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V. vulnificus is a gram-negative, halophilic, oxidase-positive, facultative anaerobic, encapsulated bacillus measuring approximately 0.5-0.8 |im in width and 1.4-2.6 |im in length with an ultrastructure typical of most other gram-negative bacteria (Fig. 1). They do not form endospores or micro-cysts, but become spherical when grown under culture conditions where low levels of nutrients are found, culminating in a viable but nonculturable dormancy state (32). To date, characterizations of V. vulnificus strains, at both the phenotypic and genotypic levels, have not recognized unique traits that can be used to estimate pathogenicity for humans. However, characterization of strains has led to the subdivision of the species into three biotypes or biogroups based on phenotypic, serological, and host range differences (12,13,33-37). Biotype 1 strains were originally thought to be pathogenic only for humans and biotype 2 strains only for eels. However, recent evidence brought forth by Hoi et al. (35) demonstrates that biotype 1 can infect eels, and evidence reported by Amaro and Biosca (38) shows that biotype 2 strains can infect humans. Biotype 3 strains have only recently been isolated (36,37,39), and little is known about their ecology, pathogenicity, or host range. We would also like to point out that mass mortalities caused by V. vulnificus have been observed among cultured shrimp species such as P. monodon and P. japonicus in Taiwan (40), and V. vulnificus has

FIGURE 1 Transmission electron photomicrograph of glutaraldehyde fixed Vibrio vulnificus cells stained with 2% sodium phosphotungstic acid, pH 6.8. Bar marker = 0.1 |m.

also been isolated from surface lesions associated with moribund black porgy fish (Acanthopagrus schlegeli) (41).

Originally, V. vulnificus strains could be phenotypically separated from the other marine vibrios by its ability to ferment lactose. With the greater use of genetic and biochemical techniques in studies designed to examine the relatedness of members of this species and those of the genus Vibrio, we now know that V. vulnificus is not the only vibrio capable of fermenting lactose; strains of Vibrio cholerae, Vibrio mimicus, Vibrio metschnikovii, Vibrio fluvialis, and Vibrio nigripulcritudo can also ferment lactose. Thus, the utilization of lactose by V. vulnificus, as suggested by Baumann et al. (17), should be regarded with caution as a taxonomic trait for this species. The phenotypic characteristics of this organism are shown in Tables 1 and 2 and indicate that V. vulnificus can be phenotypically distinguished from other marine vibrio species by a number of distinct traits. For example, V. cholerae requires only trace amounts of Na+ for growth and the fermentation of lactose

TABLE 1 Biochemical Test Results and Other Properties of Vibrio vulnificusa

Percentage

Test positiveb

Indole production (HIB, 1% NaCl) 97

Voges-Proskauer, Barritt (1% NaCl) 80

Arginine dihydrolase, Moeller's (1% NaCl) 0

Lysine decarboxylase, Moeller's (1% NaCl) 99

Ornithine decarboxylase, Moeller's (1% NaCl) 55

D-Glucose, acid production 100

D-Glucose, gas production 0 Acid production from:

l-Arabinose 0

d-Arabitol 0

Cellobiose 99

Lactose 85

Maltose 100

d-Mannitol 45

Salicin 95

Sucrose 15

Nitrate-6-nitrite 100

Oxidase 100

Lipase (corn oil) 92

ONPG test 75 Growth in nutrient growth with:

1% NaCl 99

10% NaCl 0

a Each test is part of a recommended set of tests suggested by CDC for routine identification of Vibrio species. 1% NaCl in parentheses indicates that 1% NaCl had been added to the standard media to enhance growth; HIB, heart infusion broth; ONPG, o-nitrophenyl- d-galactopyranoside. b Percentage positive after 48 h incubation at 36°C. Source: Adapted from Ref. 43.

TABLE 2 Characteristics That Distinguish Vibrio vulnificus from Other Vibrio Species and Related Genera

Other

Test

V. vulnificus

V. alginolyticus

V. parahaemolyticus

vibrios

Aeromonas

Plesiomonas

Arginine dihydrolase

-

-

-

-/ +

+ / -

Lysine decarboxylase

+

+

+

-/ +

-

Ornithine decarboxylase

_a

-

+

-/ +

+ / -

ONPG

+a

-

-

-/ +

+

Indole production

+a

+

+

+ /-

+ / -

Voges-Proskauer

-

+

-

-/ +

+ / -

Fermentation of:

Lactose

+a

-

-

-

- / +

/-

Salicin

+a

-

-

-/ +

- / +

Sucrose

-

+

-

+ /-

+ / -

Growth in 1% peptone w/:

0% NaCl

-

-

-

-/ +

+

1% NaCl

+

+

+

+

+

8% NaCl

-

+

+

-/ +

-

10% NaCl

-

-

-

-

-

Symbols: +, most strains (~75%) positive; —, most strains (~75%) negative; + / —, strains mostly positive; —/ + , strains mostly negative.

a V. vulnificus biotype 3 strains are lactose and salicin fermentative negative and negative for ONPG test; biotype 2 strains are ornithine decarboxylase negative.

Source: Adapted from Refs. 17 and 43.

is delayed; V. nigripulchritudo produces a black pigment; V. alginolyticus is negative for both the ONPG (ortho-nitrophenyl-D-galactopyranoside) test and lactose fermentation; and V. parahaemolyt-icus is positive for acid production from L-arabinose. V. fluvialis is positive for acid production from L-arabinose, arginine dihydrolase positive, and lysine decarboxylase-negative, and negative for acid production from salicin; and V. metschnikovii is VP-positive and oxidase-negative and unable to convert nitrate to nitrite (42,43). Motile strains of V. vulnificus express a polar, sheathed flagellum, and expression of lateral flagella has not been found. Although V. vulnificus strains were originally reported to be sucrose-negative by Hollis et al. (19), the CDC has observed over recent years an increase in the number of sucrose-positive strains (43). Even though it is considered to be an obligate halophile, the Na+ requirement can be satisfied through the use of NaCl in the growth medium; the optimal NaCl concentrations appears to be between 1 and 3%, although the 0.5% NaCl present in many routine laboratory media, such as blood agar, trypticase soy agar, and brain heart infusion agars, provide enough Na + for very good growth (3,44). Recently, Azanza et al. (45) recommended using 0.1% peptone in 3% NaCl as a diluent for enumeration studies because a significant loss in viability of V. vulnificus was demonstrated in both broth cultures and seeded oyster homoge-nates when PBS was used as the diluent. Our experience is that 0.9-1.0% saline or artificial seawater at 20 ppt will suffice as an adequate diluent for many studies including preparation of inocula for infectivity studies and identification using the API 20 E identification kits (46). Kelly reported that the optimal temperature for growth of V. vulnificus is 37°C (3). However, it will grow at other temperatures. Aeration by agitation will also favor increased growth (44).

Though there are other marine vibrios that are bioluminescent, such as Vibrio fischerii and Vibrio harveyi, there has only been one report of a luminescent strain of V. vulnificus that was isolated from a patient with a fatal wound infection. This is the first description of a human infection caused by a luminescent bacterium (47). Biochemically, strains of biotype 2 and 3 closely resemble strains of biotype 1. Important taxonomic and biochemical traits used to differentiate the three biotypes are shown in Table 3. Biotype 1 strains can be differentiated from biotype 2 strains based on positive reactions for ornithine decarboxylase, indole production, and mannitol fermentation, and a

TABLE 3 Biochemical Characteristics of the Three Vibrio vulnificus Biotypes

Biotype

TABLE 3 Biochemical Characteristics of the Three Vibrio vulnificus Biotypes

Biotype

Test

1

2

3

Oxidase

+

+

+

Arginine dihydrolase

-

-

-

Lysine decarboxylase

+

+

+

Ornithine decarboxylase

+

-

+

Sucrose fermentation

-

-

-

Indole production

+

-

+

d-Mannitol fermentation

+

-

-

d-Sorbitol fermentation

-

+

-

Citrate utilization (Simmons')

+

+

-

Salicin fermentation

+

+

-

Cellobiose fermentation

+

+

-

Lactose fermentation

+

+

-

ONPG test

+

+

d

+ , Most strains (~75%) positive; —, most strains (~75%) negative; d, delayed. Source: Adapted from Ref. 37.

+ , Most strains (~75%) positive; —, most strains (~75%) negative; d, delayed. Source: Adapted from Ref. 37.

negative reaction for D-sorbitol fermentation. Biotype 3 strains are indole-positive, salicin-negative, cellobiose-negative, citrate-negative, and lactose-negative and have a delayed reaction for ONPG.

Besides biochemical analyses, bacteria can be further characterized by using bacteriophage typing methods (48). Considerable information is known about the Classical, El Tor, and O139 bacteriophage (or phage particles) infectious for V. cholerae, the type species of the genus (17, 43,49). Nevertheless, little is known about phage particles infectious for V. vulnificus. To date, only three studies have been published (50-52). Pelon et al. (50) found phage particles infectious for V. vulnificus in estuarine waters, sediments, plankton, crustacean, molluscan shellfish, and the intestines of finfish of the U.S. Gulf Coast. Encapsulated strains were more susceptible to infections by the phage than unencapsulated strains, suggesting that the capsule may be involved in attachment of the phage. The identification of phage receptor(s) and their role in pathogenicity is currently not well understood. DePaola et al. (52) further characterized several phage particles isolated from a variety of econiches by transmission electron microscopy (TEM) and showed that they morphologically belonged to three different families of double-stranded DNA phage representing the Podoviri-dae, Styloviridae, and Myoviridae phage families. One morphotype, identified as a member of the Podoviridae family, was found to be ubiquitous in Gulf Coast eastern oysters. This morphotype has an elongated capsid (mean, 258 nm; standard deviation, 4 nm; n = 35) and was also found in sediment and in the water column. Oysters were found to have much greater phage densities than other habitat samples. Within oyster tissues and fluids, the lowest densities of phage particles were seen in hemolymph and mantle fluids (51). Phages were found throughout the year in Eastern oysters collected from estuaries adjacent to the Gulf of Mexico (Apalachicola Bay, FL; Mobile Bay, AL; and Black Bay, LA) and the abundance of phage particles ranged from 101 to 105 phage particles per g of oyster tissue.

Another widely used system of characterizing pathogenic bacteria is to determine plasmid diversity and molecular variation by restriction endonuclease fragmentation pattern (REFP) analysis (53,54). Marine vibrios such as Vibrio angullarium are known to carry virulence plasmids (55, 56). Davidson and Oliver (54) found that of 42 clinical and environmental V. vulnificus isolates, 12% carried plasmids. In contrast, these authors found that 20 of 32 (62.5%) unidentified lactose-fermenting Vibrio spp. possessed plasmids with masses of 2.1-150 MDa. It was concluded that V. vulnificus, unlike most other Vibrio spp., shows a general lack of these extrachromosomal DNA elements.

V. vulnificus is the leading cause of reported deaths from foodborne illness in the United States (26). Studies comparing the virulence of isolates from different sources have yielded contradictory results (6,13,57-60). V. vulnificus's ability to cause invasive disease in humans and in a variety of economically important seafood hosts demonstrates not only its unique (unknown?) qualities as a pathogen to overcome species barriers, but also its importance as a major bacterial pathogen, which greatly impacts aquaculture, marine fish farming, and public health issues.

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