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.     

Uncertainty in Model Predictions of Vibrio vulnificus Response to Climate Variability and Change: A Chesapeake Bay Case Study

The effect that climate change and variability will have on waterborne bacteria is a topic of increasing concern for coastal ecosystems, including the Chesapeake Bay. Surface water temperature trends in the Bay indicate a warming pattern of roughly 0.3–0.4°C per decade over the past 30 years. It is unclear what impact future warming will have on pathogens currently found in the Bay, including Vibrio spp. Using historical environmental data, combined with three different statistical models of Vibrio vulnificus probability, we explore the relationship between environmental change and predicted Vibrio vulnificus presence in the upper Chesapeake Bay. We find that the predicted response of V. vulnificus probability to high temperatures in the Bay differs systematically between models of differing structure. As existing publicly available datasets are inadequate to determine which model structure is most appropriate, the impact of climatic change on the probability of V. vulnificus presence in the Chesapeake Bay remains uncertain. This result points to the challenge of characterizing climate sensitivity of ecological systems in which data are sparse and only statistical models of ecological sensitivity exist.     

Spatial and Temporal Distribution of Two Diazotrophic Bacteria in the Chesapeake Bay

The aim of this study was to initiate autecological studies on uncultivated natural populations of diazotrophic bacteria by examining the distribution of specific diazotrophs in the Chesapeake Bay. By use of quantitative PCR, the abundance of two nifH sequences (907h22 and 912h4) was quantified in water samples collected along a transect from the head to the mouth of the Chesapeake Bay during cruises in April and October 2001 and 2002. Standard curves for the quantitative PCR assays demonstrated that the relationship between gene copies and cycle threshold was linear and highly reproducible from 1 to 10⁷ gene copies. The maximum number of 907h22 gene copies detected was approximately 140 ml⁻¹ and the maximum number of 912h4 gene copies detected was approximately 340 ml⁻¹. Sequence 912h4 was most abundant at the mouth of the Chesapeake Bay, and in general, its abundance increased with increasing salinity, with the highest abundances observed in April 2002. Overall, the 907h22 phylotype was most abundant at the mid-bay station. Additionally, 907h22 was most abundant in the April samples from the mid-bay and mouth of the Chesapeake Bay. Despite the fact that the Chesapeake Bay is rarely nitrogen limited, our results show that individual nitrogen-fixing bacteria have distinct nonrandom spatial and seasonal distributions in the Chesapeake Bay and are either distributed by specific physical processes or adapted to different environmental niches.     

Predicting the Distribution of Vibrio spp. in the Chesapeake Bay: A Vibrio cholerae Case Study

Vibrio cholerae, the causative agent of cholera, is a naturally occurring inhabitant of the Chesapeake Bay and serves as a predictor for other clinically important vibrios, including Vibrio parahaemolyticus and Vibrio vulnificus. A system was constructed to predict the likelihood of the presence of V. cholerae in surface waters of the Chesapeake Bay, with the goal to provide forecasts of the occurrence of this and related pathogenic Vibrio spp. Prediction was achieved by driving an available multivariate empirical habitat model estimating the probability of V. cholerae within a range of temperatures and salinities in the Bay, with hydrodynamically generated predictions of ambient temperature and salinity. The experimental predictions provided both an improved understanding of the in situ variability of V. cholerae, including identification of potential hotspots of occurrence, and usefulness as an early warning system. With further development of the system, prediction of the probability of the occurrence of related pathogenic vibrios in the Chesapeake Bay, notably V. parahaemolyticus and V. vulnificus, will be possible, as well as its transport to any geographical location where sufficient relevant data are available.     

Spatial and Temporal Aspects of Mixotrophy In Chesapeake Bay Dinoflagellates

ABSTRACT. Gymnodinium sanguineum, Gyrodinium uncatenum , and Ceratium furca are large phototrophic dinoflagellates that commonly form red tides in the mesohaline portion of Chesapeake Bay during the summer. Examination of protargol-stained specimens revealed that these dinoflagellates also feed heterotrophically as indicated by the presence of food vacuoles containing partially digested prey. Ingested prey were generally identified as nanociliates (≥20 μm) belonging to the oligotrich genera Strobilidium and Strombidium ; occasionally other small ciliates (e.g. Balanion sp. and Mesodinium sp.), dinoflagellates, and diatoms were observed in early stages of digestion. the percentage of these mixotrophs that had ingested prey was usually less than 20%, but approached 30% in some samples. Occurrence of food vacuoles in Gymno. sanguineum was positively correlated with ≤20 μm oligotrichous ciliate density; limited data for Gyro. uncatenum suggests a similar relationship, but C. furca feeding was not related to nanociliate densities.     

High Temporal but Low Spatial Heterogeneity of Bacterioplankton in the Chesapeake Bay

Compared to freshwater and the open ocean, less is known about bacterioplankton community structure and spatiotemporal dynamics in estuaries, particularly those with long residence times. The Chesapeake Bay is the largest estuary in the United States, but despite its ecological and economic significance, little is known about its microbial community composition. A rapid screening approach, ITS (internal transcribed spacer)-LH (length heterogeneity)-PCR, was used to screen six rRNA operon (16S rRNA-ITS-23S rRNA) clone libraries constructed from bacterioplankton collected in three distinct regions of the Chesapeake Bay over two seasons. The natural length variation of the 16S-23S rRNA gene ITS region, as well as the presence and location of tRNA-alanine coding regions within the ITS, was determined for 576 clones. Clones representing unique ITS-LH-PCR sizes were sequenced and identified. Dramatic shifts in bacterial composition (changes within subgroups or clades) were observed for the Alphaproteobacteria (Roseobacter clade, SAR11), Cyanobacteria (Synechococcus), and Actinobacteria, suggesting strong seasonal variation within these taxonomic groups. Despite large gradients in salinity and phytoplankton parameters, a remarkably homogeneous bacterioplankton community was observed in the bay in each season. Stronger seasonal, rather than spatial, variation of the bacterioplankton population was also supported by denaturing gradient gel electrophoresis and LH-PCR analyses, indicating that environmental parameters with stronger seasonal, rather than regional, dynamics, such as temperature, might determine bacterioplankton community composition in the Chesapeake Bay.     

Predictability of Vibrio cholerae in Chesapeake Bay

Vibrio cholerae is autochthonous to natural waters and can pose a health risk when it is consumed via untreated water or contaminated shellfish. The correlation between the occurrence of V. cholerae in Chesapeake Bay and environmental factors was investigated over a 3-year period. Water and plankton samples were collected monthly from five shore sampling sites in northern Chesapeake Bay (January 1998 to February 2000) and from research cruise stations on a north-south transect (summers of 1999 and 2000). Enrichment was used to detect culturable V. cholerae, and 21.1% (n = 427) of the samples were positive. As determined by serology tests, the isolates, did not belong to serogroup O1 or O139 associated with cholera epidemics. A direct fluorescent-antibody assay was used to detect V. cholerae O1, and 23.8% (n = 412) of the samples were positive. V. cholerae was more frequently detected during the warmer months and in northern Chesapeake Bay, where the salinity is lower. Statistical models successfully predicted the presence of V. cholerae as a function of water temperature and salinity. Temperatures above 19°C and salinities between 2 and 14 ppt yielded at least a fourfold increase in the number of detectable V. cholerae. The results suggest that salinity variation in Chesapeake Bay or other parameters associated with Susquehanna River inflow contribute to the variability in the occurrence of V. cholerae and that salinity is a useful indicator. Under scenarios of global climate change, increased climate variability, accompanied by higher stream flow rates and warmer temperatures, could favor conditions that increase the occurrence of V. cholerae in Chesapeake Bay.     

Predictability of Vibrio cholerae in Chesapeake Bay

Vibrio cholerae is autochthonous to natural waters and can pose a health risk when it is consumed via untreated water or contaminated shellfish. The correlation between the occurrence of V. cholerae in Chesapeake Bay and environmental factors was investigated over a 3-year period. Water and plankton samples were collected monthly from five shore sampling sites in northern Chesapeake Bay (January 1998 to February 2000) and from research cruise stations on a north-south transect (summers of 1999 and 2000). Enrichment was used to detect culturable V. cholerae, and 21.1% (n = 427) of the samples were positive. As determined by serology tests, the isolates, did not belong to serogroup O1 or O139 associated with cholera epidemics. A direct fluorescent-antibody assay was used to detect V. cholerae O1, and 23.8% (n = 412) of the samples were positive. V. cholerae was more frequently detected during the warmer months and in northern Chesapeake Bay, where the salinity is lower. Statistical models successfully predicted the presence of V. cholerae as a function of water temperature and salinity. Temperatures above 19 degrees C and salinities between 2 and 14 ppt yielded at least a fourfold increase in the number of detectable V. cholerae. The results suggest that salinity variation in Chesapeake Bay or other parameters associated with Susquehanna River inflow contribute to the variability in the occurrence of V. cholerae and that salinity is a useful indicator. Under scenarios of global climate change, increased climate variability, accompanied by higher stream flow rates and warmer temperatures, could favor conditions that increase the occurrence of V. cholerae in Chesapeake Bay.     

Ecology of Vibrio parahaemolyticus in Chesapeake Bay

A study of the ecology of Vibrio parahaemolyticus and related vibrios in the Rhode River area of Chesapeake Bay was carried out over the period December 1970 through August 1971. The incidence of V. parahaemolyticus and related vibrios was found to be correlated with water temperature. The vibrios could not be detected in the water column during the winter months, although they were present in sediment. From late spring to early summer, when water temperatures were 14 +/- 1 C, vibrios over-wintering in sediment were released from the bottom communities and attached to zooplankton, proliferating as the temperature rose. The number of vibrios in and on plankton was reflected in the water column bacterial population densities at water temperatures of ca. 19 C. Thus, temperature of the water column in the range of 14 to 19 C was found to be critical in the annual cycle of the vibrios. Interaction between sediment, water, and zooplankton was found to be essential in the natural estuarine ecosystem. Bacterial counts of zooplankton were found to be temperature dependent. The bacterial population associated with zooplankton was found to be predominantly on external surfaces and was specific, differing from that of the sediment. Vibrio spp. and related organisms comprised the total bacterial population associated with zooplankton in summer months. The ecological role of Vibrio spp., including V. parahaemolyticus, was found to be significant, with respect to their property of chitin digestion and in relation to the population dynamics of zooplankton in Chesapeake Bay.     

Incidence of Vibrio parahaemolyticus in Chesapeake Bay

A Bay-wide survey of the distribution of Vibrio parahaemolyticus was carried out in Chesapeake Bay during May 1972, to determine whether the annual cycle of V. parahaemolyticus which was observed to occur in the Rhode River subestuary of Chesapeake Bay took place in other parts of Chesapeake Bay. In an earlier study, April to early June, when the water temperature rises from 14 to 19 C, was found to be a critical period in the annual cycle of the organism in the Rhode River, since this is the time period when the annual cycle is initiated. Results of this study, however, revealed that V. parahaemolyticus could not be found in the water column during May 1972. Nevertheless, several samples of sediment and plankton yielded V. parahaemolyticus isolates. Comparison of data with those for the Rhode River area examined in the earlier studies of the annual cycle of V. parahaemolyticus suggests that the time of initiation of the annual cycle of V. parahaemolyticus in the open Bay proper may be influenced by various factors such as temperature and salinity, i.e., deeper water locations may show initiation of the V. parahaemolyticus annual cycle later than shallow areas. Confirmation of the presence of the organisms in the samples studied was accomplished using numerical taxonomy with 19 reference strains also included in the analyses.     

Distribution of viruses in the Chesapeake Bay.

High virus counts were found in water samples collected from the Chesapeake Bay. Viruses were enumerated by ultracentrifugation of water samples onto grids which were visualized by transmission electron microscopy. Virus counts in September 1990, April 1991, June 1991, August 1991, and October 1991 ranged between 2.6 x 10(6) and 1.4 x 10(8) viruses ml-1 with a mean of 2.5 x 10(7) viruses ml-1. Virus counts were usually at least three times higher than direct bacterial counts in corresponding samples. Virus counts in August and October were significantly higher than at the other sampling times, whereas bacterial counts were significantly lower at that time, yielding mean virus-to-bacterium ratios of 12.6 and 25.6, respectively. From analysis of morphology of the virus particles, it is concluded that a large proportion of the viruses are bacteriophages. The high virus counts obtained in this study suggest that viruses may be an important factor affecting bacterial populations in the Chesapeake Bay, with implications for gene transfer in natural aquatic bacterial populations and release of genetically engineered microorganisms to estuarine and coastal environments.     

Distribution of viruses in the Chesapeake Bay.

High virus counts were found in water samples collected from the Chesapeake Bay. Viruses were enumerated by ultracentrifugation of water samples onto grids which were visualized by transmission electron microscopy. Virus counts in September 1990, April 1991, June 1991, August 1991, and October 1991 ranged between 2.6 x 10(6) and 1.4 x 10(8) viruses ml-1 with a mean of 2.5 x 10(7) viruses ml-1. Virus counts were usually at least three times higher than direct bacterial counts in corresponding samples. Virus counts in August and October were significantly higher than at the other sampling times, whereas bacterial counts were significantly lower at that time, yielding mean virus-to-bacterium ratios of 12.6 and 25.6, respectively. From analysis of morphology of the virus particles, it is concluded that a large proportion of the viruses are bacteriophages. The high virus counts obtained in this study suggest that viruses may be an important factor affecting bacterial populations in the Chesapeake Bay, with implications for gene transfer in natural aquatic bacterial populations and release of genetically engineered microorganisms to estuarine and coastal environments.     

Diversity and Seasonality of Bioluminescent Vibrio cholerae Populations in Chesapeake Bay

Association of luminescence with phenotypic and genotypic traits and with environmental parameters was determined for 278 strains of Vibrio cholerae isolated from the Chesapeake Bay during 1998 to 2000. Three clusters of luminescent strains (A, B, and C) and two nonluminescent clusters (X and Y) were identified among 180 clonal types. V. cholerae O1 strains isolated during pandemics and endemic cholera in the Ganges Delta were related to cluster Y. Heat-stable enterotoxin (encoded by stn) and the membrane protein associated with bile resistance (encoded by ompU) were found to be linked to luminescence in strains of cluster A. Succession from nonluminescent to luminescent populations of V. cholerae occurred during spring to midsummer. Occurrence of cluster A strains in water with neutral pH was contrasted with that of cluster Y strains in water with a pH of >8. Cluster A was found to be associated with a specific calanoid population cooccurring with cyclopoids. Cluster B was related to cluster Y, with its maximal prevalence at pH 8. Occurrence of cluster B strains was more frequent with warmer water temperatures and negatively correlated with maturity of the copepod community. It is concluded that each cluster of luminescent V. cholerae strains occupies a distinct ecological niche. Since the dynamics of these niche-specific subpopulations are associated with zooplankton community composition, the ecology of luminescent V. cholerae is concluded to be related to its interaction with copepods and related crustacean species.     

Variability of haemolysin(s) produced by Vibrio vulnificus

The peptide composition and antigenic cross-reactivity of partially purified and concentrated haemolysins of 16 strains of Vibrio vulnificus were examined by SDS-PAGE and immunoblotting analysis, using a monoclonal antibody (MAb), 6F8D, raised against the haemolysin. All strains produced a common peptide of 36 kDa and the MAb reacted with this peptide. In some strains, larger molecules, including a 56 kDa peptide, were produced, but the MAb did not react with this peptide. The haemolytic activity of the strains was effectively neutralized by the MAb, except in the case of strains producing the 56 kDa peptide. These findings indicate that the 36 kDa haemolysin is common to all 16 strains and that V. vulnificus can produce a second haemolysin which differs in molecular mass and antigenicity.     

Spatial and temporal distribution of two diazotrophic bacteria in the Chesapeake Bay

The aim of this study was to initiate autecological studies on uncultivated natural populations of diazotrophic bacteria by examining the distribution of specific diazotrophs in the Chesapeake Bay. By use of quantitative PCR, the abundance of two nifH sequences (907h22 and 912h4) was quantified in water samples collected along a transect from the head to the mouth of the Chesapeake Bay during cruises in April and October 2001 and 2002. Standard curves for the quantitative PCR assays demonstrated that the relationship between gene copies and cycle threshold was linear and highly reproducible from 1 to 10(7) gene copies. The maximum number of 907h22 gene copies detected was approximately 140 ml(-1) and the maximum number of 912h4 gene copies detected was approximately 340 ml(-1). Sequence 912h4 was most abundant at the mouth of the Chesapeake Bay, and in general, its abundance increased with increasing salinity, with the highest abundances observed in April 2002. Overall, the 907h22 phylotype was most abundant at the mid-bay station. Additionally, 907h22 was most abundant in the April samples from the mid-bay and mouth of the Chesapeake Bay. Despite the fact that the Chesapeake Bay is rarely nitrogen limited, our results show that individual nitrogen-fixing bacteria have distinct nonrandom spatial and seasonal distributions in the Chesapeake Bay and are either distributed by specific physical processes or adapted to different environmental niches.     

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 H₂S 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.     

Incidence of marine bdellovibrios lytic against Vibrio parahaemolyticus in Chesapeake Bay.

The incidence of marine bdellovibrios at selected sampling sites in the Chesapeake Bay during the months of June 1978 and 1979 was studied. Bdellovibrios were isolated from eight of nine sampling stations in the bay. Higher numbers than previously reported with sea or ocean water were recovered in the midregion of the bay.     

Spatial and temporal aspects of Gyrodinium galatheanum in Chesapeake Bay: distribution and mixotrophy

Gyrodinium galatheanum (Braarud) Taylor 1995 is a common bloom-forming,potentially toxic photosynthetic dinoflagellate in ChesapeakeBay, USA. Abundance of this dinoflagellate achieved densities>4 x 10³ cells ml^–1 in the mid- and upper Bay duringlate spring and early summer of 1995 and 1996. Ingestion ofcryptophytes by this dinoflagellate was detected in most samplescollected from the Bay. During late spring and early summer,mean number of ingested cryptophytes per G.galatheanum was ashigh as 0.46 for dinoflagellate populations located in surfacewaters of the mid- and upper Bay where dissolved inorganic phosphoruswas low. Observations on the distribution of G.galatheanum inChesapeake Bay show that populations of this dinoflagellatewere usually restricted to waters with salinities ranging from7 to 18 psu, seasonally progressed up the estuary, and usuallyco-occurred with cryptophytes. Correlation analysis indicatesthat abundance of G.galatheanum and incidence of feeding wasnegatively correlated with dissolved inorganic phosphorus, andthat incidence of feeding was positively correlated with abundanceof cryptophyte prey. These results indicate that G.galatheanumis an important component of the Chesapeake Bay phytoplanktonduring the spring and summer. Our results suggest that the phagotrophiccapability possessed by this phototrophic dinoflagellate maycontribute to its success in a varying-resource environmentlike Chesapeake Bay.     

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.