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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 • Faint staining Gram Negative Rods – Safranin counterstain for >2 minutes – Substitute carbolfuschin for safranin • Serological testing useful Click icon for audio Bordetella pertussis Clinical 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 Click icon for audio Bordetella pertussis Specimen Collection • 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 pertussis Growth Characteristics • 35ºC, 5-10% CO2 for at least 7 days • Colony morphology – No growth on BAP & MAC – Bordet-Gengou – Regan-Lowe Click icon for audio Bordetella pertussis Identification • Gram stain – Small, faintly staining gram-negative coccobacilli • • • • Oxidase + Nitrate – Urea – Nonmotile Click icon for audio Bordetella pertussis Serological Testing • Direct fluorescent antibody test • Agglutination methods • DNA probes for direct detection in: – Specimen – Culture confirmation Click icon for audio Bordetella pertussis Treatment & Prevention • Erythromycin is drug of choice • Vaccination 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. Click icon for audio Francisella tularensis Clinical 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 Click icon for audio Francisella tularensis Specimen 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 Click icon for audio Francisella tularensis Specimen Processing • Requires cysteine / cystine for growth • Glucose-cystine blood (Francis’) agar • Grows on – Chocolate – BCYE – Modified TM Click icon for audio Francisella tularensis Growth Characteristics • Strict aerobic • 35°C with 5-10% CO2 for 7 days • Colony morphology – BAP & MAC = No growth – CHOC = Small, gray alpha-hemolytic colony at 2-5 days Click icon for audio Francisella tularensis Identification • Pale staining gram negative coccobacilli • Oxidase – • Catalase – to weak + • Glucose fermenter • Nonmotile Click icon for audio Francisella tularensis Serological 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 Click icon for audio Francisella tularensis Treatment & Prevention • Streptomycin is drug of choice What substance is required in culture media in order to grow Francisella? Cysteine / Cystine 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. Click icon for audio Legionella species Clinical Significance • Legionnaires’ disease • Pontiac Fever • Transmission: inhalation of the organism in aerosols • Legionella pneumophila serogroup 1 Click icon for audio Legionella species Specimen 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 Click icon for audio Legionella species Growth 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 Click icon for audio Legionella species Identification • Thin, faintly staining short to filamentous GNR • Oxidase wk + • Catalase wk + • Non-”F” • Non-”O” • Motile:polar flagella Click icon for audio Legionella species Serological 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 Click icon for audio Legionella species Treatment and Prevention • Susceptibility testing not routinely performed • Drug of choice: Erythromycin alone or with Rifampin What substance is required in culture media in order to grow Legionella? Cysteine and iron salts. 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. Click icon for audio Fastidious GNR Summary • Looked at several organisms • Clinical significance • Specimen collection, transport & processing • Growth characteristics & identification • Serological testing • Treatment and prevention Who am I? Reagin-Lowe media Gram Stain Causes Whooping Cough Bordetella pertussis Who am I? BCYE agar Gram Stain Causes Pontiac Fever Legionella species