Mycobacteria

Bacteriology MYCOBACTERIA MYCOBACTERIUM TUBERCULOSIS Morphology a. Rod with rounded ends b. True branching occurs in vitro under special culture conditions c. Acid-fast, non-sporeforming, nonencapsulated d. Gram stain poorly, but usually considered gram-positive e. Cell wall backbone contains two polymers, a peptidoglycan and arabinoglycan, covalently linked by phosphodiester bonds i. Peptidoglycan contains N-glycolylmuramic acid instead of N-acetyl derivative ii. Peptidoglycan has two types of inter-peptide linkages (a) D-ala-meso-diaminopimelic acid (DAP) (b) Meso-DAP-DAP iii. Peptidoglycan contains two amide groups f. Cell wall contains lipoproteins or glycolipoproteins essential for Tuberculin activity g. Cell wall lipids account for 60% of dry weight of cell wall i. Cord factor ii. Sulfatides iii. Mycosides Physiology a. Obligate aerobe b. Slow-growing, requiring 10-20 days at 37°C before growth visualized c. Primary isolation requires complex media containing either egg-potato base or serum-agar base d. Glycerol is preferred carbon and energy source e. Catalase and peroxidase are present f. Iron assimilation via one hydrophilic and one lipophilic transport system g. Mycobacteriophages i. Double-stranded DNA phages ii. Thus far unassociated with virulence h. Highly resistant to drying Antigenic structure Culture filtrate antigens i. Old tuberculin (a) Crude extract from G-week-old broth cultures (b) Heat-stable protein is active component (c) First described by Koch, 1881 ii. Purified protein derivative (a) Partially purified preparation of old tuberculin by ammonium sulfate fractionation (b) Mixture of small tuberculoproteins (c) PPD-S, large batch made by Seibert in 1939 iii. U.S.-Japan reference system-11 major antigens by electrophoresis including cell wall polysaccharides, glucan, and polypeptides iv. Antigen S (a) Cytoplasmic protein from M. tuberculosis and M. bovis (b) May be valuable for tuberculin testing . v. Polysaccharides (a) Immunogenic, serologically active (b) Do not elicit delayed-type hypersensitivity Phosphatidyl inosityl mannosides i. Amphipathic polar lipids in plasma membranes

ii. Immunogenic iii. Serologically active Other immunoreactive components Wax D, muramyldipeptide (a) Heterogeneous group of peptidoglycolipids from cell wall (b) Muramyldipeptide most important in adjuvant activity Determinants of pathogenicity a. Cord factor i. High correlation between virulent strains and serpentine pattern in parallel arrangement in culture ii. Correlates with presence of a glycolipid, trehalose-G, G'-dimycolate (cord factor) iii. Biologic responses (a) Inhibits polymorphonuclear leukocytic migration (b) Elicits granuloma formation (c) Attacks mitochondrial membranes Sulfatides i. Responsible for neutral red reactivity ii. Synergistic activity with cord factor iii. Inhibits secondary lysosome fusion Clinical infection Epidemiology i. More common in Southeast U.S. and Indian reservations ii. Disease activation classically associated with onset of puberty iii. More common in lower socioeconomic areas Transmission i. Primarily through droplet nuclei inhalation from coughing, sneezing, and laughing ii. Most infectious person is one with untreated cavitary pulmonary tuberculosis iii. Direct inoculation of abraded skin by laboratory personnel iv. Drinking contaminated milk (M. bovis) v. Risk of infection much greater than risk of disease Pathogenesis i. TWO IMMUNOLOGIC RESPONSES (a) Delayed hypersensitivity, usually within 3-4 weeks after infection, correlates with positive tuberculin reaction (b) Acquired cellular immunity PRIMARY INFECTION (a) Organism is inhaled, spread to alveolar macrophages, hilar lymph nodes, bloodstream, then throughout the body (b) Tubercle forms with or without caseation (c) Primary site calcified lesions referred to as Ghon complex (d) Delayed hypersensitivity develops and infection becomes quiescent in pulmonary as well as metastatic sites (e) Immunocompromised or debilitated patients may have progressive primary disease from local site or more distant sites SECONDARY INFECTION (reactivation) (a) Usually localized, particularly lung apices due to higher oxygen tension (b) Tubercle formation histologically (c) Results from either breakdown of quiescent foci or from new infection, despite acquisition of cellular immunity. Clinical manifestations

i. 85.% cases are pulmonary ii. Nonspecific constitutional symptoms iii. Miliary or disseminated mass develops anywhere, but some sites favored (a) Bone and joints (b) Meninges (c) Genitourinary tract (d) Lymph nodes (e) Peritoneum Tuberculin test i. PPD-S designated standard, containing 50,000 tuberculin units per mg of protein (Purified Protein Derivative) ii. 0.1 ml PPD is biologically equivalent to 5TU of PPD-S iii. Tuberculin tests should be read 48-72 hours after intradermal injection iv. Reading is based on diameter of induration and recorded in millimeters v. Reaction interpretation (a) More than/= 10 mm induration considered positive for M. tuberculosis infection (not necessarily active disease) (b) 5-10 mm considered doubtful signficance, may be due to other mycobacterial infection (e) < 5 mm. induration should be considered negative vi. False negative reactions usually result of tuberculin injection too deep into skin. Other causes are (a) Improper storage or handling (b) Associated viral or other infectious diseases (e) Immunosuppression or anergy vii. False positive reactions (a) Usually caused by hypersensitivity to mycobacteria other than M. tuberculosis (b) Specificity varies geographically viii. Booster effect (a) Usually in person over 55 years old (b) Results from stimulus of a retest in boosting a positive reaction in someone whose positivity has waned with time (c) Should not be interpreted as new infection Diagnosis a. Microsopic examination of specimen smear stained with Ziehl-Neelsen or Kinyoun stain b. Digestion and decontaminate culture with N-acetyl-L-cysteine and sodium hydroxide to avoid bacterial overgrowth c. Inoculate in either egg-potato-base media (Lowenstein-Jensen) or agar-base media (Middlebrook 7H10) d. Culture in 5-10% C02 atmosphere for 3-6 weeks Treatment a. Must use at least 2 drugs to prevent emergence of resistant mutants b. Must use at least 1 first-line drug i. Isoniazid (beware of resistance) ii.Rifampin iii.Streptomycin iv.Ethambutol (bacteriostatic) Prevention a. INH prophylaxis i.Household contacts of newly diagnosed active disease ii.Recent conversion (within past two years) (a) Children under 5 years old, without exception (b) Adults greater than 55 years old generally followed clinically

(c) Immunosuppressed b. BCG prophylaxis i.Affords relative rather than absolute immunity ii.Given only to PPO-negative individuals in countries where incidence of tuberculosis is high MYCOBACTERIUM BOVIS 1. Obtained through unpasteurized infected cow's milk 2. Portal of entry in humans is usually gastrointestinal tract 3. Extrapulmonary lesions common a. Cervical and mesenteric lymph nodes b. Bones and joints NONTUBERCULOUS MYCOBACTERIA-based on Runyon's classification General characteristics a. No known primary animal host; usually occur in the soil b. No evidence for human-to-human transmission c. Ubiquitous but endemic in some areas Clinical manifestations a. Pulmonary disease i. Usually in older white men with chronic bronchitis and emphysema ii. Pathogens include M. kansasii, M. avium-intracellulare, M. fortuitum complex b. Lymphadenitis i. Usually occurs in children ii. M. SCROFULACEUM most common cause c. Cutaneous disease i. M. MARINUM in U.S. ii. M. ULCERANS in Africa, Southeast Pacific iii. Injection abscesses with M. fortuitum, M. chelonei d. Disseminated disease i. M. KANSASII ii. M. AVIUM-INTRACELLULARE, particularly in patients with acquired immunodeficiency syndrome (AIDS) Treatment a. M. kansasii-standard antituberculous therapy b. Others-standard antituberculous therapy usually ineffective, multiple drugs in various combinations usually tried Slow growers (Groups I, II, III) M. kansasii i. Photochromogenic, Group I ii. Antigenically more similar to M. tuberculosis iii. Clinically similar to pulmonary tuberculosis iv. Common isolate in Louisiana, Chicago, Texas, northeast London; more frequent in urban areas M. marinum i. Photochromogenic, Group I ii. Associated with swimming pools, tropical fish aquariums, beaches, freshwater lakes iii. May heal spontaneously M. scrofulaceum i. Scotochromogenic, Group II ii. Important cause of cervical lymphadenitis; portal of entry is oropharynx

iii. May colonize old lung tuberculosis cavities M. avium-intracellulare i. Nonphotochromogenic, Group III ii. Ubiquitous in distribution (a) Soil, water (b) Dairy products (c) Bird and mammal tissue iii. Clustering of cases in AIDS patients iv. Highly resistant to antituberculous drugs Rapid growers-M. fortuitum-chelonei complex a. Most are environmental saprophytes b. Isolated from soil, sputum of healthy persons, operating room scrub sink c. M. fortuitum and M. chelonei-two well-defined species but have similar metabolic characteristics d. Must examine cultures weekly because M. fortuitum colonies will resemble M. tuberculosis if not examined until 3-4 weeks e. Clinically manifests as abscess at trauma site, usually from injection f. Other clinical manifestations i. Corneal ulcers ii. Pulmonary infection iii. Surgical wounds g. Treatment usually combines multiple drugs with surgical drainage Mycobacterium ulcerans a. Produces chronic ulcerating skin disease in the tropics, near rivers, swamps b. Usually on legs and arms c. Treatment requires wide surgical excision Mycobacterium leprae Morphology, physiology i. Thus far the organism has not been successfully cultured in vitro ii. Acid-fastness removable by pretreatment with pyridine, distinguishing it from most other mycobacteria iii. Presence of a phenolase separates it from other mycobacteria Experimental disease i. Can be grown only in mice, rats, armadillos, and hedgehogs ii. During logarithmic growth phase, has generation time of 12 days Clinical infection (Hansen's disease) i. Humans the only natural host ii. Endemic in Africa, South and Southeast Asia, and South America iii. Small endemic areas in U.S. (a) Hawaii (b) Texas (c) California (d) Louisiana (e) Florida iv. Transmission is through contact with organisms from nasal secretions and ulcer exudates v. Wild armadillos nClturally infected with organisms indistinguishable from M. leprae vi. Ridley and Jopling classification into five disease forms (a) Tuberculoid leprosy -Mature granuloma in dermis, containing epithelioid cells, giant cells, lymphocytes -Acid-fast bacilli usually not found -Schwann cells selectively colonized -Nonspecific nerve damage

(b) Borderline tuberculoid (c) Borderline (d) Borderline lepromatous (e) Lepromatous leprosy -Foamy histiocytes; epithelioid and giant cells absent; lymphocytes absent -Numerous acid-fast organisms present -May have constant bacillemia and reticuloendothelial system invasion -Schwann cells infected, but less nerve damage than in tuberculoid vii. Immunity (a) Disease of low infectivity; disease more likely with defective cell-mediated immunity (b) Tuberculoid leprosy -Strong delayed -type hypersensitivity -Poor humoral response (c) Lepromatous leprosy -Loss of cell -mediated immunity -Loss of delay -type hypersensitivity -High serologic response: polyclonal hypergammaglobulinemia; antibodies cross react with other mycobacterial disease; antibodies are not protective viii. Lepromin test-determines immunologic response of patient; not for diagnostic use (a) Fernandez reaction (early) -Reaction within 48 hours -Indicates delayed hypersensitivity response by previously sensitized individual -Usually positive in tuberculoid, negative in lepromatous leprosy (b) Mitsuda reaction (late) -Development of indurated nodule within 3-4 weeks -Corresponds with ability to produce immunologic granuloma in presence of whole bacteria -Usually positive in tuberculoid, negative in lepromatous leprosy (c) Lacks specificity ix. Erythema nodosum leprosum (ENl) (a) Occurs in > 50% patients within first year of effective therapy (b) Probably precipitated by released antigenic material from organism degradation Treatment (a) Dapsone-mainstay but increasing resistance (b) Rifampin (c) Clofazimine (particularly for lepromatous leprosy)