11-4-09 Introduction to Mycobacteria Become familiar with the following characteristics of mycobacteria: 1.

Structure Intracellular Non-motile Non-spore forming Aerobic Rod-shaped Complex lipid-rich cell wall Makes the cell wall hydrophobic responsible for slow growth provides resistance to detergents and antibiotics (has antigenicity) cord formation Acid-fast (has resistance to decolorizing acids due to mycolic acid; stain with ZiehlNeelsen) Slow-growing: 12-24 hours/cell division 100 human disease-causing species identified Most well known: M. tuberculosis (TB) M. leprae (leprosy aka Hansens disease) M. avium (severe systemic infection in AIDS) M. fortuitum, M. chelonae, and M. abscessus (all are rapid growing but rarely cause disseminated disease; they have been implicated in subQ tissue infection after trauma, IV catheters, contaminated wound dressings, and heart valves) M. kansasii (pulmonary TB) 2. Classification Three criteria for classifying something as mycobacteria: 1. acid-fast 2. high mycolic acid content in cell wall 3. high G+C content in DNA 3. Methods of Identification Three main methods of IDing mycobacteria 1. acid-fast. This is a surefire way of identifying mycobacteria from other geni, but not amongst individual mycobacterial species. To differentiate amongst species, additional methods are needed. a. stain with Ziel-Neelsen or Kinyoun, both using a red carbofuchsin dye b. the dye does not wash off for acid-fast mycobacteria c. Nocardia and Gordona spp are partially acid-fast, but much less so than mycobacteria 2. slow growth rate. Pathogenic species are the slowest! A 6-8 week period is needed for species determination, so this molecular probe methods are probably more realistic.

molecular probe methods. Takes only 1 day and are used for 4 mycobacterial species that make up 95% of all clinical isolates. a. DNA hybridization: uses DNA probes that are species-specific for mycobacterial rRNA b. High performance liquid chromatography (HPLC): profiles mycolic acid, which varies from one mycobacterial species to another c. PCR: rapid, specific, and the preferred method for IDing M. tubercuosis d. Enzyme Immunoassay (ELIZA): detects mycobacterial antigens, used because it is fast. Less specific than other molecular probes. 4. PPD (purified protein derivative) tuberculin skin test: specific for TB, inject and wait for 48 hours; induration of 10mm or more is considered positive. Note that immunocompromised patients may have a negative response regardless of exposure. a. a positive test does not imply that the individual has active disease. It also does not mean that the individual has immunity to TB. Rather, it demonstrates that the patient has been infected in the past and continues to carry viable mycobacteria with the potential for reactivation. Remember that over 90% of infected individuals are asymptomatic. b. A negative test indicates that the individuals has never been exposed. S/he can be infected in the future.
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4. Pathogenesis and host immune response Host immune response 1. Innate immune response: consists of NK cells and phagocytes a. NK cells: NK cells are stimulated by IL-12 and in turn produce IFN-gamma, which activates macrophages and promotes phagocytosis. NK cells are the early defense against mycobacteria, before adaptive immunity kicks in. b. Neutrophils and macrophages: phagocytose mycobacteria but fail to destroy them because they are resistant to degradation due to unique lipid-rich cell wall c. Innate immunity limits the growth of mycobacteria but cannot eradicate it. Eradication requires adaptive immunity. 2. Adaptive immune response: a. CD4 T cells produce CD40 ligand, TNF-alpha, and IFN-gamma, resulting in the killing of phagocytosed bacteria b. CD8 T cells lyse infected cells c. AIDS patients lack the adaptive immune response and are therefore susceptible to infections with intracellular bacteria such as M. tuberculosis. d. The adaptive immune response can also cause pathologies. Since mycobacteria are very resistant to destruction from phagocytosis, the T cell response can be chronically initiated again and again, resulting in the formation of granulomas. Granulomatous inflammation is a form of delayed-type hypersensitivity (DTH) that results in fibrosis from healing. i. Recall that TB is associated with granulomas! Much of the respiratory difficulty in TB is due to the replacement of normal tissue with scarring. Pathogenesis (of TB)

Transmission: infection occurs via the respiratory route and is transmitted by airborne droplets from person-to-person Primary infection: M. tuberculosis bacilli multiply slowly in lungs and cause only mild inflammation; 90% of infected patients are asymptomatic, while the bacteria survive in the lungs and can be activated The intracellular M. tuberculosis survives and replicates within phagocytes, evading circulating antibodies (their elimination requires cellular immunity). Thus, they are considered facultative intracellular bacteria that can live inside and outside of cells. Dendritic cells then help prime T cells, which recruit and activate macrophages to contain bacterial multiplication. Failure of containment results in primary TB infection. Viable bacteria persist in two locations: 1) focal lesions walled off from healthy tissue by fibrosis/calcification (caseation and liqueafaciton occurs within) and 2) outside of lesions, held in check with immune response Primary disease lesions occur anywhere in the lung, while post-primary lesions most commonly appear in apical regions

5. Epidemiology Humans are the only natural reservoir for M. tuberculosis Its estimated that 1/3 of the worlds population is infected, with 10 million in the US Consequently, eradication of TB is highly unlikely Focus is on control via Active surveillance Prophylactic and therapeutic intervention: vaccination with attenuated M. bovis (BCG) common in some countries where TB incidence is high (i.e. NOT the US); cannot be used in immunocompromised (i.e. not useful in African nations with high AIDS incidence) Careful case monitoring Treatments Three regimens available. Must keep in mind that there are high levels of antibiotic resistance, so patients must take multiple antibiotics for 6-9 months to prevent development of further resistance 1) isoniazid for 9 mo 2) rifampin for 4 mo 3) rifampin and pyrazinamide for 2 mo For drug resistant M. tuberculosis strains Pyrazinamide + either ethambutol or levofloxacin for 6-12 months 6. Clinical Syndromes Leprosy caused by M. leprae Tuberculoid leprosy: strong cellular response but weak humoral antibody response resulting in infected tissues with few bacteria, tons of lymphocytes, and many granulomas. Theres lots of IFN-gamma and IL-12 being made to clear out bacteria, while granulomas are shielded from such a response.

Lepromatous leprosy: strong humoral antibody response but weak cellular response resulting in high levels of bacteria within dermal macrophages and Schwann cells of peripheral nerves.