Mycobacteria

Question 1

Discuss the unique cell wall structure of Mycobacterium:

Answer 1

Cell wall is comprised of peptidoglycan complex and 60% lipids. The 60% lipids are compromised of mycelia acids, cord factor, and wax D.

Mycolic acid is a unique alpha branching lipid that is highly hydrophobic creating a lipid shell

Cord factor is a serpentine cord that is toxic and causes inhibition of PMNs

Wax D is the cell envelope

Question 2

Describe the temperature pattern commonly seen with tuberculosis infections in humans:

Skin -
Lungs -

Answer 2

Skin - 30°C
Lungs - 37°C

42°C for rare Mycobacterium species (xenopi)

Question 3

Name two acid-fast organisms and explain what is meant by the phrase “acid fast”:

Answer 3

M. tuberculosis and M. avium

Acid-Fast: resists decolorization with up to 3% hydrochloric acid and some also resist decolorization with 95% ethanol

Question 4

Compare and contrast the Ziehl-Neelsen and the Kinyon methods of staining for acid fast bacilli:

Answer 4

Both methods utilize carbon fuchsin solution for the primary stain the difference between the two methods is how they get the primary stain into the cell wall of the organism. The Ziehl-Neelson method uses heat where as the Kinyon method uses the detergent phenol

3% acid - alcohol as a decolorizing agent
The counter (2nd) stain used is Methylene blue counter stain

Question 5

Define Multiple Drug Resistant TB:

Answer 5

This organism is resistant to at least one of the two of the anti TB drugs either ionized or rifampin or both and it may be necessary to use amikacin, streptomycin and ciprofloxacin

Question 6

Discuss treatment options for TB:

Answer 6

First line: Isoniazid, Rifampin, Rifapentine

Second line: Clycloserine, Ethionamide, Levofloxacin, Gatifloxacin, Streptomycin

Initial treatment is a 8 week regiment of Isoniazid, rifampin and pyrazinamide

Question 7

M. tuberculosis

Colony Morphology:
Growth Rate:
Heat Stable Catalase:
Nitrate:
Niacin:
NaCl:
Tween 80 Hydrolysis:
Urease:

Answer 7

Colony Morphology: rough cords
Growth Rate: slow
Heat Stable Catalase: neg
Nitrate: pos
Niacin: pos
NaCl: neg
Tween 80 Hydrolysis: neg
Urease: pos

Question 8

M. avium complex

Colony Morphology:
Growth Rate:
Heat Stable Catalase:
Nitrate:
Niacin:
NaCl:
Tween 80 Hydrolysis:
Urease:

Answer 8

Colony Morphology: smooth rough translucent
Growth Rate: slow
Heat Stable Catalase: w pos
Nitrate: pos
Niacin: neg
NaCl: neg
Tween 80 Hydrolysis: neg
Urease: pos

Question 9

M. kansasii

Colony Morphology:
Growth Rate:
Heat Stable Catalase:
Nitrate:
Niacin:
NaCl:
Tween 80 Hydrolysis:
Urease:

Answer 9

Colony Morphology: smooth rough
Growth Rate: slow
Heat Stable Catalase: pos
Nitrate: pos
Niacin: neg
NaCl: neg
Tween 80 Hydrolysis: neg
Urease: pos

Question 10

M. foriticum

Colony Morphology:
Growth Rate:
Heat Stable Catalase:
Nitrate:
Niacin:
NaCl:
Tween 80 Hydrolysis:
Urease:

Answer 10

Colony Morphology: smooth
Growth Rate: rapid
Heat Stable Catalase: pos
Nitrate: pos
Niacin: neg
NaCl: pos
Tween 80 Hydrolysis: pos
Urease: pos

Question 11

M. leprae Identification in the Lab

Answer 11

Cannot grow in the Lab, skin biopsy

Question 12

What disease is caused by Hansen’s bacillus?

Answer 12

Leprosy - M. leprae Tuberculoid and Lepromatous

Question 13

What is the purpose and type of stain for auramine-rhodamine florochrome stain?

Answer 13

Is more sensitive than the carbofuschin stains
Can be screened on a lower magnification
minimum of 300 fields
Orgs appear yellow-orange against a dark background

Question 14

What is the BACTEC method for culturing Mycobacterium? PCR method?

Answer 14

BACTEC - radiometric culture system, isolation, susceptibility testing and an inhibitor to only allow M. tuberculosis to be detected. the org may be isolated for ID within 3 weeks instead of the traditional 6 weeks

PCR technique - now becoming available for use

Question 15

What are the criteria for specimen collection for M. tuberculosis?

Answer 15

Resp. secretions most common, sputum, first morning specimens most concentrated. Collect first in morning

Gastric, urine, stool, blood, CSF and tissue samples are also possible.

Res secretions digestion and decontamination is necessary

Question 16

What is the clinical significance and method of differentiation for the following Non-tuberculosis mycobacterium?

M. kansasii-

M. fortuitum-chelonae-

Answer 16

M. kansasii- pulmonary disease of upper lobes, usually with cavitation and scarring. photochromogen, cat pos, nitrate pos

M. fortuitum-chelonae- variety of infections of skin, lungs, bone, CNS, both are rapid growers, nitrate neg

Question 17

What is the clinical significance and method of differentiation for the following Non-tuberculosis mycobacterium?

M. bovis-

M. avium -

Answer 17

M. bovis- disease manifestation looks like M. tuberculosis in pt., grows more slowly, niacin neg, nitrate neg, don’t grow in TCH, in cattle

M. avium - usually appears as a lung disease, disseminated in immunosuppressed pts. swine poultry

Question 18

Discuss media used for the isolation of Mycobacterium:

Answer 18

Media: Egg based media, Lowenstein-Jensen, American thoracic society (ATS), Petragnan. They contain malachite green to inhibit normal flora, pH between 6.5 to 6.8, and need an increased humidity

Serum or agar based: 7H10 and 7H11 agars
Liquid: BACTEC systems

Question 19

Principle of the acridine orange test

Answer 19

emits green fluorescence when bound to dsDNA(at 520) and red fluorescence when bound to ssDNA or RNA(at 650 nm)

Question 20

Describe and identify the general characteristics of the Runyon groups of Mycobacterium.

Photochromogen

Scotochromogens

Nonchromogenic

Rapid growers

Answer 20

Photochromogen - little or no pigment when grown in the dark but becomes highly pigmented when grown in light.

Scotochromogens - slow-growing and produce a yellow-orange pigment regardless of whether they are grown in the dark or the light.

Nonchromogenic - Nonphotoreactive; pertains to lack of pigment production when colonies are exposed to light.

Rapid growers - grow rapidly

Question 21

Which of the following Mycobacterium spp. can be differentiated by the rapid hydrolysis of Tween 80?

Answer 21

Mycobacterium kansasii