Clinical Laboratory Medicine
2nd Edition

MISCELLANEOUS FACULTATIVELY ANAEROBIC GRAM-NEGATIVE BACILLI
Microbiology
The genera and species discussed in this section are usually slow growing; may not grow on MacConkey, blood, or chocolate agar; and often grow better in a CO2-enriched atmosphere. They include Gardnerella, Cardiobacterium, Chromobacterium, Pasteurella, Actinobacillus, Kingella, Capnocytophaga, Streptobacillus, Suttonella, and CDC group EF-4.
G. vaginalis was previously classified as Haemophilus vaginalis or Corynebacterium vaginale. The cell wall is similar to but much thinner than gram-positive organisms. G. vaginalis is a small (0.5 × 1.5 μm), pleomorphic, fastidious, facultatively anaerobic, nonmotile, oxidase- and catalase-negative, gram-negative to gram-variable rod that does not grow on MacConkey agar.
Cardiobacterium hominis and Suttonella indologenes are a facultatively anaerobic, slow-growing, fastidious, nonmotile, oxidase-positive, catalase-negative, pale-staining gram-negative rods. They do not grow on MacConkey agar, and growth is enhanced by humidity and CO2.
The genus Chromobacterium contains two species, but only C. violaceum is isolated from clinical specimens. It is a facultatively anaerobic, motile, oxidase- and catalase-positive, gram-negative rod that usually produces a violet pigment. Unlike the other organisms in this section, C. violaceum grows on most routine laboratory media in ambient air.
The genus Pasteurella contains 20 recognized species and is similar to Actinobacillus based on moles percent G + C of their chromosomal DNA and phenotypic characteristics. P. multocida is the most common human pathogen of the genus. The other species are only rarely isolated from clinical specimens. P. multocida is a facultatively anaerobic, nonmotile, catalase- and oxidase-positive, gram-negative rod that may exhibit bipolar staining and does not grow on MacConkey agar.
The genus Actinobacillus contains six species, A. ureae, A. lignieresii, A. equuli, A. suis, A. hominis, and A. pleuropneumoniae. The Actinobacillus species are primarily animal pathogens. A. actinomycetemcomitans is closely related to H. aphrophilus and H. segris. These latter three species do not belong in the genus Haemophilus or Actinobacillus and probably will be classified in a new genus in the future. A. actinomycetemcomitans is a facultatively
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anaerobic, fastidious, slow-growing, small (0.5 × 1.5 μm), nonmotile, catalase-positive, gram-negative coccobacillary rod that does not grow on MacConkey agar and grows best in humidity and CO2.
The genus Kingella consists of three species: K. kingae, K. denitrificans, and K. oralis. Kingella species are facultatively anaerobic, nonmotile, catalase-negative, oxidase-positive, gram-negative rods that do not grow on MacConkey agar.
Seven species of Capnocytophaga are recognized: C. ochracea, C. gingivalis, C. sputigena, C. canimorsus, C. cynodegmi, C. haemolytica, and C. granulosa. They are facultatively anaerobic, fastidious, capnophilic, oxidase-variable, catalase-negative, gram-negative fusiform rods that may exhibit gliding motility and do not grow on MacConkey agar. C. canimorsus is a weakly oxidase-positive, catalase-positive, nonmotile, fermentative, long, thin, gram-negative fusiform rod that does not grow on MacConkey agar and grows poorly on other laboratory media (86).
CDC group EF-4 is an oxidase- and catalase-positive, nonmotile, fermentative (for glucose), gram-negative rod that grows poorly on MacConkey agar.
Spectrum of Disease
G. vaginalis is associated with BV (nonspecific vaginitis), which is characterized by a malodorous vaginal discharge and overgrowth with various anaerobic bacteria. Extragenital infections include bacteremia, vaginal abscesses, neonatal bacteremia, and soft-tissue infections. C. hominis and S. indologenes have been implicated in cases of endocarditis and bacteremia (87,88). C. violaceum is associated with urinary tract infections, localized abscesses, and fulminant sepsis with abscesses in multiple organs.
P. multocida is associated with three broad categories of disease: (a) localized infection after an animal bite or scratch (usually from a cat), (b) chronic pulmonary disease, particularly in patients with underlying pulmonary disease, and (c) bacteremia with or without metastatic foci. The latter group includes meningitis; brain, liver, and renal abscesses; otitis; mastoiditis; sinusitis; endocarditis; septic arthritis; and peritonitis. Spontaneous peritonitis and septicemia occur most often in patients with cirrhosis.
Actinobacillus is associated with juvenile periodontitis; endocarditis (particularly damaged or prosthetic heart valves); and infrequent reports of meningitis, brain abscess, osteomyelitis, urinary tract infection, pericarditis, and pulmonary infections.
Kingella infections include endocarditis, meningitis, bacteremia, septic arthritis, osteomyelitis, intervertebral disk infections, and infections of the eye. K. kingae infections occur primarily in children and young adults. K. denitrificans causes endocarditis in adults.
Capnocytophaga is associated with juvenile periodontitis, bacteremia, endocarditis, osteomyelitis, septic arthritis, sinusitis, pulmonary infections, conjunctivitis, and keratitis. Most infections occur in immunocompromised patients with severe granulocytopenia and oral ulcerations. C. canimorsus and C. cynodegma are associated with animal bites.
C. canimorsus causes two patterns of disease: (a) shock and disseminated intravascular coagulation seen in splenectomized patients, and (b) a milder form of disease in patients with a spleen. Infections include septicemia, meningitis, endocarditis, pneumonia, necrotizing wound infections, brain abscess, cellulitis, septic arthritis, corneal ulceration, and glomerulonephritis. Predisposing factors include splenectomy and a history of alcoholism.
CDC group EF-4 is associated with dogs and cats and cause wound infections and rarely bacteremia after bites, scratches, or contamination of a preexisting wound.
Epidemiology
G. vaginalis is part of the endogenous flora of the human genital tract and is associated with bacterial vaginosis (BV), the most common cause of vaginal discharge in sexually active women. BV is associated with preterm birth, premature rupture of membranes, and chorioamnionitis (89). Sexual transmission of the disease is not proven, but a high proportion of the male sex partners of women with the syndrome carry G. vaginalis on their urethral mucosa.
C. hominis, S. indologenes, A. actinomycetemcomitans, Kingella species, and Capnocytophaga species are part of the oropharyngeal flora in humans. Most infections result from endogenous spread of the bacterium, often after dental manipulations or oral disease such as periodontitis.
C. violaceum inhabits soil and water, particularly in warmer climates such as the southeastern United States, Southeast Asia, or South America. Infections generally occur after contamination of wounds with soil or water, or rarely after ingestion of contaminated water.
P. multocida, C. canimorsus, C. cynodegma, and CDC group EF-4 are found in the upper respiratory tract of dogs and cats. C. canimorsus, C. cynodegma, and EF-4a infections in humans generally follow dog bites or exposure to dog secretions. C. canimorsus, C. cynodegma, and EF-4b are also associated with cat bites and scratches. The reservoir for P. multocida is the upper respiratory tract of cats, dogs, other mammals, birds, and probably humans. The frequency of carriage varies with the species but is most common in cats. Transmission to humans occurs after bites, scratches, or exposure to secretions.
Diagnostic Procedures
Microscopy
BV can be presumptively diagnosed by Gram stain of vaginal secretions that demonstrate numerous clue cells (vaginal epithelial cells covered by gram-variable coccobacilli) associated with a mixed flora of large numbers of gram-negative to gram-variable coccobacilli in the absence of lactobacilli (90). The syndrome is best diagnosed by clinical criteria, a Gram stain or wet mount preparation of discharge, vaginal pH, and 10% KOH preparation of discharge rather than by culture of G. vaginalis.
The organisms discussed in this section are gram-negative rods that cannot be definitely identified by direct examination of the clinical specimen. C. hominis is a pale-staining pleomorphic (sometimes teardrop-shaped) rod; P. multocida is a small coccobacillus that may exhibit bipolar staining; A. actinomycetemcomitans
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is a small coccobacillus; Kingella are small coccobacilli arranged in pairs or chains; Capnocytophaga are thin fusiform rods; and C. canimorsus is a filamentous, sometimes curved fusiform rod. Both C. hominis and Kingella have a tendency to retain the primary stain.
Culture Techniques
G. vaginalis is most easily isolated on human blood bilayer Tween agar (HBT), vaginalis agar (V-agar), or Columbia CNA. Media are incubated at 35° to 37°C in a humid atmosphere containing 5% CO2 for 2 days. After 48 hours, colonies are approximately 0.5 mm in diameter and gray and exhibit diffuse β hemolysis on human but not on sheep blood. On routine sheep blood agar, colonies are pinpoint to barely visible.
C. hominis produces pinpoint colonies on chocolate or blood agar after 2 to 3 days incubation in a moist, 5% CO2 atmosphere. A slight green to brown color may develop around the colony.
C. violaceum grows well on blood and MacConkey agar in 24 hours at 35° to 37°C in air. Most colonies exhibit a characteristic violet pigment (produced optimally at 22°C) and often produce an odor of cyanide (almond).
Actinobacillus and Capnocytophaga grow best on blood or chocolate agar incubated in high humidity and in a 5% to 10% CO2-enriched atmosphere. After 2 to 3 days of incubation, colonies of Actinobacillus are small (0.5 mm), punctate, and adherent with a mixed morphology. On continued incubation, a starlike, opaque center may be observed under 100× magnification (Fig. 51.16). Capnocytophaga produces yellow nonhemolytic colonies with fingerlike projections and an almond odor after 2 to 3 days of incubation. Some strains will grow only anaerobically on initial isolation. Colonies may pit the agar.
FIGURE 51.16. Colony of A. actinomycetemcomitans on heart infusion agar. Note the central star-shaped structure. (From Baron EJ, Finegold SM. Bailey and Scott’s diagnostic microbiology, St. Louis: Mosby, 1990, with permission.)
Kingella grows in air or CO2 (may enhance growth) but grows poorly under anaerobic conditions. Colonies are 0.5 to 1 mm in diameter after 48 hours of incubation at 35°C and may pit the agar or produce a “fried-egg” appearance. K. denitrificans grows on Martin-Lewis agar and may be confused with N. gonorrhoeae.
Pasteurella grows well on nonselective media such as blood or chocolate agar at 35°C in air or CO2. Colonies are usually smooth, gray and 1 to 2 mm after 24 hours of incubation, have a musty odor, and may produce a green to brown discoloration on blood agar.
C. canimorsus grows slowly (2 to 4 days) on enriched chocolate agar incubated at 35°C in a humid, CO2-enriched atmosphere. Growth is poor under anaerobic conditions.
EF-4 grow well on nonselective media (e.g., blood or chocolate agar) within 24 hours of incubation at 35°C. Colonies are small and yellow-orange and produce a popcornlike odor.
Identification Methods
G. vaginalis can be identified presumptively on the basis of negative catalase and oxidase tests, β-hemolytic colonies on HBT agar, and a typical Gram-stain morphology. Confirmatory tests include starch and hippurate hydrolysis, α- and β-glucosidase tests, or use of some commercial systems for identification of Corynebacterium.
C. hominis and S. indologenes are oxidase positive and catalase negative and are differentiated from similar organisms (e.g., Eikenella, Kingella, and H. aphrophilus) by the production of indole (Table 51.25). Serum may have to be added to carbohydrate tests to ensure growth.
TABLE 51.25. DIFFERENTIATION OF SELECTED GENERA AND SPECIES OF GRAM-NEGATIVE BACILlI

Organism Fusiform Shape Catalase Oxidase Indole Motility Nitrate Reduction Glucose Fermentation Growth on MacConkey

Cardiobacterium + + + v
Chromobacterium violaceum + v a + + + +
Capnocytophaga + v v + +
Actinobacillus actinomycetemcomitans + v + +
Pasteurella multocida + + + + +
Eikenella corrodens + +
Kingella + v v + v
Haemophilus aphrophilus v + +
CDC Group EF4a + + + + v

+, >90% positive; –, 90% negative; v, variable reaction; aSome nonpigmented stains are positive.
C. violaceum is oxidase and catalase positive and motile, reduces nitrate, and ferments glucose. It can be distinguished from other genera such as Aeromonas, Plesiomonas, and Vibrio by negative tests for indole, ornithine, and lysine decarboxylase reactions and lack of fermentation of mannitol and maltose.
Actinobacillus may be differentiated from similar genera with a minimum number of tests, but the definitive identification of a suspected isolate requires a more comprehensive battery.
Kingella species are catalase negative, oxidase positive, and nonmotile (Table 51.25). K. kingae is distinguished from the other two species by β hemolysis on sheep blood agar and the absence of indole production, nitrate reduction, and growth on Martin-Lewis agar.
Pasteurella species are generally catalase negative, oxidase positive, and nonmotile (Table 51.25). Other characteristic reactions for P. multocida include a positive ornithine decarboxylase and indole reaction.
Characteristics useful for the identification of the genus Capnocytophaga are listed in Table 51.25. Commercial identification systems are also available. C. canimorsus often requires the addition of rabbit serum to obtain adequate growth in the biochemical tests. It is weakly oxidase (positive) and catalase positive; indole, urease, and nitrate-reduction negative; and arginine dihydrolase positive.
CDC group EF-4 strains are oxidase and catalase positive, reduce nitrate, and are negative for urease and indole production.
Direct Antigen to Nucleic Acid Detection
No commercial systems are available for the direct detection of any of these bacteria in clinical specimens.
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Serologic Procedures
No routine serologic tests are available for the diagnosis of infections caused by this group of organisms.
Interpretation of Results
With the exception of G. vaginalis, these bacteria are seldom isolated from clinical specimens. Therefore, their isolation is clinically significant until proven otherwise. The isolation of any members of the HACEK group (Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, Kingella) from blood is highly suspicious of endocarditis, especially if the patient has oral trauma or poor dentition. The presence of large numbers of G. vaginalis organisms in vaginal secretions is suggestive of BV but does not prove that G. vaginalis causes the syndrome.
Therapeutic Considerations
Standardized susceptibility tests are often difficult to perform because of the growth requirements of some of these bacteria. Oral metronidazole or clindamycin treatment is the treatment of choice for BV.
Cardiobacterium and Suttonella are susceptible to β-lactam agents, tetracycline, chloramphenicol, and aminoglycosides but variably resistant to clindamycin, erythromycin, oxacillin, and vancomycin.
C. violaceum should be tested for susceptibility to antimicrobial agents. Most isolates are susceptible to tetracycline, chloramphenicol, erythromycin, and trimethoprim-sulfamethoxazole with variable resistance to aminoglycosides. They are often resistant to the β-lactam antibiotics and clindamycin.
P. multocida is susceptible to penicillin, cephalothin, trimethoprim-sulfamethoxazole, and tetracycline. Limited experience is available for the treatment of infections with the newer antimicrobial agents.
Actinobacillus is generally susceptible to chloramphenicol, tetracycline, carbenicillin, and streptomycin, and is variably susceptible to penicillin. In vitro tests indicate susceptibility to the newer cephalosporins, rifampin, trimethoprim-sulfamethoxazole, and fluoroquinolones. Combined penicillin-aminoglycoside therapy has been recommended for endocarditis.
Kingella are generally susceptible to the β-lactams (K. denitrificans may be moderately resistant to penicillin), cephalothin, aminoglycosides, tetracycline, chloramphenicol, and sulfonamides.
Capnocytophaga are susceptible to penicillin, extended-spectrum cephalosporins, chloramphenicol, tetracycline, erythromycin, clindamycin, quinolones, and are resistant to vancomycin, aminoglycosides, metronidazole, and trimethoprim-sulfamethoxazole.
CDC group EF-4a organisms appear susceptible to the β-lactams, chloramphenicol, and tetracycline based on limited studies.
Summary and Conclusions
The miscellaneous facultatively anaerobic gram-negative bacilli discussed in this section are usually inhabitants of the upper respiratory tract of humans (e.g., Actinobacillus, Kingella, Cardiobacterium, and Capnocytophaga), the genital tract (e.g., Gardnerella), the upper respiratory tract of animals (e.g., Pasteurella, C. canimorsus, and CDC group EF-4), or soil and water (e.g., Chromobacterium). Infections usually follow the spread of the bacterium from a colonized site or after exposure to animal bites, scratches, or secretions. These species cause a variety of diseases ranging from localized soft-tissue infections to systemic life-threatening infections. Most of the organisms are fastidious, do not grow on MacConkey agar, and require increased humidity and CO2-enriched atmosphere for growth. Chromobacterium grows well on most routine media. C. violaceum is resistant to penicillin and A. actinomycetemcomitans is moderately resistant; the other species are susceptible to penicillin.