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

2. 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

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

4. 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. paratuberculosis
M. canetti

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

6. 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”

7. 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

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

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

10. Mycobacterium tuberculosis (cont’d)
Interferon-Gamma Release Assays
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

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

12. Other Mycobacteria Mycobacterium bovis
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

13. Other Mycobacteria
MOTT (Mycobacteria Other Than Tubercle Bacillus) or NTM (Nontuberculous mycobacteria)
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

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

15. 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)

16. Mycobacterium leprae (cont’d)

17. 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
PPD/ Mantoux test

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

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

20. 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

21. Specimen Collection and Processing (cont’d)
Sputum
Collect in a wide-mouth container to avoid aerosols
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

22. 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 midstream preferred
Requires 15 mL minimum
Pool if necessary, not to exceed hours

23. 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
Tissues should be ground

24. 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

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

26. Digestion & Decontamination of Specimens
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

27. Digestion & Decontamination of Specimens
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

28. Concentration
After decontamination and digestion, the specimen is centrifuged in a closed, vented centrifuge for g to concentrate the organisms

29. 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

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

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

32. 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
BACTEC broth contains 14C-labeled substrate
When organisms grow, 14C in the form of 14CO2 is released and detected radiometrically

33. 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

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

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

36. 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
Photoreactivity

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

38. 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

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

40. References
Centers for Disease Control. (n.d.). Tuberculosis. Retrieved from
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 (4th ed.). Maryland Heights, MO: Saunders.