Chapter 43: Mycobacterium B.

Nontuberculosis mycobacteria (NTM’s)

 Categorization of species
General Characteristics o Runyon Classification of NTM
1. Slender, slightly curved or straight rods
2. Extremely high lipid content a.) Slow Growing NTM
- cord factor wax D and mycolic acid >7 days growth and synthesize carotenoids
3. Acid fast
- Stained by basic fuchsin dye 1. Photochromogens
- resist decolorization by 3% HCl - Develop yellow pigment following exposure to light
4. Slow growth a. Mycobacterium asiaticum
- 2 to 6 weeks at optimum temperature - Rare pulmonary infection
b. Mycobacterium kansasii
A. MTC: Mycobacterium tuberculosis complex - “Yellow bacillus”, second most common NTM in lungs
- Chronic pulmonary and extrapulmonary disease
1. Mycobacterium tuberculosis c. Mycobacterium marinum
a. Epidemiology - “of the sea”
- >1 billion persons are infected c. Mycobacterium simiae
- 8-10 million new cases per year - Rare pulmonary infection
- 15-20% infected develops the disease 2. Scotochromogens
- Transmission - Develop yellow to orange pigment in the dark or light
- Inhalation of aerosols or close contact a. Mycobacterium gordonae
b. Spectrum of Disease - “Tap water bacillus”, rarely implicated in disease
a. Tuberculosis b. Mycobacterium szulgai
- Primary / Reactivation - Pulmonary and extrapulmonary infection
i. Tubercles or granuloma  - Both a photochromogen (22ºC) and scotochromogen
granulomatous lesions (37ºC)
formed from c. Mycobacterium scrofulaceum
ii. Caseation  - Cervical lymphadenitis in children
formation of cheese like d. Mycobacterium xenopi
masses from break down - Pulmonary and extrapulmonary infections
of tubercles. - Grows best 42°C
- “Bird’s nest” in cornmeal agar
Extrapulmonary TB (Miliary TB) 3. Nonphotochromogens
 kidney , GUT - Nonpigmented (white to tan)
 Lymph nodes a. Mycobacterium avium complex “Battey bacillus”
 CNS (meningitis) - Most commonly isolated NTM
 Arthritis - Most common cause of systemic bacterial infection in
 Body cavities AIDS patients
 Larynx b. Mycobacterium avium subspecies paratuberculosis
 Bone (Pott disease ) - Associated with chronic diarrhea
(e.g. Johne’s disease and Crohn’s disease)
 Identification of Mycobacterium tuberculosis c. Mycobacterium celatum
- Raised with a dry, rough appearance, buff colored - Frequent isolate in respiratory specimen
- Grows in 2-3 weeks days on LJ and Middlebrook or 5- d. Mycobacterium genavense
10 days in Middlebrook 7H10 and 7H11 - Associated with infections of AIDS patients
e. Mycobacterium haemophilium
2. Mycobacterium bovis and BCG - Requires hemoglobin and hemin
a. Pathogenesis f. Mycobacterium malmoense
- M. bovis - Coccobacilli without cross-bands
- Ingestion of milk from infected cattle g. Mycobacterium terrae complex
- M. bovis (bacille Calmette-Guerin) - “Radish bacillus”
- Immunization of immunocompromised h. Mycobacterium ulcerans
individuals - Third most common Mycobacteria
b. Colonial Appearance - Buruli ulcers
- Small, granlar, rounded white colonies with irregular
margins (resembles “water droplets”) in Middlebrook b.) Rapidly Growing NTM (<7 days growth)
c. Laboratory Diagnosis 1. Rapid Growers
a. Mycobacterium fortuitum – abscessus group
• Localized cutaneous infections
• Non Photochromogen
b. Mycobacterium chelonae
• Disseminated infections in
immunocompromised patient
• Non Photochromogen
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c. Mycobacterium smegmatis 4. Staining for Acid-Fast Bacilli
• Rare cause of infections  Acid-Fast Stain (AFB Stain)
• Schotochromogen 1. Ziehl-Neelsen (hot stain) and Kinyuon (cold stain)
 Auramine-Rhodamine Fluorochrome Stain
 Noncultivatable 2. Can be examined at (250x to 400x). More
1. Mycobacterium leprae sensitive
- Hansen’s Disease - Reporting of Smears
a. Tuberculoid Leprosy
o Skin lesions and peripheral nerve involvement
b. Lepromatous leprosy
o Extensive skin lesions & symmetric nerve
damage

 Isolation and Identification of Mycobacteria

1. Laboratory Safety Considerations

o Specimen is collected in sterile, leak proof
5. Culture Media and Isolation Methods
containers.
o Biosafety Level 2 practices for preparing AFB
A. Solid Media
smears and culture
- Incubated at 35°C in the dark in 5-10% CO2 and
o Biosafety Level 3 practices for propagation
↑ humidity
o All aerosol-generating procedures must be
- Most isolates appear at ≥17 daysS
performed in Class II or III BSC
1. Egg Based
- Lowenstein-Jensen and Petragnani
2. Specimen Collection
- Gruft modified Lowenstein-Jensen
a. Sputum and Other secretions
2. Agar based
- Sputum: Early morning specimens collected in
- Middlebrook 7H10 and 7H11
three (3) consecutive days
- Selective Middlebrook 7H10 and 7H11
i. Transtracheal aspiration
(Mitchison’s)
ii. Bronchoscopy
iii. Laryngeal swabbing
B. Liquid Media
b. Gastric Aspirates and Washings
- Incubated at 35°C in the dark in 5-10% CO2 and
i. Three specimens are collected within 3
high humidity
days using a Levine tube
- Most isolates appear at 10 days
ii. 20-25 ml in a 50 ml conical tube
- Examples:
c. Urine
 BACTEC 12B medium
- Early morning urine in 3 consecutive days
 Middlebrook 7H9 broth
- Collected by midstream clean catch
 Septi-Chek AFB
d. Stool
- For isolation of disseminated M. avium complex
6. Laboratory Identification
in AIDS patients.
I. Tuberculin Skin Testing
e. Blood
o Mantoux test, Pirquet test, PPD test
- For isolation of disseminated M. avium complex
 PPD is intracutaneously injected
in AIDS patients.
 Positive: erythema (redness) and
- Recovered by radiometric BACTEC 13A vial or
induration (firmness) in 48 to 72 hours
by Isolator lysis centrifugation system
f. Tissue and Body Fluids
II. Conventional Phenotypic Tests
- CSF (tuberculosis meningitis)
- Pleural, pericardial and peritoneal fluid
1. Growth Rate and Characteristics
3. Digestion and Decontamination of Specimens a) Photoreactivity
a. n-Acetyl-L-cysteine (NALC) or dithioreitol Determine the ability of Mycobacteria to produce
- NALC liquefy specimen (splits disulfide bonds) carotenoids
b. 2-4% NaOH b) Photochromogen
- Acts as both digestant (mucolytic) and Unpigmented when grown in the dark and develop
decontaminating (antibacterial) agent pigment after light exposure
c. Benzalkonium chloride (Zephiran) and Trisodium c) Schotochromogen
phosphate (Z-TSP) Pigmented when grown in the dark but may/may not
- Z-TSP acts as digestant and Zephiran acts as intensify w/ exposure to light
decontaminating agent d) Nonphotochromogen
d. Oxalic Acid Nonpigmented when grown in the dark and exposed
For Pseudomonas contaminated samples to light

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III. Biochemical testing
e) Tellurite reduction
a) Niacin (Accumulation) Test

f) Arylsulfatase

b) Nitrate reduction

g) Growth Inhibition by TCH

c) Semiquantitative Catalase Test

h) Tolerance to 5% Salt
o Positive – M. triviale
i) Iron Uptake: ferric ammonium citrate  iron oxide
o Positive – M-fortuitum
o Negative – M. chelonei
c.1) Heat stable (68ºC) Catalase Test j) Urease
o Positive – M. scrofulaceum, M. bovis, M. gastri,
M. fortuitum
o Negative – M.gordonae, M. avium-intracellulare
k) Growth in MacConkey
o Positive – M. fortuitum-chelonae
l) Pyrazinamidase (4 d): pyrazinamide  pyrazinoic acid
o Positive – M. tuberculosis
o Negative – M. bovis

 Key Biochemical Reactions

d) 5 Days Tween 80 Hydrolysis

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