Bact

• Mycobacterium


  Genus of bacteria belonging to the family Mycobacteriaceae
• Over 80 named species of mycobacteria
• Mycobacterium


  Meaning 'fungus like bacterium' derived from the mold-like appearance of Mycobacterium tuberculosis when growing in liquid media
• Nontuberculous mycobacteria (NTM)


  • Also called mycobacteria other than M. tuberculosis
  • Cause a resurgence of diseases in immunocompromised individuals worldwide
• Atypical mycobacteria


  • Also called mycobacteria other than the tubercle bacillus
• Mycobacteria


  • Slender, slightly curved or straight, rod-shaped organisms, 0.2 to 0.6 µm × 1 to 10 µm in size
  • Nonmotile and do not form spores
  • Cell wall has extremely high lipid content, resist staining with commonly used basic aniline dyes
  • Referred to as acid fastness, hence the term acid-fast bacilli (AFB)
  • Strictly aerobic, but increased carbon dioxide (CO2) concentration will enhance the growth of some species
• Pathogenic mycobacteria


  • Grow more slowly than most other bacteria pathogenic to humans
  • Most mycobacteria associated with disease require 2 to 6 weeks of incubation on complex media at specific optimal temperatures
  • M. leprae fails to grow in vitro
  • Rapidly growing species generally grow on simple media in 2 to 3 days at temperatures of 20° to 40° C
• AFB staining


  1. Carbolfuchsin stains include the Ziehl-neelsen and Kinyoun, which use carbolfuchsin as the primary stain, acid alcohol for the decolorizing agent, and a methylene blue counterstain

  2. Ziehl-neelsen stain utilizes heat to drive the stain into the mycobacterial cell wall

  3. Kinyoun stain is a "cold" variation of the stain

  4. Stained smears are viewed with a light microscope

  5. Acid-fast organisms stain pink or red against a blue background, nonacid-fast organisms stain blue
• Fluorochrome staining


  1. Auramine and auramine-rhodamine stains are very sensitive and require a fluorescence microscope

  2. Acid-fast organisms fluoresce a yellow-orange against a dark background
• Mycobacterial culture


  1. Common specimen is respiratory secretions (sputum)

  2. Mycobacteria can be recovered from virtually any body site

  3. Nonsterile sites require digestion and decontamination before inoculation of growth media

  4. Decontamination and digestion agents include sodium hydroxide, N-acetyl-l-cysteine, benzalkonium chloride, oxalic acid

  5. Treatment with mucolytic agents, such as NALC, splits mucoprotein, allowing greater sedimentation

  6. Suspension is diluted with buffer and centrifuged to concentrate any organisms present, sediment is inoculated onto mycobacterial growth media and used to make a smear for acid-fast staining
• Runyon classification system


  • Designed to classify the species within the genus Mycobacterium
  • Three groups based on photosensitivity: photochromogens (Group I), scotochromogens (Group II), and nonphotochromogens (Group III)
  • "Rapid growers" are Group IV
  • The species within the MTb group, although nonchromogens, are categorized separately
• Determining pigment groups or photoreactivity

  1. Inoculate specimen to two tubes or plates of mycobacterial media

  2. One tube is incubated in the light, the other in the dark

  3. Following growth, pigment production of each is noted

  4. The medium initially incubated in the dark is incubated in bright light for several hours, and pigment production, if any, is noted

  5. Nonphotochromogenic (nonchromogenic) mycobacteria do not produce pigment in either light or dark

  6. Photochromogens produce pigment in light, whereas scotochromogens produce pigment in the light or dark

  7. Pigment color may range from light yellow to a dark orange
• Pigment groups


  • Photochromogens (Group I)
  • Scotochromogens (Group II)
  • Nonphotochromogens (Group III)
  • Rapid growers (Group IV)
• Niacin accumulation


  Detected by measuring nicotinic acid, which reacts with cyanogen bromide in the presence of aniline to form a yellow compound
• Nitrate reduction


  Performed as with the method used for Enterobacteriaceae, a positive test is a red pigment
• Catalase


  All mycobacteria are catalase-positive, quantity of catalase and production of heat-stable catalase are species-specific
• Semiquantitative catalase production


  Determined by measuring the height of the column of bubbles when hydrogen peroxide and Tween 80 are added to a deep with mycobacterial growth
• Catalase heat stability


  Determined by heating the specimen to 68°C for 20 minutes prior to the addition of hydrogen peroxide, production of bubbles is a positive reaction
• Tween hydrolysis


  Measures the presence of a lipase, causes a pink color change
• NaCl tolerance


  Determined by inoculating an egg-based media with 5% NaCl and observing growth or no growth following incubation
• Iron uptake


  Determined by adding ferric ammonium citrate to the mycobacterial colonies, dusty-brown colonies are a positive reaction for iron uptake and the formation of iron oxide
• Arylsulfatase activity


  Detected by adding phenolphthalein to the colony/substrate mixture and observing the formation of a pink color
• Urease test


  Detects an organism's ability to produce urease and hydrolyze urea to form ammonia, which produces an alkaline reaction, the increase in pH is detected by a change of color to pink or red
• Susceptibility to thiophen-2-carboxylic acid hydrazide (TCH)


  Determined by observing growth or no growth of mycobacteria
• Nucleic acid amplification


  Available for identification of some species of mycobacteria, including MTB complex and MAC complex
• Gas-liquid chromatography and high-performance liquid chromatography


  Methods to analyze mycobacterial lipids
• Mycobacterium tuberculosis


  • First described by Robert Koch in 1882, one of the oldest documented communicable diseases
  • Pulmonary infection transmitted by the inhalation of droplet nuclei
  • Slender, straight, or slightly curved rod with rounded ends, about 3 µm × 0.3 µm, in pairs or as small clumps
  • Nonmotile, nonsporing, noncapsulated and acid-fast
  • Unsaponifiable wax (mycoloic acid) or to a semipermeable membrane around the cell causes acid fastness
  • Beaded or barred forms are frequently seen, M. bovis appear straighter, stouter and shorter
• Mycobacterium tuberculosis


  • Obligate aerobe while M. bovis is microaerophilic on primary isolation, becoming aerobic on subculture
  • Optimal growth temperature is 35°C to 37°C, M. marinum grows best at 30°C, whereas M. xenopi grows best at 42°C
  • Optimum pH is 6.4–7.0
  • Bacilli grow slowly with a generation time in vitro being 14–15 hours
  • Colonies appear in about two weeks and may sometimes take up to eight weeks
  • Able to grow on a wide range of enriched culture media and do not have exacting growth requirements
• Mycobacterium tuberculosis on solid media

  • Lowenstein-Jensen (L-j) medium is most widely employed for routine culture
  • Consists of coagulated hens' egg, mineral salt solution, asparagine and malachite green
  • Egg acts as a solidifying agent, addition of 0.5% glycerol improves the growth of M. tuberculosis (no effect on M. Bovis), sodium pyruvate helps the growth of both types
  • Forms dry, rough, raised, irregular colonies with a wrinkled surface, are creamy white, becoming yellowish or buff colored on further incubation
  • Tenacious and not easily emulsified (M. bovis is flat, smooth, moist, white and break up easily when touched)
  • Has a luxuriant growth (eugenic growth) as compared to M. Bovis which grows poorly on LJ glycerol medium (dysgonic growth), growth of M. Bovis is much better on LJ pyruvate medium
• Mycobacterium tuberculosis


  • Agar-based media are variations of middlebrook 7H10 medium, all contain some inhibitory agents to suppress the growth of contaminating bacteria
• Primary tuberculosis


  The initial infection, the mycobacterium is eradicated by the host cellular immune response or walled off in a granuloma in the lung
• Secondary tuberculosis


  Reactivation of latent infections can occur in immunocompromised individuals
• Miliary TB


  A disseminated infection with multiple organ involvement
• Pott's disease


  Miliary tuberculosis in the bones or spine
• Tuberculin test


  Used to detect MTB-infected individuals, a purified protein derivative (PPD) is the MTB antigen, a hypersensitivity reaction at the injection site within 72 hours is a positive test, a positive skin test does not distinguish patients with active disease from those with latent infections
• Tuberculin testing methods


  • Mantoux test
  • Heaf test
• Agar-based media


  Variations of middlebrook 7H10 medium, contain some inhibitory agents to suppress the growth of contaminating bacteria
• Pulmonary Tuberculosis


  Primary tuberculosis is the initial infection, the mycobacterium is eradicated by the host cellular immune response or walled off in a granuloma in the lung, Reactivation of latent infections can occur in immunocompromised individuals and cause secondary tuberculosis, Miliary TB is a disseminated infection with multiple organ involvement, Pott's disease is military tuberculosis in the bones or spine
• Tuberculin test


  Used to detect mtb-infected individuals, A purified protein derivative (ppd) is the mtb antigen, Hypersensitivity reaction at the injection site within 72 hours is a positive test, A positive skin test does not distinguish patients with active disease from those with latent infections, Purified protein derivative (ppd) is the skin test reagent that is primarily used to detect hypersensitivity, Many methods such as: mantoux test and heaf test had been described for tuberculin testing
• Multidrug Resistant Mycobacterium Tuberculosis (MDR-TB)


  WHO defines it as one that is at least resistant to rifampicin(R) and isoniazid (H), Drug resistance is usually acquired by spontaneous mutations as a result of the inappropriate use of antimicrobial agents and the lack of patient compliance, Requires an extended treatment period compared with drug susceptible isolates, DOT is a method now being widely followed for treatment of cases of tuberculosis