Incidence and Abundance of Pathogenic Vibrio Species in the Great Bay Estuary, New Hampshire

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1 Incidence and Abundance of Pathogenic Vibrio Species in the Great Bay Estuary, New Hampshire Jones Steve 1 *; Striplin Megan 2, Mahoney Jennifer 2, Cooper Vaughn 2, Whistler Cheryl 2 1 University of New Hampshire, Jackson Estuarine Laboratory, 85 Adams Point Rd., Durham, NH USA 2 University of New Hampshire, Department of Molecular, Cellular and Biomedical Sciences, Durham, NH USA Pathogenic Vibrios have emerged as a serious and global threat to human health. Of particular concern are Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae, which cause diarrhea, gastroenteritis and septicemia. Of increased concern is the expanding range of detection in areas not naturally endemic to pathogenic Vibrio species, i.e., northern temperate and colder climates. The purpose of this study was to combine culturebased detection with molecular typing methods to identify what pathogenic Vibrio species are present in the Great Bay Estuary, NH and to determine their virulence potential. V. parahaemolyticus, V. vulnificus, and, for the first time, V. cholerae, were all identified from oyster, water and sediment samples during , though no isolates contained clinical/human virulence genes. V. parahaemolyticus was detected most frequently, all Vibrio were more abundant in oysters and sediments than in water, and V. cholerae was rarest. V. parahaemolyticus abundance was most associated with temperature because of wide seasonal differences during the study, but did not vary among sample sites despite varying environmental conditions. The threat of human vibrio infections in the Gulf of Maine from exposure to shellfish and coastal waters under expected global warming conditions is a growing concern. Keywords: Vibrio parahaemolyticus/ Vibrio vulnificus/ Vibrio cholerae/virulence markers/estuarine ecosystem * Corresponding author: shj@unh.edu Introduction Pathogenic vibrios have emerged as a serious and global threat to human health. The incidence of infections has risen steeply worldwide with the appearance of pandemic clones of greater infective ability (Thompson et al. 2004). Of particular concern are Vibrio parahaemolyticus (Vp) (Yeung and Boor, 2004), Vibrio vulnificus (Vv) (Linkous and Oliver, 1999; Gulig et al. 2005) and Vibrio cholerae (Vc) (Colwell 2004). All cause diarrhea and gastroenteritis, but Vv, and more rarely Vp, may also cause septicemia through wound and intestinal infections (Thompson et al. 2004). Vp is one of the leading causes of foodborne illness in countries such as Japan and Taiwan (Pan et al. 1997; Wong et al. 2000) and is the most frequent cause of foodborne vibrioassociated gastroenteritis in the United States (Daniels et al. 2000), with an estimated 2,800 cases each year from the consumption of raw oysters (US FDA 2001). Of increased concern is the expanding range of detection in areas not naturally endemic to pathogenic Vibrio species (Jones 2009). Vibrio disease outbreaks are generally associated with 1

2 harvested shellfish from areas with warm water, like the Gulf of Mexico and the waters of Southeast Asia (DePaola et al. 2003; Thompson et al. 2004; Zimmerman et al. 2007; MartinezUrtaza et al. 2008) while they are detected less often (Jones and SummerBrason 1998; Bauer et al. 2006) or absent (Wilson and Moore 1996) in northern temperate and colder climates. This general trend has been belied by several significant outbreaks of Vp in north temperate areas in recent years, most recently in Alaska in 2004 (McLaughlin et al. 2005). Some climate change models indicative of significant seawater warming suggest a scenario in which new ecological niches, ideal for growth of human pathogens, are created. In cooler, more northern waters, small, typically nonvirulent Vibrio populations may expand to include more virulent strains as water temperatures increase. In addition, other climate change effects may alter other environmental conditions that are key to Vibrio presence and abundance. While temperature and nutrients strongly correlate with all populations of Vibrio spp, salinity serves as an additional predictor of both general and specific populations. Other factors like dissolved organic carbon (DOC), suspended solids and plankton and copepods may also play a role (Jones and Summer Brason 1998; Gugliandolo et al. 2005). The climatic variables of higher temperature and increased flow/lower salinity are predicted conditions associated with global climate change (Louis et al. 2003). Whereas the individual effects of these physical variables are documented, their combined or synergistic effects are not understood. The inherent seasonal and spatial variability of climate, its effect on estuarine nutrients and water conditions (e.g. rainfall/salinity, ph, temperature, dissolved oxygen) makes shellfishgrowing waters ideal sites for genetic diversity and evolution of resident and pollutant bacterial species that may harbor pathogenic strains. Previous studies show Vv and Vp are present in the Great Bay Estuary of New Hampshire, although in a spatially and temporally heterogeneous manner (O'Neill et al. 1990; O'Neill et al. 1992; Jones and SummerBrason 1998). These Vibrio species are detectable only from late May to early November, and are distributed consistently throughout the tributaries of the estuary but less so in the larger bays. No study thus far in the region has attempted to determine the pathogenic potential of these resident Vibrios. In this first survey that combines culturebased methods with molecular typing, we identified Vc for the first time, and both Vv and Vp once again, in NH waters. We also examined the environmental factors that may explain their distribution and abundance. Further, analysis for virulence factors helps to frame the status of the potential for infections associated with shellfish and estuarine water in a north temperate estuary where no information on virulent strains is currently available. 1. Materials And Methods 1.1. Sampling sites Sample sites were chosen based on differences in water quality and successful culture of the targeted Vibrio species in previous studies (Jones and SummerBrason 1998). The first is an oyster bed located near Nannie Island in Great Bay that is within an area classified as approved for shellfish harvesting (Fig. 1). The second, an oyster bed located in the Oyster River is within an area classified as prohibited because of its proximity to the Durham, NH wastewater treatment facility effluent outfall. An additional site, Fox Point, is located across from the mouth of the Oyster River in an approved area. It was sampled once to collect colocated oysters, blue mussels and soft shell clams. 2

3 Fig 1. The sampling locations and shellfish harvest classifications in the lower Great Bay Estuary, New Hampshire, USA Sampling procedures Water and oyster (Crassostrea virginica) samples were collected on the same day approximately every two weeks during June to December 2007 and May to December 2008 from the Nannie Island and Oyster River sites. Samples of oysters, soft shell clams (Mya arenaria) and blue mussels (Mytilus edulis) were collected from Fox Point on July 30, Oysters were sampled using oyster tongs at locations within the same general area of the oyster beds each time. Live animals were separated and external debris was removed on site. Clams were dug using a clam rake while mussels were gathered from rocky outcrops along the low intertidal. Aquatic macrophytes samples were collected from rocks and the sediment using sterile knives and placed into sterile WhirlPak bags. Sediments were collected with a van Veen grab sampler. The top 2 cm of sediment were scooped into sterile plastic bags. Water samples were collected on site by filling and then capping sterile 1 L plastic bottles at ~30 cm below the water surface. All shellfish, plant, sediment and water samples were immediately stored in coolers containing ice packs and brought back to the laboratory for analysis. A YSI 85 (Yellow Springs Inc., Yellow Springs, OH) was used to measure water temperature, salinity, ph and dissolved oxygen on site at each sample event. 3

4 1.3. Bacterial analysis Shellfish were cleaned of debris and shucked using aseptic procedures. Tissue was quantitatively transferred to sterile beakers and weighed, then diluted 1:1, 1:2, 1:3 or 1:4, depending on the sample size and shellfish species, with alkaline peptone water (APW, ph 8.6, 1% NaCl) prior to homogenization for 60 s in a Waring blender. Sediment samples were homogenized by hand and 1.0 g wet weight was transferred to 9.0 ml of APW for further dilution. Macrophyte samples were rinsed with sterile APW, homogenized within the WhirlPak bags, then a 1.0 g wet subsample was transferred to a sterile glass tube containing 9.0 ml APW and ground with a tissue grinder for 60 seconds. Water samples were shaken for 20 s. Following homogenization, 10, 1.0, 0.1 and/or ml volumes of shellfish and plant homogenates and water samples were inoculated into multiple tube fermentation (MPN) analysis series with APW selective enrichment. Turbid MPN tubes were streaked onto thiosulfatecitratebile saltssucrose (TCBS) (Kaysner and DePaola 2004) and colistinpolymixin Bcellobiose (CPC) (Warner and Oliver 2007) agar plates. Following incubation, all colonies with the correct colony morphology type were counted from each dilution plate, but only a single representative isolate of each colony morphology type recognized as either Vp, Vc or Vv on differential medium (TCBS or CPC media) from each environmental sampler were restreaked for isolation and characterized by molecular typing. Colonies putatively designated as Vv, Vc or Vp were further characterized by multiplex PCR using protocols previously published (Panicker et al. 2004). This multiplex PCR assay confirms the species identification of the isolates (using vvh for Vv, toxr (all serotypes) for Vc and tlh for Vp) and simultaneously identifies the presence of pathogen specific genes (including viub for Vv, tdh, trh and the pandemic clone marker ORF8 for Vp, and tcpi, ompu, hyla for Vc) allowing identification of human pathogenic strains. For each isolate, whole cells from the colony were suspended in the PCR reaction buffer using a sterile toothpick. Following amplification, the PCR products were separated and visualized on a 1% agarose gel in TAE, and compared to positive controls that included Vv strain MO624, and Vp strains F113A (ORF8 negative) and serotype 03:K6 BAC (ORF8 positive), and a negative control of water. For any isolate whose amplification resulted in a faint band, the reaction was repeated. Only isolates confirmed as Vv or Vp based on molecular typing were used for the calculation of MPN data Data analysis Concentration data for the pathogenic Vibrio species were expressed as the highest concentration measured for cases where more than one strain was isolated from a sample. For comparisons of monthly data, geometric means were calculated for all concentrations from a given sample type or sample location within each month from May to December. ANOVA was performed on Vp concentrations and sample matrix, and between site differences in salinity and temperature. Correlation analysis of the relationship between Vibrio concentrations and both water temperature and salinity were conducted using the water quality data collected at the time of sampling. 2. Results Vv, Vc and Vp were detected during the study (Table 1). Vp was detected most frequently, in 66% of all samples, with relatively even frequency of detection in water and oysters, much 4

5 higher detection in sediments, and similar frequencies of detection at the two sites and between years. Vv was detected much less frequently (8% of all samples), only in oysters during 2007, only in water and sediments at Oyster River in 2008 and in water and oysters at % INCIDENCE 2007 NI N Vp Vv Vc Water 12 50% 0% 0% Oysters 12 58% 25% 0% OR Water 12 58% 0% 0% Oysters 12 67% 17% 0% 2008 NI Water 19 74% 11% 16% Oysters 14 57% 7% 14% Sediment 10 80% 0% 0% OR Water 19 63% 11% 16% Oysters 14 71% 0% 7% Sediment 10 80% 10% 0% TOTAL % 8% 7% % VIRULENCE Vp Vv Vc TOTAL 0% 0% 0% Table 1. The % incidence and isolate virulence for V. parahaemolyticus (Vp), V. vulnificus (Vv) and V. chloerae (Vc) in water, oysters and sediments at Nannie Island (NI) and in the Oyster River (OR) in 2007 and Nannie Island in Vc was also less frequently detected (7% of all samples). It was not detected during 2007 and was found in 10% of the 2008 samples. Of the 156 isolates confirmed to be Vp, Vv and Vc by molecular typing (130 Vp, 14 Vv and 12 Vc), none harbored the virulence markers tdh, trh, viub or tcpi used to distinguish clinical/human pathogenic isolates from environmental isolates (Panicker et al. 2004). Concentrations of Vp in water samples ranged from 3 to 1210 MPN/100 ml, from 69 to 44,000 MPN/100 g in shellfish tissue and from 74 to 12,100 MPN/g in sediment. For samples at either of the two main study sites, Vp concentrations (MPN/100 ml) were significantly lower in water than concentrations (MPN/100 g sample) in both sediments and oyster tissue (Table 2). The concentration ranges for Vv were similar to those for Vp, from 20 to 930 MPN/100 ml water, 150 to 11,000 MPN/100 g oyster tissue and, for one positive sample, >11,000 MPN/100 g sediment. Vc concentrations were relatively lower than for the other Vibrios, ranging from 3.6 to 240 MPN/100 ml water and MPN/100 g oyster tissue. In shellfish samples collected from Fox Point on July 30, 2007 Vp was detected in oysters, mussels and clams at relatively high (36,000 to 44,000 MPN/100 g) levels, and Vv was detected at 7200 MPN/100 g in oyster tissue. 5

6 A. OR Water Oyster Sediment Water X X Oyster X ns Sediment X ns NI Water Oyster Sediment Water X X Oyster X ns Sediment X ns B Both years Temperature ns ns X Salinity ns X X Table 2. ANOVA for V. parahaemolyticus concentrations in water, oysters and sediment and water quality parameters. A. Within site comparisons of geometric mean V. parahaemolyticus concentrations in different sampling matrices; B. Differences between sites for water quality parameters during 2007 and *see 2008 data, the only year in which sediments were sampled the legend is not clear The geometric mean monthly concentrations of Vp detected in 2007 and 2008 were highest during the warm summertime from July to September and declined in water and oysters as water temperatures decreased at both study sites (Fig. 2). Vp in sediment, however, remained detectable at ~100 MPN/g through December at both sites. The average salinity and water temperatures were significantly higher at Nannie I. Compare d to the Oyster River (Table 2B) Nannie Island Water Oysters Sediment Fig 2. Monthly geometric mean Vp levels in the three matrices and average monthly water temperatures at Nannie Island (upper panel) and Oyster River (lower panel): June19 C July21 C Aug22 C Sept16 C Oct14 C Nov7 C Dec0 C Month and Average Temperature ( C) Oyster River Water Oysters Sediment June20 C July22 C Aug22 C Sept19 C Oct14 C Nov7 C Dec1 C Month and Average Temperature ( C) 6

7 Despite these environmental differences and some monthtomonth differences, the geometric mean monthly Vp concentrations were relatively similar at Nannie I. and Oyster River in all three sample matrices, except Vp was detected in water from Oyster River into December while it declined to nondetectable levels at Nannie I. (Fig. 2). No geometric mean monthly data are available for Vv and Vc; both were detected only during June to September. The decline in Vp concentrations following warm summer months, and the differences observed between Nannie I. and Oyster River samples suggests environmental factors may have affected Vp concentrations. Correlation analysis using all data for 2007 and 2008 showed temperature to be a significant factor, with Vp concentrations increasing with increasing temperature (Table 3). This affect was mostly a factor of conditions at Oyster River, as there were no significant effects of temperature on Vp concentrations at Nannie I. The relationships between Vp concentrations and salinity were not significant except in the 2008 sediments and for oysters and water together in 2007 (Table 3). Table 3. Relationships between Vp concentration and environmental conditions (salinity, temperature) in G reat Bay Estuary oysters, sediments, overlying water and all matrices: 2007 and X designates a significant (P< 0.05) relationship. ND= no data. *see 2008 results, the only year in which sediments were sampled. Matrix 2007 Oysters Water Sediment All Temperature All data X ND X NI ND OR X ND X Salinity All data ND NI ND X OR ND 2008 Temperature All data X X NI OR X X Salinity All data NI X OR Both Years Temperature All data X X * NI * OR X X * Salinity All data * NI * X X X OR * X 7

8 3. Discussion Detection of the three Vibrio species was successful from both study sites and in all ecosystem matrices, i.e., water, oysters and sediments. Vp was detected much more frequently than Vv and Vc. The low frequency of detection for Vv was unexpected, based on previous studies in the same study area (Jones and SummerBrason 1998). The reason for this difference, be it climate change, other changes in ecosystem conditions or simply environmental variability within a larger context for the two years of this study compared to longterm conditions is not known. The lack of differences in Vp levels between sites suggests Vibrio levels were independent of shellfish harvesting classification, which is based mainly on fecal pollution levels. Other significant differences in environmental conditions exist between these two sites (Jones and SummerBrason 1998) that have been correlated with differences in Vibrio levels. For example, temperature was positively associated with Vp abundance and there were significant differences in temperature and salinity between the two sites for The lack of differences in Vp levels between sites appears to reflect overwhelming influences of seasonal temperature variation rather than sitespecific effects. The seasonal differences in water temperature limited detection of Vp to the months of Mayearly December, and only from July to September for Vv and Vc in this study. All three Vibrio species concentrations were relatively high in oysters and sediments and low in water. This may be a function of the oysters and sediments acting as sinks from the water column via sedimentation and uptake. Conversely, they may also be sources to the water column via sediment resuspension and release from shellfish. The levels of Vc were relatively lower in oysters and water compared to detected levels of Vp and Vv. Vc is commonly associated with lower salinity conditions, and their incidence in this study may be a function of downstream transport from lower salinity areas, though a study with only two sites is inadequate to determine this. Although genes commonly associated with Vibrio pathogenicity were not detected in our sampling, this does not preclude the possible contribution of these populations to future infections and even epidemics in our region. First, strains lacking hemolysin may nonetheless be pathogenic (Meador et al. 2007; Han et al. 2009; Harth et al. 2009). Second, and perhaps more importantly in light of climate change, nonpathogenic strains that persist in large numbers may serve as a reservoir for the emergence of new pathogens when conditions become favorable. Such emergence may be facilitated by horizontal genetic exchange among Vibrios and between Vibrios and other estuarine bacteria; given that all three major Vibrio pathogens are found locally, each is therefore a potential source or donor of pathogenic genes. The continued presence of Vp and Vv, and the newly discovered presence of Vc in the Great Bay Estuary of New Hampshire and Maine is a concern for public health officials. Trends in climate change suggest warmer conditions will probably evolve in the Gulf of Maine, and the threat of human Vibrio infections from exposure to shellfish and coastal waters will probably increase. Ongoing studies are focusing on understanding the genetic relatedness of Vp strains isolated from the Great Bay Estuary and determining other phenotypes that may be associated with virulence. 8

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