Mycobacterium Flashcards
1
Q
Mycobacteria General Characteristics
A
- Acid fast
- Aerobic
- Non spore forming
- Non motile
- Have a high lipid cell wall content
- Temperature Requirements
- Most mycobacteria can be isolated at 36 o C with 7 10% CO 2
- There are several species which require a significantly lower primary recovery temperature
- 30 to 32 o C.
- There are also some which require a higher temperature for recovery as well.
2
Q
Group I Photochromogens
A
- They are defined as colonies which develop a yellow orange pigment on exposure to the light of a 60 watt bulb placed 6 inches above the tube for 60 minutes. They take longer than 7 days to appear on solid media.
- Members include:
- M. kansasii , M. simiae , M. marinum , M. asiaticum
3
Q
Group II Scotochromogens
A
- They are defined as colonies that develop a yellow-orange pigment in the dark or light and take longer than 7 days to appear on solid media.
- Members of this group include
- M. gordonae
- M. scrofulaceum
- M. szulgai (at 37 o C)
4
Q
Group III Nonphotochromogens
A
- They are defined as colonies that are non-pigmented regardless of whether they are grown in the dark or light and take longer than 7 days to appear on solid media.
- Members include:
- M. avium complex
- M. intracellulare
- M. xenopi
- M. ulcerans
- M. malmoense
5
Q
Group IV Rapid Growers
A
- They are defined as colonies that grow on solid media in less than 7 days.
- Mycobacteria forming colonies within 7 days are termed rapid growers, those requiring longer periods are termed, slow growers
- Inoculate well isolated colony of organism to 7H9 broth containing Tween 80
- Incubate broth for several days until medium faintly turbid
- Dilute broth 1:100, streak inoculate to Middlebrook 7H10 agar plate. Observe cultures at 5 to 7 days and (if no growth) weekly thereafter for visible colonies
- Members of this group include:
- M. fortuitum
- M. chelonae
- M. peregrinum
- M. abscessus
- M. thermoresistibile
6
Q
M. leprae General
A
- Cause of Hansen’s Disease or Leprosy
- Discovered by the Norwegian G. H. Armauer Hansen in 1873
- 2 3 million cases worldwide
- WHO listed 91 countries where it is endemic
- In 2004 there were 407,791 new cases world wide
- In 2015 the CDC reported 178 cases in the United States
- There are 4 strains of M. leprae
7
Q
M. leprae Clinical Picture
A
- Causes severe, disfiguring skin sores and nerve damage in the arms, legs and skin areas around the body.
- It can also attack the eyes and the thin mucus lining of the nose.
- The main symptom is disfiguring skin sores, lumps or bumps that do not go away after several weeks or months.
- Skin sores are pale colored.
- Nerve damage is also common.
8
Q
M. leprae Laboratory Testing
A
- There are no serological or skin tests available.
- Skin biopsy needed for the definitive diagnosis.
- PCR for M. leprae DNA may be needed in special circumstances.
- Cardinal signs along with skin biopsy is the most common way to diagnosis leprosy.
9
Q
Leprosy Treatment
A
- Treatment is done as a multi drug regimen.
- Treatment for paucibacillary leprosy:
- 6 month regimen
- Adult: 100 mg Dapsone/daily & 600 mg
- Rifampicin/monthly
- Treatment for multibacillary leprosy:
- 12 month regimen
- Adult: Dapsone 100 mg/daily & Clofazimine 300 mg/monthly & Rifampicin 600 mg/monthly or 50 mg/daily
10
Q
M. Tuberculosis Complex
A
- M. tuberculosis
- M. africanum
- M. bovis & the Bacillus Calmette Guerin strain (humans +)
- M. microti
- M. canetti
- M. caprae
- M. pinnipedii
- M. suricattae
- M. mungi
11
Q
M. Tuberculosis Complex Symptoms & Transmission
A
- Symptoms:
- Early symptoms include
- Weight loss
- Fever
- Night sweats
- Loss of appetite
- Early symptoms include
- Transmission:
- Aerosol from one person to another usually thru coughing or sneezing even singing!
- Extrapulmonary TB
- M. tuberculosis can infect any organ of the body
- Symptoms vary by site of disease
- Pulmonary TB
- Cough >2 weeks
- often productive (sputum), can be bloody
- Fever
- Night sweats
- Weight loss
- Chest pain
12
Q
Diagnosis of TB Disease
A
- Signs and Symptoms consistent with TB
- Chest X ray
- Clinical Judgment
- Bacteriology
- AFB smear microscopy
- Nucleic Acid Amplification Testing
- Culture and Identification
- Drug susceptibility testing
13
Q
TB Specimens for Testing
A
- Sputum 3 first morning collections
- Bronchial washes
- Stool
- Biopsies
- CSF
- Gastric washings
- Urine
14
Q
TB Primary Isolation Medias
A
- Lowenstein Jensen (LJ’s) slants
- Lowenstein Jensen Gruft’s
- Middlebrook 7H9 broth
- Middlebrook 7H10 plates
- Middlebrook 7H11 plates
15
Q
Digestion Methods
A
- 3 most common methods for sputum are;
- NAOH method (sodium hydroxide)
- Zephiran trisodium phosphate method
- NALC NaOH method (N-Acetyl L-cysteine)
16
Q
Identification by Biochemical Procedures
A
- The following biochemical procedures are run when identifying unknown mycobacteria:
- Growth rate
- Colony morphology
- Photochromogenicity
- Nitrate Reduction
- Niacin Accumulation
- Arylsulfatase
- 68 o C Catalase
- Semi quantitative catalase
- Tween 80 hydrolysis
- Tellurite Reduction
17
Q
Treatment of TB
A
- Active TB
- Isoniazid (INH)
- Rifampin (RIF)
- Ethambutol (EMB)
- Pyrazinamide (PZA)
18
Q
Drug-Resistant TB
A
- Defined as TB bacteria that are resistant to at least one first line anti TB drug.
- Multidrug resistant TB (MDRTB) is resistant to more than one anti TB drug and at least Isoniazid (INH and Rifampin (RIF).
- Extensively drug resistant TB (XDRTB) is a rare type of MDRTB that is resistant to Isoniazid and Rifampin, plus any fluoroquinolone and at least one of the three
- injectable second line drugs.
- Second line drugs include: Amikacin, Kanamycin or Capreomycin.
19
Q
CCR 5199 f (4)(F) Safe Procedures
A
- Establish safe handling and prohibit practices, such as sniffing in vitro cultures, that myincrease employee exposure to infectious agents”
-
CCR 5199 f (4)(F) Decon
- “Establish effective decontamination anddisinfection procedures
20
Q
Respiratory (Pulmonary) Specimens
A
- Sputum
- Most frequent specimen submitted for Mycobacteriological testing
- Expectorated
- Induced
- Most frequent specimen submitted for Mycobacteriological testing
- Bronchoalveolar lavage
- Bronchial wash
- Bronchial brush
- Transtracheal aspirate
21
Q
Extrapulmonary Specimens
A
- Non Respiratory Samples
- Tissue, Body fluids, Stool, Gastric Lavage
- Two groups
- Aseptically collected specimens
- Specimens known to contain contaminating flora
22
Q
Culture of acid-fast bacilli
A
- Egg based medium (Lowenstein Jensen)
- Agar and broth based medium (Middlebrook 7H10, Dubos Tween Albumen Broth)
23
Q
Lowenstein-Jensen Egg Base Medium
A
- Coagulated whole eggs
- Potato flour
- Glycerol
- Defined salts
- Malachite Green (0.025 g/100 mL) (Petragnani 0.052 g/100 mL) (ATS 0.020 g/100 mL)
24
Q
Middlebrook Agar Base 7H10 Medium
A
- Defined salts
- Vitamins and Cofactors
- Oleic acid
- Albumin
- Catalase
- Glycerol
- Dextrose
- Malachite Green (0.0025g/100 mL)
25
Q
Middlebrook Agar Base 7H11 Medium
A
- The same composition as Middlebrook 7H10 except for 0.1% casein hydrolysate added for enhanced recovery of fastidious isoniazid-resistant Mycobacterium tuberculosis
- Selective 7H11 contains carbenicillin, amphotericin B, polymixin B, and trimethoprim to inhibit oropharyngeal commensals
26
Q
Identification of Mycobacterium tuberculosis Complex by AccuP robe
A
- Acridinium ester labeled DNA probes utilized that hybridize to Mycobacterium tuberculosis complex specific 16S rRNA (AccuProbe, GenProbe Inc., San Diego,
- Target 16S rRNA released by sonication of organisms recovered by culture
- Acridinium ester is chemiluminescent, and DNA probe 16S rRNA hybrids emit light when acridinium ester hydrolyzed to ground state by alkaline peroxidation
- Chemiluminescence measured in a luminometer
- Amount of light emitted proportional to amount of DNA RNA hybrids formed
- Total time for AccuProbe test is 2 hours
- Available for TB, M. avium cplx, M. gordonae, M. kansasii
- Positive identification for all members of the Mycobacterium tuberculosis complex, including Mycobacterium tuberculosis and Mycobacterium bovis (including attenuated BCG)
- In clinical setting where invasive BCG infection possible (adjuvant therapy for bladder cancer) biochemical identification of BCG required
27
Q
Biochemial identification of Mycobacterium Bovis BCG
A
- Growth on T2H
- Niacin accumulation
- Nitrate reduction
- Pyrazinamidase
- BCG is Neg for all
- TB is POS for all
28
Q
Biochemical Tests for the Identification of Mycobacteria
A
- Niacin accumulation
- Nitrate reduction
- Pyrazinamidase
- Tween 80 hydrolysis
- Urease
- Arylsulfatase
- Iron uptake
29
Q
Niacin Accumulation
A
- Niacin (nicotinic acid) is a precursor in the biosynthesis of NAD and NADP
- All mycobacteria produce nicotinic acid, but some species have a block in the NAD scavenging pathway and excrete niacin
- Free niacin was detected with a cyanogen halide in the presence of a primary amine producing a yellow-colored product
- Niacin paper strip test is commercially available (BD and Remel)
30
Q
Nitrate Reduction
A
- Species of mycobacteria differ quantitatively in the ability to reduce nitrate to nitrite
- Nitrate substrate broth heavily inoculated with organism
- After 2 hr at 37 o C sulfanilamide and N-napthylethylenediamine added
- Nitrite forms pink-red product (no color add zinc powder to confirm negative)
31
Q
Pyrazinamidase
A
- Enzyme pyrazinamidase hydrolyzes pyrazinamide (PZA) to ammonia and pyrazinoic acid
- Agar with Dubos broth containing PZA heavily inoculated with organism and incubated at 37 o C for 4 days
- 1% ferrous ammonium sulfate added and agar observed after 4 hr
- Pink band forms in agar from reaction of ferrous ammonium sulfate with pyrazinoic acid in a positive reaction
32
Q
Tween 80 hydrolysis
A
- Lipases produced by several mycobacterial species hydrolyze the detergent polyoxyethylene sorbitan monooleate (Tween 80) into oleic acid and polyoxyethylene sorbitol
- Tween 80 substrate solution inoculated with organism contains neutral red which is bound to Tween 80 producing an amber color
- With hydrolysis of Tween 80 neutral red is released and produces a red color
- A change in the color of Tween 80 from amber to pink or red after 24 h, 5 days, or 10 days is a positive result for Tween 80 hydrolysis
33
Q
Urease
A
- Urease production useful in identification of scotochromogens and nonchromogens
- Christensen’s urea agar with phenol red pH indicator inoculated with organisms visually read after 1, 3, and 5 days of incubation
- Urease hydrolyzes urea to ammonia and CO 2 increasing agar pH to alkaline values
- Positive reaction is a pink to red color of phenol red at an alkaline pH
34
Q
Arylsulfatase
A
- Arylsulfatase hydrolyzes the sulfur ester bond linking the aromatic rings of tripotassium phenolphthalein disulfate, releasing free phenolphthalein
- Although arylsulfatase activity can be detected with all mycobacteria with prolonged incubation, a 3 day incubation identifies several species. We also do the 14 day arylsulfatase test
- Dubos liquid medium containing tripotassium phenolphthalein disulfate is inoculated, incubated 3 days, and Na 2 CO 3 added to alkalinize the medium
- Free phenolphthalein turns a pink color at alkaline pH, and development of a pink color with addition of Na 2 CO 3 is a positive reaction
35
Q
Iron uptake
A
- Iron uptake test utilized to identify rapidly growing mycobacteria capable of converting ferric ammonium citrate to an iron oxide
- LJ slant inoculated with the organism incubated until visible growth develops, aqueous ferric ammonium citrate added, and the slant incubated for up to 21 days at 37 o C
- Development of reddish-brown color in the colonies indicates production of iron oxide and is a positive result
- Rapid Growers
- Fortuitum = POS
- Chelonae = NEG
