Lec 8: Mycobacteria and Tuberculosis (Last Lec Before Exam)

Mycobacteria: Overview

  • Tuberculosis (TB) is a leading cause of mortality from an infectious agent worldwide.

    • Death toll: 2 mill per year (approx.)

  • In the United States (US):

    • In 2021, 7{,}860 persons infected

    • Approx. 10\% will develop TB disease

    • Total infected in US: 13{ million}

  • Humans are the only natural reservoir for TB in many contexts.

  • Transmission: person-to-person spread by infectious aerosols (droplets nuclei).

  • Diagnosis relies on isolation of the organism from the host along with clinical presentation.

General characteristics of Mycobacteria

  • Morphology: Rod-shaped, non-spore-forming, non-encapsulated, acid-fast.

  • Complex cell wall with high lipid content.

  • Acid-fast bacilli (AFB).

  • Very slow growth: generation times of 12>20{ hours}

  • >150 species exist; only a few groups cause human disease.

Mycobacterium cell wall components

  • Complex, lipid-rich wall:

    • Arabinogalactan attached to murein

    • Mycolic acids: ~60% of wall

    • Wax D-glycolipid: ~15–20 mycolic acids

    • Cord factor: disaccharide glycolipid + 2 mycolic acids

    • Other glycolipids: Lipoarabinomannan (LAM), Phosphatidylinositol mannosides (PIM)

Acid-fast bacterial cell envelope

  • Unique properties:

    • Resists Gram staining due to lipid coat

    • Stains strongly with acid-fast stain (Carbol-Fuchsin, red); counterstain with methylene blue

    • Nickname: RED SNAPPERS

    • Chemically resistant to disinfectants, detergents, and many antibiotics

    • Slow growth often due to low nutrient permeability

Acid fast bacterial cell wall structure (detailed)

  • Outer lipids (waxes)

  • Mycolic acid

  • Polysaccharides (arabinogalactan)

  • Peptidoglycan

  • Plasma membrane

  • Lipoarabinomannan (LAM)

  • Phosphatidylinositol mannoside (PIM)

Some clinically important mycobacteria

  • M. tuberculosis

  • M. leprae – leprosy

  • M. avium complex (MAC): M. avium, M. intracellulare

  • M. kansasii – isolated in Kansas (geographic note)

  • M. marinum – associated with seawater

  • M. fortuitum complex (M. fortuitum, M. chelonae, M. abscessus)

  • Other miscellaneous species (e.g., love water)

Classification of Mycobacteria

  • Based on growth properties and culture colonial morphology:

    • M. tuberculosis complex – slow growing

    • Other slow-growing nontuberculous mycobacteria (NTMs) or MOTTs (mycobacteria other than TB)

    • Rapidly growing mycobacteria

  • Runyon classification – rate of growth and pigment

    • Pigmented species produce yellow carotenoids

    • “Photochromogens” – pigment when exposed to light

    • “Scotochromogens” – pigment in the absence of light

Traditional Runyon classification

  • I. Slow growing “photochromogens”

    • examples: M. kansasii, M. marinum

  • II. Slow growing “scotochromogens”

    • example: M. gordonae

  • III. Slow-growing non-pigmented

    • examples: M. avium, M. intracellulare, M. tuberculosis

  • IV. Rapidly growing mycobacteria

    • examples: M. fortuitum, M. chelonae, M. abscessus

    • Generally grow in less than a week

Mycobacteria and clinical disease

  • MTB complex includes: M. tuberculosis, M. leprae (noncultivatable), M. bovis, M. africanum

    • DNA probe tests and PCR tests may not distinguish all members; biochemical testing differentiates species

  • MAC: M. avium and M. intracellulare

  • Other notable species: M. kansasii, M. scrofulaceum, M. marinum, M. fortuitum complex (fortuitum, chelonae, abscessus)

    • M. kansasii: pulmonary infection resembling classic TB; may disseminate; requires different treatment

    • M. scrofulaceum: cervical lymphadenitis in children; resistance to traditional TB treatment

    • M. marinum: cutaneous infection after seawater exposure; skin and soft tissue infection

Pathogenesis of M. tuberculosis

  • Intracellular pathogen

    • can establish lifelong infection

  • Entry: inhaled into airways and reach alveoli

  • Phagocytosed by alveolar macrophages

  • Key evasion: prevents fusion of phagosome with lysosome (Cord factor involved)

    • Cord factor contributes to evasion and pathogenesis

  • Macrophages release cytokines, including TNF and IL-4, which can promote Th2 responses

    • T cells and NK cells are recruited to the infection site

      • May produce granulomas as containment

Immune response to mycobacteria

  1. MTB is intracellular; phagocytosed by macrophages but not killed by unstimulated cells

  2. Prevention of phagosome-lysosome fusion via cord factor

    • Growth occurs in vacuoles

  3. Cell-mediated immunity (CMI) is required for control:

    • CD4+ T cells

    • IFN-γ activates macrophages

    • TNF-α maintains macrophage function (TH1 pathway)

      • TH2 is IL pathway which happens after awhile when infection isn’t rid of quick enough

  4. Granuloma formation as containment mechanism

  5. Large numbers of CD4+ T cells are present during active infection

    • Delayed-type hypersensitivity (DTH) to mycobacterial antigens

TB pathogenesis: antigen burden and tissue damage

  • Small antigen burden may allow destruction of bacteria with minimal tissue damage

  • Large antigen burden triggers an intense CMI response, causing tissue necrosis

    • “Cellular mediated immunity - Tcells”

  • Granuloma: mass of bacteria surrounded by wall of CD4+, CD8+, NK T cells, and macrophages

  • Bacteria may be killed or remain dormant within granulomas and reactivate later

TB epidemiology and transmission

  • Principal risk factor: breathing (airborne transmission)

  • Human pathogen; transmission by droplet nuclei

  • Predisposing factors:

    1. Immunosuppression

    2. Increased travel

    3. Homelessness

    4. IV drug use

    5. Decreased public health vigilance

TB clinical disease: general notes

  • In immunocompetent individuals, most infections are pulmonary and confined to lungs

    • Middle and lower lobes are common sites for replication (more perfusion, less O2 retention)

      • for chronic = upper lungs

  • Immune response often leads to cessation of bacterial replication after ~3–6 weeks

  • Disease progression risk: $5 progress to active disease within 2 years; an additional 5 – 10 % later in life

Factors governing progression to active disease

  • Initial infectious dose

  • Patient's immune competence

  • TB can infect any organ

  • Immunosuppression with low CD4 count markedly increases risk: about 10\% within 1 year; higher risk of extrapulmonary disease

Clinical presentation and history

  • Insidious onset (slow):

    • malaise, weight loss, cough, night sweats

  • Historically called “consumption” due to progressive wasting

  • Initial diagnosis may rely on:

    • Symptoms

    • Radiography (main source)

    • Positive tuberculin skin test (PPD) and laboratory detection of mycobacteria

  • Extrapulmonary disease can spread hematogenously (classic route)

Pulmonary TB: radiography findings

  • Chest X-ray may show cavitary lesions with granulomas

  • Cavitary disease contributes to transmission

  • Autopsy may show caseous necrosis in lung tissue

  • “caseous necrosis = TB”

Extrapulmonary TB

  1. Hematogenous spread (primary TB) can seed any organ

  2. Predisposing factors: immunosuppression (including HIV), chronic illness, diabetes

  3. Common organ targets: brain, heart, kidney, lymph nodes; spine affecting bones (Pott’s disease)

Tuberculosis: cervical lymphadenitis

  • TB can involve cervical lymph nodes (lymphadenitis)

    • “lymphangitis is the infection/block of lymph pathways”

TB of the hip

  • TB of the hip can require surgical drainage and may show caseous necrosis

Spinal tuberculosis

  • Referred to as Pott’s disease

M. tuberculosis: Prevention

  • No vaccine in the United States currently in routine use

  • BCG vaccine used in Eastern Europe, Asia, etc. (not widely used in the US)

  • Screening tools:

    • Tuberculin skin test (PPD): very positive for active disease; positive if granulomas or if previously vaccinated with BCG

    • In vitro IFN-γ release assays (e.g., Quantiferon TB):

      • measure T cell response to TB-specific proteins; not confounded by BCG; often used in screening children

      • Quantiferon TB test specific for TB antigens

M. avium-intracellulare complex (MAC)

  • Most commonly encountered MAC species in clinical labs

  • Pulmonary manifestations similar to MTB; non-pigmented

  • Slow growing

  • Frequently recovered from blood cultures

  • General resistance to anti-TB medications

M. avium complex (MAC): distribution and acquisition

  • Worldwide distribution

  • Acquired via ingestion of contaminated water or food

  • Inhalation of infectious aerosols plays a minor role in transmission

  • Pre-HIV era: recovery often represented transient colonization unless AIDS present

MAC disease and epidemiology

  • Disease seen in older men with smoking history and underlying pulmonary disease (COPD)

  • In AIDS patients, MAC infections are typically disseminated with organ involvement

  • Organism may be grown from blood cultures in MAC infections

M. fortuitum complex

  • Members: M. fortuitum, M. chelonae, M. abscessus

    • “Rapid growers” (growth in 3–7 days)

    • Generally low virulence; rarely disseminated disease

    • Often associated with trauma or IV catheter infections

  • M. chelonae and M. abscessus commonly cause clinical disease

  • M. fortuitum often a fortuitous finding

M. gordonae

  • Usually does not cause clinical disease

  • Common contaminant in clinical samples due to presence in water

  • Dialysis patients may become colonized

  • Slow grower that becomes pigmented; differentiation from pathogens is a diagnostic issue

M. leprae: leprosy (Hansen’s disease)

  • Global prevalence declined dramatically; infection of skin, mucous membranes, and peripheral nerves

  • 90% of cases in Brazil, Madagascar, Mozambique, Tanzania, Nepal

  • <100 cases annually in the US; most occur in California and Texas

  • Immigrants from Mexico, Asia, Africa, and Pacific Islands

  • Endemic in armadillos; found in TX, LA

  • Spread by person-to-person contact

  • Cannot be cultured in vitro

  • Diagnosis by histopathology, skin test reactivity, and AFB in lesions

Leprosy: clinical spectrum

  • Chronic infection affecting skin and peripheral nerves (neuropathy)

  • Spectrum influenced by patient’s immune status

  • Tuberculoid form: milder disease with hypopigmented skin macules

  • Lepromatous form: disfiguring skin lesions, nodules, plaques, thickened dermis, nasal mucosa involvement

Laboratory diagnosis of mycobacteria

  • Stains: Kinyoun acid-fast stain; Auramine fluorochrome stain

  • Nucleic acid amplification tests (NAATs)

  • Culture

  • Species-specific molecular probes

Specimen processing and culture

  • Respiratory specimens: usually contain many bacteria

    • decontamination necessary to prevent overgrowth by normal flora

  • Decontamination: 2% NaOH

  • Digestion: liquefy sputum to release organisms trapped in mucus/cells

  • Concentration: centrifugal processing

  • Two smear preparations (smears) and inoculation onto liquid and selective solid media

Laboratory diagnosis: culture and probes

  • Culture: hold cultures for 6 weeks

  • Species-specific molecular probes (4 main targets):

    • MTB

    • MAC

    • M. kansasii

    • M. gordonae

Treatment, prevention, and control

  • Slow-growing mycobacteria are resistant to many antibiotics used for other bacteria

  • Emergence of extensively drug-resistant (XDR) TB worldwide

    • XDR-TB defined as MDR-TB resistant to fluoroquinolones and at least one of the second-line drugs (kanamycin, amikacin, capreomycin)

    • XDR-TB is potentially untreatable in many cases

  • ON YOUR BOARDS:

  • Standard treatment regimens (initial phase):

    • 2 months of isoniazid (INH), ethambutol, pyrazinamide, and rifampin

    • Followed by 4–6 months of INH and rifampin or alternative drugs