1. Chapter 22 – Mycobacterium tuberculosis & Other Nontuberculous Mycobacteria
MLAB 2434 – Clinical Microbiology
Cecile Sanders & 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

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

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)
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
After decontamination, the agent must be neutralized so that it will not eventually kill the Mycobacterium

14. 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

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

16. 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

17. Culture Media and Isolation Methods
Mycobacterium are strictly aerobic
Slow growers; cultures held for 6 weeks before calling negative
Media
Lowenstein-Jensen (LJ) media – egg based
Middlebrook 7H10 and 7H11 agar – serum based
Middlebrook 7H9 broth

18. 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

19. Laboratory Levels or Extents of Service for Mycobacterium
American Thoracic Society levels
1. Specimen collection only
2. Acid-fast stains and/or inoculation only
3. Isolation and presumptive identification of Mycobacterium species
4. Definitive identification and antibiotic sensitivity testing

20. Identification of Mycobacterium
Colony Morphology
Either smooth and soft or rough and friable
Growth rate
Rapid growers – colonies in < 7 days
Slow growers – colonies in > 7 days
Temperature

21. 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

22. 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

23. 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

24. 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

25. 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. 670

26. 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”

27. 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

28. Mycobacterium tuberculosis (cont’d)
PPD Test

29. Mycobacterium tuberculosis (cont’d)
PPD Test (cont’d)
Positive Test

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

31. 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

32. Mycobacterium tuberculosis (cont’d)

33. Other Mycobacteria
Mycobacterium bovis – primarily in cattle, dogs, cats, swine, parrots and human; disease in humans closely resembles M. tuberculosis
MOTT (Mycobacteria Other Than Tubercle Bacillus) or NTM (Nontuberculous mycobacteria)
Most found in soil and water
Chronic pulmonary disease resembling TB

34. Other Mycobacteria (cont’d)
NTM (cont’d)
M. avium Complex (MAC)
M. kansasii
Mycobacterium fortiutum-chelonei Complex
M. marinum
Etc., etc.

35. 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

36. Mycobacterium leprae (cont’d)