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Fastidious Gram Negative Rods Respiratory Culture Unit

Please click audio icon to hear Carol’s narration. Fastidious Gram Negative Rods Respiratory Culture Unit. Division of Medical Technology Carol Larson MSEd, MT(ASCP). Click icon for audio. General Information. Fastidious Complex / extensive nutrient requirements

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Fastidious Gram Negative Rods Respiratory Culture Unit

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  1. Please click audio icon to hear Carol’s narration Fastidious Gram Negative RodsRespiratory Culture Unit Division of Medical Technology Carol Larson MSEd, MT(ASCP)

  2. Click icon for audio General Information • Fastidious • Complex / extensive nutrient requirements • Faint staining Gram Negative Rods • Safranin counterstain for >2 minutes • Substitute carbolfuschin for safranin • Serological testing useful

  3. Click icon for audio Bordetella pertussisClinical Significance • Causes Pertussis / Whooping cough • Spread by airborne droplets • Virulence factors • Attachment to ciliated epithelium of the upper respiratory tract • Exotoxin – tracheal cytotoxin • Exotoxin - Pertussis toxin • Cell wall endotoxin

  4. Nasopharyngeal swab or aspirate Plate at bedside Regan-Lowe media Bordet-Gengou media Methicillin or cephalexin added to media to inhibit normal flora Make smear for DFA screening Click icon for audio Bordetella pertussisSpecimen Collection

  5. Click icon for audio Bordetella pertussisGrowth Characteristics • 35ºC, 5-10% CO2 for at least 7 days • Colony morphology • No growth onBAP & MAC • Bordet-Gengou • Regan-Lowe

  6. Click icon for audio Bordetella pertussisIdentification • Gram stain • Small, faintly staining gram-negative coccobacilli • Oxidase + • Nitrate – • Urea – • Nonmotile

  7. Click icon for audio Bordetella pertussisSerological Testing • Direct fluorescent antibody test • Agglutination methods • DNA probes for direct detection in: • Specimen • Culture confirmation

  8. Click icon for audio Bordetella pertussisTreatment & Prevention • Erythromycin is drug of choice • Vaccination

  9. What media is used to culture Bordetella pertussis? Regan-Lowe media and Bordet-Gengou media. Bordetella pertussis will not grow on BAP or MAC. Methicillin or cephalexin can be added to the media to inhibit normal flora.

  10. Click icon for audio Francisella tularensisClinical Significance • Causes Tularemia – an acute febrile, HIGHLY INFECTIOUS disease • Acquire by: • Direct contact with infected animals (rabbits) • Bite from an insect • Inhalation of infectious aerosols

  11. Click icon for audio Francisella tularensisSpecimen Collection • Inflammatory material from infected site • Wear gloves and use biosafety hood • Do not aerosolize or allow contact with skin or mucous membranes • CDC: Biosafety Level 2 pathogen

  12. Click icon for audio Francisella tularensisSpecimen Processing • Requires cysteine / cystine for growth • Glucose-cystine blood (Francis’) agar • Grows on • Chocolate • BCYE • Modified TM

  13. Click icon for audio Francisella tularensisGrowth Characteristics • Strict aerobic • 35°C with 5-10% CO2 for 7 days • Colony morphology • BAP & MAC = No growth • CHOC = Small, gray alpha-hemolytic colonyat 2-5 days

  14. Click icon for audio Francisella tularensisIdentification • Pale staining gram negative coccobacilli • Oxidase – • Catalase – to weak + • Glucose fermenter • Nonmotile

  15. Click icon for audio Francisella tularensisSerological Testing • Most cases diagnosed serologically • DFA tests may be performed on specimen • ELISA and agglutination tests • Four-fold rise in titer is diagnostic • Single titer of >=1:160 is presumptive

  16. Click icon for audio Francisella tularensisTreatment & Prevention • Streptomycin is drug of choice

  17. What substance is required in culture media in order to grow Francisella? Cysteine / Cystine

  18. What is the best method for determining if a patient has Tularemia and Why? Serological testing is best. To actually culture the organism in the laboratory has a high risk for laboratory personnel becoming infected.

  19. Click icon for audio Legionella speciesClinical Significance • Legionnaires’ disease • Pontiac Fever • Transmission: inhalation of the organism in aerosols • Legionella pneumophila serogroup 1

  20. Click icon for audio Legionella speciesSpecimen Collection • BAL, BW, lung biopsy, pleural fluid • Avoid aerosolization • Transport ambient temperature • Requires cysteine and iron salts for growth • Buffered Charcoal Yeast Extract agar • Selective media: BCYE + antibiotics

  21. Click icon for audio Legionella speciesGrowth Characteristics • Aerobic • 35°C in 5-10% CO2 for 10 days • Colony morphology • BAP & MAC = no growth • CHOC = grows slowly • BCYE = convex, grayish white, glistening with an entire edge at 2-4 days

  22. Click icon for audio Legionella speciesIdentification • Thin, faintly staining short to filamentous GNR • Oxidase wk + • Catalase wk + • Non-”F” • Non-”O” • Motile:polar flagella

  23. Click icon for audio Legionella speciesSerological Testing • Specimen screen & Isolate ID • DFA stain and DNA probe • IFA test of choice (serum) • Four-fold rise in titer to at least 1:128

  24. Click icon for audio Legionella speciesTreatment and Prevention • Susceptibility testing not routinely performed • Drug of choice: Erythromycin alone or with Rifampin

  25. What substance is required in culture media in order to grow Legionella? Cysteine and iron salts.

  26. What populations are most prone to Legionella infections? Men, cigarette smokers, people with underlying disease, immunocompromised/immunosuppressed patients, people who drink alcohol and nosocomial infections.

  27. Click icon for audio Fastidious GNRSummary • Looked at several organisms • Clinical significance • Specimen collection, transport & processing • Growth characteristics & identification • Serological testing • Treatment and prevention

  28. Who am I? Reagin-Lowe media Gram Stain Causes Whooping Cough Bordetella pertussis

  29. Who am I? BCYE agar Gram Stain Causes Pontiac Fever Legionella species

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