Ecological determinants of the occurrence and dynamics of Vibrio parahaemolyticus in offshore areas

The life cycle of Vibrio parahaemolyticus has been conventionally associated with estuarine areas characterized by moderate salinity and warm seawater temperatures. Recent evidence suggests that the distribution and population dynamics of V. parahaemolyticus may be shaped by the existence of an oceanic transport of communities of this organism mediated by zooplankton. To evaluate this possibility, the presence of V. parahaemolyticus in the water column of offshore areas of Galicia was investigated by PCR monthly over an 18-month period. Analysis of zooplankton and seawater showed that the occurrence of V. parahaemolyticus in offshore areas was almost exclusively associated with zooplankton and was present in 80% of the samples. The influence of environmental factors assessed by generalized additive models revealed that the abundance and seasonality of V. parahaemolyticus in zooplankton was favoured by the concurrence of downwelling periods that promoted the zooplankton patchiness. These results confirm that offshore waters may be common habitats for V. parahaemolyticus, including strains with virulent traits. Additionally, genetically related populations were found in offshore zooplankton and in estuaries dispersed along 1500 km. This finding suggests that zooplankton may operate as a vehicle for oceanic dispersal of V. parahaemolyticus populations, connecting distant regions and habitats, and thereby producing impacts on the local community demography and the spread of Vibrio-related diseases.     

Variability of Total and Pathogenic Vibrio parahaemolyticus Densities in Northern Gulf of Mexico Water and Oysters

Vibrio parahaemolyticus is indigenous to coastal environments and a frequent cause of seafood-borne gastroenteritis in the United States, primarily due to raw-oyster consumption. Previous seasonal-cycle studies of V. parahaemolyticus have identified water temperature as the strongest environmental predictor. Salinity has also been identified, although it is evident that its effect on annual variation is not as pronounced. The effects of other environmental factors, both with respect to the seasonal cycle and intraseasonal variation, are uncertain. This study investigated intraseasonal variations of densities of total and pathogenic V. parahaemolyticus organisms in oysters and overlying waters during the summer of 2004 at two sites in the northern Gulf of Mexico. Regression analyses indicated significant associations (P < 0.001) between total V. parahaemolyticus densities and salinity, as well as turbidity in water and in oysters at the Mississippi site but not at the Alabama site. Pathogenic V. parahaemolyticus organisms in Mississippi oyster and water samples were detected in 56% (9 out of 16) and 78% (43 out of 55) of samples, respectively. In contrast, 44% (7 out of 16) of oyster samples and 30% (14 out of 47) of water samples from Alabama were positive. At both sites, there was greater sample-to-sample variability in pathogenic V. parahaemolyticus densities than in total V. parahaemolyticus densities. These data suggest that, although total V. parahaemolyticus densities may be very informative, there is greater uncertainty when total V. parahaemolyticus densities are used to predict the risk of infection by pathogenic V. parahaemolyticus than previously recognized.     

Modulation of Responses of Vibrio parahaemolyticus O3:K6 to pH and Temperature Stresses by Growth at Different Salt Concentrations

Vibrio parahaemolyticus inhabits marine, brackish, and estuarine waters worldwide, where fluctuations in salinity pose a constant challenge to the osmotic stress response of the organism. Vibrio parahaemolyticus is a moderate halophile, having an absolute requirement for salt for survival, and is capable of growth at 1 to 9% NaCl. It is the leading cause of seafood-related bacterial gastroenteritis in the United States and much of Asia. We determined whether growth in differing NaCl concentrations alters the susceptibility of V. parahaemolyticus O3:K6 to other environmental stresses. Vibrio parahaemolyticus was grown at a 1% or 3% NaCl concentration, and the growth and survival of the organism were examined under acid or temperature stress conditions. Growth of V. parahaemolyticus in 3% NaCl versus that in 1% NaCl increased survival under both inorganic (HCl) and organic (acetic acid) acid conditions. In addition, at 42°C and −20°C, 1% NaCl had a detrimental effect on growth. The expression of lysine decarboxylase (encoded by cadA), the organism''s main acid stress response system, was induced by both NaCl and acid conditions. To begin to address the mechanism of regulation of the stress response, we constructed a knockout mutation in rpoS, which encodes the alternative stress sigma factor, and in toxRS, a two-component regulator common to many Vibrio species. Both mutant strains had significantly reduced survival under acid stress conditions. The effect of V. parahaemolyticus growth in 1% or 3% NaCl was examined using a cytotoxicity assay, and we found that V. parahaemolyticus grown in 1% NaCl was significantly more toxic than that grown in 3% NaCl.Vibrio parahaemolyticus is a Gram-negative bacterium that inhabits coastal waters worldwide. Vibrio parahaemolyticus grows optimally in warmer waters and is most commonly isolated during the summer months, often in association with plankton, crustaceans, mollusks, and fish (16, 17). During the winter months, the organism is typically scarce and usually is isolated from sediment samples (16). While V. parahaemolyticus has been shown to be the etiological agent of disease in several kinds of crustaceans and shellfish, it is most notably a pathogen of humans (17). Vibrio parahaemolyticus was first discovered in Japan during an outbreak of gastroenteritis in 1950 (12). It is the leading cause of seafood-related bacterial gastroenteritis in the United States and much of Asia (6, 39). Infection is most frequently associated with the consumption of oysters harvested from warm waters, particularly along the U.S. Gulf Coast, where vibrios grow to high levels during the summer months (6, 7, 42). Newly released data from the CDC comparing the incidence rates of laboratory-confirmed infections by gastrointestinal pathogens in 1996 to 2008 revealed an increase of 47% for Vibrio infections, of which V. parahaemolyticus accounted for 55%, while rates for all other enteric pathogens decreased or remained the same (5). An outbreak of V. parahaemolyticus infections which caused rapid hospitalization of those infected occurred in India in 1995 (28). These infections were caused by a single serogroup, a new, highly virulent O3:K6 strain, which has now disseminated globally (1, 6, 20, 26, 34, 38). Recent studies report the recovery of O3:K6 isolates from the water in southern Chile, a region that previously was considered too cold to support the growth of this organism (4, 11, 13).All V. parahaemolyticus strains inhabit marine, brackish, and estuarine waters, where fluctuations in salinity pose a constant challenge to the adaptive response of the organism. Vibrio parahaemolyticus is moderately halophilic in nature and requires a minimum of 0.086 M (0.5%) NaCl for growth (29). It has also been demonstrated that this organism has the ability to grow in medium containing NaCl concentrations upwards of 1.5 M, making V. parahaemolyticus more osmotolerant than many other Vibrio species, such as V. cholerae, V. vulnificus, and V. fischeri, which occupy similar niches (27). In a recent study, we examined the genome of V. parahaemolyticus O3:K6 (designated RIMD2210633) and identified homologues of ectoine and betaine synthesis genes, as well as homologues of four single-component compatible solute transporters and two multicomponent compatible solute transporters (27). The large compendium of compatible solute systems in V. parahaemolyticus suggests that they might play an additional role(s) in survival.Within offshore waters, V. parahaemolyticus is generally faced with NaCl concentrations of 3.5% salinity (35 ppt), but in estuarine systems and within oysters (which are osmoconformers), it must adapt to changes in salinity. In addition, as a human pathogen, once inside the human host, like most enteric pathogens, V. parahaemolyticus must overcome the inorganic-pH challenge presented by gastric acid from the stomach and organic acids found within the intestine, as well as decreasing salinity (salinity in the intestine is approximately 300 mM NaCl). Organic acids have the ability to cross the cell membrane and enter the cytoplasm of the cell, whereas inorganic acids remain in the extracellular environment. Once in the cells, the organic acids can disassociate, decreasing the cytoplasmic pH and increasing the turgor pressure within the cell due to increases in anions from the acids (9). Thus, inorganic and organic acids can affect cells very differently.We suggest that the ability to grow at different NaCl concentrations, such as those vibrios would encounter in estuarine environments, allows V. parahaemolyticus to adapt more effectively to other environmental stresses (temperature fluctuations) and to the challenges that occur upon invasion of the human host (low pH). In this study, we show that V. parahaemolyticus RIMD2210633 cells grown at 3% NaCl are more resistant to acid and temperature stresses than cells grown at 1% NaCl. We demonstrate that V. parahaemolyticus grown in 3% NaCl is better able to survive sublethal and lethal acid shock conditions, as well as persistent high- and low-temperature conditions. We determined possible regulatory mechanisms involved in stress responses by examining the global regulator genes toxRS and rpoS. Last, we examined how changing environmental conditions, such as high and low NaCl and low pH, might affect the virulence of V. parahaemolyticus by determining its cytotoxicity toward human intestinal (Caco-2) cells.     

Antimicrobial Susceptibilities of Vibrio parahaemolyticus and Vibrio vulnificus Isolates from Louisiana Gulf and Retail Raw Oysters

The antimicrobial susceptibilities of 168 Vibrio parahaemolyticus and 151 Vibrio vulnificus isolates recovered from 82 Louisiana Gulf and retail oysters in 2005 and 2006 were determined. Overall, the two vibrios remained susceptible to the majority of antimicrobials tested; reduced susceptibility was detected only in V. parahaemolyticus for ampicillin (81%; MIC ≥ 16 μg/ml). Additionally, V. parahaemolyticus displayed significantly higher MICs for cefotaxime, ciprofloxacin, and tetracycline than V. vulnificus.     

Defining the Niche of Vibrio parahaemolyticus During Pre- and Post-Monsoon Seasons in the Coastal Arabian Sea

The bacteria Vibrio parahaemolyticus is an important component of coastal ecosystems worldwide, and in recent years, V. parahaemolyticus has caused several cases of food-borne gastroenteritis. However, research investigating which parameters are important in regulating V. parahaemolyticus abundance in tropical areas with relatively stable temperatures and salinity are largely lacking. The objective here was to investigate which environmental forces are driving elevated abundances of V. parahaemolyticus in a tropical oligotrophic coastal area in the Arabian Sea. We analysed a large number of environmental parameters in parallel with cell densities of V. parahaemolyticus and Vibrio spp. Abundance data was obtained using real-time PCR, during two different sampling periods, representative for two distinct seasons. Water temperature and salinity were stable during and between sampling periods, but V. parahaemolyticus abundances were on average six times higher during the first sampling period in December, compared to the second period in February–March. V. parahaemolyticus abundance was found to be positively correlated to inorganic phosphate concentration and copepod abundance. We thus hypothesise that these are important factors regulating V. parahaemolyticus abundance in coastal tropical areas during these periods.     

Relationships between Environmental Factors and Pathogenic Vibrios in the Northern Gulf of Mexico

Although autochthonous vibrio densities are known to be influenced by water temperature and salinity, little is understood about other environmental factors associated with their abundance and distribution. Densities of culturable Vibrio vulnificus containing vvh (V. vulnificus hemolysin gene) and V. parahaemolyticus containing tlh (thermolabile hemolysin gene, ubiquitous in V. parahaemolyticus), tdh (thermostable direct hemolysin gene, V. parahaemolyticus pathogenicity factor), and trh (tdh-related hemolysin gene, V. parahaemolyticus pathogenicity factor) were measured in coastal waters of Mississippi and Alabama. Over a 19-month sampling period, vibrio densities in water, oysters, and sediment varied significantly with sea surface temperature (SST). On average, tdh-to-tlh ratios were significantly higher than trh-to-tlh ratios in water and oysters but not in sediment. Although tlh densities were lower than vvh densities in water and in oysters, the opposite was true in sediment. Regression analysis indicated that SST had a significant association with vvh and tlh densities in water and oysters, while salinity was significantly related to vibrio densities in the water column. Chlorophyll a levels in the water were correlated significantly with vvh in sediment and oysters and with pathogenic V. parahaemolyticus (tdh and trh) in the water column. Furthermore, turbidity was a significant predictor of V. parahaemolyticus density in all sample types (water, oyster, and sediment), and its role in predicting the risk of V. parahaemolyticus illness may be more important than previously realized. This study identified (i) culturable vibrios in winter sediment samples, (ii) niche-based differences in the abundance of vibrios, and (iii) predictive signatures resulting from correlations between environmental parameters and vibrio densities.Vibrio spp. occur naturally in estuarine and marine environments, and two species of this genus, V. vulnificus and V. parahaemolyticus, are responsible for the majority of reported vibrio illnesses in the United States (2). V. vulnificus infections are most commonly associated with the Gulf of Mexico, either via consumption of raw oysters harvested from these waters or wound infections following exposure to seawater. On average, about 50 cases of V. vulnificus septicemia are reported in the United States each year, with a case fatality rate of approximately 50% (31), the highest of any food-borne pathogen. In contrast, V. parahaemolyticus is the most common cause of seafood-associated bacterial gastroenteritis in the United States, with an estimated annual rate of 4,500 cases per year according to the Centers for Disease Control and Prevention. V. parahaemolyticus also causes wound infections, though these are less frequent and less severe compared to those caused by V. vulnificus (5). Primary septicemia can occur following V. parahaemolyticus infection, but it is relatively rare for this pathogen. In the United States, V. parahaemolyticus illness most often results from consumption of raw or undercooked seafood, particularly oysters.It is well established that vibrio densities correlate strongly with sea surface temperature (SST), with densities increasing as temperatures increase; however, with the exception of salinity, little is definitively known about the influence of other environmental parameters, such as turbidity and chlorophyll a (22, 33). Consequently, while SST has been estimated to explain approximately 50% of the annual variation of V. parahaemolyticus abundance in oysters harvested from the northern Gulf of Mexico (40), a considerable amount of variation remains unexplained. It is of interest to delineate the effects of other environmental parameters independent of SST, as these parameters may be associated with spatial and temporal variation of vibrio densities within seasonal periods when SST is relatively constant and risk of human exposure and illness is high. Moreover, the majority of what is known about V. parahaemolyticus in the environment is based on total populations; little information is available on the pathogenic subpopulations. Isolates containing genetic markers for pathogenicity factors, including the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) typically constitute <1% of the population in marine or postharvest oyster samples, but they account for >90% of clinical isolates (12). The basis for V. vulnificus pathogenicity remains unclear, as few pathogenicity factors have been described definitively (31). To address these data gaps, we monitored densities of culturable V. vulnificus containing vvh (the V. vulnificus hemolysin gene) and V. parahaemolyticus containing tlh (the thermolabile hemolysin gene, ubiquitous in V. parahaemolyticus), tdh, and trh in water, oysters, and sediment collected from coastal waters of Mississippi and Alabama. Associations between bacterial densities and environmental parameters were analyzed by regressing observations against sea surface temperature, chlorophyll a, turbidity, and salinity.     

Seasonal Abundance of Total and Pathogenic Vibrio parahaemolyticus in Alabama Oysters

Recent Vibrio parahaemolyticus outbreaks associated with consumption of raw shellfish in the United States focused attention on the occurrence of this organism in shellfish. From March 1999 through September 2000, paired oyster samples were collected biweekly from two shellfish-growing areas in Mobile Bay, Ala. The presence and densities of V. parahaemolyticus were determined by using DNA probes targeting the thermolabile hemolysin (tlh) and thermostable direct hemolysin (tdh) genes for confirmation of total and pathogenic V. parahaemolyticus, respectively. V. parahaemolyticus was detected in all samples with densities ranging from <10 to 12,000 g⁻¹. Higher V. parahaemolyticus densities were associated with higher water temperatures. Pathogenic strains were detected in 34 (21.8%) of 156 samples by direct plating or enrichment. Forty-six of 6,018 and 31 of 6,992 V. parahaemolyticus isolates from enrichments and direct plates, respectively, hybridized with the tdh probe. There was an apparent inverse relationship between water temperature and the prevalence of pathogenic strains. Pathogenic strains were of diverse serotypes, and 97% produced urease and possessed a tdh-related hemolysin (trh) gene. The O3:K6 serotype associated with pandemic spread and recent outbreaks in the United States was not detected. The efficient screening of numerous isolates by colony lift and DNA probe procedures may account for the higher prevalence of samples with tdh⁺ V. parahaemolyticus than previously reported.     

Environmental Investigations of Vibrio parahaemolyticus in Oysters after Outbreaks in Washington, Texas, and New York (1997 and 1998)

Total Vibrio parahaemolyticus densities and the occurrence of pathogenic strains in shellfish were determined following outbreaks in Washington, Texas, and New York. Recently developed nonradioactive DNA probes were utilized for the first time for direct enumeration of V. parahaemolyticus in environmental shellfish samples. V. parahaemolyticus was prevalent in oysters from Puget Sound, Wash.; Galveston Bay, Tex.; and Long Island Sound, N.Y., in the weeks following shellfish-associated outbreaks linked to these areas. However, only two samples (one each from Washington and Texas) were found to harbor total V. parahaemolyticus densities exceeding the level of concern of 10,000 g⁻¹. Pathogenic strains, defined as those hybridizing with tdh and/or trh probes, were detected in a few samples, mostly Puget Sound oysters, and at low densities (usually <10 g⁻¹). Intensive sampling in Galveston Bay demonstrated relatively constant water temperature (27.8 to 31.7°C) and V. parahaemolyticus levels (100 to 1,000 g⁻¹) during the summer. Salinity varied from 14.9 to 29.3 ppt. A slight but significant (P < 0.05) negative correlation (−0.25) was observed between V. parahaemolyticus density and salinity. Based on our data, findings of more than 10,000 g⁻¹ total V. parahaemolyticus or >10 g⁻¹ tdh- and/or trh-positive V. parahaemolyticus in environmental oysters should be considered extraordinary.     

Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters

Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >10⁴ CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh⁺ and trh⁺ strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.     

Rapid Proliferation of Vibrio parahaemolyticus,Vibrio vulnificus,and Vibrio cholerae during Freshwater Flash Floods in French Mediterranean Coastal Lagoons

Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae of the non-O1/non-O139 serotype are present in coastal lagoons of southern France. In these Mediterranean regions, the rivers have long low-flow periods followed by short-duration or flash floods during and after heavy intense rainstorms, particularly at the end of the summer and in autumn. These floods bring large volumes of freshwater into the lagoons, reducing their salinity. Water temperatures recorded during sampling (15 to 24°C) were favorable for the presence and multiplication of vibrios. In autumn 2011, before heavy rainfalls and flash floods, salinities ranged from 31.4 to 36.1‰ and concentrations of V. parahaemolyticus, V. vulnificus, and V. cholerae varied from 0 to 1.5 × 10³ most probable number (MPN)/liter, 0.7 to 2.1 × 10³ MPN/liter, and 0 to 93 MPN/liter, respectively. Following heavy rainstorms that generated severe flash flooding and heavy discharge of freshwater, salinity decreased, reaching 2.2 to 16.4‰ within 15 days, depending on the site, with a concomitant increase in Vibrio concentration to ca. 10⁴ MPN/liter. The highest concentrations were reached with salinities between 10 and 20‰ for V. parahaemolyticus, 10 and 15‰ for V. vulnificus, and 5 and 12‰ for V. cholerae. Thus, an abrupt decrease in salinity caused by heavy rainfall and major flooding favored growth of human-pathogenic Vibrio spp. and their proliferation in the Languedocian lagoons. Based on these results, it is recommended that temperature and salinity monitoring be done to predict the presence of these Vibrio spp. in shellfish-harvesting areas of the lagoons.     

Population Structure of Clinical and Environmental Vibrio parahaemolyticus from the Pacific Northwest Coast of the United States

Vibrio parahaemolyticus is a common marine bacterium and a leading cause of seafood-borne bacterial gastroenteritis worldwide. Although this bacterium has been the subject of much research, the population structure of cold-water populations remains largely undescribed. We present a broad phylogenetic analysis of clinical and environmental V. parahaemolyticus originating largely from the Pacific Northwest coast of the United States. Repetitive extragenic palindromic PCR (REP-PCR) separated 167 isolates into 39 groups and subsequent multilocus sequence typing (MLST) separated a subset of 77 isolates into 24 sequence types. The Pacific Northwest population exhibited a semi-clonal structure attributed to an environmental clade (ST3, N = 17 isolates) clonally related to the pandemic O3:K6 complex and a clinical clade (ST36, N = 20 isolates) genetically related to a regionally endemic O4:K12 complex. Further, the identification of at least five additional clinical sequence types (i.e., ST43, 50, 65, 135 and 417) demonstrates that V. parahaemolyticus gastroenteritis in the Pacific Northwest is polyphyletic in nature. Recombination was evident as a significant source of genetic diversity and in particular, the recA and dtdS alleles showed strong support for frequent recombination. Although pandemic-related illnesses were not documented during the study, the environmental occurrence of the pandemic clone may present a significant threat to human health and warrants continued monitoring. It is evident that V. parahaemolyticus population structure in the Pacific Northwest is semi-clonal and it would appear that multiple sequence types are contributing to the burden of disease in this region.     

Identification of a DNA Methyltransferase Gene Carried on a Pathogenicity Island-Like Element (VPAI) in Vibrio parahaemolyticus and Its Prevalence among Clinical and Environmental Isolates

In this study we identified a putative virulence-associated DNA methyltransferase (MTase) gene carried on a novel 22.79-kb pathogenicity island-like element (VPAI) in V. parahaemolyticus. The V. parahaemolyticus MTase gene was shown by PCR to be prevalent (>98%) in pandemic thermostable direct hemolysin gene-positive isolates, which suggests that VPAI may confer unique virulence traits to pandemic strains of V. parahaemolyticus.     

Isolation and Characterization of Bacteriophages from Inland Saline Aquaculture Environments to Control Vibrio parahaemolyticus Contamination in Shrimp

Among the various bacterial pathogens associated with the aquaculture environment, Vibrio parahaemolyticus the important one from shrimp and human health aspects. Though having been around for several decades, phage-based control of bacterial pathogens (phage therapy) has recently re-emerged as an attractive alternative due to the availability of modern phage characterization tools and the global emergence of antibiotic-resistant bacteria. In the present study, a total of 12 V. parahaemolyticus specific phages were isolated from 264 water samples collected from inland saline shrimp culture farms. During the host range analysis against standard/field isolates of V. parahaemolyticus and other bacterial species, lytic activity was observed against 2.3–45.5% of tested V. parahaemolyticus isolates. No lytic activity was observed against other bacterial species. For genomic characterization, high-quality phage nucleic acid with concentrations ranging from 7.66 to 220 ng/µl was isolated from 9 phages. After digestion treatments with DNase, RNase and S1 nuclease, the nature of phage nucleic acid was determined as ssDNA and dsDNA for 7 and 2 phages respectively. During transmission electron microscopy analysis of phage V5, it was found to have a filamentous shape making it a member of the family Inoviridae. During efficacy study of phage against V. parahaemolyticus in shrimp, 78.1% reduction in bacterial counts was observed within 1 h of phage application. These results indicate the potential of phage therapy for the control of V. parahaemoyticus in shrimp.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-021-00934-6.     

Genes Similar to the Vibrio parahaemolyticus Virulence-Related Genes tdh,tlh, and vscC2 Occur in Other Vibrionaceae Species Isolated from a Pristine Estuary

Detection of the human pathogen Vibrio parahaemolyticus often relies on molecular biological analysis of species-specific virulence factor genes. These genes have been employed in determinations of V. parahaemolyticus population numbers and the prevalence of pathogenic V. parahaemolyticus strains. Strains of the Vibrionaceae species Photobacterium damselae, Vibrio diabolicus, Vibrio harveyi, and Vibrio natriegens, as well as strains similar to Vibrio tubiashii, were isolated from a pristine salt marsh estuary. These strains were examined for the V. parahaemolyticus hemolysin genes tdh, trh, and tlh and for the V. parahaemolyticus type III secretion system 2α gene vscC2 using established PCR primers and protocols. Virulence-related genes occurred at high frequencies in non-V. parahaemolyticus Vibrionaceae species. V. diabolicus was of particular interest, as several strains were recovered, and the large majority (>83%) contained virulence-related genes. It is clear that detection of these genes does not ensure correct identification of virulent V. parahaemolyticus. Further, the occurrence of V. parahaemolyticus-like virulence factors in other vibrios potentially complicates tracking of outbreaks of V. parahaemolyticus infections.     

Presence of pathogenic Vibrio Parahaemolyticus in waters and seafood from the Tunisian Sea

The occurrence of the hemolysin genes, tdh and trh, in Vibrio parahaemolyticus strains isolated from environmental samples collected from various exported seafood products comprising of fishes and shellfish (Mytilus edulis and Crassostrea gigas) or seawater, was studied. Eight strains were confirmed as V. parahaemolyticus by toxR -based polymerase chain reaction and only one strain out of these 8 strains was positive for tdh and trh genes. Toxigenic V. parahaemolyticus isolates are present in Tunisian coastal areas and they may also be present in Tunisian exported seafood products.     

Prevalence of Pandemic Thermostable Direct Hemolysin-Producing Vibrio parahaemolyticus O3:K6 in Seafood and the Coastal Environment in Japan

Although thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus has caused many infections in Asian countries, the United States, and other countries, it has been difficult to detect the same pathogen in seafoods and other environmental samples. In this study, we detected and enumerated tdh gene-positive V. parahaemolyticus in Japanese seafoods with a tdh-specific PCR method, a chromogenic agar medium, and a most-probable-number method. The tdh gene was detected in 33 of 329 seafood samples (10.0%). The number of tdh-positive V. parahaemolyticus ranged from <3 to 93/10 g. The incidence of tdh-positive V. parahaemolyticus tended to be high in samples contaminated with relatively high levels of total V. parahaemolyticus. TDH-producing strains of V. parahaemolyticus were isolated from 11 of 33 tdh-positive samples (short-necked clam, hen clam, and rock oyster). TDH-producing strains of V. parahaemolyticus were also isolated from the sediments of rivers near the coast in Japan. Representative strains of the seafood and sediment isolates were examined for the O:K serovar and by the PCR method specific to the pandemic clone and arbitrarily primed PCR and pulsed-field gel electrophoresis techniques. The results indicated that most O3:K6 tdh-positive strains belonged to the pandemic O3:K6 clone and suggested that serovariation took place in the Japanese environment.     

Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the Coastal and Estuarine Waters of Louisiana,Maryland, Mississippi,and Washington (United States)

Vibrio parahaemolyticus and Vibrio vulnificus, which are native to estuaries globally, are agents of seafood-borne or wound infections, both potentially fatal. Like all vibrios autochthonous to coastal regions, their abundance varies with changes in environmental parameters. Sea surface temperature (SST), sea surface height (SSH), and chlorophyll have been shown to be predictors of zooplankton and thus factors linked to vibrio populations. The contribution of salinity, conductivity, turbidity, and dissolved organic carbon to the incidence and distribution of Vibrio spp. has also been reported. Here, a multicoastal, 21-month study was conducted to determine relationships between environmental parameters and V. parahaemolyticus and V. vulnificus populations in water, oysters, and sediment in three coastal areas of the United States. Because ecologically unique sites were included in the study, it was possible to analyze individual parameters over wide ranges. Molecular methods were used to detect genes for thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) as indicators of V. parahaemolyticus and the hemolysin gene vvhA for V. vulnificus. SST and suspended particulate matter were found to be strong predictors of total and potentially pathogenic V. parahaemolyticus and V. vulnificus. Other predictors included chlorophyll a, salinity, and dissolved organic carbon. For the ecologically unique sites included in the study, SST was confirmed as an effective predictor of annual variation in vibrio abundance, with other parameters explaining a portion of the variation not attributable to SST.     

Comparison of Free-Living Amoebae in Hot Water Systems of Hospitals with Isolates from Moist Sanitary Areas by Identifying Genera and Determining Temperature Tolerance

Legionella-contaminated hot water systems and moist sanitary areas in six hospitals were sampled for amoebae by following a standardized collection protocol. Genus identifications and temperature tolerance determinations were made. Amoebae identified as Hartmannella vermiformis (65%), Echinamoebae spp. (15%), Saccamoebae spp. (12%), and Vahlkampfia spp. (9%) were detected in 29 of 56 (52%) hot water samples. Twenty-three of 49 (47%) swabs obtained from moist areas were amoeba positive. The following genera were identified: Acanthamoeba (22%), Naegleria (22%), Vahlkampfia (20%), Hartmannella (15%), and Vanella (7%). The temperature tolerance of amoebae from hot water systems was strikingly different from that of amoebae from moist areas. At 44°C on agar, 59% of amoebic isolates sampled from hot water systems showed growth. The corresponding value for isolates from moist areas was only 17%. Six Acanthamoeba isolates from the moist areas were considered potential pathogens. Four Hartmannella and two Saccamoeba isolates from hot water could be cultured at 53°C.     

Occurrence, Diversity, and Pathogenicity of Halophilic Vibrio spp. and Non-O1 Vibrio cholerae from Estuarine Waters along the Italian Adriatic Coast

The occurrence, diversity, and pathogenicity of Vibrio spp. were investigated in two estuaries along the Italian Adriatic coast. Vibrio alginolyticus was the predominant species, followed by Vibrio parahaemolyticus, non-O1 Vibrio cholerae, and Vibrio vulnificus. By using a biochemical fingerprinting method, all isolates were grouped into nine phenotypes with similarity levels of 75 to 97.5%. The production of toxins capable of causing cytoskeleton-dependent changes was detected in a large number of Vibrio strains. These findings indicate a significant presence of potentially pathogenic Vibrio strains along the Adriatic coast.     

Dynamics of Clinical and Environmental Vibrio parahaemolyticus Strains during Seafood-Related Summer Diarrhea Outbreaks in Southern Chile

Seafood consumption-related diarrhea became prevalent in Chile when the pandemic strain of Vibrio parahaemolyticus serotype O3:K6 reached a region in the south of Chile (Region de los Lagos) where approximately 80% of the country''s seafood is produced. In spite of the large outbreaks of clinical infection, the load of V. parahaemolyticus in shellfish of this region is relatively low. The pandemic strain constitutes a small but relatively stable group of a diverse V. parahaemolyticus population, composed of at least 28 genetic groups. Outbreaks in Region de los Lagos began in 2004 and reached a peak in 2005 with 3,725 clinical cases, all associated with the pandemic strain. After 2005, reported cases steadily decreased to a total of 477 cases in 2007. At that time, 40% of the clinical cases were associated with a pandemic strain of a different serotype (O3:K59), and 27% were related to V. parahaemolyticus isolates unrelated to the pandemic strain. In the results published here, we report that in the summer of 2008, when reported cases unexpectedly increased from 477 to 1,143, 98% of the clinical cases were associated with the pandemic strain serotype O3:K6, a change from 2007. Nevertheless, in 2009, when clinical cases decreased to 441, only 64% were related to the pandemic strain; the remaining cases were related to a nonpandemic tdh- and trh-negative strain first identified in shellfish in 2006. Overall, our observations indicate that the pandemic strain has become a relatively stable subpopulation and that when the number of diarrhea cases related to the pandemic strain is low, previously undetected V. parahaemolyticus pathogenic strains become evident.Diarrhea associated with seafood consumption is caused primarily by pathogenic V. parahaemolyticus. This species includes marine bacterial strains, only a few of which are pathogenic in humans (13). The load of pathogenic strains in shellfish depends on physical environmental variables, such as temperature and salinity, and on biological variables including the presence of protozoan predators, competing nonpathogenic bacteria, and bacteriophages capable of killing V. parahaemolyticus (21). Therefore, diarrhea outbreaks caused by V. parahaemolyticus are mainly an environmental problem. Records of the Public Health Institute of Chile indicate that from 1992 to 1997 diarrhea cases related to seafood consumption were not widespread in Chile in spite of the large consumption of raw shellfish. Cases of seafood-related diarrhea increased greatly with the arrival of the pandemic strain O3:K6, originally observed in Southeast Asia (9). This strain corresponds to a clonal complex. The clonal nature of the V. parahaemolyticus pandemic isolates obtained worldwide has been ascertained by the high degree of similarity among their genomes. This comparison includes the presence of specific genetic markers and similarity of the restriction patterns of their genomes, demonstrated by genome restriction fragment length polymorphism-pulsed-field gel electrophoresis (22), direct genome restriction enzyme analysis (DGREA) (8), arbitrarily primed PCR (15, 18), and multilocus sequence typing (6, 10). Characteristics of isolates of the O3:K6 pandemic clone are the O3:K6 antigens, a distinctive toxRS sequence (toxRS_new) (15), orf8 (17) and tdh genes, and the absence of the trh gene found in some pathogenic strains. However, numerous serovariants have apparently emerged since 1996 (16). Genome sequencing of the RIMD 2210633 pandemic strain revealed two sets of gene clusters encoding a type III secretion system apparatus, one in each of its two chromosomes (14).Since 2004, we have characterized the strains of V. parahaemolyticus in both clinical cases and shellfish in a southern region of Chile (Region de los Lagos) in an effort to understand the proliferation of the pathogenic strains in the environment (7, 8, 11). Region de los Lagos extends from 40°13′S to 44°3′S and produces approximately 80% of the seafood in Chile (Anuario 2008 Sernapesca [http://www.sernapesca.cl]). It is generally accepted that the seafood from this region causes most of the clinical cases of V. parahaemolyticus-associated diarrhea observed in the entire country. The large diarrhea outbreaks related to seafood consumption started in this region in 2004. In 2005, cases reported by the Ministry of Health reached a peak of 3,600 and 10,984 in Region de los Lagos and the whole country, respectively. Since then, the number of cases has oscillated between 450 and 1,100 cases annually in Region de los Lagos and between 1,500 and 3,500 in the country as a whole (19).Until 2007, more than 95% of the cases were related to the classical pandemic V. parahaemolyticus strain O3:K6 (7, 8). Variants of the pandemic strain were recovered in the summer of 2007, when the outbreaks diminished to 477 reported cases in Region de los Lagos. That year, many cases were caused by a new serovar of the pandemic strain, O3:K59 (11). This same year, a larger percentage of cases analyzed (27%) were due to nonpandemic strains. Some of these last cases corresponded to a strain apparently generated by transference of the pathogenicity island containing the type III secretion island from the pandemic clone to an indigenous V. parahaemolyticus strain (11). Another example of interactions between the pandemic strain and native microflora is the finding of variants containing a 42-kb plasmid corresponding to a telomeric temperate phage (24). The observations in 2007 suggested that the changes in the epidemiology of seafood-related diarrhea represented an inflection point in outbreak trends and a decreased prevalence of the pandemic strain in clinical cases. We present here the results of the analysis of V. parahaemolyticus in clinical cases and shellfish samples obtained during the summer of 2008, when reported cases unexpectedly increased from 477 to 1,143, and the summer of 2009, when clinical cases decreased to 441 (http://epi.minsal.cl/epi/html/elvigia/elvigia.htm). The number of cases observed in 2009 was the lowest since the beginning of large outbreaks in 2004. Overall, our observations illustrate the dynamics of V. parahaemolyticus population in outbreaks of diarrhea. They show the following: (i) that the pandemic strain has become a relatively stable subpopulation of the V. parahaemolyticus population in shellfish, (ii) that pandemic strain variants have emerged, and (iii) that V. parahaemolyticus pathogenic strains unrelated to the pandemic strains become evident when the number of diarrhea cases due to the pandemic strain are low. These data will be helpful in the understanding of V. parahaemolyticus ecology and improving the risk analysis of seafood related diarrhea.