Mycobacterium tuberculosis

Mycobacterium tuberculosis (the tubercle bacillus, discovered by Robert Koch in 1882) is the cause of tuberculosis (TB), still among the world's leading infectious killers and the single highest-yield organism in NEET PG microbiology, with overlapping questions in pathology, pharmacology, medicine and community medicine. These notes consolidate the staining, culture, immunology, clinical syndromes, diagnostics and chemotherapy you must own cold.

The organism — structure & classification

M. tuberculosis is a slender, slightly curved, non-motile, non-sporing, aerobic bacillus (0.2–0.6 × 1–10 µm). It belongs to the M. tuberculosis complex (MTC) which also contains M. bovis, M. africanum, M. microti, M. caprae and the vaccine strain M. bovis BCG.

The cell wall is the key to almost every exam fact. It is uniquely rich in lipids (~60% dry weight) including:

• Mycolic acids — long-chain (C60–C90) fatty acids responsible for acid-fastness, hydrophobicity and resistance to drying/disinfectants.
• Cord factor (trehalose-6,6'-dimycolate) — correlates with virulence; causes serpentine "cord" growth of virulent strains and inhibits neutrophil migration / mitochondrial respiration.
• Wax-D — adjuvant component, the active ingredient of Freund's complete adjuvant.
• Sulfatides (sulfolipids) — inhibit phagosome–lysosome fusion, aiding intracellular survival.
• LAM (lipoarabinomannan) — analogous to endotoxin; basis of the urinary LAM antigen test in HIV-TB.

High-yield: Acid-fastness is due to mycolic acid in the cell wall. M. tuberculosis is acid-fast AND modified acid-fast; it is weakly Gram-positive (often "ghost cells").

Feature	M. tuberculosis	M. bovis
Niacin production	Positive	Negative
Nitrate reduction	Positive	Negative
Oxygen requirement	Aerobic	Microaerophilic
Pyrazinamide susceptibility	Susceptible	Resistant (inherently)
Glycerol utilisation	Eugonic (luxuriant) growth	Dysgonic (poor) growth
Classic transmission	Inhalation (droplet nuclei)	Unpasteurised milk → GI/cervical TB

High-yield: M. bovis is intrinsically resistant to pyrazinamide — therefore M. bovis (and BCG-related) disease is treated without PZA.

Staining — Ziehl–Neelsen & beyond

The hallmark laboratory feature is acid-fastness: once stained with hot carbol fuchsin, the bacilli resist decolourisation by acid–alcohol.

Ziehl–Neelsen (ZN) hot method — stepwise:

Carbol fuchsin (with heat) → 20% sulphuric acid (decolouriser) → methylene blue (counterstain).

AFB appear bright red/pink rods against a blue background.

• Kinyoun stain = "cold" ZN (higher phenol concentration, no heat).
• Auramine–rhodamine fluorochrome stain → bacilli fluoresce golden-yellow under UV; more sensitive, used for screening high loads of slides; counterstain is potassium permanganate/acridine orange.
• Decolouriser for M. tuberculosis is 20–25% H₂SO₄; for weakly acid-fast organisms (Nocardia, M. leprae) only 1–5% H₂SO₄ is used (modified ZN).

High-yield: Smear positivity requires ≈ 10,000 bacilli/mL of sputum. ZN smear grading (RNTCP/NTEP) ranges from scanty to 3+. A smear is reported negative only after examining ≥ 100–300 fields.

Culture — the gold standard

Despite molecular tests, culture remains the gold standard for diagnosis and the only way to confirm phenotypic drug susceptibility and viability.

Medium	Type	Key facts
Lowenstein–Jensen (LJ)	Solid, egg-based	Most used; coagulated egg + mineral salts + glycerol + malachite green (selective, inhibits contaminants). Colonies in 2–8 weeks (slow growth, generation time ~15–20 h).
Middlebrook 7H10 / 7H11	Solid, agar-based	Transparent → earlier colony detection.
Middlebrook 7H9	Liquid	Used in automated systems.
MGIT / BACTEC	Liquid automated	Fastest culture (1–2 weeks); detects growth by O₂ consumption (fluorescence) or radiometric CO₂.

Colonies on LJ are classically dry, rough, raised, buff-coloured ("rough, tough and buff") with a cauliflower/breadcrumb appearance.

High-yield: LJ medium = standard solid culture; malachite green is the selective agent; MGIT (liquid) gives the fastest result. M. bovis shows dysgonic (sparse) growth on LJ because glycerol does not enhance its growth.

Pathogenesis & Koch's phenomenon

TB is acquired by inhaling droplet nuclei (1–5 µm) reaching alveoli. Bacilli are ingested by alveolar macrophages but survive by inhibiting phagosome–lysosome fusion (sulfatides). Tissue damage is immune-mediated (type IV hypersensitivity), not toxin-mediated — M. tuberculosis produces no classical exotoxin or endotoxin.

Sequence: inhalation → alveolar macrophage infection → lymphohaematogenous spread → CMI develops in 3–8 weeks (tuberculin conversion) → granuloma (tubercle) formation with caseous necrosis → containment (latent TB) or progression.

Koch's phenomenon describes the altered reaction of a previously infected/sensitised host to reinjected tubercle bacilli:

• Primary infection (naïve host): no immediate reaction; slow ulcer, regional node enlargement, dissemination — the Ghon (primary) complex.
• Reinfection (sensitised host): rapid (1–2 days) localised induration → necrosis/sloughing, little or no lymphatic/systemic spread — demonstrates acquired CMI but also tissue-destructive hypersensitivity.

High-yield: Ghon focus = subpleural parenchymal lesion (usually lower part of upper lobe / upper part of lower lobe). Ghon (primary) complex = Ghon focus + draining lymphangitis + hilar lymphadenopathy. A healed, calcified complex is the Ranke complex.

Clinical features

Pulmonary TB

• Post-primary (reactivation/secondary) TB classically affects the apical/posterior segments of the upper lobes (high O₂ tension favours the obligate aerobe).
• Symptoms: cough > 2 weeks, evening-rise low-grade fever, night sweats, weight loss/anorexia, haemoptysis.
• Cavitation, fibrosis, bronchiectasis, and aspergilloma (fungal ball in old cavity) are sequelae.

Extrapulmonary TB (EPTB)

• Lymph node TB (scrofula) — commonest EPTB, esp. cervical; matted nodes, cold abscess, sinus.
• Pleural effusion — exudative, lymphocyte-predominant, raised ADA (> 40 U/L), low glucose.
• Spinal TB (Pott's disease) — commonest skeletal site, thoracolumbar; gibbus deformity, cold (psoas) abscess.
• TB meningitis — basal meningitis, cranial nerve palsies; CSF: high protein, low glucose, lymphocytic pleocytosis, cobweb clot.
• Genitourinary TB — "sterile pyuria"; Miliary TB — millet-seed nodules from haematogenous dissemination, classic choroid tubercles on fundoscopy.

The tuberculin (Mantoux) test & IGRA

The Mantoux test uses 0.1 mL of 5 TU PPD (purified protein derivative) injected intradermally on the flexor forearm; read at 48–72 hours, measuring the transverse diameter of INDURATION (not erythema) in mm. It detects type IV hypersensitivity = prior exposure/sensitisation, NOT active disease.

Induration	Interpreted positive in…
≥ 5 mm	HIV, recent close contacts, organ transplant/immunosuppressed (≥ 15 mg/day prednisolone), fibrotic changes on CXR
≥ 10 mm	Recent immigrants from high-burden areas, IV drug users, healthcare workers, children < 4 yr, high-risk comorbidities (diabetes, silicosis)
≥ 15 mm	Persons with no risk factors

False negatives: miliary/overwhelming TB, HIV/immunosuppression, sarcoidosis, very young/very old, recent live vaccination, faulty technique. False positives: BCG vaccination, exposure to environmental NTM.

High-yield: IGRA (QuantiFERON-TB Gold, T-SPOT.TB) measures interferon-γ release to M. tuberculosis-specific antigens ESAT-6 and CFP-10 — these are absent in BCG and most NTM, so IGRA is not confounded by prior BCG. Like Mantoux, IGRA cannot distinguish latent from active TB.

Diagnosis — investigation of choice

High-yield (most-tested): The initial diagnostic test of choice under NTEP is now a rapid molecular NAAT — Cartridge-Based NAAT (CBNAAT / GeneXpert MTB/RIF) on sputum: detects M. tuberculosis AND rifampicin resistance in ~2 hours. Culture remains the gold standard; smear (ZN) is the cheapest screening test.

Diagnostic flow (presumptive pulmonary TB):

1. Two sputum samples (spot + morning) → CBNAAT/Truenat as upfront test.
2. If MTB detected + rifampicin-sensitive → start first-line ATT.
3. If rifampicin-resistant → presumed MDR → Line Probe Assay (LPA) + culture/DST + start MDR regimen.
4. CXR, ADA (effusions), histopathology (caseating granuloma with Langhans giant cells), MGIT culture as adjuncts.

• Truenat MTB/Rif — chip-based portable NAAT, point-of-care.
• LPA (Line Probe Assay / Hain test) — molecular DST: first-line LPA detects rpoB (rifampicin), katG & inhA (isoniazid); second-line LPA detects fluoroquinolone and aminoglycoside/injectable resistance.
• Urinary LAM — useful in advanced HIV (CD4 low).

Drug resistance — MDR / XDR / pre-XDR (memorise definitions)

Category	Definition
Mono/poly-resistant	Resistance to one (or several) first-line drugs but not both H and R
MDR-TB	Resistance to at least isoniazid (H) AND rifampicin (R)
RR-TB	Rifampicin resistance (with/without other resistance) — managed as MDR
Pre-XDR-TB	MDR/RR-TB plus resistance to any fluoroquinolone
XDR-TB (2021 WHO def.)	MDR/RR + FQ resistance + resistance to ≥1 Group A drug (bedaquiline or linezolid)

High-yield: Remember "MDR = H + R". Rifampicin resistance is the sentinel marker (rpoB gene) detected by CBNAAT, because isolated R-resistance is rare and usually signals MDR.

Management — first-line chemotherapy (DOC)

Standard regimen for drug-sensitive TB = 2 months intensive (HRZE) + 4 months continuation (HR) = total 6 months, given daily (NTEP now uses daily fixed-dose combinations, weight-band based).

HRZE → 2 months → HR → 4 months.

Drug	Key action	Signature toxicity / fact
Isoniazid (H)	Inhibits mycolic acid synthesis (needs katG activation)	Peripheral neuropathy (give pyridoxine/B6), hepatitis, SLE-like, gives B6 deficiency
Rifampicin (R)	Inhibits DNA-dependent RNA polymerase (rpoB)	Orange-red secretions, hepatotoxic, potent CYP450 inducer (fails OCPs, warfarin), flu-like syndrome
Pyrazinamide (Z)	Active at acidic pH in macrophages	Hyperuricaemia/gout, hepatotoxic; best sterilising drug early
Ethambutol (E)	Inhibits arabinosyl transferase (cell wall)	Optic neuritis (red-green colour blindness) — dose-related, reversible
Streptomycin	Aminoglycoside, blocks 30S	Ototoxic (vestibular), nephrotoxic; avoid in pregnancy

High-yield: Rifampicin is the most potent sterilising/bactericidal first-line drug and the key to short-course therapy; isoniazid is the most bactericidal against rapidly dividing bacilli. Pyridoxine (B6) co-prescribed with INH prevents neuropathy.

Drug-resistant TB (newer, all-oral regimen): The BPaLM regimen — Bedaquiline + Pretomanid + Linezolid + Moxifloxacin for 6 months — is now WHO-preferred for MDR/RR and pre-XDR-TB, replacing long injectable-based regimens. Bedaquiline inhibits mycobacterial ATP synthase (caution: QT prolongation).

Latent TB infection (LTBI) / TB preventive therapy: options include 6 months isoniazid (6H), 3 months weekly isoniazid + rifapentine (3HP), or 3 months daily HR (3HR).

BCG vaccine (quick capsule)

Live attenuated M. bovis strain (Calmette–Guérin), given intradermally over left deltoid at birth in India. Best protection is against disseminated childhood TB — miliary TB and TB meningitis; protection against adult pulmonary TB is variable. Causes Mantoux positivity (a key confounder distinguishing it from IGRA).

Complications

• Massive haemoptysis (Rasmussen aneurysm — eroded pulmonary artery in a cavity).
• Fibrosis, bronchiectasis, cor pulmonale, aspergilloma in old cavity.
• Empyema, bronchopleural fistula, laryngeal TB.
• Pott's spine → paraplegia; TBM → hydrocephalus, infarcts, SIADH.
• Drug toxicities: hepatitis (H, R, Z), optic neuritis (E), neuropathy (H).
• IRIS (immune reconstitution) after starting ART in HIV-TB co-infection.

Key differentials

• Pulmonary cavity: lung abscess, necrotising pneumonia (Klebsiella), Wegener's/GPA, squamous cell carcinoma, fungal (aspergillosis, histoplasmosis).
• Granuloma with caseation: TB is classic; non-caseating → sarcoidosis, Crohn's, berylliosis.
• Cervical lymphadenopathy: lymphoma, NTM (esp. children, M. avium), cat-scratch disease, metastasis.
• Acid-fast bacilli that are NOT MTB: M. leprae, Nocardia (partially), Rhodococcus, Legionella micdadei, Cryptosporidium/Cyclospora/Isospora oocysts (modified ZN).

High-yield: Caseating granuloma with Langhans giant cells (peripheral horseshoe nuclei) = histological hallmark of TB.

Mnemonics & eponyms

• First-line ATT = "RIPES" → Rifampicin, Isoniazid, Pyrazinamide, Ethambutol, Streptomycin.
• Toxicity recall: Red urine (Rifampicin), INH → neuropathy, PZA → uric acid (gout), Ethambutol → Eyes (optic neuritis), Streptomycin → ototoxic.
• Eponyms: Ghon focus/complex, Ranke complex (healed), Pott's spine, Rasmussen aneurysm, Simon focus (apical reactivation), Assmann focus (early subclavicular post-primary), Langhans giant cells, Rich focus (subependymal tubercle rupturing → TBM).

Recently asked / exam angle

• CBNAAT/GeneXpert as the upfront/initial diagnostic test detecting rifampicin resistance — repeatedly asked; do not confuse "gold standard (culture)" with "investigation of choice/initial (NAAT)".
• Updated XDR-TB (WHO 2021) definition involving bedaquiline/linezolid (Group A) — replace older "second-line injectable + FQ" definition.
• BPaLM regimen and bedaquiline mechanism (ATP synthase) — newer pharmacology favourite.
• IGRA antigens ESAT-6 & CFP-10 absent in BCG → not affected by vaccination.
• Mantoux: read induration at 48–72 h; 5/10/15 mm cut-offs by risk group.
• Cord factor = trehalose dimycolate (virulence); niacin test positive distinguishes MTB from most other mycobacteria; PZA resistance is intrinsic in M. bovis.
• LJ medium components and malachite green as selective agent — classic one-liner.

Rapid revision

1. Acid-fastness is due to mycolic acid; ZN uses carbol fuchsin → 20% H₂SO₄ → methylene blue.
2. LJ medium (egg-based, malachite green selective); colonies in 2–8 weeks, rough, tough, buff; MGIT liquid is fastest culture.
3. Niacin positive, nitrate positive for MTB; M. bovis is niacin negative and PZA-resistant.
4. Cord factor (trehalose-6,6'-dimycolate) = virulence; sulfatides block phagolysosome fusion.
5. Ghon complex = Ghon focus + lymphangitis + hilar nodes; healed = Ranke complex.
6. Post-primary TB → upper lobe apical/posterior segments (aerobe loves high O₂).
7. Mantoux: intradermal 5 TU PPD, read induration at 48–72 h; 5/10/15 mm cut-offs.
8. IGRA uses ESAT-6 & CFP-10, unaffected by BCG; neither IGRA nor Mantoux distinguishes latent from active TB.
9. CBNAAT/GeneXpert = initial test (detects R-resistance, rpoB); culture = gold standard.
10. MDR = H + R resistance; XDR (2021) = MDR + FQ + bedaquiline/linezolid resistance.
11. First-line ATT = 2 HRZE + 4 HR; Rifampicin → orange urine + CYP inducer; Ethambutol → optic neuritis; INH → neuropathy (give B6).
12. BCG (live M. bovis) protects best against childhood miliary TB and TB meningitis; BPaLM is preferred for MDR/RR-TB.