Effects of temperature and salinity on the survival of Vibrio vulnificus in seawater and shellfish.

Sterilized seawater was used to assess the effects of temperature and salinity on the survival of Vibrio vulnificus. In the temperature range of 13 to 22 degrees C, numbers of V. vulnificus increased during the 6-day incubation. Temperatures outside this range reduced the time of V. vulnificus survival in sterile 10-ppt seawater. At these restrictive temperatures, V. vulnificus numbers were reduced by 90% after 6 days of incubation. Incubation between 0.5 and 10.5 degrees C demonstrated that V. vulnificus survives poorly below 8.5 degrees C. At salinities between 5 and 25 ppt and at 14 degrees C, V. vulnificus numbers actually increased or remained unchanged after 6 days of incubation. At salinities of 30, 35, and 38 ppt, numbers of V. vulnificus decreased 58, 88, and 83%, respectively. V. vulnificus could not be recovered from deionized water, indicating lysis. When a rifampin-resistant strain of V. vulnificus was used to inoculate sterilized and unsterilized seawater (20 ppt, 20 degrees C), numbers increased in sterile seawater but decreased to undetectable levels in 14 days in the unsterilized seawater, indicating that biological factors may play a role in the survival of V. vulnificus in the environment. Since our studies demonstrated sensitivity to low temperatures, the survival of V. vulnificus in naturally contaminated oysters at temperatures of 0, 2, and 4 degrees C was also determined. Numbers of endogenous V. vulnificus in oyster shellstock increased by more than 100-fold in shellstock stored at 30 degrees C but were reduced approximately 10- and 100-fold after 14 days at 2 to 4 degrees C and 0 degrees C, respectively. We conclude that both biological and physicochemical factors are important to the survival of V. vulnificus in the environment and that temperature is critical to controlling its growth in oyster shellstock.     

Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light.

Vibrio vulnificus is an estuarine bacterium which can cause opportunistic infections in humans consuming raw Gulf Coast oysters, Crassostrea virginica. Although V. vulnificus is known as a ubiquitous organism in the Gulf of Mexico, its ecological relationship with C. virginica has not been adequately defined. The objective of the present study was to test the hypothesis that V. vulnificus is a persistent microbial flora of oysters and unamenable to traditional methods of controlled purification, such as UV light depuration. Experimental depuration systems consisted of aquaria containing temperature-controlled seawater treated with UV light and 0.2-microns-pore-size filtration. V. vulnificus was enumerated in seawater, oyster shell biofilms, homogenates of whole oyster meats, and tissues including the hemolymph, digestive region, gills, mantle, and adductor muscle. Results showed that depuration systems conducted at temperatures greater than 23 degrees C caused V. vulnificus counts to increase in oysters, especially in the hemolymph, adductor muscle, and mantle. Throughout the process, depuration water contained high concentrations of V. vulnificus, indicating that the disinfection properties of UV radiation and 0.2-microns-pore-size filtration were less than the rate at which V. vulnificus was released into seawater. Approximately 10(5) to 10(6) V. vulnificus organisms were released from each oyster per hour, with 0.05 to 35% originating from shell surfaces. These surfaces contained greater than 10(3) V. vulnificus organisms per cm2. In contrast, when depuration seawater was maintained at 15 degrees C, V. vulnificus was not detected in seawater and multiplication in oyster tissues was inhibited.     

Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light.

Vibrio vulnificus is an estuarine bacterium which can cause opportunistic infections in humans consuming raw Gulf Coast oysters, Crassostrea virginica. Although V. vulnificus is known as a ubiquitous organism in the Gulf of Mexico, its ecological relationship with C. virginica has not been adequately defined. The objective of the present study was to test the hypothesis that V. vulnificus is a persistent microbial flora of oysters and unamenable to traditional methods of controlled purification, such as UV light depuration. Experimental depuration systems consisted of aquaria containing temperature-controlled seawater treated with UV light and 0.2-microns-pore-size filtration. V. vulnificus was enumerated in seawater, oyster shell biofilms, homogenates of whole oyster meats, and tissues including the hemolymph, digestive region, gills, mantle, and adductor muscle. Results showed that depuration systems conducted at temperatures greater than 23 degrees C caused V. vulnificus counts to increase in oysters, especially in the hemolymph, adductor muscle, and mantle. Throughout the process, depuration water contained high concentrations of V. vulnificus, indicating that the disinfection properties of UV radiation and 0.2-microns-pore-size filtration were less than the rate at which V. vulnificus was released into seawater. Approximately 10(5) to 10(6) V. vulnificus organisms were released from each oyster per hour, with 0.05 to 35% originating from shell surfaces. These surfaces contained greater than 10(3) V. vulnificus organisms per cm2. In contrast, when depuration seawater was maintained at 15 degrees C, V. vulnificus was not detected in seawater and multiplication in oyster tissues was inhibited.     

Ecology of Vibrio vulnificus in estuarine waters of eastern North Carolina

While several studies on the ecology of Vibrio vulnificus in Gulf Coast environments have been reported, there is little information on the distribution of this pathogen in East Coast waters. Thus, we conducted a multiyear study on the ecology of V. vulnificus in estuarine waters of the eastern United States, employing extensive multiple regression analyses to reveal the major environmental factors controlling the presence of this pathogen, and of Vibrio spp., in these environments. Monthly field samplings were conducted between July 2000 and April 2002 at six different estuarine sites along the eastern coast of North Carolina. At each site, water samples were taken and nine physicochemical parameters were measured. V. vulnificus isolates, along with estuarine bacteria, Vibrio spp., Escherichia coli organisms, and total coliforms, were enumerated in samples from each site by using selective media. During the last 6 months of the study, sediment samples were also analyzed for the presence of vibrios, including V. vulnificus. Isolates were confirmed as V. vulnificus by using hemolysin gene PCR or colony hybridization. V. vulnificus was isolated only when water temperatures were between 15 and 27 degrees C, and its presence correlated with water temperature and dissolved oxygen and vibrio levels. Levels of V. vulnificus in sediments were low, and no evidence for an overwintering in this environment was found. Multiple regression analysis indicated that vibrio levels were controlled primarily by temperature, turbidity, and levels of dissolved oxygen, estuarine bacteria, and coliforms. Water temperature accounted for most of the variability in the concentrations of both V. vulnificus (47%) and Vibrio spp. (48%).     

Offshore suspension relaying to reduce levels of Vibrio vulnificus in oysters (Crassostrea virginica).

Oysters naturally contaminated with 10(3) to 10(4) most probable numbers (MPN) of Vibrio vulnificus per g were relayed to offshore waters (salinity, 30 to 34 ppt), where they were suspended in racks at a depth of 7.6 m. V. vulnificus counts in oysters were reduced to < 10 MPN/g within 7 to 17 days in five of the six studies. At the end of the studies (17 to 49 days), V. vulnificus levels were reduced further and ranged from a mean of 0.23 to 2.6 MPN/g. Oyster mortalities during relaying were < 6%. The reduction of V. vulnificus in relayed oysters is associated with exposure to high-salinity environments essentially devoid of V. vulnificus. Offshore suspension relaying may be a method that industry can employ to reduce V. vulnificus levels in raw Gulf Coast oysters.     

Temporal and Spatial Variability in the Distribution of Vibrio vulnificus in the Chesapeake Bay: A Hindcast Study

Vibrio vulnificus, an estuarine bacterium, is the causative agent of seafood-related gastroenteritis, primary septicemia, and wound infections worldwide. It occurs as part of the normal microflora of coastal marine environments and can be isolated from water, sediment, and oysters. Hindcast prediction was undertaken to determine spatial and temporal variability in the likelihood of occurrence of V. vulnificus in surface waters of the Chesapeake Bay. Hindcast predictions were achieved by forcing a multivariate habitat suitability model with simulated sea surface temperature and salinity in the Bay for the period between 1991 and 2005 and the potential hotspots of occurrence of V. vulnificus in the Chesapeake Bay were identified. The likelihood of occurrence of V. vulnificus during high and low rainfall years was analyzed. From results of the study, it is concluded that hindcast prediction yields an improved understanding of environmental conditions associated with occurrence of V. vulnificus in the Chesapeake Bay.     

Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment.

Using plate counts, total cell counts, and direct viable counts, we examined the fate of cells of Vibrio vulnificus placed into natural estuarine waters during both winter and summer months. Cells inoculated into membrane diffusion chambers and placed into estuarine waters entered into a viable but nonculturable (VBNC) state in January and February, when the water temperatures were low (average, < 15 degrees C). In contrast, when cells in the VBNC state were placed into the same waters in the warmer months of August through November (average water temperature of ca. 21 degrees C), the cells appeared to undergo a rapid (typically, within 24 h) resuscitation to the fully culturable state. These results were independent of whether the cells were in the logarithmic or stationary phase and whether they were encapsulated or not. This study indicates that the inability to isolate V. vulnificus from cold estuarine sites may be accounted for by entrance of the cells into a VBNC state and that recovery from this state in natural environments may result from a temperature upshift.     

Distribution of Vibrio vulnificus in the Chesapeake Bay.

Vibrio vulnificus is a potentially lethal human pathogen capable of producing septicemia in susceptible persons. Disease is almost always associated with consumption of seafood, particularly raw oysters, or with exposure of wounds to seawater. An oligonucleotide DNA probe (V. vulnificus alkaline phosphatase-labeled DNA probe [VVAP]), previously shown to be highly specific for V. vulnificus, was used to enumerate this species in environmental samples collected from the Chesapeake Bay between April 1991 and December 1992. Total aerobic, heterotrophic, culturable bacteria were enumerated by plate counts on nonselective medium. The number of V. vulnificus organisms was determined by colony lifts of spread plates for subsequent hybridization with VVAP. V. vulnificus was not detected in any samples collected during February and March (water temperature of < 8 degrees C) but was found in 80% of the water samples collected during May, July, September, and December (water temperature of > 8 degrees C), with concentrations ranging from 3.0 x 10(1) to 2.1 x 10(2)/ml (ca. 8% of the total culturable heterotrophic bacteria). In a multiple regression analysis, increased V. vulnificus concentrations were correlated with lower salinities and with isolation from samples collected closer to the bottom. Isolation from oysters was demonstrable when water temperatures were 7.6 degrees C, with concentrations ranging from 1.0 x 10(3) to 4.7 x 10(4)/g (ca. 12% of total culturable bacteria). In samples collected in May and July, V. vulnificus was identified in seven of seven plankton samples and four of nine sediment samples. Our data demonstrate that V. vulnificus is a widespread and important component of the bacterial population of the Chesapeake Bay, with counts that are comparable to those reported from the Gulf of Mexico.     

Effects of temperature and salinity on Vibrio vulnificus population dynamics as assessed by quantitative PCR

The abundance of Vibrio vulnificus in coastal environments has been linked to water temperature, while its relationship to salinity is less clear. We have developed a culture-independent, most-probable-number quantitative PCR approach to examine V. vulnificus population dynamics in Barnegat Bay, N.J. Based on the combined analysis of our results from Barnegat Bay and from the literature, the present data show that (i) V. vulnificus population dynamics are strongly correlated to water temperature and (ii) although the general trend is for V. vulnificus abundance to be inversely correlated with salinity, this relationship depends on salinity levels. Irrespective of temperature, high abundances of V. vulnificus are observed at 5 to 10 ppt, which thus appears to be the optimal salinity regime for their survival. At 20 to 25 ppt, V. vulnificus abundances show a positive correlation to salinity. Unsuccessful attempts to resuscitate V. vulnificus, combined with our inability to detect cells during the winter despite an assay adapted to detect viable but nonculturable (VBNC) cells, suggest that the decline and eventual disappearance of V. vulnificus from the water column during the winter months is due primarily to a significant reduction in population size and is not only the consequence of cells entering the VBNC state. These findings are in line with the hypothesis that the sediment serves as a refuge for a subpopulation of V. vulnificus over the winter and weather-driven mixing events during the spring initiate a summer bloom in the water column.     

Effects of temperature abuse on survival of Vibrio vulnificus in oysters.

Opaque and translucent morphotypes of a TnphoA-containing strain of Vibrio vulnificus were fed to oysters, which were subsequently stored at temperatures ranging from 0.5 to 22 degrees C for 10 days. Samples of oysters were homogenized and plated at intervals to determine the cell density of V. vulnificus and total aerobic population of bacteria present. At all temperatures, the numbers of V. vulnificus (both morphotypes) declined over the 10-day study period. The same observation was made with a lower inoculum of V. vulnificus. Identical experiments with shucked oysters showed a more rapid decrease in V. vulnificus. Identical experiments with shucked oysters showed a more rapid decrease in V. vulnificus to levels below limits of detection. Little change in the total bacterial counts was observed in shellstock oysters at any of the test temperatures, whereas incubation at the higher temperatures (17 and 22 degrees C) resulted in large increases in total counts in shucked oysters. These data suggest that temperature abuse of oysters may not be a factor in increasing the public health risk of V. vulnificus through raw oyster consumption. However, the data also suggest that even with proper storage, indigenous levels of V. vulnificus may remain sufficiently higher in shellstock oysters to produce infection in compromised hosts.     

Development and validation of a predictive model for the growth of Vibrio vulnificus in postharvest shellstock oysters

Postharvest growth of Vibrio vulnificus in oysters can increase risk of human infection. Unfortunately, limited information is available regarding V. vulnificus growth and survival patterns over a wide range of storage temperatures in oysters harvested from different estuaries and in different oyster species. In this study, we developed a predictive model for V. vulnificus growth in Eastern oysters (Crassostrea virginica) harvested from Chesapeake Bay, MD, over a temperature range of 5 to 30°C and then validated the model against V. vulnificus growth rates (GRs) in Eastern and Asian oysters (Crassostrea ariakensis) harvested from Mobile Bay, AL, and Chesapeake Bay, VA, respectively. In the model development studies, V. vulnificus was slowly inactivated at 5 and 10°C with average GRs of -0.0045 and -0.0043 log most probable number (MPN)/h, respectively. Estimated average growth rates at 15, 20, 25, and 30°C were 0.022, 0.042, 0.087, and 0.093 log MPN/h, respectively. With respect to Eastern oysters, bias (B(f)) and accuracy (A(f)) factors for model-dependent and -independent data were 1.02 and 1.25 and 1.67 and 1.98, respectively. For Asian oysters, B(f) and A(f) were 0.29 and 3.40. Residual variations in growth rate about the fitted model were not explained by season, region, water temperature, or salinity at harvest. Growth rate estimates for Chesapeake Bay and Mobile Bay oysters stored at 25 and 30°C showed relatively high variability and were lower than Food and Agricultural Organization (FAO)/WHO V. vulnificus quantitative risk assessment model predictions. The model provides an improved tool for designing and implementing food safety plans that minimize the risk associated with V. vulnificus in oysters.     

Effects of temperature abuse on survival of Vibrio vulnificus in oysters.

Opaque and translucent morphotypes of a TnphoA-containing strain of Vibrio vulnificus were fed to oysters, which were subsequently stored at temperatures ranging from 0.5 to 22 degrees C for 10 days. Samples of oysters were homogenized and plated at intervals to determine the cell density of V. vulnificus and total aerobic population of bacteria present. At all temperatures, the numbers of V. vulnificus (both morphotypes) declined over the 10-day study period. The same observation was made with a lower inoculum of V. vulnificus. Identical experiments with shucked oysters showed a more rapid decrease in V. vulnificus. Identical experiments with shucked oysters showed a more rapid decrease in V. vulnificus to levels below limits of detection. Little change in the total bacterial counts was observed in shellstock oysters at any of the test temperatures, whereas incubation at the higher temperatures (17 and 22 degrees C) resulted in large increases in total counts in shucked oysters. These data suggest that temperature abuse of oysters may not be a factor in increasing the public health risk of V. vulnificus through raw oyster consumption. However, the data also suggest that even with proper storage, indigenous levels of V. vulnificus may remain sufficiently higher in shellstock oysters to produce infection in compromised hosts.     

Densities of Vibrio vulnificus in the intestines of fish from the U.S. Gulf Coast.

Densities of Vibrio vulnificus in the intestinal contents of various finfish, oysters, and crabs and in sediment and waters of the U.S. Gulf Coast were determined by the most probable number procedure. Species were identified by enzyme immunoassay. During the winter, densities of V. vulnificus were low, and the organism was isolated more frequently from sheepshead fish than from sediment and seawater. From April to October, V. vulnificus densities were considerably higher (2 to 5 logs) in estuarine fish than in surrounding water, sediment, or nearby oysters and crustacea. Highest densities were found in the intestinal contents of certain bottom-feeding fish (10(8)/100 g), particularly those that consume mollusks and crustaceans. Densities of V. vulnificus in fish that feed primarily on plankton and other finfish were similar to those in oysters, sediment, and crabs (10(5)/100 g). V. vulnificus was found infrequently in offshore fish. The presence of high densities of V. vulnificus in the intestines of common estuarine fish may have both ecological (growth and transport) and public health (food and wound infections) implications.     

Effect of low temperature on starvation-survival of the eel pathogen Vibrio vulnificus biotype 2.

At present, no reports exist on the isolation of the eel pathogen Vibrio vulnificus biotype 2 from water samples. Nevertheless, it has recently been demonstrated that this biotype can use water as a route of infection. In the present study, the survival of this pathogen in artificial seawater (ASW) microcosms at different temperatures (25 and 5 degrees C) was investigated during a 50-day period, with biotype 1 as a control, V. vulnificus biotype 2 was able to survive in the culturable state in ASW at 25 degrees C in the free-living form, at least for 50 days, entering into the nonculturable state when exposed to low temperature. In this state, this microorganism survived with reduced rates of activity, showing marked changes in size and morphology. The rate at which cells became nonculturable was dependent on their physiological age. The capsule seems not to be necessary for the survival of biotype 2 in aquatic environments as a free-living organism. Culturability remained the highest on modified salt water yeast extract agar, which is closer in salt and nutrient composition to ASW than heart infusion agar. Biotype 2 cells recovered culturability on solid media after an increase of incubation temperature from 5 to 25 degrees C. Culturable cells of this bacterium maintained infectivity for either eel or mice, while dormant cells seemed to lose their virulence. The former finding suggests that the aquatic environment is a reservoir and vehicle of transmission of this pathogen.     

Changes in membrane fatty acid composition during entry of Vibrio vulnificus into the viable but nonculturable state

Vibrio vulnificus, a Gram-negative bacterium found in estuarine waters, is responsible for over 95% of all seafood-related deaths in the United States. As a result of a temperature downshift to 5 degrees C, this organism enters the viable but nonculturable (VBNC) state. Changes in the membrane fatty acid (FA) composition of V. vulnificus may be a contributing factor to the ability of this organism to enter into and survive in the VBNC state. This hypothesis was tested by incubating the organism at 5 degrees C in artificial sea water and analyzing the cells' FAs during the initial hours of temperature and nutrient down-shift. Prior to downshift, the predominant FAs were 16:0, 16:1 and 18:0. During the first four hours of downshift, statistically significant changes occurred in 15:0, 16:1, 16:0, 17:0, and 18:0. These results indicate that changes in FA composition occur prior to entry of V. vulnificus into the VBNC state, suggesting that the ability to maintain membrane fluidity may be a factor in this physiological response. Cells in which fatty acid synthesis was inhibited did not survive, indicating that active fatty acid metabolism is essential for entry of cells into the VBNC state.     

Apparent loss of Vibrio vulnificus from North Carolina oysters coincides with a drought-induced increase in salinity

Despite years of successful isolation of Vibrio vulnificus from estuarine waters, beginning in 2007, it was extremely difficult to culture V. vulnificus from either North Carolina estuarine water or oyster samples. After employing culture-based methods as well as PCR and quantitative PCR for the detection of V. vulnificus, always with negative results, we concluded that this pathogen had become nearly undetectable in the North Carolina estuarine ecosystem. We ensured that the techniques were sound by seeding North Carolina oysters with V. vulnificus and performing the same tests as those previously conducted on unadulterated oysters. V. vulnificus was readily detected in the seeded oysters using both classes of methods. Furthermore, oysters were obtained from the Gulf of Mexico, and V. vulnificus was easily isolated, confirming that the methodology was sound but that the oysters and waters of North Carolina were lacking the V. vulnificus population studied for decades. Strikingly, the apparent loss of detectable V. vulnificus coincided with the most severe drought in the history of North Carolina. The drought continued until the end of 2009, with an elevated water column salinity being observed throughout this period and with V. vulnificus being nearly nonexistent. When salinities returned to normal after the drought abated in 2010, we were again able to routinely isolate V. vulnificus from the water column, although we were still unable to culture it from oysters. We suggest that the oysters were colonized with a more salt-tolerant bacterium during the drought, which displaced V. vulnificus and may be preventing recolonization.     

Occurrence, seasonality and genetic diversity of Vibrio vulnificus in coastal seaweeds and water along the Kii Channel, Japan

Vibrio vulnificus is a ubiquitous toxigenic bacterium found in a coastal environment but little is known about its occurrence and seasonality among seaweeds, which are widely consumed as seafood in Japan. Therefore, we have observed the bacterium's abundance in seawater and seaweed samples from three areas of the Kii Channel, Japan, during June 2003 to May 2004. A total of 192 samples were collected: 24 from each source in summer, autumn, winter and spring. The samples were selectively cultivated following the most probable number (MPN) technique. Vibrio vulnificus population ranged from 0 to 10(3) MPN 100 mL(-1) seawater or 10 g seaweeds; higher counts were observed during summer. The optimum temperature, salinity and pH for the bacterium were 20-24 degrees C, 24-28 p.p.t. and 7.95-8.15, respectively. However, seaweeds always contained higher V. vulnificus than seawater. Among 280 V. vulnificus strains, detected by species-specific colony hybridization and PCR, 78, 74, 11 and 16 were from seaweeds and 46, 42, 2 and 11 were from seawater during summer, autumn, winter and spring, respectively. Ribotyping of 160 selected strains revealed a higher genotypic diversity (18 patterns) among strains from seaweeds than from seawater (10 patterns). Seaweeds can thus act as a potential habitat for V. vulnificus and are more unsafe for consumption during summer.     

Distribution ofVibrio cholerae in two Florida estuaries

The distribution ofVibrio cholerae was examined in 2 Florida estuaries, Apalachicola and Tampa Bay.Vibrio cholerae serotype non-01 was the most abundant serotype, being isolated from 45% of the oyster samples, 30% of the sediments, 50% of the waters, and 75% of the blue crabs.Vibrio cholerae serotype 01 was isolated from only one oyster sample. Strong linear correlations betweenV. cholerae and temperature, salinity, or the other physical/chemical parameters measured,Escherichia coli, or fecal coliforms were not observed, but a range of temperatures and salinities appeared relevant to the distribution of the organism. The organism was present in the highest concentrations when salinities were 10‰–25‰ and temperatures were 20?C–35?C.In vitro growth curves of 95V. cholerae environmental isolates further supported that 10‰–25‰ was an ideal salinity range for the organisms. The results suggest thatV. cholerae is a widely distributed organism in the nutrient-rich warm waters of the Gulf Coast estuaries.     

Growth response of Vibrio cholerae and other Vibrio spp. to cyanobacterial dissolved organic matter and temperature in brackish water

Environmental control of growth and persistence of vibrios in aquatic environments is poorly understood even though members of the genus Vibrio are globally important pathogens. To study how algal-derived organic matter and temperature influenced the abundance of different Vibrio spp., Baltic Sea microcosms inoculated with Vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus and native bacterioplankton, were exposed to different temperatures (12-25 degrees C) and amended with dissolved organic matter from Nodularia spumigena (0-4.2 mg C L(-1)). Vibrio abundance was monitored by culture-dependent and molecular methods. Results suggested that Vibrio populations entered a viable but nonculturable state during the incubations. Abundance of Vibrio spp. and total bacterioplankton were orders of magnitude higher in microcosms amended with organic matter compared with reference microcosms. Vibrio cholerae abundances ranged from 0.9 to 1.9 x 10(5) cells mL(-1) in treatments amended with 4.2 mg C L(-1). Vibrio cholerae abundance relative to total bacterioplankton and other Vibrio spp. also increased >10-fold. In addition, V. vulnificus abundance increased in mesocosms with the highest organic matter addition (0.9-1.8 x 10(4) cells mL(-1)). Temperature alone did not significantly affect abundances of total bacterioplankton, total Vibrio spp. or individual Vibrio populations. By contrast, cyanobacterial-derived organic matter represented an important factor regulating growth and abundance of V. cholerae and V. vulnificus in brackish waters.     

Distribution of Vibrio vulnificus and other lactose-fermenting vibrios in the marine environment

During the summer of 1981, 3,887 sucrose-negative vibrios were isolated from seawater, sediment, plankton, and animal samples taken from 80 sites from Miami, Fla., to Portland, Maine. Of these, 4.2% were able to ferment lactose. The lactose-positive strains isolated from the various samples correlated positively with pH and turbidity of the water, vibrios in the sediment and oysters, and total bacterial counts in oysters. Negative correlations were obtained for water salinity. Numerical taxonomy was performed on 95 of the lactose-fermenting environmental isolates and 23 reference strains. Five clusters resulted, with the major cluster containing 33 of the environmental isolates and all of the Vibrio vulnificus reference strains. The 33 isolates, which produced an acid reaction in lactose broth within hours of initial inoculation, represented 20% of all lactose-fermenting vibrios studied. These isolates were nearly identical phenotypically to clinical strains of V. vulnificus studied by the Centers for Disease Control, Atlanta, Ga., and by our laboratory, and their identification was confirmed by DNA-DNA hybridization studies. V. vulnificus was isolated from all sample types and from Miami to Cape Cod, Mass., and comparison of the environmental parameters of the eight subsites yielding this species with those of all 80 subsites revealed no significant differences. The majority of the isolates were obtained from animals, with clams providing most (84%) of these. On injection into mice, 82% of the V. vulnificus isolates resulted in death. Members of the remaining four clusters contained strains which differed from V. vulnificus in such phenotypic traits as luminescence and in urease or H(2)S production. None of the other reference cultures, including nine other Vibrio species, were contained in the remaining clusters, and these isolates could not be identified. Most of these were also lethal for mice. Phenotypic differences, potential pathogenicity, and geographic distribution of the five clusters were examined. It is concluded that V. vulnificus is a ubiquitous organism, both geographically and in a variety of environmental sources, although it occurs in relatively low numbers. The public health significance of this organism and of the other unidentified lactose-fermenting Vibrio species is discussed.