General Characteristics

The organisms discussed in this chapter are considered together because they are all oxidase-positive, glucose-fermenting, gram-negative bacilli capable of growth on MacConkey agar. Their individual morphologic and physiologic features are presented later in this chapter in the discussion of laboratory diagnosis. Other halophilic organisms, such as Halomonas venusta and Shewanella algae, require salt but do not ferment glucose, as do the halophilic Vibrio spp.

Aeromonas spp. are gram-negative straight rods with rounded ends or coccobacillary facultative anaerobes that occur singly, in pairs, or in short chains. They are typically oxidase and catalase positive and produce acid from oxidative and fermentative metabolism. Chromobacterium violaceum is a facultative anaerobic, motile, gram-negative rod or cocci.

The family Vibrionaceae includes six genera, three of which are discussed in this chapter. The Photobacterium and Grimontia each include a single species. The genus Vibrio consists of 10 species of gram-negative, facultative anaerobic, curved or comma-shaped rods. Most species are motile and are catalase and oxidase positive except Vibrio metschnikovii. All Vibrio spp. require sodium for growth and ferment glucose.

Epidemiology

Many aspects of the epidemiology of Vibrio spp., Aeromonas spp., and C. violaceum are similar (Table 26-1). The primary habitat for most of these organisms is water; generally, brackish or marine water for Vibrio spp., freshwater for Aeromonas spp., and soil or water for C. violaceum. Aeromonas spp. may also be found in brackish water or marine water with a low salt content. None of these organisms are considered part of the normal human flora. Transmission to humans is by ingestion of contaminated water, fresh produce, meat, dairy products, or seafood or by exposure of disrupted skin and mucosal surfaces to contaminated water.

The epidemiology of the most notable human pathogen in this chapter, Vibrio cholerae, is far from being fully understood. This organism causes epidemics and pandemics (i.e., worldwide epidemics) of the diarrheal disease cholera. Since 1817 the world has witnessed seven cholera pandemics. During these outbreaks the organism is spread among people by the fecal-oral route, usually in environments with poor sanitation.

The niche that V. cholerae inhabits between epidemics is uncertain. The form of the organism shed from infected humans is somewhat fragile and cannot survive long in the environment. However, evidence suggests that the bacillus has survival, or dormant, stages that allow its long-term survival in brackish water or saltwater environments during interepidemic periods. These dormant stages are considered viable but nonculturable. Asymptomatic carriers of V. cholerae have been documented, but they are not thought to be a significant reservoir for maintaining the organism between outbreaks.

Pathogenesis and Spectrum of Disease

As a notorious pathogen, V. cholerae elaborates several toxins and factors that play important roles in the organism’s virulence. Cholera toxin (CT) is primarily responsible for the key features of cholera (Table 26-2). Release of this toxin causes mucosal cells to hypersecrete water and electrolytes into the lumen of the gastrointestinal tract. The result is profuse, watery diarrhea, leading to dramatic fluid loss. The fluid loss results in severe dehydration and hypotension that, without medical intervention, frequently lead to death. This toxin-mediated disease does not require the organism to penetrate the mucosal barrier. Therefore, blood and the inflammatory cells typical of dysenteric stools are notably absent in cholera. Instead, “rice water stools,” composed of fluids and mucous flecks, are the hallmark of cholera toxin activity.

V. cholerae is divided into three major subgroups; V. cholerae O1, V. cholerae O129, and V. cholerae non-O1. The somatic antigens O1 and O139 associated with the V. cholerae cell envelope are positive markers for strains capable of epidemic and pandemic spread of the disease. Strains carrying these markers almost always produce cholera toxin, whereas non-O1/non-O139 strains do not produce the toxin and hence do not produce cholera. Therefore, although these somatic antigens are not virulence factors per se, they are important virulence and epidemiologic markers that provide important information about V. cholerae isolates. The non-O1/non-O139 strains are associated with nonepidemic diarrhea and extraintestinal infections.

V. cholerae produces several other toxins and factors, but the exact role of these in disease is still uncertain (see Table 26-2). To effectively release toxin, the organism first must infiltrate and distribute itself along the cells lining the mucosal surface of the gastrointestinal tract. Motility and chemotaxis mediate the distribution of organisms, and mucinase production allows penetration of the mucous layer. Toxin coregulated pili (TCP) provide the means by which bacilli attach to mucosal cells for release of cholera toxin.

Depending on the species, other vibrios are variably involved in three types of infection: gastroenteritis, wound infections, and bacteremia. Although some of these organisms have not been definitively associated with human infections, others, such as Vibrio vulnificus, are known to cause fatal septicemia, especially in patients suffering from an underlying liver disease.

Aeromonas spp. are similar to Vibrio spp. in terms of the types of infections they cause. Although these organisms can cause gastroenteritis, most frequently in children, their role in intestinal infections is not always clear. Therefore, the significance of their isolation in stool specimens should be interpreted with caution. Severe watery diarrhea has been associated with Aeromonas strains that produce a heat-labile enterotoxin and a heat-stable enterotoxin. In addition to diarrhea, complications of infection with Aeromonas spp. include hemolytic-uremic syndrome and kidney disease.

C. violaceum is not associated with gastrointestinal infections, but acquisition of this organism by contamination of wounds can lead to fulminant, life-threatening systemic infections.

Because no special considerations are required for isolation of these genera from extraintestinal sources, the general specimen collection and transport information provided in Table 5-1 is applicable. However, stool specimens suspected of containing Vibrio spp. should be collected and transported only in Cary-Blair medium. Buffered glycerol saline is not acceptable, because glycerol is toxic for vibrios. Feces is preferable, but rectal swabs are acceptable during the acute phase of diarrheal illness.

Direct Detection Methods

V. cholerae toxin can be detected in stool using an enzyme-linked immunosorbent assay (ELISA) or a commercially available latex agglutination test (Oxoid, Inc., Odgensburg, New York), but these tests are not widely used in the United States.

Microscopically, vibrios are gram-negative, straight or slightly curved rods (Figure 26-1). When stool specimens from patients with cholera are examined using dark-field microscopy, the bacilli exhibit characteristic rapid darting or shooting-star motility. However, direct microscopic examination of stools by any method is not commonly used for laboratory diagnosis of enteric bacterial infections.

Aeromonas spp. are gram-negative, straight rods with rounded ends or coccobacilli. No molecular or serologic methods are available for direct detection of Aeromonas spp. Cells of C. violaceum are slightly curved, medium to long, gram-negative rods with rounded ends. A polymerase chain reaction (PCR) amplification assay has been developed for identification of C. violaceum.

Cultivation

Media of Choice

Stool cultures for Vibrio spp. are plated on the selective medium thiosulfate citrate bile salts sucrose (TCBS) agar. TCBS contains 1% sodium chloride, bile salts that inhibit the growth of gram-positive organisms, and sucrose for the differentiation of the various Vibrio spp. Bromothymol blue and thymol blue pH indicators are added to the medium. The high pH of the medium (8.6) inhibits the growth of other intestinal flora. Although some Vibrio spp. grow very poorly on this medium, those that grow well produce either yellow or green colonies, depending on whether they are able to ferment sucrose (which produces yellow colonies). Alkaline peptone water (pH 8.4) may be used as an enrichment broth for obtaining growth of vibrios from stool. After inoculation, the broth is incubated for 5 to 8 hours at 35°C and then subcultured to TCBS.

Chromogenic Vibrio agar, which was developed for the recovery of Vibrio parahaemolyticus from seafood, supports the growth of other Vibrio spp. Colonies on this agar range from white to pale blue and violet.

Aeromonas spp. are indistinguishable from Yersinia enterocolitica on modified cefsulodin-irgasan-novobiocin (CIN) agar (4 µg/mL of cefsulodin); therefore, it is important to perform an oxidase test to differentiate the two genera. Aeromonas agar is a relatively new alternative medium that uses D-xylose as a differential characteristic. These organisms typically grow on a variety of differential and selective agars used for the identification of enteric pathogens. They are also beta-hemolytic on blood agar.

C. violaceum grows on most routine laboratory media. The colonies may be beta-hemolytic and have an almondlike odor. Most strains produce violacein, an ethanol-soluble violet pigment.

All of the genera considered in this chapter grow well on 5% sheep blood, chocolate, and MacConkey agars. They also grow well in the broth of blood culture systems and in thioglycollate or brain-heart infusion broths.

Incubation Conditions and Duration

These organisms produce detectable growth on 5% sheep blood and chocolate agars when incubated at 35°C in carbon dioxide or ambient air for a minimum of 24 hours. MacConkey and TCBS agars only should be incubated at 35°C in ambient air. The typical violet pigment of C. violaceum colonies (Figure 26-2) is optimally produced when cultures are incubated at room temperature (22°C).

Colonial Appearance

Table 26-3 describes the colonial appearance and other distinguishing characteristics (e.g., hemolysis and odor) of each genus on 5% sheep blood and MacConkey agars. The appearance of Vibrio spp. on TCBS is described in Table 26-4 and shown in Figure 26-3.

TABLE 26-3

Colonial Appearance and Characteristics

Organism	Medium	Appearance
Aeromonas spp.	BA Mac

Large, round, raised, opaque; most pathogenic strains are beta-hemolytic except A. caviae, which is usually nonhemolytic

Both NLF and LF

Chromobacterium violaceum

BA

Mac

Round, smooth, convex, some strains are beta-hemolytic; most colonies appear black or very dark purple; cultures smell of ammonium cyanide (almond-like)

NLF

Vibrio spp. and Grimontia hollisae

BA

Mac

Medium to large, smooth, opaque, iridescent with a greenish hue; V. cholerae, V. fluvialis, and V. mimicus can be beta-hemolytic

NLF except V. vulnificus, which may be LF

P. damsela

BA

Mac

Medium to large, smooth, opaque, iridescent with a greenish hue; may be beta-hemolytic

NLF

BA, 5% sheep blood agar; Mac, MacConkey agar; LF, lactose fermenter, NLF, non–lactose fermenter.

TABLE 26-4

Key Biochemical and Physiologic Characteristics of Vibrio spp. and Grimontia hollisae

				FERMENTATION OF
Species	Oxidase	Indole	Gas from Glucose	Lactose	Sucrose	Lysine Decarboxylasea	Arginine Dihydrolasea	Ornithine Decarboxylasea	Growth in 0% NaClb	Growth in 6% NaClb	TCBSc Growth	Colony on TCBSc
Grimonti hollisae	+	+	−	−	−	−	−	−	−	+	Very poor	Green
Vibrio alginolyticus	+	v	−	−	+	+	−	v	−	+	Good	Yellow
Vibrio cholerae	+	+	−	v	+	+	−	+	+	v	Good	Yellow
Vibrio cincinnatiensisd	+	v	−	−	+	v	−	−	−	+	Very poor	Yellow
P. damsela	+	−	−	−	−	v	+	−	−	+	Reduced at 36°C	Greene
Vibrio fluvialis	+	v	−	−	+	−	+	−	−	+	Good	Yellow
Vibrio furnissi	+	v	+	−	+	−	+	−	−	+	Good	Yellow
Vibrio harveyi	+	+	−	−	v	+	−	−	−	+	Good	Yellow
Vibrio metschnikovii	−	v	−	v	+	v	v	−	−	v	May be reduced	Yellow
Vibrio mimicus	+	+	−	v	−	+	−	+	+	v	Good	Green
Vibrio parahaemolyticus	+	+	−	−	−	+	−	+	−	+	Good	Greenf
Vibrio vulnificus	+	+	−	(+)	−	+	−	+	−	+	Good	Greeng

V, Variable; +, >90% of strains are positive; −, >90% of strains are negative; (+), delayed.

^a1% NaCl added to enhance growth.

^bNutrient broth with 0% or 6% NaCl added.

^cThiosulfate citrate bile salts sucrose agar.

^e5% yellow.

^f1% yellow.

^g0% yellow.

^dFerments myoinositol.

Approach to Identification

The colonies of these genera resemble those of the family Enterobacteriaceae but can be distinguished notably by their positive oxidase test result (except for V. metschnikovii, which is oxidase negative). The oxidase test must be performed from 5% sheep blood or another medium without a fermentable sugar (e.g., lactose in MacConkey agar or sucrose in TCBS), because fermentation of a carbohydrate results in acidification of the medium, and a false-negative result may occur if the surrounding pH is below 5.1. Likewise, if the violet pigment of a suspected C. violaceum isolate interferes with performance of the oxidase test, the organism should be grown under anaerobic conditions (where it cannot produce pigment) and retested.

The reliability of commercial identification systems has not been widely validated for identification of these organisms, although most are listed in the databases of several systems. The API 20E system (bioMérieux, St. Louis, Missouri) is one of the best for vibrios. Because the inoculum is prepared in 0.85% saline, the amount of salt often is enough to allow growth of the halophilic (salt-loving) organism.

The ability of most commercial identification systems to accurately identify Aeromonas organisms to the species level is limited and uncertain, and with some kits, difficulty arises in separating Aeromonas spp. from Vibrio spp. Therefore, identification of potential pathogens should be confirmed using conventional biochemical tests or serotyping. Tables 26-4 and 26-5 show several characteristics that can be used to presumptively group Vibrio spp., Aeromonas spp., and C. violaceum.

Comments Regarding Specific Organisms

V. cholerae and Vibrio mimicus are the only Vibrio spp. that do not require salt for growth. Therefore, a key test for distinguishing the halophilic species from V. cholerae, V. mimicus, and Aeromonas spp. is growth in nutrient broth with 6% salt. Furthermore, the addition of 1% NaCl to conventional biochemical tests is recommended to allow growth of halophilic species.

The string test can be used to differentiate Vibrio spp. from Aeromonas spp. In this test, organisms are emulsified in 0.5% sodium deoxycholate, which lyses Vibrio cells, but not those of Aeromonas spp. Cell lysis releases DNA, which can be pulled up into a string with an inoculating loop (Figure 26-4).

A Vibrio static test using 0/129 (2,4-diamino-6, 7-diisopropylpteridine)–impregnated disks also has been used to separate vibrios (susceptible) from other oxidase-positive glucose fermenters (resistant) and to differentiate V. cholerae O1 and non-O1 (susceptible) from other Vibrio spp. (resistant). However, recent strains of V. cholerae O139 have demonstrated resistance, so the dependability of this test is questionable.

Serotyping should be performed immediately to further characterize V. cholerae isolates. Toxigenic strains of serogroup O1 and O139 can be involved in cholera epidemics. Strains that do not type in either antiserum are identified as non-O1. Although typing sera are commercially available, isolates of V. cholerae are usually sent to a reference laboratory for serotyping.

Identification of V. cholerae or V. vulnificus should be reported immediately because of the life-threatening nature of these organisms.

Aeromonas spp. and C. violaceum can be identified using the characteristics shown in Table 26-5. Aeromonas spp. identified in clinical specimens should be identified as A. hydrophilia, A. caviae complex, or A. veronii complex.

Pigmented strains of C. violaceum are so distinctive that a presumptive identification can be made based on colonial appearance, oxidase, and Gram staining. Nonpigmented strains (approximately 9% of isolates) may be differentiated from Pseudomonas, Burkholderia, Brevundimonas, and Ralstonia organisms based on glucose fermentation and a positive test result for indole. Negative lysine and ornithine reactions are useful criteria for distinguishing C. violaceum from Plesiomonas shigelloides. In addition to the characteristics listed in Table 26-5, failure to ferment either maltose or mannitol also differentiates C. violaceum from Aeromonas spp.

Serodiagnosis

Agglutination, vibriocidal, or antitoxin tests are available for diagnosing cholera using acute and convalescent sera. However, these methods are most commonly used for epidemiologic purposes. Serodiagnostic techniques are not generally used for laboratory diagnosis of infections caused by the other organisms discussed in this chapter.

Antimicrobial Susceptibility Testing and Therapy

Two components of the management of patients with cholera are rehydration and antimicrobial therapy (Table 26-6). Antimicrobials reduce the severity of the illness and shorten the duration of organism shedding. The drug of choice for cholera is tetracycline or doxycycline; however, resistance to these agents is known, and the use of other agents, such as chloramphenicol, ampicillin, or trimethoprim-sulfamethoxazole, may be necessary. The Clinical and Laboratory Standards Institute (CLSI) has established methods for testing for V. cholerae, and the CLSI document should be consulted for this purpose.

The need for antimicrobial intervention for gastrointestinal infections caused by other Vibrio spp. and Aeromonas spp. is less clear. However, extraintestinal infections with these organisms and with C. violaceum can be life-threatening, and directed therapy is required. C. violaceum is often resistant to β-lactams and colistin.

Antimicrobial agents with potential activity are listed, where appropriate, in Table 26-6. It is important to note these organisms’ ability to show resistance to therapeutic agents; especially noteworthy is the ability of Aeromonas spp. to produce various beta-lactamases.

Prevention

No cholera vaccine is available in the United States. Two oral vaccines are available outside the United States, although the World Health Organization no longer recommends immunization for travel to or from cholera-infected areas. Individuals who have recently shared food and drink with a patient with cholera (e.g., household contacts) should be given chemoprophylaxis with tetracycline, doxycycline, or trimethoprim-sulfamethoxazole. However, mass chemoprophylaxis during epidemics is not indicated. No approved vaccines or chemoprophylaxis exists for the other organisms discussed in this chapter.