Mycobacterium species &
Other Nontuberculous
Mycobacteria
MLAB 2434 – Microbiology
Keri Brophy-Martinez
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
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
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. canetti
M.
paratuberculosis
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

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”

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
Mycobacterium
tuberculosis (cont’d)

PPD Test-
Mycobacterium
tuberculosis (cont’d)

PPD Test (cont’d)
Positive Test

Detects patients
cell-mediated
immune response
to bacterial
antigens
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
Mycobacterium
tuberculosis (cont’d)

Extrapulmonary tuberculosis
Spleen
 Liver
 Lungs
 Bone marrow
 Kidney
 Adrenal gland
 Eyes

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
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
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
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)
Mycobacterium leprae
(cont’d)
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
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
Safety Considerations
(cont’d)

Use of Proper Disinfectant
Bactericidal for mycobacteria
 Also called “tuberculocidal”


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
 Swabs are discouraged due to
decreased recovery

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

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 12-24
hours
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
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
Digestion & Decontamination of
Specimens (cont’d)

Purposes
To liquefy the sample to clear
proteinaceous material
 Agent kills nonmycobacterial
organisms

Digestion & Decontamination
of Specimens

Decontamination


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 (used with Ps. aeruginosa)

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 for 15 minutes @ 3000g
to concentrate the organisms
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 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
 5-10% CO2
 35-37oC
 Slow growers; cultures held for 6
weeks before calling negative

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
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

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

New Techniques for
Identification
Automated culture system, such as
BACTEC
 Nucleic acid probes with PCR
 Gas Liquid chromatography
 High-performance liquid
chromatography

Identification of
Mycobacteria

Traditional characteristics used to
identify Mycobacterium






Rate of growth
Colony morphology
Pigment production
Nutritional requirements
Optimum incubation temperature
Biochemical test results
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
Identification of
Mycobacterium (cont’d)

Biochemical Identification
•
•
•
•
•
•
•
•
•
Niacin accumulation
Nitrate reduction
Catalase
Iron uptake
Arylsulfatase
Pyrainamidase
Telluride reduction
Urease
Hydrolysis of Tween 80
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
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
References



Centers for Disease
Control. (n.d.). Tuberculosis. Retrieved from
http://www.cdc.gov/tb/publications/LTBI/diagnosis.h
tm#4
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.