Mycobacterium & Nontuberculous Mycobacteria: Characteristics & Safety

1. Chapter 26 – Mycobacterium tuberculosis & Other Nontuberculous Mycobacteria MLAB 2434 – Clinical Microbiology Keri Brophy-Martinez

2. General Characteristics Slender, slightly curved or straight rod-shaped organisms Non-motile Do not form spores Cell wall with extremely high lipid content Staining requires longer time or application of heat Once stained, resist decolorization with acid-alcohol (acid-fast)

3. General Characteristics (cont’d) Strictly aerobic Grow more slowly than most bacteria Traditional characteristics used to identify Mycobacterium Rate of growth Colony morphology Pigment production Nutritional requirements Optimum incubation temperature Biochemical test results

4. General Characteristics (cont’d) Newer techniques Automated culture system, such as BACTEC Nucleic acid probes with PCR Thin-layer chromatography GLC High-performance liquid chromatography

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

6. Safety Considerations (cont’d) Skin test Also called “Mantoux test” and PPD Those with positive skin test must be advised to have chest X-ray Proper Ventilation Separate from other parts of lab Negative air pressure (6 to 12 room air changes/hour)

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

8. 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 Sputum is most common; should be collected in a wide-mouth container to avoid aerosols Swabs are discouraged due to low volume

9. Specimen Collection and Processing (cont’d) Sputum Number of specimens needed is inversely related to the frequency of smear positivity Should be from a deep cough or expectorated sputum induced by neubulization Bronchial washings or lavages may be collected

10. Specimen Collection and Processing (cont’d) Gastric aspirates Used to recover mycobacterium that may have been swallowed during the night Only used when patient is unable to produce a good quality sputum specimen Urine First morning preferred 3 consecutive days Pool if necessary, not to exceed 12-24 hours

11. 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 – need a fairly large volume of CSF, since number of organisms in that site are rare

12. 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

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

14. Digestion & Decontamination of Specimens (cont’d) Decontamination Specimen from non-sterile site is mixed with an agent that will kill non-mycobacterium bacteria Common decontamination agents NaOH is most common Benzalkonium chloride (Zephiran) Oxalic acid (used with Ps. aeruginosa) After decontamination, the agent must be neutralized so that it will not eventually kill the Mycobacterium

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

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

17. 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 Ziehl-Neelsen – uses heat to drive the color into the lipids of the cell wall; decolorized with acid-alcohol Kinyoun – cold stain Auramine or auramine-rhodamine fluorochrome stain – more sensitive After staining, a minimum of 300 oif are examined

18. Culture Media and Isolation Methods Mycobacterium are strictly aerobic 5-10% CO2 35-37oC Slow growers; cultures held for 6 weeks before calling negative

19. Culture Media and Isolation Methods Media- 3 types Egg-Based with Malachite green (inhibits bacteria) Lowenstein-Jensen (LJ) Serum albumin agar (promotes early growth) Middlebrook 7H10 and 7H11 agar – serum based Liquid Media Middlebrook 7H9 Broth

20. Culture Media and Isolation Methods (cont’d) Labs with large volumes of Mycobacterium cultures use an automated reader (BACTEC) BACTEC broth contains 14C-labeled substrate When organisms grow, 14C in the form of 14CO2 is released and detected radiometrically

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

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

23. Identification of Mycobacterium (cont’d) Photoreactivity Photochromogens – produce carotene pigment upon exposure to light Scotochromogens – produce pigment in light or dark Nonchromogenic – no pigment; these colonies are a buff color

24. Identification of Mycobacterium (cont’d) Biochemical Identification Most labs now use nucleic acid probes with or without PCR Older tests Niacin accumulation Nitrate reduction Catalase Hydrolysis of Tween 80 Iron uptake Arylsulfatase

25. Identification of Mycobacterium (cont’d) Older tests (cont’d) Pyrainamidase Urease Inhibitory tests NAP TCH Growth in 6.5% NaCl Tellurite reduction Growth on MacConkey

26. Antibiotic Sensitivity Testing for Mycobacterium These tests must be performed with great attention to detail, because Mycobacterium is fairly resistant and only a few organisms left can cause reinfection Development of drug-resistance Common antibiotics (usually two or more are given) Isoniazid Rifampin Ethambutol Streptomycin Pyrazinamide

27. Mycobacterium Infections Truly pathogenic M. tuberculosis M. bovis M. ulcerans Potential pathogens M. kansasii M. marinum Other possible pathogens and rare pathogens listed on p. 696

28. Mycobacterium tuberculosis Primarily a pathogen of the respiratory tract (“TB”) One of the oldest communicable diseases Over 1 billion cases worldwide, with 8 to 10 new cases each year and 3 million deaths per year Once called “consumption”

29. Mycobacterium tuberculosis (cont’d) Primary tuberculosis 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

30. Mycobacterium tuberculosis (cont’d) PPD Test-

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

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

33. Mycobacterium tuberculosis (cont’d) Identification 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

34. Mycobacterium tuberculosis (cont’d)

35. Treatment Isoniazid and rifampin 9- month course

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

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

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

39. 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) Diagnosis made from finding acid-fast bacilli in scrapings from lesions Not culturable, except in mouse foot pads

40. Mycobacterium leprae (cont’d)