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Corynebacterium (1)


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Corynebacterium (1)

  1. 1. Corynebacterium MycobacteriumMary Joyce Saborrido-Teoxon, RMT, MD Dept. of Microbiology and Parasitology
  2. 2. GENUS: CORYNEBACTERIUM• Gram-positive, pleomorphic rods• Nonspore-forming, nonmotile, non- encapsulated• Aerobic
  3. 3. Corynebacterium diphtheriae• Distinguishing Characteristics: – Kleb Loeffler’s Bacillus – Club-shaped Gram-positive rods arranged in V , L, X, Y shapes – Granules (Babes Ernst) produced on Loeffler’s coagulated serum medium stain metachromatically
  4. 4. Corynebacterium diphtheriae• Transmission – Bacterium or phage via respiratory droplets from oropharynx of infected person• Pathogenesis – Organism not invasive; colonizes epithelium of oropharynx or skin in cutaneous diphtheria. – Diphtheria toxin (A-B component) – inhibits protein synthesis by adding ADP-ribose to EF-2. – Effect on oropharynx: – Dirty gray pseudomembrane (made up of dead cells and fibrin exudates bacterial pigment) – Extension into larynx/trachea → obstruction – Effect of systemic circulation → heart & nerve damage.
  5. 5. Diphtheria
  6. 6. Corynebacterium diphtheriae• LABORATORY DIAGNOSIS• 1. DME (G/S, LAMB)• 2. CULTURE – Loeffler’s serum agar slant – Pai coagulated egg – Tinsdale (black  dark brown halos) – Tellurite blood agar – Cystine tellurite blood agar (black  gray)
  7. 7. Corynebacterium diphtheriae• LABORATORY DIAGNOSIS• 3. Catalase test (+)• 4. Urease test (-)• 5. Toxigenicity test – Elek test (in vitro) – Animal inoculation test (in vivo)
  8. 8. Corynebacterium diphtheriae• Treatment – Erythromycin and antitoxin• Prevention – Toxoid vaccine (formaldehyde-modified toxin is still immunogenic but with reduced toxicity), part of DtaP, DTP, or Td
  9. 9. Corynebacterium minutissimum• Agent of ERYTHRASMA• “coral red fluorescence” on Wood’s light – Presence of porphyrin
  10. 10. Diphtheroid• C. pseudodiphthericum• Hoffman’s Bacillus• Causes diphtheria like disease
  11. 11. GENUS: MYCOBACTERIUM• Acid fast rods with waxy cell wall• Obligate aerobe• Non-sporeforming, Non-encapsulated• Slow-growers (except: M. fortuitum, M. chelonei)• Granules (Much)
  12. 12. GENUS: MYCOBACTERIUMThree Groups:• M. tuberculosis complex- cause TB – M. tuberculosis – pulmunonary tuberculosis – M. bovis – intestinal tuberculosis – M. africanum – pulmonary tuberculosis ( Africa)• MOTT• M. leprae
  13. 13. Mycobacterium tuberculosis• Distinguishing Characteristics – Koch Bacillus – Acid fast – Aerobic, require CO2 – slow growing – Produces niacin – Produces a heat-sensitive catalase: • Catalase negative at 68°C (standard catalase test) – (other mycobacterial catalase are heat insensitive) • Catalase active at body temperature
  14. 14. Mycobacterium tuberculosis• Reservoir – Human lungs• Transmission – Respiratory droplets and droplet• Predisposing Factor – For active disease is poverty, HIV infections, or any CMI system immunosuppression.
  15. 15. Mycobacterium tuberculosis• Pathogenesis – Facultative Intracellular Organism – Sulfatides (sulfolipids in cell envelope) • Inhibit the phagosome-lysosomal fusion allowing intracellurlar survival. (If fusion occurs, waxy nature of cell envelope reduces killing effect.) – Cord factor (trehalose di-myoclate) » Causes serpentine growth in vitro » Inhibits leukocyte migration; disrupts mitochondrial respiration and oxidative phosphorylation • Tuberculin (surface protein) along with mycolic acid → delayed hypersensitivity and CMI – Granulomas and caseation mediated by cell- mediated immunity (CMI) – No exotoxins nor endotoxin; damage done by immune system
  16. 16. Mycobacterium tuberculosisDisease• Tuberculosis• Causative agents: Mycobacterium tuberculosis , M. bovis, and M. africanum• Complex disease: pulmonary, urinary tract, and organ or military (disseminated)• Primary infection: organisms replicate in naïve macrophages, killing macrophages until CMI is set up.• Most people heal without disease; some organisms walled off in the Ghon complex remain viable unless treated.• Post primary (reactivational TB) erosion of granulomas into airways (high oxygen) later in life under conditions of reduced T-cell immunity leads to mycobacterial replication and disease symptoms
  17. 17. SPECIMEN PROCESSING: Specimen Sterile Nonsterile
  19. 19. 1.) Liquefy• NALC• Dithiothreitol (sputolysin)• Enhance by mixing with a vortex type of mixer in a closed container, stand 15 mins
  20. 20. 2.) Decontaminate• NaOH• Zephiran-trisodium• 6% Oxalic acid (g-, Pseudomonas, Proteus)
  21. 21. 3.) Neutralize• Buffer• H2O
  22. 22. Mycobacterium tuberculosis• LABORATORY DIAGNOSIS• 1. Gram stain – to qualify specimen• 2. Acid Fast Stain – Fuchsin stain – Fluorochrome
  23. 23. Acid Fast Reporting0 No AFB seen1-2 / 300 fields Doubtful; request another specimen1-9/ 100 fields +11-9/ 10 fields +21-9/ field +3>9 +4
  24. 24. Mycobacterium tuberculosis• 3. CultureA. Agar Base Media: 1. Duboi’s Oleic Acid Albumin medium 2. Mitchison’s medium 3. Middlebrook 7H10 – 7H11 – ASTB. Egg-Base Media: malachite green 1. Petragnani medium 2. Lowenstein-Jensen medium 3. American Thoracic Society medium 4. Dorset Egg mediumC. Liquid Media: Bactec 12B, Septi-Chek AFB, Middlebrook 7H9
  25. 25. M. tuberculosis on Lowenstein-Jensen(LJ) agar.Coagulated eggs, glycerol, potato flour, and salts, Malachite green.
  26. 26. Young colonies of M. tuberculosis on(10 days)Middlebrook 7H11 agar viewed microscopically. Beginning of cording characteristic of M.tb
  27. 27. M. tuberculosis exhibiting cauliflower colonies
  28. 28. M. Tuberculosis on Middlebrook 7H11 agar. Cream-colored, dry, and wrinkled colonies. Contains caseinhydrolysates that improve recovery of INH resistant strains of M.tb and shorten incubation time for M. avium complex
  29. 29. Biochemical Tests1. NIACIN TEST  principle: NIACIN + NIACIN RIBONUCLEOTIDE + ANILINE DYE + CYANOGEN BROMIDE  M. tuberculosis = positive (yellow)  M. bovis = negative
  30. 30. Biochemical Tests2. Catalase test: -medium: TWEEN 80 -reagent: 30 % H2O2 -all Mycobacteria (+) types: a. Semi-quantitative test - column of bubbles b. Heat stable catalase test - 68 oC – denature enzyme -M. tb. = negative (+) M. kansasii
  31. 31. Biochemical Tests3. Nitrate reduction test:  nitroreductase  detected by:a. HCLb. sulfanilamidec. alpha napthyl amine  (+) result = pink color (+) M.tb (-) M.avium
  32. 32. Biochemical Tests4. ARYLSULFATASE TEST: – Detects rapid growers – Principle: – Tripotasium Arylsulfatase Free Phenolphthalein Phenolphthalein Disulfide/sulfate (END PRODUCT) – RESULT: (+) Red/ Pink – Strongly (+)  M. fortuitum-chelonei – (-)  M-avium
  33. 33. Biochemical Tests5. TWEEN 80 HOH test:Principle:Tween 80 hydrolysis of tween 80(polyoxyethelene (oleic acid +Sorbitan polyoxyethylatedMonooleate) sorbitol)(+) red = M. kansasii(-) no red = M. avium
  34. 34. Biochemical Tests6. Tellurite reduction test:Px; Telurite --- black metallic tellurium used to ID M. avium (+) ; M. kansasii (-)
  35. 35. Biochemical Tests7. TCH Susceptibility test (+) susceptible = M. bovis (-) resistant = M. tbTCH  Thiophene-2-carboxylic acid hydrazide
  36. 36. Automated test for Mycobacterium1. Bactec 460 Middlebrook 7H12 (RIA based) Principle : 14C palmitic acid + orgs= 14 CO2 Result (+) : more than 10 growth index2. Mycobacteria Growth Indicator Tube (MGIT) – Fluorometric based3. Bactec 12B + NAP – P-nitro acetylamino beta hydroxypropiophenone (NAP)AST = disk elution using S-I-R-E disks
  37. 37. • Diagnosis – PPD skin test (Mantoux): – >5 mm in HIV+ or anyone with recent TB exposure; AIDS patients have reduced ability to mount skin test. – >10 mm in high-risk population: IV drug abusers, people living in poverty, or immigrants from high TB area. – >15 mm in low-risk population – Positive skin test indicates only exposure but not necessarily active disease.
  38. 38. • Treatment – Multiple drugs critical to treat infection – Standard observed short-term therapy for uncomplicated pulmonary TB (rate where acquired <4%): • First 2 months: isoniazid + rifampin + pyrazinamide • Next 4 months: isoniazid and rifampin – Ethambutol or streptomycin added for possible drug- resistant cases until susceptibility tests are back (if area acquired has >4% DRM TB
  39. 39. • Prevention – Isoniazid taken for 6-9 months can prevent TB in persons with infection but not clinical symptoms. – Bacille-Calmette-Guerin (BCG) vaccine contains live, attenuated organisms may prevent disseminated disease. Not commonly used in the U.S. – UV lights or HEPA filters used to treat potentially contaminated air
  40. 40. Mycobateria Other Than Tuberculosis (MOTTS)• (MOTTS) = Non-tuberculous Mycobacteria = atypical Mycobacteria• Non-contagious!• Found in surface waters, soil, cigarettes; most common in southeastern U.S.
  41. 41. Table I. Runyon Grouping of MOTTSRunyun Runyon Group Dark Light GrowthGroup # NameI Photochromogen - + Slow (+) Cream/buff Orange/yellow in 10-21 daysII Scotochromogen + + Slow (+) Orange/ Yellow 10- 21 daysIII Non- - - Slow photochromogen Cream buff in 10-21 daysIV Rapid growers Fast < 7days
  42. 42. Table I. Runyon Grouping of MOTTS RUNYON’S Genus & specieCLASSIFICATIONPhotochromogen M. kansasii M. marinum M. asiaticum M. simiaeScotochromogen M. scrofulaceum (scrofula) M. szulgai M. gordonae (tap H2O bacillus)Non- M. avium orPhotochromogen M. intracellulare (battey bacillus) M. Ulcerans (Buruli) M. xenopi ( hot ,cold H2o taps) M. triviale M.haemophilum M. malmoense
  43. 43. Table I. Runyon Grouping of MOTTS RUNYON’S CLASSIFICATION Genus & specieRapid growers M. fortuitum M. chelonei M. phlei M. smegmatis
  44. 44. Mycobateria Other Than Tuberculosis (MOTTS)• Disease – Pulmonary/Gastrointestinal/Disseminated – Patients: AIDS (prophylaxis <75 CD4+ cells/mm3), cancer, chronic lung disease – M. avium-intracellulare, M. kansasii. – Mycobacterial lymphadenitis – Usually solitary cervical lymph nodes (surgically removed) in kids.• M. scrofulaceum. – Soft-Tissue Infections• M. marinum: cutaneous granolomas in tropical fish enthusiast (fist tank granuloma) or scuba divers from abrasions on coral
  45. 45. Mycobacterium leprae• Distinguishing Characteristics – Acid fast rods (seen in punch biopsy) – Cigarette-packet/picket-fence – Can hydrolyze 3,4-dihydroxy-phenylalanine (DOPA) – Obligate intracellular parasite (cannot be cultured in vitro) – Optimal growth at less than body temperature• Reservoir – Human mucosa, skin, and nerves are the only significant reservoir. – Some infected armadillon in Texas and Lousiana• Transmission – Nasal discharge from untreated lepromatous leprosy patients
  46. 46. Mycobacterium leprae• Pathogenesis – Obligate intracellular parasite – Cooler parts of body e.g., skin, mucous membranes, and peripheral nerves• Disease – Leprosy (Hansen’s) A continuum of disease, which usually start out with an indeterminate stage called “borderline “
  47. 47. Mycobacterium leprae Tuberculoid LepromatousCell-mediated immune Strong CMI Weak CMIsystemLepromin skin test Lepromin test + Lepromin test -Number of organisms Low B High (foam cells totally filled)in tissue oDamage form Immune response r Large number of intracellular (CMI killing infected d organisms cells) e Nerve damage from overgrowth Granuloma formation r of bacteria in cells → nerve l Loss of sensation → burns and enlargement/damage i trauma Loss of sensation → n burns and trauma eNumber of lesions and Fewer lesions: Numerous lesions becomingother syndromes macular; nerve nodular; loss of eyebrows; enlargement, destruction of nasal septum paresthesia Paresthesia Leonine facies
  48. 48. Mycobacterium leprae• Laboratory Diagnosis – Punch biopsy or nasal scrapings; acid fast stain – Lepromin skin test is positive in the tuberculoid but not in the lepromatous form. – No cultures• Treatment – Multiple-drug therapy with dapsone and rifampin, with clofazimineadded for lepromatous• Prevention – Dapsone for close family contacts