General Characteristics

The genera described in this chapter are all catalase-negative, non-spore-forming, gram-positive rods; some may exhibit rudimentary branching. Erysipelothrix rhusiopathiae is one of three species in the genus, but it is considered the only human pathogen. E. rhusiopathiae consists of several serovars based on peptidoglycan structure. The serovars most commonly associated with human infection include serovars 1 and 2. Arcanobacterium spp. demonstrate irregular, gram-positive rods on Gram stain. Gardnerella sp. fermentation byproducts include acetic and lactic acid. The cell wall of Gardnerella sp. is significantly thinner and contains less peptidoglycan than the typically gram-positive bacteria. Weissella confusa, formerly classified as Lactobacillus confusus, is included in Tables 18-3 and 18-4 because it is easily confused on culture media with the organisms included in this chapter, and in rare cases it has been isolated associated with bacteremia and endocarditis.

Epidemiology

Erysipelothrix spp. are found worldwide in a variety of vertebrate and invertebrate animals, including mammals, birds, and fish. Other domestic animals that may be infected include sheep, rabbits, cattle, and turkeys. The organism may be transmitted through direct contact or ingestion of contaminated water or meat. Arcanobacterium spp. are normal inhabitants of the mucosal membranes of cattle, sheep, dogs, cats, and pigs. The organisms listed in Table 18-1 include those that are closely associated with animals and are contracted by humans through animal exposure (e.g., E. rhusiopathiae and Arcanobacterium pyogenes) and those that are part of the normal human flora (e.g., Lactobacillus spp. and Gardnerella vaginalis).

Pathogenesis and Spectrum of Disease

G. vaginalis and Lactobacillus spp. (Table 18-2) are natural inhabitants of the human vagina. Vaginal infections with G. vaginalis are often found in association with a variety of mixed anaerobic flora. Extravaginal infections are uncommon but have been identified associated with postpartum endometritis, septic abortion, and cesarean birth.

Generally, no special considerations are required for specimen collection and transport of the organisms discussed in this chapter. Of note, skin lesions for Erysipelothrix should be collected by biopsy of the full thickness of skin at the leading edge of the discolored area. Refer to Table 5-1 for other general information on specimen collection and transport.

Direct Detection Methods

Gram staining of Arcanobacterium spp. demonstrates delicate, curved, gram-positive rods with pointed ends and occasional rudimentary branching. This branching is more pronounced after these organisms have been cultured anaerobically. Arcanobacterium spp. stain unevenly after 48 hours of growth on solid media and also exhibit coccal forms.

Lactobacillus is highly pleomorphic, occurring in long chaining rods and in coccobacilli and spiral forms (Figure 18-1).

E. rhusiopathiae stains as both short rods and long filaments. These morphologies correspond to two colonial types: (1) rough colonies that contain slender, filamentous, gram-positive rods with a tendency to overdecolorize and appear gram negative and (2) smooth colonies that contain small, slender rods. This variability in staining and colonial morphology may be mistaken for a polymicrobial infection both on direct examination and culture.

Gardnerella organisms are small, pleomorphic gram-variable or gram-negative coccobacilli and short rods. Wet mount and Gram staining of vaginal secretions are key tests for diagnosing bacterial vaginosis caused by G. vaginalis. A wet mount prepared in saline reveals the characteristic “clue cells,” which are large, squamous epithelial cells with numerous attached small rods. A Gram-stained smear of the discharge shows the attached organisms to be gram-variable coccobacilli. In bacterial vaginosis, clue cells are typically present, and large numbers of other gram-positive rods (i.e., lactobacilli), representing normal vaginal flora, are absent or few in number. In addition, the BDaffirm vaginal DNA probe (VDP) may be used for direct detection from genital specimens. Special vials containing transport reagent are used to stabilize the organism’s nucleic acids prior to testing (Becton, Dickinson and Company Franklin Lakes, NJ).

Cultivation

Media of Choice.

All the genera described in this chapter grow on 5% sheep blood and chocolate agars. They do not grow on MacConkey agar but do grow on Columbia colistin-nalidixic acid (CNA) agar. CNA agar is a nutritional base that may include 5% sheep blood to enhance the growth of fastidious organisms. The antibiotics colistin and nalidixic acid prevent the overgrowth of gram-negative organisms. All genera except Gardnerella sp. grow in commercially available blood culture broths. Gardnerella organisms are inhibited by sodium polyanetholsulfonate (SPS), which currently is used as an anticoagulant in most commercial blood culture media. An SPS-free medium or a medium with SPS that is supplemented with gelatin should be used when G. vaginalis sepsis is suspected.

Isolation of G. vaginalis from female genital tract specimens is best accomplished using the selective medium human blood bilayer Tween agar (HBT). HBT is CNA agar with amphotericin B added to prevent the growth of yeasts and filamentous fungi. Human blood is layered over the top to enhance the beta-hemolytic pattern of G. vaginalis.

Incubation Conditions and Duration

Detectable growth of these organisms should occur on 5% sheep blood and chocolate agars, CNA, and HBT incubated at 35°C in 5% to 10% carbon dioxide (CO₂) within 48 hours of inoculation.

Colonial Appearance

Table 18-3 describes the colonial appearance and other distinguishing characteristics (e.g., hemolysis) of each genus on sheep blood agar. G. vaginalis produces small, gray, opaque colonies surrounded by a diffuse zone of beta-hemolysis on HBT agar (Figure 18-2).

TABLE 18-3

Colonial Appearance on 5% Sheep Blood Agar and Other Characteristics

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Organism	Appearance
Arcanobacterium spp.	Small to large colonies with various appearances, including smooth, mucoid, and white and dry, friable, and gray; may be surrounded by narrow zone of beta-hemolysis
Erysipelothrix rhusiopathiae	Two colony types: large and rough or small, smooth, and translucent; shows alpha-hemolysis after prolonged incubation
Gardnerella vaginalis	Pinpoint; nonhemolytic