Transcript Fastidious Gram Negative Rods

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Fastidious Gram Negative Rods
Respiratory Culture Unit
Division of Medical Technology
Carol Larson MSEd, MT(ASCP)
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General Information
• 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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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
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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
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Bordetella pertussis
Growth Characteristics
• 35ºC, 5-10% CO2 for at least 7 days
• Colony morphology
– No growth on
BAP & MAC
– Bordet-Gengou
– Regan-Lowe
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Bordetella pertussis
Identification
• Gram stain
– Small, faintly staining gram-negative
coccobacilli
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•
•
•
Oxidase +
Nitrate –
Urea –
Nonmotile
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Bordetella pertussis
Serological Testing
• Direct fluorescent antibody test
• Agglutination methods
• DNA probes for
direct detection in:
– Specimen
– Culture confirmation
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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.
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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 colony
at 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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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.
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Legionella species
Clinical Significance
• Legionnaires’ disease
• Pontiac Fever
• Transmission: inhalation of the
organism in aerosols
• Legionella pneumophila serogroup 1
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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
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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
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Legionella species
Identification
• Thin, faintly staining short to filamentous
GNR
• Oxidase wk +
• Catalase wk +
• Non-”F”
• Non-”O”
• Motile:polar
flagella
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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
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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.
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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