MYCOBACTERIA

• Mycobacteria


  • Slender, slightly curved or straight, rod-shaped organisms
  • Nonmotile and do not form spores
  • Cell wall has extremely high lipid content, resist staining with basic aniline dyes
  • Take up dye with increased staining time or application of heat but resist decolorization with acid-ethanol
  • Strictly aerobic, but increased carbon dioxide (CO2) will enhance the growth of some species
• Mycobacterium tuberculosis complex


  • M. tuberculosis
  • M. bovis
  • M. africanum
  • M. canettii
  • M. microti
  • M. africanum
• Mycobacterium tuberculosis


  • TB is usually a disease of the respiratory tract
  • Tubercle bacilli are acquired from persons with active disease who are excreting viable bacilli by sneezing or talking
  • Hard tubercle or granuloma may be formed
  • Granuloma is an organization of lymphocytes, macrophages, fibroblasts, and capillaries
  • With granuloma formation, healing occurs, as well as fibrosis, encapsulation, and calcification, with scar formation as a reminder of the past infection
  • In infected individuals, there is a potential for reactivation of TB
• Miliary TB


  Seeding of many organs outside the pulmonary tree with AFB through hematogenous spread
• Extrapulmonary tuberculosis


  • Pleurisy
  • Lymphadenitis
  • Genitourinary TB
  • Skeletal TB of the spine (Pott disease)
  • Meningitis
• Mycobacterium tuberculosis colonies


  • Typically raised, with a dry, rough appearance
  • Nonpigmented and classically described as being buff-colored
  • Elaboration of cord factor can result in characteristic cord formation
  • Optimal growth occurs at 35° C to 37° C
  • Positive for niacin accumulation
  • Reduction of nitrate to nitrite
  • Production of catalase
  • Grows on thiophene-2-carboxylic acid hydrazide (T2H)
• Treatment for pulmonary TB


  • 9-month course of therapy with isoniazid and rifampin, usually once per day the first month and twice a week thereafter
  • 2- to 8-week initial course of streptomycin or ethambutol
  • Pyrazinamide (PZA) may be added to the regimen if there is a suspicion of lowered cellular immunity and a need to obtain bactericidal levels of antimycobacterial activity intracellularly in macrophages
• MDR-TB


  Resistance to at least isoniazid and rifampin
• XDR-TB


  Resistance to isoniazid and rifampin plus resistance to any fluoroquinolone and at least one of three injectable second-line anti-TB drugs; the aminoglycosides amikacin, kanamycin, or capreomycin
• Mycobacterium bovis


  • TB primarily in cattle but also in other ruminants, as well as in dogs, cats, swine, parrots, and humans
  • The disease in humans closely resembles that caused by M. tuberculosis and is treated similarly
  • 21 days of incubation at 37° C
  • Niacin-negative
  • Do not reduce nitrate
  • Do not grow in the presence of (T2H) thiophene-2-carboxylic acid hydrazide
• Slowly growing nontuberculous mycobacteria


  • Mycobacterium avium Complex
  • Mycobacterium avium subsp. paratuberculosis
  • Mycobacterium genavense
  • Mycobacterium haemophilum
  • Mycobacterium kansasii
  • Mycobacterium malmoense
  • Mycobacterium scrofulaceum
  • Mycobacterium simiae
  • Mycobacterium ulcerans
  • Mycobacterium marinum
  • Mycobacterium xenopi
• Rapidly growing nontuberculous mycobacteria

  • Mycobacterium chelonae–Mycobacterium abscessus Group
  • Mycobacterium fortuitum Group
  • Mycobacterium smegmatis Group
• Mycobacterium leprae


  • Causative agent of Hansen disease (leprosy), an infection of the skin, mucous membranes, and peripheral nerves
  • The two major forms of the disease are tuberculoid leprosy and lepromatous leprosy
  • Symptoms of tuberculoid leprosy include skin lesions and nerve involvement that can produce areas with loss of sensation
  • Patients with lepromatous leprosy if untreated, life-threatening
  • Characterized by skin lesions and progressive, symmetric nerve damage
  • Lesions of the mucous membranes of the nose may lead to destruction of the cartilaginous septum, resulting in nasal and facial deformities
  • Combination of diaminodiphenylsulfone (dapsone), clofazimine, and rifampin
  • M. leprae has not been grown on artificial media
  • Mouse footpad
  • The length of the bacillus is at least five times the width of the bacillus
• Classification of mycobacteria


  • TB Complex
  • Photochromogens
  • Scotochromogens
  • Nonchromogens
  • Rapid growers
• The most commonly recommended container for specimen collection is a sterile, wide-mouth cup with a tightly fitted lid
• Sputum and Other Respiratory Secretions specimen collection

  1. Early-morning specimen should be collected on three consecutive days

  2. If at least two of the first three sputum direct smears are positive, then three specimens are often sufficient to confirm a diagnosis

  3. When none, or only one, of the first three sputum smears is positive, additional specimens are needed for culture confirmation

  4. 5 to 10 mL of sputum produced by a deep cough of expectorated or induced by inhalation of an aerosol of hypertonic saline should be used
• SSON 6: MYCOBACTERIA LABORATORY DIAGNOSIS
• Professor: Ana Blezilda R. Arca | 2nd Semester | S.Y. 2023-2024
• Transcribed by: Roldan, Keigan R.
• Specimen collection


  The most commonly recommended container is a sterile, wide-mouth cup with a tightly fitted lid
• Specimen types


  • Sputum and Other Respiratory Secretions
  • Bronchial washing, Bronchoalveolar lavage (BAL), or transbronchial biopsy specimens
  • Gastric aspirates
  • Urine
  • Stool
  • Blood
  • Tissue or body fluids
• Sputum collection


  1. An early-morning specimen should be collected on three consecutive days

  2. 5 to 10 mL of sputum produced by a deep cough of expectorated or induced by inhalation of an aerosol of hypertonic saline should be used

  3. Children have difficulty producing sputum, gastric aspiration is recommended

  4. Gastric aspirates should be obtained in the morning after an overnight fast

  5. Sterile water, 30 to 60 mL, is instilled orally or via nasogastric tube aspiration

  6. Specimen processing should be done expeditiously, or the specimen should be neutralized with sodium carbonate or another buffer to pH 7.0 as soon as possible after specimen collection
• Urine collection


  1. A first morning midstream specimen is preferred

  2. A minimum of 15 mL, is collected in a sterile container
• Tissue or body fluid collection

  1. 10 to 15 mL of sterile saline should be added to prevent dehydration

  2. 2mL for CSF, 3 to 5mL for exudates and pericardial and synovial fluids, and 10 to 15 mL for abdominal and chest fluids
• When tissue is collected, histologic evaluation may reveal caseating or noncaseating granuloma formation with the presence of multinucleated giant cells
• Digestion and decontamination of specimens

  • To liquefy the sample through digestion of the proteinaceous material
  • To allow the chemical decontaminating agent to contact and kill the nonmycobacterial organisms
• Specimen types requiring digestion and decontamination

  • Sputum
  • Gastric washing
  • BAL
  • Bronchial washing
  • Transtracheal aspirate
• Specimen types requiring decontamination


  • Voided urine
  • Autopsy tissue
  • Abdominal fluid
  • Any contaminated fluid
• Digestion and decontamination agents

  • Sodium Hydroxide
  • N-Acetyl-L-cysteine
  • Benzalkonium Chloride
  • Oxalic Acid
• Sodium Hydroxide


  Usual concentration 2%, 3%, or 4%; serves as a digestant and decontaminating agent
• 1. Acetyl-L-cysteine (NALC)


  • A combination of a liquefying agent, plus NaOH is commonly used
  • Treatment with mucolytic agents such as NALC splits mucoprotein, allowing greater sedimentation
• Benzalkonium Chloride


  • Another digestant-decontamination agent
  • Combined with trisodium phosphate (Z-TSP)
  • TSP liquefies sputum rapidly
  • Benzalkonium chloride shortens the exposure time and effectively destroys many contaminants, with little bactericidal effect on the tubercle bacilli
• Oxalic Acid


  5%, is used to decontaminate specimens contaminated with Pseudomonas aeruginosa, such as sputum specimens from patients with Cystic fibrosis
• Acid-fast staining


  • The conventional acid-fast staining methods, Ziehl-Neelsen and Kinyoun stains, use carbolfuchsin as the primary stain, acid-alcohol as a decolorizing agent, and a methylene blue counterstain
  • The Ziehl-Neelsen staining procedure involves the application of heat with the carbolfuchsin stain, whereas the Kinyoun acid-fast stain is a cold stain
  • Slides are examined using a ×100 oil immersion objective on a light microscope for 15 minutes, viewing a minimum of 300 fields before a slide is called negative
• Mycobacteria


  • Strictly aerobic and grow more slowly
  • The generation time of mycobacteria is longer than 12 hours; M. tuberculosis has the longest replication time, at 20 to 22 hours
  • 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
  • M. genavense, does not grow on media used routinely to isolate mycobacteria and requires extended incubation (6 to 8 weeks), whereas M. leprae fails to grow on artificial media
• Culture media


  • Egg-based media (Löwenstein-Jensen (LJ), Petragnani, and American Thoracic Society (ATS) media)
  • Agar-based media (Middlebrook 7H10 and 7H11 agars)
  • Liquid media (Middlebrook 7H9 broth and Dubos Tween albumin)
• Egg-based media


  • Nonselective egg-based media containing 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
  • Selective media that contain antimicrobial agents, such as the Gruft modification of LJ and Mycobactosel are sometimes used in combination with nonselective media to increase the isolation of mycobacteria from contaminated specimens
• Agar-based media


  • Serum albumin agar media, such as Middlebrook 7H10 and 7H11 agars
  • Middlebrook 7H11 medium also contains 0.1% casein hydrolysate, which improves the recovery of isoniazid-resistant strains of M. tuberculosis
  • Drug susceptibility tests may be performed on agar-based media without altering drug concentrations, which occurs with egg-based media
• Liquid media


  • Mycobacterium spp. grow more rapidly in liquid medium
  • Middlebrook 7H9 broth and Dubos Tween albumin are nonselective liquid media used for subculturing stock strains
• Preliminary identification of mycobacteria


  • Colony morphology
  • Growth rate
  • Niacin accumulation
  • Nitrate reduction
  • Catalase
  • Hydrolysis of Tween 80
  • Iron uptake
  • Arylsulfate
  • Pyrazinamidase
  • Tellurite reduction
  • Urease