Microbiology lectures- mycobacteria

Dr. Abdullah Alhatami

MYCOBACTERIA
Mycobacteria fungus like.. described by Robert Koch 1882 (human bacilli) Johns- 1885 isolated M.paratuberculosis There are more than 100 species of Mycobacteria. Classification 1. Cultivable mycobacteria a) Typical mycobacteria M tuberculosis- infect primates and human M bovis-Cattle, ruminants,pigs, dogs, cats, wild animals and humans M.microti-Vole tuberculosis b) Atypical Mycobacteria ( - Photochromogens, produce pigment in light -Scotochromogens, produce pigment regardless of exposure to light - Nonchromogens- M avium complex , Fowl, pigs, horse immunocompromised man TB M.avium paratuberculosis- Johne's disease in cattle -Rapid grower and saprophytic Mycobacteria (within 7 days) Non-cultivable Mycobacteria ...Mycobacterium leprae

2.

General properties of Mycobaceria Aerobic, rod-shaped 0.5 m in wide and variable in length. Non-sporing, nonmotile nor capsulated. Poorly stained with Gram stain and they are better stained by hot Carbol-fuchsin, and once stained, they resist decolorization with 3% HCl in ethanol (acid-alcohol), therefore, referred to Acid-Fast bacilli (AFB). This property is due to high content of lipid in cell wall. Structure of cell wall About 60% of cell wall is lipid that account for acid fastness and pathogenic and immunogenic properties 1. peptidoglycan layer 2. arabinogalactan layer 3. mycolic acids layer .... acid fastness Mycolic acids are components of a variety of lipids found only in Mycobacteria, Nocardia and Corynebacteria. The chain length of these mycolic acids is longest in mycobacteria, intermediate in nocardia and shortest in corynebacteria. This explains why mycobacteria are generally acid fast; nocardia less acid fast; and corynebacteria are non-acid fast 1

Microbiology lectures- mycobacteria

Dr. Abdullah Alhatami

4. Mycosides surface layer ... glycolipid and peptidoglycolipid... colony morphology, serologic reactions, intracellular survival M.tuberculosis and M.bovis

Test or character Oxygen preference Growth Temperature Action of glycerol in media Colony Biochemical reactions Niacin production Nitrate reduction Animal pathogenicity rabbit Guinea pig

M.tuberculosis Strict aerobic eugonic 35-37 Growth enhanced Rough, raise or wrinkled surface. white or buff + + mild + progressive and ultimately fatal

M.bovis microaerophilic dysgonic The same Growth inhibited smooth, flat and white colonies, easily break up when touched Generalized progressive lesions Similar

Culture media Complex organic media are used for cultivation of TB bacilli like LowensteinJensen's media which contains eggs, potato flour and malachite green to inhibit contaminants. Oleic acid-albumin media such as Middlebrook's also used. Generation time of tubercle bacilli is about 12 hrs and more, therefore, culture usually appear after 2-3 weeks. Viability Tubercle bacilli survive exposure to 1N NaOH or HCl for 15-30 min. They susceptible to phenolic compounds. They resist drying and survive for long periods in soil. They killed by pasteurization, sunlight and UV radiation. Epidemiology The source of tubercle bacilli is tuberculous host.

Microbiology lectures- mycobacteria

Dr. Abdullah Alhatami

Transmission Infection takes place by inhalation or ingestion of contaminated airborne droplet nuclei, urine, milk of infected animal or contaminated feed or water. Determinants of pathogenicity 1. Cord factor is a glycolipid derivatives of mycolic acid
a) it inhibit migration of polymorphnuclear leukocytes and elicit granuloma formation and cause mitochondrial disruption. b) Corde factor cause bacilli to grow in liquid culture in serpentine "cord". This is indicative of pathogenicity c) It is immunogenic

2. Sulfatides, glycolipids located on the surface of Mycobacteria, inhibit phagolysosome formation....permit intracellular survival 3. Wax-D has adjuvant activity, activates macrophage ...... induce granuloma formation

Pathogenesis of tuberculosis Mycobacterium tuberculosis infects the lung, and is distributed systemically within macrophages and survives intracellularly. Inhibition of phagosome-lysosome fusion and resistance to lysosomal enzymes have both been suggested to play a role. Cellmediated immunity develops which causes infiltration of macrophages and lymphocytes with development of granulomas (tubercles). Other pathogenesis factors (of considerably less importance than delayed hypersensitivity) include mycobactin (a siderophore) and cord factor which damages mitochondria. Immunological aspects Effective immune response is of cell-mediated immunity. Infected macrophage secretes IL-12 that stimulates CD4+ TH1 to release gamma interferon which activate resting macrophage and increase its intracellular killing activity. The activation of CMI is responsible for formation of granulomatous lesions characteristic of TB infection. Vaccination The BCG vaccine (Bacillus Calmette Guerin, an attenuated strain of M. bovis) is used for human immunization.

Laboratory diagnosis 1. Sample collection. Tracheobronchial and gastric lavages;lymph node;urine;feces; and biopsy. Homogenization of tissues then decontamination with sodium hydroxide 4% or 5% oxalic acid. The suspension is centrifuged, 3

Microbiology lectures- mycobacteria

Dr. Abdullah Alhatami

the supernatant is discarded, and the sediment is used for culture and microscopic examination. 2. Direct examination. Ziehl-Neelsen stain, the bacilli appear as pink short straight uniform staining. 3. Isolation and identification. LJ media 4. Animal inoculation (Rabbit, guinea pig) 5. DNA probe and PCR 6. Tuberculin test The disease can be diagnosed by skin testing for delayed hypersensitivity with tuberculin (also know as purified protein derivatives from Mycobacterium tuberculosis, PPD). A positive test does not indicate active disease; merely exposure to the organism. 7. Serologic tests note useful in disgnosis Treatment Human infection therapy involve first-line drugs: streptomycin, rifampin, isoniazid, and ethambutol. Second-line drugs are pyrazinamide, para-aminosalicylic acid, kanamycin, cycloserine, capreomycin, and ethionamide. Infected animals not treated to avoid emergence of resistance and spread of infection. Mycobacterium avium avium
infect animals( fowl tuberculosis, pigs and immunocompromised patients (AIDS). Non-chromogens slow grower, produce eugenic growth grow at 25C-45C colony is dome shaped

Mycobacterium avium ssp. paratuberculosis

Cause enteritis in cattle and ruminants and the disease is called Johne's diseae their protein derivatives called johnin and the skin test is called johnin test one member of atypical Mycobacteria, non-chromogens group require mycobactin (an iron-acqiring lipid) for their growth, therefore can be differentiate by culturing on Herrold's egg yolk medium... 8-12 weeks to produce visible colonies treatment ... clarithromycin

Mycobacterium leprae M. leprae is the causative agent of leprosy, a chronic disease often leading to disfigurement.. It is rarely seen in the U.S. but common in the third world. The organism infects the skin, because of its growth at low temperature. It also has a strong affinity for nerves. The organism does not grow in culture media. However, it grows well in the armadillo (which has a low body temperature), allowing production of M. leprae antigens and pathogenesis studies.