Unit 15 Mycobacteria Flashcards by Gabrielle Baldwin

Explain the differences between a typical bacteria and mycobacteria. (4)

High lipid content and mycolic acid in cell wall creates hydrophobic permeability barrier
Resist decolorization with acidified alcohol (3%) when strained with carbolfuchsin
Grows more slowly because of hydrophobic cell surface
Require increased CO2

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What is the purpose of collecting early morning specimens in 3 successive days when dealing with specimens such as sputum, urine, and gastric washings?

Secretions pool overnight so the morning specimens have the highest concentration

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Explain the reason for the decontamination process and what types of specimens require decontamination?

Nonsterile sites; These sites contain mixed flora and decontamination reduces bacterial overgrowth and breaks up the mucus without killing/hurting the mycobacterium

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Explain thoroughly the NALC-NaOH procedure used to decontaminate/concentrate patient specimens for acid fast bacilli.

1) 2% NaOH and N-acetyl-L cysteine are mixed (must be used within 24 hours). Sputum is added to tube. NALC is added to for 1:1 ratio.
2) Vortex for 10 sec
3) Sit for 15 minutes (too long it will kill myco, not enough and not all the flora is killed)
4) Phosphate buffer is added to the 50 mL mark and tilted to neutralize.
5) Centrifuge with separate screw tops for 15 min.
6) Open under hood and decant supernatant. Lip cannot tough bucket. Leave at least 1mL supernatant.
7) Vortex ready to inoculate

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List the 3 reagents used in the NALC-NaOH concentration method and describe their functions.

2% NaOH- decontaminating agent
N-acetyl-L cysteine- mucolytic agent
Phosphate buffer- “washes” the specimen, neutralizes the digestion mixture and lowers the specific gravity

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List 2 advantages of the Auramine O fluorescent stain for the demonstration of AFB in direct smears of clinical specimens compared with either the Ziehl-Nielsen or Kinyoun’s procedures.

1) Dead mycobacterium will still stain

2) Anti-TB meds not clear yet

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What is the “tap water bacillus”?

M. gordonae

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What is considered the optimal environment (temperature and atmosphere) for isolation of most mycobacterium species?

35-37C For most (some 30C and one 42C)

3-11% CO2

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Discuss the general characteristics of the four Runyon groups.

1) Photochromogen- forms pigment after light exposure
2) Scotochromogen- bright yellow/orange in dark or light
3) Nonphotochromogen- No pigment at all; buff
4) Rapid growers

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Describe in detail the principle of the niacin test. Include in your answer what a positive niacin test indicates.

Those that lack the enzyme to convert niacin into niacin ribonucteotide will have niacin accumulation; chemical reaction released cyogen bromide gas causes the strip to discolor to brown OR fluid will turn yellow for accumulation

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Special temperature requirements or other specific growth requirements: M. marinum

25-30C

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Special temperature requirements or other specific growth requirements: M. xenopi

42C

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Special temperature requirements or other specific growth requirements: M. haemophilum

requires hemin to grow; CHOC @ 25-30C

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Special temperature requirements or other specific growth requirements: M. ulcerans

25-30C

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Rapid grower, nitrate negative, arylsulfatase positive within 3 days, sodium chloride positive.

M. chelonae

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Rapid grower, nitrate positive, arylsulfatase positive within 3 days, sodium chloride positive.

M. fortuitum

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infection in AIDS patients

M. avian complex

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cutaneous lesions known as “Bairnsdale ulcer”-

M. ulcerans

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skin infections; associated with surgical wounds, skin trauma, or needle injections:

M. fortuitum/chelonae

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highly communicable respiratory infection-

M. tuberculosis

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pulmonary disease; noncommunicable from person to person-

M. kansasii

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lymphadenitis; commonly affecting children during “teething” years-

M. crofulaceum

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What is the drug(s) of choice to treat a Mycobacterial infection? Primary

Primary:
			Streptomycin
			Isoniazide-most common prophylaxis 
			Rifampin
			Ethambutol

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What is the drug(s) of choice to treat a Mycobacterial infection? Secondarys

Secondary:
Pyrazinamide
Clarithromycin
Fluoroquinolones

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Key tests that will help to differentiate each of the following pairs of mycobacteria: M. tuberculosis vs. M. bovis

1. TB is Niacin and Nitrate POS | 2. TB grows on T2H/TCH

Key tests that will help to differentiate each of the following pairs of mycobacteria: M. fortuitum vs. M. chelonei

1. MF is Nitrate POS

Key tests that will help to differentiate each of the following pairs of mycobacteria: M. gordonae vs. M. scrofulaceum

MG Tween 80 hydrolysis POS

Key tests that will help to differentiate each of the following pairs of mycobacteria: M. kansasii vs. M. marinum

1. MK Nitrate POS 2. MK Urease POS 3. MK is catalase POS

What is the problem in attempting to recover mycobacteria from a swab specimen?

Hydrophobic so it sticks to swab/hard to get on media

What does TB look like on Kinyoun stain?

Serpentine cords

Which is the yellow bacillus?

M. kansasii

Safety precautions

HEPA filter Gown and gloves Strong disinfectant/ Mycobactericidil Annual PPD to process Laminated floor/ no grooves in floor or ceiling Neg. air pressure-read and monitored daily

Nonselective media name

Lowenstein-Jensen

NS media: 2 components and purpose

1) egg based- albumin makes them stick | 2) anilin dye=malachite green- inhibiting agent

Selective medias

LJ Gruft LJ Mycobactosel Selective Middlebrook 7H10 Selective Middlebrook 7H11

NS vs selective

Selective have 2+ antibiotics

Selective Middlebrook 7H11: alternative name and use

Mitchison's Medium used to set up stools; most antibiotics

When is Selective Middlebrook 7H9 broth used?

Only for swabs

4 components of MGIT broth

- 7H9 broth - albumin - glucose - flueorescent oxygen sensor

Why must you do an AFB stain on POS MGIT samples?

Must prove acid fast- S. aureus will also grow (contaminant)

What is the only mycobacterium that requires 42C?

M. xenopi

What type of mycobacterium require 25-30C?

Skin/subcutanous specimens

How does the MIGT incubator work?

If it is present oxygen will be depleted Indicator lights up Instrumer reads qty w/ UV light periodically

What are the 2 carbolfuchsin (+carboxylic acid) stains and how do they differ?

Ziehl-Nelson (hot stain)-flood on filter paper and heat underneath slide until steaming Kinyoun (cold stain)

What is the counter stain in carbolfuchsin stains?

Methylene blue

Pro to using heat in carbolfuchsin stains?

Heat makes cell wall more permeable

What type of water when stain and why?

DI b/c mycobacteria live in tap

How often do we check in incubator?

Weekly

In what position are tubes incubated?

On side

Most common fluorochrome stain? Ingredients?

Auromine O: 3% acid alcohol Potassium permanganate

How do you confirm a positive fluorochrome stain?

Must verify it is acid fast (Pseudo will grow) | Kinyoun right on top (cannot do in reverse)

Typical GENERAL colony morphology of all mycobacteria

rough with feathered edge

Photochromogen

pigment forms after light exposure

Scotochromogen

bright yellow/ orange in light/dark

POS nitrate reduction?

red/pink

2 types of catalase test

1) Semiquantitative | 2) Heat stable @ 68C

Growth inhibition by T2H/TCH

Growth=resistant

Tween 80 hydrolysis

Some mycobacteria produce lipase Tween 80+neutral red will be converted to stuff with lower pH in presence of lipase POS=red

Tellurite reduction

K tellurite production --> POS=brown/black

Arylsulfatase

POS=pink/red 3-4 days

Special MAC

NO crystal violet | Rapid growers only

Why mycobacterium is most likely a contaminant?

M. gordonae

What combination of patient+organism allows for the use of stool specimens?

MAC+AIDS patients

Which mycobacterium grows on algae in stagnant water?

M. ulcerans

Which mycobacterium tend to clump?

M. ulcerans

Which mycobacterium are found in breast implants?

M. fotuitum/chelonae

General way of treating?

1 or 2 primary first, then if really resistant, secondary

What are the only 2 FDA-approved automated methods for susceptibility testing?

MGIT | VersaTrek

Issues with tuberculin skin test(TST)?

very subjective | not senstiive

PPD: what does it stand for?

Purified Protein Derivative skin test

2 immunodiagnostic tests for LATENT tuberculosis

1) Quantiferon gold in-tube | 2) T spot (ELISA) time consuming

4 other ID methods

1) Gene sequencing: Gen-Probe and GenXpert 2) HPLC (high performance liquid chromatography)-reference lab/state health dept 3) DNA microarrays 4) Mass Spec: MALDI-TOF