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Fastidious Gram Negative RodsRespiratory II
Clinical Laboratory Science Program
Carol Larson MSEd, MT(ASCP)
Please click audio iconto hear Carol’s narration
• Fastidious– Complex / extensive nutrient requirements
• Faint staining Gram Negative Rods– Safranin counterstain for >2 minutes– Substitute carbolfuschin for safranin
• Serological testing useful
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General Information
• 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
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Bordetella pertussis
Clinical Significance
• Nasopharyngeal swab or aspirate
• Plate at bedside– Regan-Lowe media– Bordet-Gengou media– Methicillin or cephalexin added to media
to inhibit normal flora
• Perform DNA probe testing on aspirate
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Bordetella pertussis
Specimen Collection
• 35ºC, ambient air for at least 7 days
• Colony morphology– No growth on
BAP & MAC– Bordet-Gengou– Regan-Lowe
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Bordetella pertussis
Growth Characteristics
• Gram stain– Small, faintly staining gram-negative
coccobacilli
• Oxidase +
• Nitrate –
• Urea –
• Nonmotile
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Bordetella pertussis
Identification
• DNA probes for direct detection in:– Specimen– Culture confirmation
• Direct fluorescent antibody test
• Agglutination methods
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Bordetella pertussis
Serological Testing
• Erythromycin is drug of choice
• Vaccination
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Bordetella pertussis
Treatment & Prevention
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.
• Causes acute respiratory infection
• Grows on BAP and sometimes on MAC
• Bordet-Gengou media
• Oxidase negative
• Urease positive (24 hours)
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Bordetella parapertussis
• Immunocompromised patients
• Grows on BAP and MAC
• Bordet-Gengou media
• Oxidase positive
• Urease positive (3-4 hours)
• Motile
• Nitrate positive
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Bordetella bronchiseptica
How can you differentiate between Bordetella pertussis and B. parapertussis?
Colony morphology on Bordet-Gengou media, growth on BAP/MAC, oxidase, and urease.
Capnocytophaga species
Clinical Significance• Normal oropharyngeal flora
• Associated with:– Periodontal disease– Sepsis
• Granulocytopenia• Malignancies
– Endocarditis– Animal bites
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Capnocytophaga species
Specimen Collection• Respiratory specimens
• Capnophilic
• Facultative anaerobe
• Set up BAP, CHOC
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• BAP colony morphology– Slight yellow, nonhemolytic, spreading over
agar surface (at 48 hrs)– Center of colony moist, mottled
• No growth on MAC
• Only grows in presence of CO2 (microaerophilic and anaerobic)
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Capnocytophaga species
Growth Characteristics
Capnocytophaga species
Identification• Fusiform, filamentous gram-negative
rod
• Oxidase –
• Catalase –
• Motility = gliding, no flagella so ittwitches
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What atmosphere does Capnocytophaga require to grow?
Higher concentrations of CO2 (capnophilic) – microaerophilic or anaerobic conditions.
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
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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
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Francisella tularensis
Specimen Processing• Requires cysteine / cystine for growth
• Glucose-cystine blood (Francis’) agar
• Grows on– Chocolate– BCYE– Modified TM
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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 colonyat 2-5 days
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Francisella tularensis
Identification• Pale staining gram negative coccobacilli
• Oxidase –
• Catalase – to weak +
• Glucose fermenter
• Nonmotile
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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
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• Streptomycin is drug of choice
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Francisella tularensis
Treatment & Prevention
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.
• Legionnaires’ disease
• Pontiac Fever
• Transmission: inhalation of the organism in aerosols
• Legionella pneumophila serogroup 1
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Legionella species
Clinical Significance
• 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
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Legionella species
Specimen Collection
• 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
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Legionella species
Growth Characteristics
• Thin, faintly staining short to filamentous GNR
• Oxidase wk +
• Catalase wk +
• Non-”F”
• Non-”O”
• Motile:polar flagella
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Legionella species
Identification
• Specimen screen & Isolate ID– DFA stain and DNA probe
• IFA test of choice (serum)– Four-fold rise in titer to at least 1:128
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Legionella species
Serological Testing
• Susceptibility testing not routinely performed
• Drug of choice: Erythromycin alone or with Rifampin
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Legionella species
Treatment and Prevention
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.
• Looked at several organisms
• Clinical significance
• Specimen collection, transport & processing
• Growth characteristics & identification
• Serological testing
• Treatment and prevention
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Fastidious GNR
Summary
Who am I?
Reagin-Lowe media Gram Stain
Bordetella pertussis
Causes Whooping Cough
Who am I?
BCYE agar Gram Stain
Legionella species
Causes Pontiac Fever