Corynebacterium (1)
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    Corynebacterium (1) Corynebacterium (1) Presentation Transcript

    • Corynebacterium MycobacteriumMary Joyce Saborrido-Teoxon, RMT, MD Dept. of Microbiology and Parasitology
    • GENUS: CORYNEBACTERIUM• Gram-positive, pleomorphic rods• Nonspore-forming, nonmotile, non- encapsulated• Aerobic
    • 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
    • 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.
    • Diphtheria
    • 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)
    • Corynebacterium diphtheriae• LABORATORY DIAGNOSIS• 3. Catalase test (+)• 4. Urease test (-)• 5. Toxigenicity test – Elek test (in vitro) – Animal inoculation test (in vivo)
    • 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
    • Corynebacterium minutissimum• Agent of ERYTHRASMA• “coral red fluorescence” on Wood’s light – Presence of porphyrin
    • Diphtheroid• C. pseudodiphthericum• Hoffman’s Bacillus• Causes diphtheria like disease
    • GENUS: MYCOBACTERIUM• Acid fast rods with waxy cell wall• Obligate aerobe• Non-sporeforming, Non-encapsulated• Slow-growers (except: M. fortuitum, M. chelonei)• Granules (Much)
    • GENUS: MYCOBACTERIUMThree Groups:• M. tuberculosis complex- cause TB – M. tuberculosis – pulmunonary tuberculosis – M. bovis – intestinal tuberculosis – M. africanum – pulmonary tuberculosis ( Africa)• MOTT• M. leprae
    • 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
    • Mycobacterium tuberculosis• Reservoir – Human lungs• Transmission – Respiratory droplets and droplet• Predisposing Factor – For active disease is poverty, HIV infections, or any CMI system immunosuppression.
    • 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
    • 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
    • SPECIMEN PROCESSING: Specimen Sterile Nonsterile
    • SPECIMEN PROCESSING: NONSTERILE LIQUEFICATION DECONTAMINATION NEUTRALIZATION CENTRIFUGATION
    • 1.) Liquefy• NALC• Dithiothreitol (sputolysin)• Enhance by mixing with a vortex type of mixer in a closed container, stand 15 mins
    • 2.) Decontaminate• NaOH• Zephiran-trisodium• 6% Oxalic acid (g-, Pseudomonas, Proteus)
    • 3.) Neutralize• Buffer• H2O
    • Mycobacterium tuberculosis• LABORATORY DIAGNOSIS• 1. Gram stain – to qualify specimen• 2. Acid Fast Stain – Fuchsin stain – Fluorochrome
    • 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
    • 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
    • M. tuberculosis on Lowenstein-Jensen(LJ) agar.Coagulated eggs, glycerol, potato flour, and salts, Malachite green.
    • Young colonies of M. tuberculosis on(10 days)Middlebrook 7H11 agar viewed microscopically. Beginning of cording characteristic of M.tb
    • M. tuberculosis exhibiting cauliflower colonies
    • 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
    • Biochemical Tests1. NIACIN TEST  principle: NIACIN + NIACIN RIBONUCLEOTIDE + ANILINE DYE + CYANOGEN BROMIDE  M. tuberculosis = positive (yellow)  M. bovis = negative
    • 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
    • Biochemical Tests3. Nitrate reduction test:  nitroreductase  detected by:a. HCLb. sulfanilamidec. alpha napthyl amine  (+) result = pink color (+) M.tb (-) M.avium
    • 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
    • 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
    • Biochemical Tests6. Tellurite reduction test:Px; Telurite --- black metallic tellurium used to ID M. avium (+) ; M. kansasii (-)
    • Biochemical Tests7. TCH Susceptibility test (+) susceptible = M. bovis (-) resistant = M. tbTCH  Thiophene-2-carboxylic acid hydrazide
    • 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
    • • 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.
    • • 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
    • • 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
    • 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.
    • 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
    • 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
    • Table I. Runyon Grouping of MOTTS RUNYON’S CLASSIFICATION Genus & specieRapid growers M. fortuitum M. chelonei M. phlei M. smegmatis
    • 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
    • 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
    • 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 “
    • 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
    • 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