FASTIDIOUS GRAM-NEGATIVE RODS

Objectives: After review of the Clinical Microbiology Study Manual and completion of the clinical
rotation, the Clinical Laboratory Science/Medical Technology student will be able to:

1. Define the term fastidious, as used in clinical microbiology.

2. Discuss appropriate specimen collection for the isolation of Bordetella pertussis.

3. Evaluate the following media, including purpose, proper use, inhibitory or selective properties
and colonial appearance:
a. Blood cysteine c. Bordet-Gengou
b. Regan-Lowe d. Buffered charcoal yeast extract (BCYE)

4. Describe specific specimen processing requirements, including special media, incubation

times and any characteristic colonial morphology for each of the following:
a. Brucella species d. Legionella species
b. Francisella species e. Capnocytophaga species
c. Bordetella species f. Eikenella corrodens

5. List the organisms referred to as the HACEK group.

6. Discuss the following techniques used to enhance Gram stain morphology:

a. Extended safranin
b. Substitution of carbolfuschin

7. Assess the use of immunologic methods for direct specimen detection and/or organism
identification of the following organisms:
a. Francisella tularensis c. Brucella species
b. Bordetella pertussis d. Legionella species

8. Identify Eikenella corrodens based on the following characteristics:

a. Colony morphology c. Oxidase reaction
b. Glucose utilization d. Catalase reaction

9. Identify Capnocytophaga species based on the following characteristics:

a. Colony morphology
b. Gram stain morphology

10. Correlate the clinical, epidemiological and laboratory findings associated with the following
infections:
a. Francisella tularensis
1) Tularemia
b. Bordetella pertussis
1) Pertussis (whooping cough)
c. Brucella species
1) Brucellosis (undulant fever)
d. Legionella species
1) Legionnaire’s disease
2) Pontiac fever
e. Eikenella corrodens
1) Human bite wound
f. HACEK group
1) Subacute bacterial endocarditis

11. Evaluate the appropriateness of susceptibility testing of the organisms listed in objective #4.

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 FASTIDIOUS GRAM NEGATIVE BACILLI

General Information

• Fastidious = an organism with complex or extensive nutritional requirements

• Organisms appear as faintly staining gram negative rods
• Two methods to enhance the gram reaction
• Extend time for safranin counterstain to at least 2 minutes
• Substitute carbolfuschin for safranin
• Serologic methods are useful in direct detection, identification and diagnosis of disease
• Fluorescent antibody
• Direct agglutination
• DNA probes
• Enzyme immunoassay

Genus Bordetella (Mahon, 2nd edition, pages 457-462)

Bordetella pertussis
Clinical Significance
• Bordetella pertussis causes Whooping Cough or Pertussis in man
• Strict human pathogen, spread by airborne droplets
• Lives in ciliated epithelium of the upper respiratory tract
• Produces an exotoxin and has a cell wall endotoxin
• The major virulence factor is the pertussis toxin

Specimen collection, transport, and processing

• Nasopharyngeal swab or aspirate is the specimen of choice
• Specimen should be plated at bedside and a smear made for DFA screening
**The number of organisms present in the secretions limits sensitivity of the screen
• If the specimen must be transported, Regan-Lowe media is recommended for transport. It
contains charcoal, horse blood and cephalexin
• The organism is a fastidious obligate aerobe
• Need special media for isolation:
o Bordet-Gengou = contains potato-glycerol-blood agar
o Regan-Lowe = contains charcoal and horse blood
o Methicillin or cephalexin can be added to inhibit normal flora
o Media should be incubated in a humid environment at 35ºC with 5-10% CO2 for at least 7
days

Laboratory identification
• Colony morphology:
• Bordet-Gengou = slightly beta hemolytic smooth, shiny, resembling a mercury droplet
• Regan-Lowe = domed and shiny with a white mother-of-pearl opalescence
• BAP = no growth
• MAC = no growth
• Gram Morphology: small faintly staining gram negative coccobacilli
• Oxidase = positive
• Nitrate = negative
• Urea = negative
• Nonmotile

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Serologic identification
• Usually identified by fluorescent antibody
• Agglutination methods are also available
• Probes are available for direct detection in the specimen and for culture confirmation

Treatment & Prevention

• Erythromycin is the drug of choice for treatment
• Vaccination is the best protection

Bordetella parapertussis
• Found on the mucous membranes of humans
• Causes acute respiratory tract infection resembling mild whooping cough in man

Bordetella bronchiseptica
• Found on the mucous membranes of animals and occasionally man
• Causes respiratory infections in animals
• Human infection seen primarily in immunocompromised patients = wound, blood, respiratory

Bordetella Bordetella Bordetella

pertussis parapertussis bronchiseptica
Gram stain morphology Faint staining GNCB Faint staining GNR Faint staining GNR
Growth on Bordet- “Mercury droplet” Gray-brown Large, flat, dull
Gengou Beta hemolytic Beta hemolytic Beta hemolytic
(3-4 days) (2-3 days) (24 hrs)
Growth on Regan Lowe Shiny white Similar to Similar to
“Mother of pearl” Bordet-Gengou Bordet-Gengou
BAP No growth Growth Growth
MAC No growth Variable Growth
Oxidase + - +
Urea - + (24 hr) + (3-4 hrs)
Motility - - +
Nitrate - - +
Carbohydrate utilization - - -

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Genus Brucella (Mahon, 2nd edition, pages 443-444)
Clinical significance
• Four species are pathogenic to man:
B. abortus (cattle), B. canis (dogs), B. melitensis (goats), B. suis (pigs)
• Brucella causes brucellosis (undulant fever/Malta fever), a relapsing febrile illness
• Transmission is via: Direct contact with infected animals
Ingestion of contaminated meat or dairy products
Inhalation of the aerosolized organism
• Incidence in US has declined due to vaccination of animals and pasteurization processes
Imported cheeses and candies have been implicated in US cases
• Brucella is a facultative intracellular organism

Specimen collection, transport, and processing

• Specimen of choice is blood, bone marrow or tissue
• There is a risk of laboratory acquired infection
Always work in a hood
• Grows on BAP, CHOC, and BCYE (used for isolation of Legionella)
• Blood culture mediums support growth of Brucella
• Media should be incubated at 35ºC with 5-10% CO2 with a humid atmosphere
• Organism is very slow to grow – hold cultures at least 21 days

Laboratory identification
• Colony morphology: smooth glistening, translucent colonies that become brown with age
• Gram Morphology: tiny faint staining gram negative coccobacilli
• Oxidase: positive
• Nitrate: positive
• Catalase: positive
• Glucose oxidizer
• Non-motile

Brucella Brucella Brucella Brucella

abortus melitensis suis canis
CO2 requirement + - - -
H2S (Pb acetate strip) + - + -
Urea + (>2 hr) Variable (>2 + (0-30 min) + (0-30min)
hrs)
Growth on dye medium:
Basic fuchsin Growth Growth No growth No growth
Growth on dye medium:
Thionine No growth Growth Growth Growth

Serologic identification
• Tube agglutination test most commonly used
• Single titer of >=1:160 or a fourfold rise in titer between acute and convalescent specimens is
considered significant
• Does not detect Brucella canis

Treatment & Prevention

• Recommended treatment: doxycycline and rifampin for 6 weeks

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Genus Capnocytophaga (Mahon, 2nd edition, page 440)
Clinical Significance
• Part of normal oropharyngeal flora
• Associated with periodontal disease, sepsis in patients with granulocytopenia and
malignancies, endocarditis, and animal bites

Specimen Collection, Transport and Processing

• Most frequently isolated from respiratory specimens
• Requires CO2 for growth (capnophilic)
• Facultative anaerobe

Laboratory Identification
• Colony morphology
• BAP & CHOC = slight yellow, nonhemolytic, spreading over agar surface (at 48 hrs),
center of colony has moist, mottled appearance
• MAC = no growth
• Gram stain morphology = fusiform, filamentous gram neg. rod, size and shape vary with
age
• Oxidase: negative
• Catalase: negative
• Motility: “gliding motility” = do not have flagella but move by twitching

Genus Eikenella (Mahon, 2nd edition, page 439)

Clinical significance
• Only one species: Eikenella corrodens
• Normal flora of mouth, respiratory tract and GI tract of man.
• Opportunistic pathogen in man
• Associated with dental/periodontal and head/neck infections/abscesses, human bite wounds,
septicemia following tooth extraction and endocarditis

Specimen Collection, Transport and Processing

• No special collection requirements
• Needs hemin in media for growth

Laboratory identification:
• BAP & CHOC = tiny colonies at 48 hrs
• MAC = no growth
• Colonies "pit" (corrode) the agar and are often sunk into small craters in the agar
• Usually have a pale yellow pigment
• May have greening around colony
• May have a characteristic bleach odor
• Gram morphology: small, slender GNR
• Oxidase: positive
• Glucose non-oxidizer (asaccharolytic)
• Catalase: negative
• Nitrate positive
• Non-motile

Treatment & Prevention

• Susceptibility testing is not routinely performed
• Treat with penicillins, 3rd generation cephalosporins, tetracycline, and quinolones

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Genus Francisella (Mahon, 2nd edition, page 444)
Clinical Significance
• Francisella tularensis 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, Transport and Processing

• Specimen should be inflammatory material from the infected site (lesion, lymph node
aspirate, sputum, tissue biopsy)
• Wear gloves and perform all work in a biosafety hood
• Do not aerosolize or allow contact with skin or mucous membranes
• Organism can penetrate unbroken skin---be careful!!!
• Requires cysteine / cystine for growth
• Glucose-cystine blood (Francis’) agar preferred for isolation
• Grows on Chocolate agar, buffered charcoal yeast extract (BCYE) and modified Thayer-
Martin (MTM)
• Incubate at 35 C with 5-10% CO2 for 7 days (strict aerobe)

Laboratory identification
• Colony morphology
• BAP = No growth
• Choc = Small, gray alpha hemolytic (if on blood containing media) colony at 2-5 days
• MAC = No growth
• Gram morphology: pale staining gram negative coccobacilli
• Oxidase: negative
• Catalase: negative-weak pos
• Ferments glucose
• Non-motile

**NOTE: Usually identified by DFA or direct agglutination tests due to risk of laboratory acquired
infection. Biochemical identification is usually performed in reference laboratories.

Serologic testing
• Most cases diagnosed serologically
• DFA tests may be performed on specimen
• Antibody titers can be determined by ELISA and agglutination tests
• A four-fold rise in titer between acute and convalescent specimen is considered diagnostic
• A single acute phase titer of 1:160 or greater is considered presumptive

Treatment & Prevention

• Streptomycin is drug of choice for treatment.

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Genus LEGIONELLA (Mahon, 2nd edition, pages 447-456)
Clinical Significance
• There are two forms of Legionellosis:
• Legionnaires’ disease Characterized by malaise, myalgia with rapid onset of a dry
cough and fever, and development of pneumonia
Illness is fatal in 15-30% of cases not treated
• Pontiac Fever Characterized by fever, headache, myalgia and malaise
Lacks symptoms of pneumonia and is not fatal
• Disease occurs most frequently in men, cigarette smokers, people with underlying disease,
immunosuppressed/immunocompromised, people who drink alcohol
• Major cause of nosocomial pneumonia
• Organism exists in natural/man-made water systems and in soil
• Transmission: inhalation of the organism in aerosols
• Legionella pneumophila serogroup 1 causes most human infection

Specimen Handling and Processing

• Appropriate specimens: BAL, bronchial washing, lung biopsy, pleural fluid
• Avoid aerosolization and transport ambient temperature
• Organism requires cysteine and iron salts for growth
• Buffered Charcoal Yeast Extract agar (BCYE) is most widely used
• BCYE + antibiotics is used as a selective media for contaminated specimens
• Incubate at 35 C in 5-10% CO2 with increased humidity for 10 days
• Organisms usually take 2-4 days to grow

Laboratory Identification
• Colony Morphology:
• BAP & MAC = no growth
• CHOC = may grow very slowly
• BCYE = convex, grayish white, glistening with an entire edge at 2-4 days
When viewed with dissecting microscope they have a "ground glass" appearance with a
pink or blue-green tint
• Gram stain morphology: thin, faintly staining gram negative short to filamentous rods
• Oxidase and Catalase: weakly positive
• Gelatin: positive (most species)
• Motility: positive by polar flagella
• Does not oxidize or ferment carbohydrates = biochemically inert
• Some species fluoresce under UV light, color varies depending on the species

Serologic diagnosis
• Direct screen of specimen can be done with DFA stain and DNA probe
• Identify isolate with DFA or DNA probe
• IFA test of choice
• Need to demonstrate a four-fold rise in titer to at least 1:128
• Draw acute specimen within 7 days of onset
• Draw convalescent specimen 3-5 weeks after onset

Treatment & Prevention

• Susceptibility testing is not routinely performed
• Drug of choice: Erythromycin alone or with Rifampin

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HACEK Group (Mahon, 2nd edition, pages 436-440)
Organisms include
Haemophilus aphrophilus
Actinobacillus actinomycetemcomitans
Cardiobacterium hominis
Eikenella corrodens
Kingella kingae

Clinical Significance
Infective endocarditis
Usually diagnosed in Blood Cultures
Hold cultures for extended period beyond 1 week and make blind subcultures to several enriched
media, including buffered charcoal-yeast extract

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 GRAM NEGATIVE ROD
(GNCB)

Glucose fermentation

(+) (-)

Oxidase Glucose oxidation

(+) (-)

OX MOT MAC
Aeromonas + + + Enterobacteriaceae
Vibrio + + +
Plesiomonas + + +
Pasteurella + - -
Actinobacillus +/- - +/-

(+) (-)

OX MOT MAC OX MOT MAC

Pseudomonas + + + Eikenella + - -
**Flavobacterium + - +/- Moraxella + - -
Acinetobacter - - + Alcaligenes + + +
Stenotrophomonas - + + Acinetobacter lwoffii - - +

**Some species capable of weak glucose fermentation

Gram Negative Bacilli (GNCB) Flowchart Page 9