Chapter 22 –
Mycobacterium
tuberculosis & Other
Nontuberculous
Mycobacteria
MLAB 2434 – Clinical Microbiology
Cecile Sanders & Keri Brophy-Martinez
General Characteristics
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Slender, slightly curved or straight
rod-shaped organisms
Non-motile
Do not form spores
Cell wall with extremely high lipid
content
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Staining requires longer time or
application of heat
Once stained, resist decolorization
with acid-alcohol (acid-fast)
General Characteristics
(cont’d)
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
Strictly aerobic
Grow more slowly than most bacteria
Traditional characteristics used to
identify Mycobacterium
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Rate of growth
Colony morphology
Pigment production
Nutritional requirements
Optimum incubation temperature
Biochemical test results
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

Safety Considerations

Mycobacteriology workers are three
times more likely to seroconvert
(develop positive skin test)
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Adequate safety equipment
Safe laboratory procedures training
Information on hazards
Preparations for unexpected accidents
Staff must be monitored regularly by
medical personnel
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)

Safety Considerations
(cont’d)
Biological Safety Cabinet
 Use of Proper Disinfectant

Bactericidal for mycobacteria
 Also called “tuberculocidal”
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Other precautions
Disposables
 Protective clothing, face masks

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

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

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
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Urine – First morning preferred
Specimen Collection and
Processing (cont’d)
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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
Digestion & Decontamination
of Specimens
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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)

Decontamination
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Specimen from non-sterile site is mixed
with an agent that will kill nonmycobacterium 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
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
Concentration

After decontamination and
digestion, the specimen is
centrifuged in a closed, vented
centrifuge to concentrate the
organisms
Acid Fast Stains
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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 acidalcohol
 Kinyoun – cold stain
 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
 Slow growers; cultures held for 6
weeks before calling negative
 Media
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Lowenstein-Jensen (LJ) media – egg
based
 Middlebrook 7H10 and 7H11 agar –
serum based
 Middlebrook 7H9 broth

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

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

Identification of
Mycobacterium
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Colony Morphology
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Either smooth and soft or rough and
friable
Growth rate
Rapid growers – colonies in < 7 days
 Slow growers – colonies in > 7 days
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Temperature
Identification of
Mycobacterium (cont’d)
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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

Identification of
Mycobacterium (cont’d)

Biochemical Identification
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Most labs now use nucleic acid probes with
or without PCR
Older tests
•
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Niacin accumulation
Nitrate reduction
Catalase
Hydrolysis of Tween 80
Iron uptake
Arylsulfatase
Identification of
Mycobacterium (cont’d)
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Older tests (cont’d)
• Pyrainamidase
• Urease
• Inhibitory tests
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NAP
TCH
Growth in 6.5% NaCl
Tellurite reduction
Growth on MacConkey
Antibiotic Sensitivity
Testing for Mycobacterium
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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
Mycobacterium Infections
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Truly pathogenic
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Potential pathogens
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M. tuberculosis
M. bovis
M. ulcerans
M. kansasii
M. marinum
Other possible pathogens and rare
pathogens listed on p. 670
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 (cont’d)

Primary tuberculosis
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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
Mycobacterium
tuberculosis (cont’d)

Extrapulmonary tuberculosis
Spleen
 Liver
 Lungs
 Bone marrow
 Kidney
 Adrenal gland
 Eyes

Mycobacterium
tuberculosis (cont’d)
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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

Mycobacterium
tuberculosis (cont’d)
Other Mycobacteria
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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)
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Most found in soil and water
Chronic pulmonary disease resembling TB
Other Mycobacteria
(cont’d)
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NTM (cont’d)
M. avium Complex (MAC)
 M. kansasii
 Mycobacterium fortiutum-chelonei
Complex
 M. marinum
 Etc., etc.
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Mycobacterium leprae
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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
Mycobacterium leprae
(cont’d)