Mycobacterium

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1

Mycobacteria

slender, slow growing bacilli; cell wall rich in lipids (cord factor wax D and mycolic acid); agents of tuberculosis and other chronic infections

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acid fast bacilli

once stained, cannot be decolorized even with acid-alcohol as the decolorizer

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Mycobacterium tuberculosis (MTBC) complex

clinically significant human pathogens capable of causing tuberculosis; includes M. tuberculosis, bovis, africanum, carnetti, and microti

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Nontuberculosis mycobacterium (NTB)

atypical mycobacterium other than tubercule; Runyan groups classified based on production of pigment and growth characteristics

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pulmonary tuberculosis

TB most frequently involves the lung; sputum specimen: deep cough secretions collected on 3 consecutive days, preferably in morning or gastric lavage for children; urine (3 consecutive days); sterile sites: CSF, synovial, pericardial or peritoneal fluids; blood; tissue; stool

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decontaminate, digest, and centrifuge

specimen processing of sputum sample

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decontaminate

removes normal flora and bacterial contaminants from sputum

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digestion

splits disulfide bonds in mucoid of sputum to release the organism

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centrifuge

concentrates mycobacteria to concentrate specimen to improve recovery

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2% NaOH and benzalkonium

weak bases used to decontaminate sputum

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n-Acetyl-L-cysteine (NALC) and dithiothreitol

used to digest sputum

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acid-fast stain

used in preliminary identification of mycobacteria; positive is presumptive; negative does not rule out

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acid-fast method

carbolfuchsin, decolorize, and counterstain with methylene blue

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carbolfuschin

binds to mycolic acid in cell wall; cannot be removed with acid alcohol

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Ziehl-Neelsen

hot method

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Kinyoun

cold method

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methylene blue

counterstain in acid-fast method

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fluorochrome stains

used to stain for AFB using auramine O and rhodamine; greater sensitivity than carbolfuchsin but very labor intensive and costly

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Auramine O

stains mycobacteria yellow-green

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rhodamine

counterstains red fluorescent color

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nonselective culture media

requires whole egg for growth, incubated 6-10 weeks and examined weekly; includes Lowenstein-Jensen, Petragnani, and American Thoracic Society

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agar-based culture media

transparent, growth can be detected earlier than nonselective; includes Middlebrook 7H10 and 7H11

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selective culture media

antibiotics incorporated to inhibit bacteria and fungi; may enhance recovery of mycobacteria, but also inhibits their growth; includes Gruft modified Lowenstein-Jensen slant and selective Middlebrook 7H11 agar

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cap loosened

important for first few weeks to allow CO2 to circulate inside media

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rapid and slow growers

growth rate

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whole egg

required for growth in nonselective media

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photochromogens, scotochromogens, and nonchromogens

pigment production

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growth rate, pigment production, colonial morphology, and growth temperature

identification

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35C +/- 2C

M. tuberculosis complex growth temperature

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30 +/- 2C

skin pathogen (M. marinum and ulcerans) growth temperature

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niacin

produced by all mycobacterium

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yellow

color produced if niacin is being produced

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M. tuberculosis

positive (yellow) nitrate reduction test meaning nitrate is built up

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M. bovis

negative nitrate reduction test meaning nitrate is reduced to nitrate ribonucleotide

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nitrate reduction

nitroreductase converts nitrates (NO3) to nitrites (NO2)

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catalase positive

catalase reaction of most mycobacterium; not all for heat-stable catalase

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photochromogens

mycobacterium develop a yellow to orange only when exposed to light

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scotochromogens

yellow to deep orange pigment in the dark which intensifies to an orange to red when exposed to a constant light source

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nonchromogens

do not develop pigment

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molecular techniques

uses nucleic acid probes, DNA sequencing, fingerprinting, and strain typing leading to direct identification

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M. tuberculosis

only mycobacterium that cannot convert niacin to niacin ribonucleotide due to lacking the enzyme; the niacin builds up

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lungs

most common spectrum of the disease

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Tuberculin Skin Test (TST)

used to determine if individual has been exposed

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Purified Protein Derivative (PPD)

screen test for exposure

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tuberculosis, ulcerans, bovis, and leprae

the most clinically significant mycobacterium

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thin, slightly curved, strongly acid-fast bacillus

staining morphology

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nonpigmented buff to tan colored on LJ and Middlebrook

colonial morphology color

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slow-growing, rough colonies resembling bread crumbs

colonial morphology

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niacin, nitrate, and catalase positive

major biochemical reactions

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Rifampin

most common first line drug

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Buruli ulcers

cutaneous lesions under the skin found in M. ulcerans

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M. ulcerans

produces Buruli ulcers; primarily found in central and west Africa, Mexico, and Australia; carried from soil into draining systems

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M. bovis

tuberculosis in cattle and other warm-blooded animals; niacin and nitrate negative

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M. leprae

agent of leprosy/Hansen's disease; cannot be cultured in vitro; infection of skin, mucous membranes, and peripheral nerves spread through direct contact

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NTM

includes photochromogens

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M. kansasii

yellow bacillus photochromogens; chronic pulmonary disease

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M. marinum

inadequately chlorinated fresh water or salt water; swimming pool granuloma; skin infection

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M. gordonae

tap water bacillus; smooth, deep yellow-orange pigment

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Battey Bacillus

outbreak of M. avium-intracellulare complex; nonphotochromogen

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M. fortuitum and chelonei

rapidly growing - less than 7 days and are not pigmented