Mycobacterium species & Other Nontuberculous Mycobacteria

MLAB 2434 – Microbiology Keri Brophy-Martinez

General Characteristics

Slender, slightly curved or straight rod-shaped organisms

Non-motileDo not form sporesStrictly aerobicVarious 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. paratuberculosis

M. canetti

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

SpleenLiverLungsBone marrowKidneyAdrenal glandEyes

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 labNonrecirculating ventilation

systemsNegative 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 mycobacteriaAlso called “tuberculocidal”

Other precautionsDisposablesProtective 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)

PurposesTo 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 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

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 acid-alcohol

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 basedLiquid 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

marrowBACTEC broth contains 14C-labeled

substrateWhen 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 organismsAdvantages include yielding

isolated colonies, quantification of mycobacteria, shorter recovery times

New Techniques for Identification

Automated culture system, such as BACTEC

Nucleic acid probes with PCRGas Liquid chromatographyHigh-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.htm#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.