Mycobacterium species Other Nontuberculous Mycobacteria MLAB 2434

Mycobacterium species & Other Nontuberculous Mycobacteria MLAB 2434 – Microbiology Keri Brophy-Martinez

General Characteristics ¢ Slender, slightly curved or straight rod-shaped organisms ¢ Non-motile ¢ Do not form spores ¢ Strictly aerobic ¢ Various species found in the soil and water

General Characteristics: Cell Wall ¢ ¢ Extremely high lipid content l Mycolic acid l Waxy substances l Assists in resisting harsh environments l Assists in penetrating host immune system Consequences of high lipid content l Staining requires longer time or application of heat l Once stained, resist decolorization with acid-alcohol (acid-fast) l Long generation time

Mycobacterium Infections M. tuberculosis complex Photochromogens Scotochromogens Nonphotochromogens Rapid Growers M. tuberculosis M. kansasii M. scrofulaceum M. avium complex M. fortuitum M. bovis M. marinum M. szulgai M. xenopi M. chelonae M. africanum M. simiae M. gordonae M. mamoense M. abscessus M. microti M. canetti M. paratuberculosis

Classification of Mycobacterium ¢ Photoreactivity Photochromogens – produce carotene pigment upon exposure to light l Scotochromogens – produce carotene pigment in light or dark l Nonphotochromogenic – no pigment; these colonies are a buff color l

Mycobacterium tuberculosis Primarily a pathogen of the respiratory tract (“TB”) ¢ One of the oldest communicable diseases ¢ Over 9 million cases worldwide, and 2 million deaths per year ¢ Once called “consumption” ¢

Mycobacterium tuberculosis (cont’d) ¢ Primary tuberculosis l l Spread by coughing, sneezing, or talking Inhaled into alveoli, where the organisms are phagocytized If the organism does not cause immediate infection, the organism can be “walled off” in a granuloma Granulomas can break down in future and the organisms can cause infection later

Mycobacterium tuberculosis (cont’d) ¢ PPD Test-

Mycobacterium tuberculosis (cont’d) ¢ PPD Test (cont’d) Positive Test l Detects patients cell-mediated immune response to bacterial antigens

Mycobacterium tuberculosis (cont’d) ¢ Interferon-Gamma Release Assays l l Blood test Measure person’s immune reactivity to specific mycobacterial antigens Advantages • Single patient visit • No booster phenomenon • Less reader bias in interpretation Disadvantages/Limitations • Sample must be processed within 8 -16 hours • Limited data on certain populations

Mycobacterium tuberculosis (cont’d) ¢ Extrapulmonary tuberculosis Spleen l Liver l Lungs l Bone marrow l Kidney l Adrenal gland l Eyes l

Other Mycobacteria ¢ Mycobacterium bovis l l l Primarily in cattle, dogs, cats, swine, parrots and human; disease in humans Slow grower Small, granular, rounded white colonies with irregular margins Nonpigmented Similar to M. tuberculosis

Other Mycobacteria ¢ MOTT (Mycobacteria Other Than Tubercle Bacillus) or NTM (Nontuberculous mycobacteria) l l l Most found in soil and water Chronic pulmonary disease resembling TB, skin infections, chronic lymphadenitis Opportunistic pathogen in patients with liver disease, immunocompromised, percutaneous trauma

Other Mycobacteria (cont’d) ¢ NTM l Photochromogens • M. kansasii • M. marinum l Scotochromogens • M. gordonae • M. scrofulaceum l Nonphotochromogens • M. avium Complex (MAC) l Rapid Growers • Mycobacterium fortiutum-chelonei Complex

Mycobacterium leprae ¢ ¢ Causes leprosy or Hansen’s Disease Infection of the skin, mucous membranes and peripheral nerves Most cases are from warm climates Bacteria infect the cooler areas of the body (ears, nose, eyebrows, fingers, toes)

Mycobacterium leprae (cont’d)

Safety Considerations ¢ Mycobacteriology workers are three times more likely to seroconvert (develop positive skin test) l l l Adequate safety equipment Safe laboratory procedures training Information on hazards Preparations for unexpected accidents Staff must be monitored regularly by medical personnel • PPD/ Mantoux test

Safety Considerations (cont’d) ¢ Proper Ventilation Separate from other parts of lab l Nonrecirculating ventilation systems l Negative air pressure l • Air flows from clean areas to less clean areas • 6 to 12 room air changes/hour ¢ Biological Safety Cabinet

Safety Considerations (cont’d) ¢ Use of Proper Disinfectant Bactericidal for mycobacteria l Also called “tuberculocidal” l ¢ Other precautions Disposables l Protective clothing, face masks l

Specimen Collection and Processing ¢ Variety of clinical specimens, including respiratory, urine, feces, blood, CSF, tissues, and aspirations ¢ Should be collected before antibiotic therapy and processed ASAP ¢ Swabs are discouraged due to decreased recovery

Specimen Collection and Processing (cont’d) ¢ Sputum Collect in a wide-mouth container to avoid aerosols l Number of specimens needed is inversely related to the frequency of smear positivity l Should be from a deep cough or expectorated sputum induced by neubulization l Bronchial washings or lavages may be collected l

Specimen Collection and Processing (cont’d) ¢ ¢ Gastric aspirates l Used to recover mycobacterium that may have been swallowed during the night l Only used when patient is unable to produce a good quality sputum specimen Urine l First morning midstream preferred l Requires 15 m. L minimum l Pool if necessary, not to exceed 12 -24 hours

Specimen Collection and Processing (cont’d) ¢ ¢ ¢ Stools – primarily collected from AIDS patients to determine Mycobacterium avium complex (MAC) Blood – most commonly from AIDS and other immunosuppressed patients Tissues and other body fluids l l Need a fairly large volume of CSF, since number of organisms in that site are rare Tissues should be ground

Digestion & Decontamination of Specimens ¢ ¢ Because Mycobacterium grow so slowly and are often collected from non-sterile body sites, they are easily overgrown by other bacteria Specimens from non-sterile sites, therefore, must be “decontaminated” Sputums or other viscous specimens also must be “digested” Specimens from sterile sites (CSF, etc. ) do not need decontamination

Digestion & Decontamination of Specimens (cont’d) ¢ Purposes To liquefy the sample to clear proteinaceous material l Agent kills nonmycobacterial organisms l

Digestion & Decontamination of Specimens ¢ Decontamination l l Specimen from non-sterile site is mixed with an agent that will kill nonmycobacterium bacteria Common decontamination agents • Na. OH is most common • Benzalkonium chloride (Zephiran) • Oxalic acid (used with Ps. aeruginosa) l After decontamination, the agent must be neutralized so that it will not eventually kill the Mycobacterium

Digestion & Decontamination of Specimens ¢ Digestion Liquefying mucus enables the mycobacterium to contact and use the nutrients in the agar medium l Common digestion agents l • N-acetyl-L-cysteine – most common • Trisodium phosphate (Z-TSP) – used with Zephiran

Concentration ¢ After decontamination and digestion, the specimen is centrifuged in a closed, vented centrifuge for 15 minutes @ 3000 g to concentrate the organisms

Acid Fast Stains ¢ ¢ ¢ After centrifugation, the button at the bottom of the tube is used to make a smear and to inoculate media Acid Fast Stains l Ziehl-Neelsen – uses heat to drive the color into the lipids of the cell wall; decolorized with acidalcohol l Kinyoun – cold stain l Auramine or auramine-rhodamine fluorochrome stain – more sensitive After staining, a minimum of 300 oif are examined

Culture Media and Isolation Methods Mycobacterium are strictly aerobic ¢ 5 -10% CO 2 ¢ 35 -37 o. C ¢ Slow growers; cultures held for 6 weeks before calling negative ¢

Culture Media and Isolation Methods ¢ Media- 3 types l Egg-Based with Malachite green (inhibits bacteria) • Lowenstein-Jensen (LJ) l Agar based • Promotes early growth • Middlebrook 7 H 10 and 7 H 11 agar – serum based l Liquid Media • Middlebrook 7 H 9 Broth

Culture Media and Isolation Methods (cont’d) ¢ Labs with large volumes of Mycobacterium cultures use an automated reader (BACTEC) Used for blood, body fluids, bone marrow l BACTEC broth contains 14 C-labeled substrate l When organisms grow, 14 C in the form of 14 CO 2 is released and detected radiometrically l

Culture Media and Isolation Methods (cont’d) ¢ Isolator-Lysis Centrifugation System Contains saponin to liberate intracellular organisms l Advantages include yielding isolated colonies, quantification of mycobacteria, shorter recovery times l

New Techniques for Identification Automated culture system, such as BACTEC l Nucleic acid probes with PCR l Gas Liquid chromatography l High-performance liquid chromatography l

Identification of Mycobacteria ¢ Traditional characteristics used to identify Mycobacterium l l l Rate of growth Colony morphology Pigment production Nutritional requirements Optimum incubation temperature Biochemical test results

Identification of Mycobacterium ¢ ¢ First Step is to confirm organism as Acid Fast Colony Morphology l Note texture, shape, pigment • Either smooth and soft or rough and friable Growth rate l Rapid growers – colonies in < 7 days l Slow growers – colonies in > 7 days Temperature l ¢ Range can vary from 20 o. C- 42 o. C Photoreactivity

Identification of Mycobacterium (cont’d) ¢ Biochemical Identification • • • Niacin accumulation Nitrate reduction Catalase Iron uptake Arylsulfatase Pyrainamidase Telluride reduction Urease Hydrolysis of Tween 80

Identification of Mycobacterium tuberculosis ¢ ¢ ¢ Slow grower Colonies are thin, flat, spreading and friable with a rough appearance May exhibit characteristic “cord” formation Grows best at 35 to 37° C Colonies are NOT photoreactive

Antibiotic Sensitivity Testing for Mycobacterium ¢ ¢ Mycobacterium is fairly resistant and only a few organisms left can cause reinfection Development of drug-resistance l l l ¢ Inadequate treatment regimes Patient noncompliance Mutations Common antibiotics (usually two or more are given) l Isoniazid l Rifampin l Ethambutol l Streptomycin l Pyrazinamide

References ¢ ¢ ¢ Centers for Disease Control. (n. d. ). Tuberculosis. Retrieved from http: //www. cdc. gov/tb/publications/LTBI/diagnosis. h tm#4 Kiser, K. M. , Payne, W. C. , & Taff, T. A. (2011). Clinical Laboratory Microbiology: A Practical Approach. Upper Saddle River, NJ: Pearson Education. Mahon, C. R. , Lehman, D. C. , & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4 th ed. ). Maryland Heights, MO: Saunders.