Fastidious Gram Negative Rods Respiratory Culture Unit Division of Medical Technology Carol Larson...

Fastidious Gram Negative RodsRespiratory Culture Unit

Division of Medical Technology

Carol Larson MSEd, MT(ASCP)

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• 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


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

• Make smear for DFA screening



Specimen Collection

• 35ºC, 5-10% CO2 for at least 7 days

• Colony morphology– No growth on

BAP & MAC– Bordet-Gengou– Regan-Lowe



Growth Characteristics

• Gram stain– Small, faintly staining gram-negative

coccobacilli

• Oxidase +

• Nitrate –

• Urea –

• Nonmotile



Identification

• Direct fluorescent antibody test

• Agglutination methods

• DNA probes for direct detection in:– Specimen– Culture confirmation



Serological Testing

• Erythromycin is drug of choice

• Vaccination



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.

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



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



Specimen Processing• Requires cysteine / cystine for growth

• Glucose-cystine blood (Francis’) agar

• Grows on– Chocolate– BCYE– Modified TM



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



Identification• Pale staining gram negative coccobacilli

• Oxidase –

• Catalase – to weak +

• Glucose fermenter

• Nonmotile



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


• Streptomycin is drug of choice



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


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


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


Legionella species

Growth Characteristics

• Thin, faintly staining short to filamentous GNR

• Oxidase wk +

• Catalase wk +

• Non-”F”

• Non-”O”

• Motile:polar flagella


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


Legionella species

Serological Testing

• Susceptibility testing not routinely performed

• Drug of choice: Erythromycin alone or with Rifampin


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


Fastidious GNR

Summary

Who am I?

Reagin-Lowe media Gram Stain


Causes Whooping Cough

Who am I?

BCYE agar Gram Stain

Legionella species

Causes Pontiac Fever

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