)
• Then group-specific and type-specific sera

• Multiplex NAAT (PCR) - identifies Shigella in stool along with other enteric pathogens simultaneously

---

Q10. MYCOBACTERIUM TUBERCULOSIS

Infections Caused

1. Pulmonary tuberculosis (primary + post-primary)
2. Miliary tuberculosis (disseminated hematogenous spread)
3. TB meningitis
4. Renal/Genitourinary TB
5. TB lymphadenitis (scrofula)
6. Bone and joint TB (Pott's spine)
7. Intestinal TB
8. TB pericarditis, pleural effusion

---

Four Methods of Detection with Principles

1. MICROSCOPY (Acid-Fast Staining)

• Ziehl-Neelsen (ZN) stain - Hot method; carbolfuchsin with heat → decolorize with acid-alcohol → counterstain with methylene blue
• Kinyoun (cold) stain - concentrated carbolfuchsin, no heat
• Truant Fluorochrome stain - Auramine-rhodamine dyes; AFB fluoresce yellow-orange under UV; more sensitive; suitable for screening

---

2. CULTURE

• Löwenstein-Jensen (LJ) medium - egg-based; colonies appear in 4-8 weeks as rough, buff, dry "cauliflower-like" colonies; malachite green inhibits contaminants
• Middlebrook 7H10/7H11 - agar-based; faster than LJ
• BACTEC MGIT 960 (broth-based) - liquid medium with fluorescent sensor; O₂ consumption → fluorescence increases as bacteria grow; detects growth in 1-2 weeks vs 4-8 weeks for solid media

---

3. TUBERCULIN SKIN TEST (TST) / MANTOUX TEST

• ≥10 mm = positive in general population
• ≥5 mm = positive in HIV/immunocompromised
• ≥15 mm = positive in low-risk individuals

---

4. NUCLEIC ACID AMPLIFICATION TESTS (NAATs) / PCR

• Detects M. tuberculosis DNA within 2 hours
• Simultaneously detects rifampicin resistance (mutations in rpoB gene)
• Uses molecular beacon probes

---

Pathogenesis of Tuberculosis

INHALATION of droplet nuclei (<5 μm) containing M. tuberculosis
        ↓
Reach alveoli → phagocytized by ALVEOLAR MACROPHAGES
        ↓
MTB PREVENTS phagosome-lysosome fusion
(blocks EEA1 bridging molecule)
        ↓
Replicates intracellularly in macrophage

PRIMARY TUBERCULOSIS (1st infection):

        ↓
Bacteremia (early, silent) → spreads to lung apices, lymph nodes, kidneys, bones
        ↓
T-cell sensitization develops (2-8 weeks)
        ↓
Delayed hypersensitivity + Cell-mediated immunity activated
        ↓
Epithelioid cell granuloma formation (Ghon focus in lung)
        ↓
Ghon focus + hilar lymph nodes = GHON'S COMPLEX (PRIMARY COMPLEX)
        ↓
In most: heals by fibrosis/calcification
In 5%: Progressive primary disease (in immunocompromised)

POST-PRIMARY (SECONDARY) TUBERCULOSIS:

Reactivation (endogenous) or Reinfection (exogenous)
        ↓
Apex of upper lobe (high O₂) - classical location
        ↓
CASEOUS NECROSIS (cheese-like soft center)
        ↓
Liquefaction → CAVITY formation
        ↓
Bacilli discharged into bronchi → sputum positivity
        ↓
Spread to other lung areas + systemic dissemination

Key Virulence Factors of MTB:

---

Q11. SPIROCHETES

Infections Caused by Spirochetes

---

Leptospirosis - Pathogenesis

LEPTOSPIRES enter through skin/mucous membranes
        ↓
PHASE 1: LEPTOSPIREMIC PHASE (Days 1-7)
Bacteremia → leptospires in blood, CSF, aqueous humor
High fever, headache, myalgia (esp. calf muscles), conjunctival suffusion
        ↓
PHASE 2: IMMUNE/LEPTOSPIRURIC PHASE (Days 7-28)
Antibody formation → leptospires cleared from blood
BUT appear in URINE (leptospiruria)
Immune complexes → organ damage
        ↓
In severe cases (WEIL'S DISEASE / Ictero-haemorrhagic leptospirosis):
- LIVER: Hepatocellular damage → jaundice (deep yellow-green)
- KIDNEY: Acute tubular necrosis → renal failure
- LUNGS: Pulmonary hemorrhage
- Blood vessels: vasculitis → hemorrhage
- MUSCLE: Myositis → elevated CPK
- Uveitis (may persist for months)

• Outer membrane lipoproteins (LipL32, LipL41)
• Endotoxin-like LPS (atypical, poor pyrogenicity)
• Sphingomyelinase - hemolysin
• Motility (hooks at both ends) - invasiveness

---

Leptospirosis - Laboratory Diagnosis

Specimen (phase dependent):

Methods:

• Dark-field microscopy of fresh blood/urine: thin, tightly coiled spirochetes with hooked ends (like "question marks")
• Silver staining (Fontana's method) - blackens spirochetes
• NOTE: Unreliable in urine (artifacts common)

• Fletcher's semi-solid medium or EMJH (Ellinghausen-McCullough-Johnson-Harris) medium
• Incubate at 28-30°C for up to 13 weeks
• Growth appears as "Dinger's ring" (subsurface disc at 1 cm depth due to microaerophilic nature)

• Microscopic Agglutination Test (MAT) - Gold standard for serology
  • Patient serum + live leptospires from different serovars
  • Agglutination seen under dark-field microscope
  • Titre ≥1:100 (single sample) or 4-fold rise (paired sera) = significant
• ELISA - IgM ELISA (Panbio Leptospira kit) - detects early infection
• Macroscopic slide agglutination test - rapid, less specific

• PCR (real-time PCR) targeting 16S rRNA or LipL32 gene - most sensitive in leptospiremic phase

• Guinea pigs inoculated with patient blood/urine - develop jaundice, hemorrhage - classic (rarely done now)

---

Syphilis - Serological Diagnosis

A. NON-TREPONEMAL TESTS (Screening)

VDRL (Venereal Disease Research Laboratory Test)

1. Heat-inactivate patient serum (56°C, 30 min) - destroys complement
2. Mix with VDRL antigen on glass slide
3. Rotate at 180 rpm for 4 minutes
4. Read under microscope: clumping = reactive

1. Screening test for syphilis (primary, secondary, latent)
2. Monitoring treatment response (titre should fall 4-fold after treatment)
3. Diagnosis of neurosyphilis (CSF-VDRL - highly specific for CNS disease)
4. Antenatal screening
5. Blood donor screening
6. Epidemiological surveys

1. Simple, inexpensive, rapid
2. Quantifiable - used to monitor treatment
3. Becomes negative after successful treatment (unlike treponemal tests)
4. Suitable for large-scale screening
5. Can be done on CSF (for neurosyphilis)

1. Biological false positives (BFP): Non-treponemal tests cannot distinguish syphilis from other conditions causing cardiolipin antibodies:
   • Acute BFP (< 6 months): Viral infections (EBV, CMV, HIV, hepatitis), malaria, pregnancy
   • Chronic BFP (> 6 months): SLE, antiphospholipid syndrome, leprosy, IV drug abuse
2. Non-specific antigen (cardiolipin, not from Treponema)
3. False negative in late syphilis (titre falls)
4. Prozone phenomenon (excess antibody → no flocculation at undiluted serum; dilute and retest)
5. Requires confirmation with treponemal tests (FTA-ABS or TPHA)

B. TREPONEMAL TESTS (Confirmatory)

---

Q12. HAEMOPHILUS

X Factor and V Factor

This is why chocolate agar (heated blood agar) is used - heating releases V factor from RBCs and destroys inhibitors of V factor.

---

Satellitism (Satellite Phenomenon)

• S. aureus produces and secretes NAD (V factor) into the surrounding medium by its own beta-lysin activity
• The V factor diffuses into the agar around S. aureus colonies
• H. influenzae (which requires both X and V factors) can use the V factor secreted by S. aureus
• X factor (hemin) is already present in blood agar
• Therefore, H. influenzae grows well only around S. aureus colonies, forming satellite colonies
• Away from S. aureus, H. influenzae grows poorly or not at all

         Blood Agar Plate
    
    ┌─────────────────────────────┐
    │                             │
    │   ● ● ● ●                   │
    │  ● ●●●●● ●                  │
    │ ●●●[S.aureus]●●●            │← Staph aureus = large colony
    │  ● ●●●●● ●                  │
    │   ● ● ● ●                   │← Satellite colonies of H. influenzae
    │                             │← (grow only near S. aureus due to V factor)
    │        ·  ·  ·              │
    │     (no/poor H. influenzae  │← Distant areas - H. influenzae absent/small
    │      growth away from       │
    │       S. aureus)            │
    └─────────────────────────────┘
    
    ● = H. influenzae satellite colonies (dense ring around S. aureus)
    · = isolated sparse/no growth away from S. aureus

---

H. influenzae - Four Lesions (Clinical Diseases)

1. Meningitis
   • Most common cause of bacterial meningitis in children (pre-vaccine era), ages 3-18 months
   • Bacteremic spread from nasopharynx to meninges
   • Caused almost exclusively by type b encapsulated strains
   • Clinical: fever, stiff neck, bulging fontanelle, Kernig/Brudzinski signs
2. Epiglottitis
   • Children 2-4 years, peak pre-vaccine era
   • Cellulitis and swelling of supraglottic tissues
   • Presents as: sore throat, fever, drooling, muffled voice, stridor
   • "Cherry-red epiglottis" on lateral X-ray (thumb sign)
   • Can cause fatal airway obstruction - emergency!
3. Cellulitis
   • Reddish-blue (violaceous) patches on cheeks or periorbital area
   • Bacteremic spread; pediatric disease
   • Pathognomonic bluish hue (due to H. influenzae bacteremia)
4. Pneumonia + Otitis Media + Sinusitis
   • Caused by nontypeable (unencapsulated) H. influenzae
   • Pneumonia: primarily in elderly with COPD
   • Otitis media + sinusitis: most common in children (along with S. pneumoniae)
   • Also causes septic arthritis (single large joint, <2 years, pre-vaccine era)

---

Q13. E. COLI

Types of E. coli Causing Diarrhoea + One Lab Test for Each

---

Enterotoxigenic E. coli (ETEC) - Detailed

1. Colonization Factor Antigens (CFA/I, CFA/II, CFA/III) - fimbriae/pili that attach to small intestinal mucosa
2. Heat-Labile Toxin (LT-I):
   • Structure: 1 A subunit + 5 B subunits (similar to cholera toxin)
   • B subunits bind to GM1 ganglioside on intestinal epithelium
   • A subunit (A1 fragment) activates adenylate cyclase → ↑cAMP
   • Result: hypersecretion of Cl⁻, Na⁺, and water → secretory diarrhea
3. Heat-Stable Toxin (ST/Sta):
   • Small peptide; resists boiling (100°C, 30 min)
   • Binds to guanylate cyclase receptor → ↑cGMP
   • Result: inhibits NaCl absorption + stimulates fluid secretion

• Profuse, watery ("rice-water") diarrhea
• Vomiting, cramping, low-grade fever
• Self-limiting (3-5 days)

---

Q14. STREPTOCOCCUS PNEUMONIAE

Morphology

• Gram-positive, lancet-shaped diplococci (arranged in pairs, with pointed ends facing outward)
• Non-motile, non-spore-forming
• Capsulated (most important virulence factor) - >92 antigenic serotypes of polysaccharide capsule
• Alpha-haemolytic on blood agar (greenish zone due to H₂O₂ production)
• Bile-soluble (unique - differentiates from Streptococcus viridans)
• Optochin-sensitive (another key differentiating feature)

        ___   ___
       /   \ /   \
      |  ● | | ● |
       \___/ \___/
      [capsule surrounds the diplococcal pair]
      
      Lancet-shaped diplococci in pairs
      Pointy ends facing AWAY from the junctional pole

---

Cultural Characteristics

• Small, mucoid (smooth, shiny "draughtsman" colonies) - due to capsule
• Alpha (α) haemolysis - partial hemolysis, green discolouration around colony (viridans-type)
• In ageing cultures: autolysis occurs → colony centre collapses → "draughtsman colonies" (raised edge, depressed centre)

• Grows well; small grey mucoid colonies

---

Pneumococcal Vaccine

1. PCV (Pneumococcal Conjugate Vaccine)

• PCV10 / PCV13 / PCV15 / PCV20 (number = serotypes covered)
• Capsular polysaccharide conjugated to a carrier protein (diphtheria toxoid CRM197 or meningococcal protein D)
• T-cell dependent response → immunological memory; effective in infants < 2 years
• Schedule: 6 weeks, 10 weeks, 14 weeks + booster at 15-18 months (India IAP schedule)
• Prevents invasive pneumococcal disease (IPD), meningitis, bacteremia

2. PPSV23 (Pneumococcal Polysaccharide Vaccine)

• 23 valent pure polysaccharide vaccine
• T-cell INDEPENDENT response → no memory, not effective in < 2 years
• Given to: Adults >65 years, immunocompromised, asplenic patients, sickle cell disease, chronic cardiopulmonary disease
• Single dose; revaccination after 5 years in high-risk groups

• Community-acquired pneumonia (most common cause requiring hospitalization)
• Bacterial meningitis (most common cause in adults)
• Acute otitis media (most common cause in children)
• Sinusitis, mastoiditis
• Bacteremia, septicemia (especially in asplenic patients)
• Endocarditis, pericarditis, peritonitis (rare)

---

Q15. ATYPICAL MYCOBACTERIA (Non-Tuberculous Mycobacteria / NTM)

Runyon's Classification

SLOW GROWERS (> 7 days)

RAPID GROWERS (< 7 days)

---

Clinical Diseases Caused by NTM:

---

Q16. MYCOBACTERIUM LEPRAE

Morphology

• Gram-positive (weakly), strongly acid-fast bacilli (AFB)
• Intracellular, non-motile rods (3-10 μm long × 0.3-0.5 μm wide)
• Obligate intracellular parasite of macrophages and Schwann cells
• Cannot be cultured on artificial media (no in vitro culture available)
• Cultured in: footpad of mice (Shepard method, 1960) or nine-banded armadillo
• In lepromatous leprosy: seen in large clumps = "Globi" (packets of bacilli within macrophages = lepra cells / Virchow cells)
• "Cigarette bundle" arrangement - bacilli in parallel bundles

---

Tuberculoid vs Lepromatous Leprosy - Four Differences

---

Lepromin Test

Description:

• Dharmendra antigen - purified, partially soluble; gives early reaction
• Mitsuda antigen - crude integral bacillary suspension; gives late reaction

1. 0.1 mL lepromin injected intradermally on forearm
2. Read at two intervals:

Four Uses of Lepromin Test:

1. Prognostic value: Positive Mitsuda reaction indicates intact CMI → good prognosis (tuberculoid end); Negative → lepromatous end, poor prognosis
2. Classification of leprosy: Helps classify patient on the Ridley-Jopling spectrum:
   • TT (tuberculoid): strongly positive
   • BT: weakly positive
   • BB (borderline): weakly positive or negative
   • BL: negative
   • LL (lepromatous): strongly negative
3. Epidemiological surveys: To determine the proportion of population with natural immunity/sensitization to M. leprae in a community
4. NOT diagnostic: Lepromin test is NOT used for diagnosis of leprosy (it is positive even in BCG-vaccinated individuals and in non-leprosy populations who have had subclinical exposure). Positive in 50-80% of healthy adults.

Note: Unlike tuberculin test (Mantoux), lepromin test is NOT a diagnostic test. It only assesses cell-mediated immunity status.

---

Summary: Infections Caused by Organisms (All Topics)

---