Mycobacteria Flashcards
What are Mycobacteria?
Aerobic, non-spore forming, nonmotile rods
Which stain is used for Mycobacteria?
Acid-fast stain: uses phenol to force cells to complex with the dye and once stained, the cells resist decolorizing even with a strong decolorizer
Gram stain crystal violet and safranin are unable to penetrate the cell wall lipids, giving a false NOS result
Which organisms are in the M. tuberculosis complex?
M. tuberculosis, M. bovis, and M. africanum
All three can cause TB
What are the three different types of tuberculosis?
Primary: an infection in a previously uninfected individual through inhalation of droplets
Secondary: an infection caused by latent organisms when a host becomes debilitated
Active: causing disease in lungs, meninges, kidneys, bones, and/or genital tract
What are the symptoms of tuberculosis?
granulomas, coughing, hemoptysis, weight loss, and fever
What are granulomas?
tumor-like inflammatory lesions that form with necrotic centers resembling soft cheese; called tubercles when cause by TB
What is miliary TB?
disseminated TB with scattered tubercles in the body
Skin test for TB
a purified protein derivative (MTB antigen) is injected into the skin and infected people will have a red, indurated area at the injection site
What is a con for the TB skin test?
It cannot distinguish between people with active disease and those with latent infections
M. avium complex diseases
M. avium and M. intracellulare are in the complex and are the most common cause of non-tuberculosis mycobacteria (NTM) infections; can cause pulmonary disease and mycobacterial lymphadenitis; can cause gastrointestinal or disseminated disease in people with AIDS
M. fortuitum complex diseases
Contains M. fortuitum, M. chelonae, and M. abscessus; can cause wound infections, abscesses, osteomyelitis, and pulmonary infections
M. haemophilum diseases
causes skin ulcers, lymphadenitis, and disseminated disease; it requires hemin, hemoglobin, or ferric ammonium citrate for growth
M. kansasii diseases
common cause of NTM pulmonary disease
M. leprae diseases
causes leprosy/Hansen’s disease which affects skin, PNS, and mucous membranes
M. leprae diagnosis
It does not grow in vitro; PCR test from tissue is available and AFB stain shows “cigar packet” appearance
M. marinum diseases
causes swimming pool granuloma when damaged skin comes in contact with fresh or salt water; leads to red, tender subcutaneous nodules that should be biopsied
M. scrofulaceum diseases
causes scrofula (inflamed neck lymph nodes)
M. ulcerans diseases
causes skin ulcers fro, mycolactones mostly in 5-15 yr olds; Africa- Buruli ulcers; Australia- Bairnsdale ulcers
Which mycobacterium is a common lab contaminant?
M. gordonae (tap-water bacillus)
What are the general steps of AFB processing?
- Concentrated w/ centrifugation (15 min @ 3000g or 1500 rpm)
- Decontamination to remove flora
- Digestion to free mycobacteria from clumps of protein and sediment during concentration
NALC-NaOH
a commonly used combination to digest and decontaminate specimen; N-acetyl-L-cysteine (NALC) digests mucus and NaOH decontaminates
A phosphate buffer is added to neutralize NaOH after decontamination
Specimen Processing Procedure
- Place 5-10 mL of specimen in a tube and add an equal amount of NALC-NaOH
- Vortex and let sit for 15 minutes
- Add phosphate buffer and centrifuge
- Decant supernatant and resuspend the sediment with saline
- Use this liquid for plates and smear
What are the different methods for AFB staining?
Ziehl-Neelsen/Hot method, Kinyoun stain/cold method, and fluorochrome stain
Ziel-Heelsen Method of Staining
- Flood smear w/ carbolfuchsin and then heat until it has steamed several minutes
- Allow the slide to cool and then rinse with water
- Decolorize with 3% acid alcohol, rinse, counterstain w/ methylene blue and rinse
- Air dry the slide
Kinyoun Method of Staining
Same steps as Ziel-Heelsen, but without heating because it has a carbolfuchsin reagent with a higher concentration of phenol and basic fuchsin
What color is AFB in the hot and cold method of staining?
AFB are red from the carbol fuchsin primary stain and non-AFB are blue from the potassium permanganate counterstain
Fluorochrome stain
Uses Auramine O
1. Flood the slide with the fluorochrome reagent and let it sit for 15 minutes
2. Rinse, decolorize w/ 0.5% acid alcohol for 2 minutes
3. Rinse, counterstain w/ potassium permanganate or acridine orange
What color is AFB in the fluorochrome method of staining?
AFB are fluorescent yellow/orange
What are the different specimen collection requirements?
- Respiratory should be sputum or BAL w/ 3-5 exporated sputum specimen from different days
- Gastric aspirates only if sputum cannot be collected and sodium bicarbonate should be added so stomach acid does not damage mycobacteria
- Urine 3-5 first morning
- Stool only for patients with AIDS and suspected M. avium complex
- Others: blood, sterile fluids, tissue, and wound aspirates
What are unacceptable specimen for AFB testing?
Swabs: difficult to dislodge mycobacteria
24 hour pooled sputum will be too contaminated
24 hour pooled urine- urine will inhibit mycobacteria with long exposure
Egg-based media vs. agar-based media
Egg-based: media with fresh eggs that have been solidified by heating
Agar-based: same as other media
Petragnani media
a type of nonselective egg-based media that has a high concentration (0.052%) of malachite green
American Thoracic Society (ATS) medium
a type of nonselective egg-based media that has a low concentration (0.02%) of malachite green; recommended for sterile fluids
Lowenstein-Jensen (LJ) medium
a type of nonselective egg-based media; mostly commonly used with a 0.025% concentration of malachite green
Malachite green
used in media to inhibit growth of contaminants
Middlebrook 7H10/7H11
a type of nonselective agar based media containing albumin, nutrients, and a low concentration of malachite green (0.0001-0.00025%)
What is a con for Middlebrook agar?
It deteriorates and produces formaldehyde when exposed to light, heat, or stored for greater than 4 weeks
BACTEC 460TB System
uses radioactive 14C to detect mycobacterial growth (contains growth factors, antimicrobial agents, and 14C-palmitic acid); if mycobacteria are present, they will metabolize 14C-palmitic acid and produce 14CO2, which is detected by the instrument
BACTEC 9000MB System
detects mycobacterial growth by monitoring the amount of dissolved oxygen in the culture media; As mycobacteria grow, they consume oxygen and a fluorescent sensor detects this change
MB-BacT System
used nonradioactive CO2 to detect mycobacterial growth; a CO2 sensor at the bottom of the bottle changes colors from green to yellow
ESP Myco System
measures the pressure changes that occur as gases are consumes, produced, or both; the bottles have cellulose sponges in modified Middlebrook broth
Other instrumentation culture systems
Septi-Chek AFB method: biphasic system
Lysis centrifugation: blood only
Why do some bottle contain cellulose sponges?
The sponges enhance growth of mycobacteria by increasing the surface area, making oxygen more accessible (mimics lungs)
Rapid growing mycobacteria vs slow growing mycobacteria
Rapid growing - 7 days or less to grow
Slow growing - greater than 7 days to grow
Photochromogens
produce a pigment when exposed to light, but not without light; Ex: M. kansasii and M. marinum
Scotochromogens
produce a pigment in both the light and dark; Ex: M. gordonae and M. scrofulaceum
Nonchromogens
do not produce a pigment; Ex: M. avium complex, M. haemophilum, and M. ulcerans
Identification of M. tuberculosis
Colonies: rough, dry, granular, and buff colored (“cauliflower”)
Microscopic: “serpentine cords” from cord factor
Positive: Niacin and nitrate; NAP susceptible; 68C catalase negative
Identification of M. bovis
colonies resemble TB
Negative: nitrate, niacin, and 68C catalase
Susceptible to NAP and TCH
- The only mycobacteria susceptible to TCH
Identification of M. kansasii
photochromogen
Microscopic: can produce strands like MTB but they are loose, broad, and banded (“cross bar stain” or “Shepherd’s crook” shape)
Hydrolyzes Tween 80, Nitrate +, high catalase producer, PZA -, niacin -
What are the key characteristics of M. kansasii?
A nitrate positive photochromogen is likely M. kansasii
Identification of M. marinum
photochromogen
Optimal temp of 30C for growth
Hydrolyzes Tween 80 and urease, low catalase producer, PZA +, nitrate -
What are the key characteristics of M. marinum?
A nitrate negative and low catalase producing photochromogen is likely M. marinum
Identification of M. gordonae
Scotochromogen
Nitrate negative and hydrolyzes Tween 80
Identification of M. scrofulaceum
Scotochromogen
Nitrate -, Tween 80 -, some strains are urease +
Identification of M. avium complex
Nonchromogen
Grows at 37C in 7-21 days with smooth creamy colonies, no cord factor for stain
low catalase producer, Tween 80 -, nitrate -, urease -, most reduce tellurite
How are the two species in the M. avium complex differentiated from each other?
Both spp. in the complex have the same biochemicals, but they can differentiated with HPLC, GLC, and nucleic acid probes
Identification of M. xenopi
Grows in 14-28 days with “bird’s nest” colonies (sticklike filaments project out)
Optimal growth at 42C, Tween 80 -, nitrate -, and urease -
Identification of M. haemophilum and M. ulcerans
Both do not grow at 37C
Optimal growth at 30C
Identification of M. fortuitum
Rapid grower
Optimal growth at 28C, but it will still grow at 37C; will grow on special MAC
Arylsulfatase +, nitrate +, takes up iron
Identification of M. chelonae and abscessus
Rapid grower
More resistant to antibacterial agents
Nitrate - and do not take up iron
How are the Rapid Growers differentiated from each other?
M. fortuitum produces arylsulfatase and grows on special MAC unlike the two other spp.
M. abscessus can grow on media with 5% NaCl and M. chelonae cannot
MTB Antimicrobial Susceptibility Tests
Portion test/ Quadrant plates: four quadrants (1 control and 3 w/ drugs) are examined over a 3 week period for growth and growth in the drug quadrants are compared to the control
BACTEC Method: a control vial and drug vials are inoculated and incubated and GI changes are monitored and compared to the control
