Fastidious Gram Negative Rods Respiratory II

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Fastidious Gram Negative RodsRespiratory II
Please click audio icon to hear Carols narration

• Clinical Laboratory Science Program
• Carol Larson MSEd, MT(ASCP)

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General Information
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• Fastidious
• Complex / extensive nutrient requirements
• Faint staining Gram Negative Rods
• Safranin counterstain for gt2 minutes
• Substitute carbolfuschin for safranin
• Serological testing useful

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Bordetella pertussisClinical Significance
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• 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 pertussisSpecimen Collection
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• 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 pertussisGrowth Characteristics
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• 35ºC, ambient air for at least 7 days
• Colony morphology
• No growth onBAP MAC
• Bordet-Gengou
• Regan-Lowe

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Bordetella pertussisIdentification
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• Gram stain
• Small, faintly staining gram-negative
  coccobacilli
• Oxidase
• Nitrate
• Urea
• Nonmotile

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Bordetella pertussisSerological Testing
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• DNA probes for direct detection in
• Specimen
• Culture confirmation
• Direct fluorescent antibody test
• Agglutination methods

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Bordetella pertussisTreatment Prevention
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• Erythromycin is drug of choice
• Vaccination

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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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Bordetella parapertussis
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• 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 bronchiseptica
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• Immunocompromised patients
• Grows on BAP and MAC
• Bordet-Gengou media
• Oxidase positive
• Urease positive (3-4 hours)
• Motile
• Nitrate positive

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How can you differentiate between Bordetella
pertussis and B. parapertussis?
Colony morphology on Bordet-Gengou media, growth
on BAP/MAC, oxidase, and urease.
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Capnocytophaga speciesClinical Significance
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• Normal oropharyngeal flora
• Associated with
• Periodontal disease
• Sepsis
• Granulocytopenia
• Malignancies
• Endocarditis
• Animal bites

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Capnocytophaga speciesSpecimen Collection
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• Respiratory specimens
• Capnophilic
• Facultative anaerobe
• Set up BAP, CHOC

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Capnocytophaga species Growth Characteristics
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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 Identification
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• 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.
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Francisella tularensisClinical Significance
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• 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 tularensisSpecimen Collection
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• 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 tularensisSpecimen Processing
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• Requires cysteine / cystine for growth
• Glucose-cystine blood (Francis) agar
• Grows on
• Chocolate
• BCYE
• Modified TM

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Francisella tularensisGrowth Characteristics
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• Strict aerobic
• 35C 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 tularensisIdentification
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• Pale staining gram negative coccobacilli
• Oxidase
• Catalase to weak
• Glucose fermenter
• Nonmotile

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Francisella tularensisSerological Testing
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• 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 gt1160 is presumptive

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Francisella tularensisTreatment Prevention
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• Streptomycin is drug of choice

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What substance is required in culture media in
order to grow Francisella?
Cysteine / Cystine
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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 speciesClinical Significance
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• Legionnaires disease
• Pontiac Fever
• Transmission inhalation of the organism in
  aerosols
• Legionella pneumophila serogroup 1

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Legionella speciesSpecimen Collection
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• 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 speciesGrowth Characteristics
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• Aerobic
• 35C 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 speciesIdentification
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• Thin, faintly staining short to filamentous GNR
• Oxidase wk
• Catalase wk
• Non-F
• Non-O
• Motilepolar flagella

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Legionella speciesSerological Testing
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• Specimen screen Isolate ID
• DFA stain and DNA probe
• IFA test of choice (serum)
• Four-fold rise in titer to at least 1128

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Legionella speciesTreatment and Prevention
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• Susceptibility testing not routinely performed
• Drug of choice Erythromycin alone or with
  Rifampin

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What substance is required in culture media in
order to grow Legionella?
Cysteine and iron salts.
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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 GNRSummary
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• Looked at several organisms
• Clinical significance
• Specimen collection, transport processing
• Growth characteristics identification
• Serological testing
• Treatment and prevention

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Who am I?
Reagin-Lowe media
Gram Stain
Causes Whooping Cough
Bordetella pertussis
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Who am I?
BCYE agar
Gram Stain
Causes Pontiac Fever
Legionella species