mycobacteria

• Mycobacteria


  • Slender, slightly curved or straight, rod-shaped organisms 0.2 to 0.6 μm × 1 to 10 μm in size
  • Non-motile and do not form spores
  • Cell wall has extremely high lipid content; thus, mycobacterial cells resist staining with commonly used basic aniline dyes, such as those used in Gram stain, at RT
  • Take up dye with increased staining time or application of heat but resist decolorization with acid-ethanol – a characteristic referred to as acid fastness – hence, the term AFB
  • Acid fastness is a basic characteristic in distinguishing mycobacteria from most other genera
  • Strictly aerobic, but increased CO2 will enhance the growth of some species
  • Pathogenic mycobacteria grow more slowly than most other bacteria pathogenic for humans
  • Rapidly growing mycobacteria generally grow on simple media in 2 to 3 days at temperatures of 20°C to 40°C
  • Most mycobacteria associated with disease require 2 to 6 weeks of incubation on complex media at specific optimal temperatures
  • One of the mycobacteria pathogenic for humans, M. leprae, fails to grow in vitro

  
• Major Groups of Mycobacteria

  • Mycobacterium tuberculosis complex
  • Mycobacterium leprae (Hansen's bacillus)
  • Atypical mycobacteria or nontuberculous mycobacteria (NTM) or mycobacteria other than tubercle bacillus (MOTT)

  
• Mycobacterium tuberculosis complex


  • Mycobacterium tuberculosis (tubercle bacilli)
  • Mycobacterium bovis (including the vaccination strain bacillus of Calmette-Guérin)
  • Mycobacterium africanum
  • Mycobacterium canettii
  • Mycobacterium microti

  
• Runyon's Classification of MOTT

  • Group I (Photochromogens)
  • Mycobacterium kansasii – also known as yellow bacilli or shepherd's crook
  • Mycobacterium marinum – causes swimming pool granuloma and are usually found in sea water
  • Mycobacterium simiae – causes pulmonary/TB-like infection in monkeys
  • Mycobacterium asiaticum

  Group II (Scotochromogens)

  • Mycobacterium scrofulaceum– causes cervical lymphadenitis, called scrofula
  • Mycobacterium szulgai
  • Mycobacterium gordonae – also called tap-water bacillus
  • Mycobacterium flavescens

  Group III (Non-photochromogens)

  • Mycobacterium avium complex – also known as Mycobacterium intracellulareor Batley's bacillus
  • Mycobacterium gastri – also called J bacillus
  • Mycobacterium terrae complex – also called radish bacillus
  • Mycobacterium terrae
  • Mycobacterium triviale
  • Mycobacterium nonchromogenicum
  • Mycobacterium xenopi – Colonies appear like bird's nest with stick-like projections in cornmeal agar, Scotochromogenic and thermophilic
  • Mycobacterium celatum
  • Mycobacterium genavense
  • Mycobacterium haemophilum
  • Mycobacterium malmoense
  • Mycobacterium ulcerans – Third most common mycobacterium after Mycobacterium tuberculosis and Mycobacterium leprae, causes Buruli ulcer in Africa and Bairnsdale ulcer in Australia

  Group IV (Rapid Growers)

  • Non-pathogenic group which grows in less than 7 days
  • Mycobacterium fortuitum– non-photochromogens
  • Mycobacterium chelonae – non-photochromogens; formerly named Mycobacterium borstolense
  • Mycobacterium smegmatis
  • Mycobacterium phlei – scotochromogen; also called Hay bacillus
  • Mycobacterium vaccae
• Gram stain


  When Gram-stained, Mycobacterium spp. stain faintly or not at all, giving a beaded appearance because of irregular uptake of the stain caused by the increased lipid content of the cell wall

  
• Acid-fast stain


  • Acid-fast smears are prepared directly from clinical specimens and from digested, decontaminated, and concentrated specimens
  • Ziehl-Neelsen (hot method)
  • Kinyoun (cold method)
  • Auramine-rhodamine (bright yellow color) – fluorescent stain
  • FITC (fluorescein isothiocyanate) – fluorescent stain
  • When examining acid fast-stained smears, examine at least 300 fields for 15 minutes

  
• Other acid-fast bacteria include Legionella micdadei, Rhodococcus, and Nocardia
• Culture Media and Isolation Methods

  • Mycobacteria are strictly aerobic and grow more slowly than most bacteria pathogenic for humans
  • M. tuberculosis has the longest replication time, at 20 to 22 hours
  • The rapidly growing species generally form colonies in 2 to 3 days, whereas most pathogenic mycobacteria require 2 to 6 weeks of incubation
  • Growth of M. tuberculosis is enhanced by an atmosphere of 5% to 10% CO2
  • Mycobacteria require a pH between 6.5 and 6.8 for the growth medium and grow better at higher humidity
  • One of the mycobacteria pathogenic for humans, M. genavense, does not grow on media used routinely to isolate mycobacteria and requires extended incubation (6 to 8 weeks)
  • M. leprae and M. microti fail to grow on artificial media

  
• Culture Media Types


  • Egg-based media
  • Serum albumin agar media
  • Liquid media
• Egg-Based Media


  • Basic ingredients in an inspissated egg medium are fresh whole eggs, potato flour, and glycerol, with slight variations in defined salts, milk, and potato flour
  • Each contains malachite green to suppress the growth of gram-positive bacteria
  • Glycerol enhances growth of Mycobacterium avium complex
  • Löwenstein-Jensen (LJ) medium is the medium most commonly used in clinical laboratories and the medium commonly used for niacin test
• Agar-Based Media


  • Serum albumin agar media, such as Middlebrook 7H10 and 7H11 agars, are prepared from a basal medium of defined salts, vitamins, cofactors, glycerol, malachite green, and agar combined with an enrichment consisting of oleic acid, bovine albumin, glucose, and beef catalase (Middlebrook OADC enrichment)
  • Middlebrook 7H11 medium also contains 0.1% casein hydrolysate, which improves the recovery of isoniazid-resistant strains of M. tuberculosis
  • The addition of antimicrobial agents to 7H10 or 7H11 makes the media more selective by suppressing the growth of contaminating bacteria
  • Advantages of clear agar-based media over opaque egg-based media: Can be examined using a dissecting microscope for early detection of growth and colony morphology, Drug susceptibility tests may be performed on agar-based media without altering drug concentrations, which occurs with egg-based media, When specimens are inoculated to Middlebrook 7H10 and 7H11 media and incubated in an atmosphere of 10% CO2 and 90% air, 99% of the positive cultures are detected in 3 to 4 weeks, earlier than for those detected on egg-based media

  
• Liquid-Based Media


  • Mycobacterium spp. grow more rapidly in liquid medium, and it can be used for both primary isolation and subculturing
  • Middlebrook 7H9 broth and Dubos Tween albumin are nonselective liquid media used for: Subculturing stock strains, Picking single colonies, Preparing inoculum for in vitro testing
  • Middlebrook 7H12 and Middlebrook 7H13

  
• Other Culture Media for Recovery of Mycobacteria

  • CHOC agar plate – should be included in the primary isolation media for skin and other body surface specimens for the recovery of M. haemophilum, which requires ferric ammonium citrate or hemin for growth, plate should be incubated at 30°C – optimal temperature for recovery of this organism and for M. marinum
  • Septi-Chek AFB – Biphasic media system for the detection and isolation of mycobacteria, consists of a bottle containing Middlebrook 7H9 broth with an atmosphere of 5% to 8% CO2, an enrichment consisting of growth-enhancing factors and antimicrobial agents, and a paddle with agar media, one side of the paddle is covered with nonselective Middlebrook 7H11 agar, one of the two sections on the reverse side of the paddle contains a modified egg-based medium for differentiating M. tuberculosis from other mycobacteria, the other side contains CHOC agar for the detection of contaminating bacteria, Biphasic media system provides for rapid growth and identification and drug susceptibility testing, without the need for routine subculturing, Biphasic media system is sensitive and does not require the use of radioactivity and a CO2 incubator, Detection times are shorter than with conventional agar but significantly longer than with the BACTEC system

  
• Isolator Lysis-Centrifugation System


  Isolator is a blood collection system that contains saponin to liberate intracellular organisms, After treatment with the saponin, the sample is inoculated onto mycobacteria media plates or tubes, Advantages: System allows for higher yields and shorter recovery times for mycobacteria than conventional blood culture methods, Yield isolated colonies, Ability to quantify mycobacteremia – useful in monitoring effectiveness of treatment (especially disseminated M. avium complex infection

  
• Preliminary Identification of Mycobacteria

  1. First step is to confirm that the isolate recovered in broth or on solid media is an acid-fast organism by performing an acid-fast stain

  2. Once the organisms are growing on solid media, the following phenotypic characteristics help speciate mycobacteria: Colony morphology, Growth rate, Optimal growth temperature, Photoreactivity

  
• Colony Morphology


  • Colonies of mycobacteria are generally distinguished as having a smooth and soft or rough and friable appearance
  • Colonies of Mycobacterium tuberculosis: Rough often exhibit a prominent patterned texture referred to as cording (curved strands of bacilli), This texture is the result of tight cohesion of the bacilli
  • Colonies of Mycobacterium avium complex (MAC) have a variable appearance, with glossy whitish colonies often occurring with smaller translucent colonies

  
• Growth Rate


  • Growth rate and recovery time depend on: Species of mycobacteria, Culture media, Incubation temperature, Initial inoculum size
  • Range in recovery time is wide, from 3 to 60 days
  • Rapid growers – visible growth in fewer than 7 days
  • Slow growers – producing colonies in more than 7 days
  • Determination of growth rate should be evaluated from the time of subculture, not the time of detection from the clinical sample
  • Inoculum should be sufficiently small to produce isolated colonies
  • Microscopic examination of agar for microcolonies allows earlier detection of growth
• Temperature


  • Optimal temperature and range at which a mycobacterial species can grow may be extremely narrow, especially at the time of initial incubation
  • M. marinum, M. ulcerans, and M. haemophilum grow best at 30°C to 32°C and poorly, if at all, at 35°C to 37°C
  • At the other extreme, M. xenopi grows best at 42°C
• Photoreactivity


  Mycobacterium spp. have traditionally been categorized into three groups according to their photoreactive characteristics: Photochromogens – species that produce carotene pigment on exposure to light, color ranges from pale yellow to orange, Scotochromogens – species that produce pigment in the light or the dark, Nonchromogenic or nonphotochromogenic – M. tuberculosis is an example, these colonies are a buff (tan) color and are non-photoreactive, exposure to light does not induce pigment formation

  
• Biochemical Tests for Mycobacteria

  Niacin Accumulation: Most mycobacteria possess the enzyme that converts free niacin to niacin ribonucleotide, Accumulation of niacin, detected as nicotinic acid, is the most commonly used biochemical test for the identification of MTB, Nicotinic acid reacts with CNBr in the presence of an amine to form a yellow-pigmented compound, Reagent-impregnated strips have eliminated the need to handle and dispose of cyanogen bromide, which is caustic and toxic, Cyanogen bromide must be alkalinized with NaOH before disposal, Niacin test may be negative when performed on young cultures with few colonies, Recommended that the test be done on egg agar cultures 3 to 4 weeks old and with at least 50 colonies, Tests that yield negative results may need to be repeated in several weeks

  
• Exposure to light does not induce pigment formation
• Niacin Accumulation


  • Most mycobacteria possess the enzyme that converts free niacin to niacin ribonucleotide
  • Accumulation of niacin, detected as nicotinic acid, is the most commonly used biochemical test for the identification of MTB
  • Nicotinic acid reacts with CNBr in the presence of an amine to form a yellow-pigmented compound

  
• Niacin Accumulation


  • Reagent-impregnated strips have eliminated the need to handle and dispose of cyanogen bromide, which is caustic and toxic
  • Cyanogen bromide must be alkalinized with NaOH before disposal

  
• Niacin test may be negative when performed on young cultures with few colonies
• Recommended that the niacin test be done on egg agar cultures 3 to 4 weeks old and with at least 50 colonies

  
• Tests that yield negative results may need to be repeated in several weeks
• Niacin test should not be performed on scotochromogenic or rapidly growing species because M. simiae, bacillus of Calmette-Guérin (BCG) strain of M. bovis, M. africanum, M. marinum, M. chelonae, and M. bovis may be positive, although this occurs rarely