Chapter 22 – Mycobacterium tuberculosis & Other Nontuberculous MycobacteriaMLAB 2434 – Clinical MicrobiologyCecile Sanders & Keri Brophy-Martinez

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 heatOnce stained, resist decolorization

with acid-alcohol (acid-fast)

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

General Characteristics (cont’d) Newer techniques

Automated culture system, such as BACTEC

Nucleic acid probes with PCRThin-layer chromatographyGLCHigh-performance liquid

chromatography

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

Safety Considerations (cont’d) Skin test

Also called “Mantoux test” and PPDThose with positive skin test must

be advised to have chest X-ray Proper Ventilation

Separate from other parts of labNegative air pressure (6 to 12

room air changes/hour)

Safety Considerations (cont’d) Biological Safety Cabinet 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

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

Urine – First morning preferred

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

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) 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

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

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 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

Culture Media and Isolation Methods (cont’d) Labs with large volumes of

Mycobacterium cultures use an automated reader (BACTEC)BACTEC broth contains 14C-

labeled substrateWhen 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 only2. Acid-fast stains and/or

inoculation only3. Isolation and presumptive

identification of Mycobacterium species

4. Definitive identification and antibiotic sensitivity testing

Identification of Mycobacterium Colony Morphology

Either smooth and soft or rough and friable

Growth rateRapid growers – colonies in < 7

daysSlow growers – colonies in > 7 days

Temperature

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

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

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

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

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

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”

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

Mycobacterium tuberculosis (cont’d) Extrapulmonary tuberculosis

SpleenLiverLungsBone marrowKidneyAdrenal glandEyes

Mycobacterium tuberculosis (cont’d) Identification

Slow growerColonies are thin, flat, spreading

and friable with a rough appearance

May exhibit characteristic “cord” formation

Grows best at 35 to 37° CColonies are NOT photoreactive

Mycobacterium tuberculosis (cont’d)

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

Other Mycobacteria (cont’d) NTM (cont’d)

M. avium Complex (MAC)M. kansasiiMycobacterium fortiutum-

chelonei ComplexM. marinumEtc., etc.

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

Mycobacterium leprae (cont’d)