Chapter 22

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Chapter 22 Mycobacterium tuberculosis Other
Nontuberculous Mycobacteria

• MLAB 2434 Clinical Microbiology
• Cecile Sanders Keri Brophy-Martinez

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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)

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General Characteristics (contd)

• 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

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General Characteristics (contd)

• 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

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Safety Considerations (contd)

• 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)

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Safety Considerations (contd)

• Biological Safety Cabinet
• Use of Proper Disinfectant
• Bactericidal for mycobacteria
• Also called tuberculocidal
• Other precautions
• Disposables
• Protective clothing, face masks

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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

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Specimen Collection and Processing (contd)

• 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

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Specimen Collection and Processing (contd)

• 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

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Specimen Collection and Processing (contd)

• 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

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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

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Digestion Decontamination of Specimens (contd)

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

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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

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Concentration

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

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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

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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

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Culture Media and Isolation Methods (contd)

• 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

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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

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Identification of Mycobacterium

• Colony Morphology
• Either smooth and soft or rough and friable
• Growth rate
• Rapid growers colonies in lt 7 days
• Slow growers colonies in gt 7 days
• Temperature

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Identification of Mycobacterium (contd)

• 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

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Identification of Mycobacterium (contd)

• 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

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Identification of Mycobacterium (contd)

• Older tests (contd)
• Pyrainamidase
• Urease
• Inhibitory tests
• NAP
• TCH
• Growth in 6.5 NaCl
• Tellurite reduction
• Growth on MacConkey

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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

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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

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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

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Mycobacterium tuberculosis (contd)

• 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

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Mycobacterium tuberculosis (contd)

• PPD Test

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Mycobacterium tuberculosis (contd)

• PPD Test (contd)
• Positive Test

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Mycobacterium tuberculosis (contd)

• Extrapulmonary tuberculosis
• Spleen
• Liver
• Lungs
• Bone marrow
• Kidney
• Adrenal gland
• Eyes

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Mycobacterium tuberculosis (contd)

• 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

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Mycobacterium tuberculosis (contd)

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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

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Other Mycobacteria (contd)

• NTM (contd)
• M. avium Complex (MAC)
• M. kansasii
• Mycobacterium fortiutum-chelonei Complex
• M. marinum
• Etc., etc.

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Mycobacterium leprae

• Causes leprosy or Hansens 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

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Mycobacterium leprae (contd)