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°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%).     

The ecology of Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus in North Carolina Estuaries

While numerous studies have characterized the distribution and/or ecology of various pathogenic Vibrio spp., here we have simultaneously examined several estuarine sites for Vibrio vulnificus, V. cholerae, and V. parahaemolyticus. For a one year period, waters and sediment were monitored for the presence of these three pathogens at six different sites on the east coast of North Carolina in the United States. All three pathogens, identified using colony hybridization and PCR methods, occurred in these estuarine environments, although V. cholerae occurred only infrequently and at very low levels. Seventeen chemical, physical, and biological parameters were investigated, including salinity, water temperature, turbidity, dissolved oxygen, levels of various inorganic nutrients and dissolved organic carbon, as well as total vibrios, total coliforms, and E. coli. We found each of the Vibrio spp. in water and sediment to correlate to several of these environmental measurements, with water temperature and total Vibrio levels correlating highly (P<0.0001) with occurrence of the three pathogens. Thus, these two parameters may represent simple assays for characterizing the potential public health hazard of estuarine waters.     

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 of pathogenic vibrios in coastal areas of France

AIMS: This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments. METHODS AND RESULTS: Samples of coastal waters and mussels collected between July and September 1999 were analysed by culture, using selective media including thiosulphate-citrate-bile salts-sucrose and modified cellobiose-polymixin B-colistin agar. Presumptive Vibrio colonies were isolated and identified using selected biochemical tests. Specific primers based on flanking sequences of the cytolysin, vvhA gene, pR72H DNA fragment and 16S-23S rRNA intergenic spacer region (ISR) were used in a polymerase chain reaction (PCR) to confirm the identification of Vibrio vulnificus, V. parahaemolyticus and V. cholerae, respectively. In this study, V. alginolyticus (99 of 189) was the predominant species, followed by V. parahaemolyticus (41 of 189), V. vulnificus (20 of 189) and non-O1/non-O139 V. cholerae (three of 189). All 20 V. vulnificus isolates showed PCR amplification of the vvhA gene, 16 of which had been isolated from estuarine water. The PCR amplification of the pR72H DNA fragment in 41 V. parahaemolyticus isolates generated two unique amplicons of 387 and 320 bp. The latter, present in 24.4% of these isolates, had not previously been found in V. parahaemolyticus strains examined to date. Amplification of the trh gene in two of the isolates suggested these to be virulent strains. Three strains identified as V. cholerae by amplification of the 16S-23S rRNA ISR were confirmed to be non-cholera (non-O1/non-O139) strains. CONCLUSIONS: The results of this study demonstrated the presence of pathogenic Vibrio species in French coastal waters. Furthermore, the PCR approach proved useful for the rapid and reliable confirmation of species identification. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate the potential sanitary risk associated with the presence of pathogenic Vibrio spp. in cultivated mussels and in the aquatic environment. The PCR can be used to detect pathogenic vibrios directly in environmental samples.     

A comparison of thiosulphate-citrate-bile salts-sucrose (TCBS) agar and thiosulphate-chloride-iodide (TCI) agar for the isolation of Vibrio species from estuarine environments

AIMS: The purpose of this study was to compare a recently described medium, thiosulphate-chloride-iodide (TCI), for the isolation of estuarine vibrios with thiosulphate-citrate-bile salts-sucrose (TCBS). METHODS: A total of 492 colonies which developed on these media from estuarine water samples taken monthly over a 10-month period were examined. RESULTS: A much larger number of colonies developed on TCBS than TCI, and minimal taxonomic criteria indicated that a higher percentage (61%) of TCBS colonies could be identified as Vibrio spp. when compared with TCI (46%). SIGNIFICANCE: This study suggests that TCBS is a superior medium when compared with TCI for the isolation of Vibrio spp. from estuarine waters. Because of the public health risk presented by V. vulnificus, V. parahaemolyticus, V. cholerae and other vibrios, the selection of the most appropriate medium for their isolation is extremely important.     

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.     

Distribution of Vibrio vulnificus phage in oyster tissues and other estuarine habitats.

Phages lytic to Vibrio vulnificus were found in estuarine waters, sediments, plankton, crustacea, molluscan shellfish, and the intestines of finfish of the U.S. Gulf Coast, but no apparent relationship between densities of V. vulnificus and its phages was observed. Phage diversity and abundance in molluscan shellfish were much greater than in other habitats. V. vulnificus phages isolated from oysters did not lyse other mesophilic bacteria also isolated from oysters. Both V. vulnificus and its phages were found in a variety of oyster tissues and fluids with lowest densities in the hemolymph and mantle fluid. These findings suggest a close ecological relationship between V. vulnificus phages and molluscan shellfish.     

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.     

Virulent strains of Vibrio vulnificus isolated from estuaries of the United States West Coast.

Vibrio vulnificus was isolated from United States West Coast estuaries at a low frequency (5.9%) from 529 samples of water, shellfish, and sediment. Four strains tested with iron-treated mice had 50% lethal dose values ranging from 7.6 to 360 CFU, compared with a 50% lethal dose of 4.9 CFU for a clinical isolate that caused the death of a septicemic patient. The presence of this pathogen may be a hazard to users of marine beaches and consumers of raw shellfish on the West Coast, especially to persons most susceptible to V. vulnificus septicemia. Species-specific antiflagellar serum and a gene probe for cytotoxin-hemolysin production were useful for screening these environmental isolates.     

Virulent strains of Vibrio vulnificus isolated from estuaries of the United States West Coast

Vibrio vulnificus was isolated from United States West Coast estuaries at a low frequency (5.9%) from 529 samples of water, shellfish, and sediment. Four strains tested with iron-treated mice had 50% lethal dose values ranging from 7.6 to 360 CFU, compared with a 50% lethal dose of 4.9 CFU for a clinical isolate that caused the death of a septicemic patient. The presence of this pathogen may be a hazard to users of marine beaches and consumers of raw shellfish on the West Coast, especially to persons most susceptible to V. vulnificus septicemia. Species-specific antiflagellar serum and a gene probe for cytotoxin-hemolysin production were useful for screening these environmental isolates.     

Methods for isolation and confirmation of Vibrio vulnificus from oysters and environmental sources: a review

The gram-negative bacterium Vibrio vulnificus is a natural inhabitant of estuarine waters and poses a significant health threat to humans who suffer from immune disorders, liver disease, or hemochromatosis (iron overload). V. vulnificus enters human hosts via wound infections or consumption of raw shellfish (primarily oysters), and infections frequently progress to septicemia and death in susceptible individuals. Prevalence in waters and shellfish is not correlated with fecal indicator organisms; therefore, species-specific detection and enumeration of V. vulnificus in the environment has become a priority for agencies that are responsible for shellfish safety. The many selective-differential media developed for isolation of Vibrio spp., and specifically for V. vulnificus detection, are reviewed here; however, none of the media developed to date combines the sensitivity to low numbers with the specificity necessary to inhibit growth of other organisms. Therefore, immunological and molecular protocols are needed for confirmation of the identity of the organism and are discussed in detail. Methods under development that hold promise for rapid, accurate, and sensitive detection and enumeration of the organism include multiplex and real-time PCR. Developing technologies that have proven useful for detection and investigation of other pathogens such as biosensors, spectroscopy and microarrays may provide the next generation of tools for investigation of the prevalence and ecology of V. vulnificus.     

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.     

Sediment Composition Influences Spatial Variation in the Abundance of Human Pathogen Indicator Bacteria within an Estuarine Environment

Faecal contamination of estuarine and coastal waters can pose a risk to human health, particularly in areas used for shellfish production or recreation. Routine microbiological water quality testing highlights areas of faecal indicator bacteria (FIB) contamination within the water column, but fails to consider the abundance of FIB in sediments, which under certain hydrodynamic conditions can become resuspended. Sediments can enhance the survival of FIB in estuarine environments, but the influence of sediment composition on the ecology and abundance of FIB is poorly understood. To determine the relationship between sediment composition (grain size and organic matter) and the abundance of pathogen indicator bacteria (PIB), sediments were collected from four transverse transects of the Conwy estuary, UK. The abundance of culturable Escherichia coli, total coliforms, enterococci, Campylobacter, Salmonella and Vibrio spp. in sediments was determined in relation to sediment grain size, organic matter content, salinity, depth and temperature. Sediments that contained higher proportions of silt and/or clay and associated organic matter content showed significant positive correlations with the abundance of PIB. Furthermore, the abundance of each bacterial group was positively correlated with the presence of all other groups enumerated. Campylobacter spp. were not isolated from estuarine sediments. Comparisons of the number of culturable E. coli, total coliforms and Vibrio spp. in sediments and the water column revealed that their abundance was 281, 433 and 58-fold greater in sediments (colony forming units (CFU)/100g) when compared with the water column (CFU/100ml), respectively. These data provide important insights into sediment compositions that promote the abundance of PIB in estuarine environments, with important implications for the modelling and prediction of public health risk based on sediment resuspension and transport.     

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.     

Study of the occurrence of Vibrio vulnificus in oysters in India by polymerase chain reaction (PCR) and heterogeneity among V. vulnificus by randomly amplified polymorphic DNA PCR and gyrB sequence analysis

The pathogenic bacterium Vibrio vulnificus is widely distributed in estuarine waters throughout the world. In this study, the presence of V. vulnificus in oysters was studied both by conventional culture and DNA-based molecular technique. Following enrichment in alkaline peptone water (APW), the bacteria were lysed and a nested polymerase chain reaction (PCR) for vvhA gene was performed. The effect of duration of enrichment on the sensitivity of detection by PCR was evaluated. The organism was isolated from 43% of samples after 18 h enrichment in APW by conventional culture method. Nested PCR amplifying a fragment of vvhA gene detected the organism in 11%, 60% and 81% of samples following 0, 6 and 18 h of enrichment. All the biochemically identified V. vulnificus strains possessed vvhA gene and belonged to biotype 1. The genetic relatedness among the strains was studied by randomly amplified polymorphic DNA (RAPD) PCR and gyrB sequence analysis. The results suggest the presence of two distinct clonal groups of V. vulnificus in oysters in India. The study demonstrates, for the first time that gyrB sequence analysis could be used to study the genetic diversity of V. vulnificus.     

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.     

Influence of aquatic microbiota on the survival in water of the human and eel pathogen Vibrio vulnificus serovar E

The eel and human pathogen Vibrio vulnificus serovar E (biotype 2) is seldom isolated from natural waters, although it can survive in sterilized artificial seawater microcosms for years. The main objective of the present study was to investigate whether aquatic microbiota can limit its survival and recovery from water samples. A set of preliminary experiments of survival in microcosms containing natural seawater and water from eel farms showed that the persistence of this pathogen was mainly controlled by grazing, and secondarily by bacterial competition. The bacterial competition was further analysed in artificial seawater microcosms co-inoculated with selected virulent serovar E (VSE) strains and potential competitors. Competitors included V. vulnificus biotype 1 isolates and strains of selected species that can grow on the selective media designed for V. vulnificus isolation from water samples. Evidences of bacterial competition that was detrimental for VSE recovery were recorded. Thus, some species produced a deleterious effect on VSE strains under starvation, and others were able to use the resources more efficiently under nutrient input. These results suggest that an overgrowth of more efficient competitor bacteria in conventional media used for isolation of V. vulnificus could mask the recovery of VSE strains and explain the scarcity of reports on the isolation of this human and eel pathogen from natural waters.     

Dynamics and predictive modelling of Vibrio spp. in the Neuse River Estuary, North Carolina, USA

Vibrio spp. are naturally occurring bacteria in marine and estuarine environments around the world. The genus includes several human and animal pathogens that can negatively impact human health, seafood and aquaculture. Vibrio spp. populations are capable of rapid adaptation in response to changing environmental conditions, making them dynamic over short-term and seasonal scales. Temperature, vertical mixing, tidal flushing, climate, precipitation and nutrient loading can change the estuarine environment and subsequently alter microbial community structure, including Vibrio spp., affecting estuarine water quality and public health. To describe these dynamics, Vibrio spp. concentrations and a range of microbial, physical and chemical measures were monitored every 2 weeks and after storm events for 19 months in the Neuse River Estuary (NRE). Results showed clear seasonal and geographic trends in Vibrio spp. abundance. Multiple regression analysis revealed a strong relationship to temperature and salinity, with additional minor influences of chlorophyll a and dissolved organic carbon. Similar models based on easily measured environmental parameters should be pursued for individual Vibrio species in the NRE and other estuarine environments. Predictive models provide useful information for managers, researchers and modellers of estuarine ecosystems.     

Temporal and spatial variability in culturable pathogenic Vibrio spp. in Lake Pontchartrain, Louisiana, following hurricanes Katrina and Rita

We investigated the abundance, distribution, and virulence gene content of Vibrio cholerae, V. parahaemolyticus, and V. vulnificus in the waters of southern Lake Pontchartrain in Louisiana on four occasions from October 2005 to September 2006, using selective cultivation and molecular assays. The three targeted pathogenic vibrios were generally below the detection level in January 2006, when the water was cold (13°C), and most abundant in September 2006, when the lake water was warmest (30°C). The maximum values for these species were higher than reported previously for the lake by severalfold to orders of magnitude. The only variable consistently correlated with total vibrio abundance within a single sampling was distance from shore (P = 0.000). Multiple linear regression of the entire data set revealed that distance from shore, temperature, and turbidity together explained 82.1% of the variability in total vibrio CFU. The log-transformed mean abundance of V. vulnificus CFU in the lake was significantly correlated with temperature (P = 0.014), but not salinity (P = 0.625). Virulence-associated genes of V. cholerae (ctx) and V. parahaemolyticus (trh and tdh) were not detected in any isolates of these species (n = 128 and n = 20, respectively). In contrast, 16S rRNA typing of V. vulnificus (n = 298) revealed the presence of both environmental (type A) and clinical (type B) strains. The percentage of the B-type V. vulnificus was significantly higher in the lake in October 2005 (35.8% of the total) than at other sampling times (P ≤ 0.004), consistent with the view that these strains represent distinct ecotypes.     

The reductase component of the chromosomally encoded benzoate dioxygenase from Pseudomonas putida C-1 is immunologically homologous with a product of the plasmid encoded xyl D gene (toluate dioxygenase) from Pseudomonas putida mt-2

Vibrio vulnificus, an autochthonous inhabitant of the estuarine environment, was detected in water and oysters from the Great Bay Estuary System of New Hampshire and Maine. Previously, it had not been detected north of Boston Harbor on the east coast of the United States. V. vulnificus was detected in water and shellfish samples at five out of ten sites, and only in areas that were not open to recreational shellfishing. Although samples were collected from May into December, V. vulnificus was only detected in shellfish in July and August. Water sampling began in August, and V. vulnificus persisted at one site into October.