Ecology of \u3ci\u3eVibrio parahaemolyticus\u3c/i\u3e and \u3ci\u3eVibrio vulnificus\u3c/i\u3e 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. parahaetnolyticus 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

A Novel Agar Formulation for Isolation and Direct Enumeration of \u3ci\u3eVibrio vulnificus\u3c/i\u3e from Oyster Tissue

A new selective and differential medium, Vibrio vulnificus X-Gal (VVX), was developed for direct enumeration of V. vulnificus (Vv) from oyster samples. This agar utilizes cellobiose and lactose as carbon sources, and the antibiotics colistin and polymyxin B as selective agents. Hydrolysis of 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (x-gal), used in the agar as a lactose analog, produces an insoluble blue dye that makes lactose positive colonies easily distinguishable from any non-lactose fermenting bacteria. Various bacterial species were spot plated onto thiosulfate-citrate-bile salts-sucrose agar (TCBS), and CHROMagar Vibrio, two vibrio-specific selective agars, nonselective agar, and VVX to compare selectivity of VVX to other widely used media. A V. vulnificus pure culture was serially diluted on VVX and non-selective agar to determine the VVX percent recovery. Water and oyster samples were spread plated on VVX agar and allowed to incubate for 16-18 h at 33 degrees C. Blue and white colonies from VVX agar were picked and screened by end point PCR for the Vv hemolysin vvhA. VVX agar showed a significant improvement over TCBS and CHROMagar at preventing non-target growth. There was an 87.5% recovery compared to non-selective plating and a 98% positivity rate of blue colonies picked from oyster tissue plating. The findings suggest that this new agar is a fast, distinctive, and accurate method for enumeration of V. vulnificus from the environment. (C) 2013 Elsevier B.V. All rights reserved

Abundance and Distribution of \u3ci\u3eVibrio cholerae\u3c/i\u3e, \u3ci\u3eV. parahaemolyticus\u3c/i\u3e, and \u3ci\u3eV. vulnificus\u3c/i\u3e Following a Major Freshwater Intrusion into the Mississippi Sound

In response to a major influx of freshwater to the Mississippi Sound following the opening of the Bonnet Carre Spillway, water samples were collected from three sites along the Mississippi shoreline to assess the impact of altered salinity on three pathogenic Vibrio species. Salinity readings across the affected area during the 2011 sample period ranged from 1.4 to 12.9 ppt (mean = 7.0) and for the 2012 sample period from 14.1 to 23.6 ppt (mean = 19.8). Analyses of the data collected in 2011 showed a reduction in densities of Vibrio parahaemolyticus and Vibrio vulnificus with a concurrent increase of Vibrio cholerae numbers, with V. cholerae becoming the only Vibrio detected once salinity readings dropped to 6 ppt. Follow-up samples taken in 2012 after recovery of the salinity in the sound showed that the relative densities of the three pathogenic vibrios had reverted back to normal levels. This study shows that although the spillway was open but a few weeks and the effects were therefore time limited, the Mississippi River water had a profound, if temporary, effect on Vibrio ecology in the Mississippi Sound

Enumeration of \u3ci\u3eVibrio parahaemolyticus\u3c/i\u3e in the Viable But Nonculturable State Using Direct Plate Counts and Recognition of Individual Gene Fluorescence \u3ci\u3eIn Situ\u3c/i\u3e Hybridization

Vibrio parahaemolyticus is a gram-negative, halophilic bacterium indigenous to marine and estuarine environments and it is capable of causing food and water-borne illness in humans. It can also cause disease in marine animals, including cultured species. Currently, culture-based techniques are used for quantification of V. parahaemolyticus in environmental samples; however, these can be misleading as they fail to detect V. parahaemolyticus in a viable but nonculturable (VBNC) state which leads to an underestimation of the population density. In this study, we used a novel fluorescence visualization technique, called recognition of individual gene fluorescence in situ hybridization (RING-FISH), which targets chromosomal DNA for enumeration. A polynucleotide probe labeled with Cyanine 3 (Cy3) was created corresponding to the ubiquitous V. parahaemolyticus gene that codes for thermolabile hemolysin (tlh). When coupled with the Kogure method to distinguish viable from dead cells, RING-FISH probes reliably enumerated total, viable V. parahaemolyticus. The probe was tested for sensitivity and specificity against a pure culture of tlh+, tdh-, trh-V. parahaemolyticus, pure cultures of Vibrio vulnificus. Vibrio harveyi, Vibrio alginolyticus and Vibrio fischeri, and a mixed environmental sample. This research will provide additional tools for a better understanding of the risk these environmental organisms pose to human health. (C) 2011 Elsevier B.V. All rights reserved

Distribution of Type III Secretion Systems in Vibrio Parahaemolyticus From the Northern Gulf of Mexico

Aims: Two well-characterized Vibrio parahaemolyticus pathogenicity factors - thermostable direct haemolysin (TDH) and TDH-related haemolysin - are produced by strains containing the tdh and trh genes, respectively. Most strains of V. parahaemolyticus contain two nonredundant type III secretion systems (T3SS), T3SS1 and T3SS2, both of which contribute to pathogenicity. Furthermore, a recent study has revealed two distinct lineages of the V. parahaemolyticus T3SS2: T3SS2 alpha and T3SS2 beta. The aim of this study was to determine the incidence of these pathogenicity factors in environmental isolates of V. parahaemolyticus. Methods and Results: We collected 130 V. parahaemolyticus isolates (TCBS agar) containing tdh and/or trh (determined by colony hybridization) from sediment, oyster and water in the northern Gulf of Mexico and screened them and 12 clinical isolates (PCR and agarose gel electrophoresis) for pathogenicity factors tdh, trh, T3SS1, T3SS2 alpha and T3SS2 beta. The majority of potential pathogens were detected in the sediment, including all tdh-/trh+ isolates. T3SS2 alpha components were detected in all tdh+/trh- isolates and zero of 109 trh+ isolates. One T3SS2 alpha gene, vopB2, was found in all tdh+/trh- clinical strains but not in any of the 130 environmental strains. Fluorescence in situ hybridization adapted for individual gene recognition (RING-FISH) was used to confirm the presence/absence of vopB2. T3SS2 beta was found in all tdh-/trh+ isolates and in no tdh+/trh- isolates. Conclusions: The combination of haemolysins found in each isolate consistently corresponded to the presence and type of T3SS detected. The vopB2 gene may represent a novel marker for identifying increased virulence among strains. Significance and Impact of the Study: This is the first study to confirm the presence of T3SS2 beta genes in V. parahaemolyticus strains isolated from the Gulf of Mexico and one of the few that examines the distribution and co-existence of tdh, trh, T3SS1, T3SS2 alpha and T3SS2 beta in a large collection of environmental strains

A Multiple Endpoint Analysis of the Effects of Chronic Exposure to Sediment Contaminated with Deepwater Horizon Oil On Juvenile Southern Flounder and Their Associated Microbiomes

Exposure to oiled sediments can negatively impact the health of fish species. Here, we examine the effects of chronic exposure of juvenile southern flounder, Paralichthys lethostigma, to a sediment-oil mixture. Oil:sediment mixtures are persistent over time and can become bioavailable following sediment perturbation or resuspension. Juvenile flounder were exposed for 32 days under controlled laboratory conditions to five concentrations of naturally weathered Macondo MC252 oil mixed into uncontaminated, field-collected sediments. The percent composition of individual polycyclic aromatic hydrocarbons (PAHs) of the weathered oil did not change after mixing with the sediment. Spiked exposure sediments contained 0.04–395 mg/kg tPAH50 (sum of 50 individual PAH concentration measurements). Mortality increased with both exposure duration and concentration of sediment-associated PAHs, and flounder exposed to concentrations above 8 mg/kg tPAH50 showed significantly reduced growth over the course of the experiment. Evident histopathologic changes were observed in liver and gill tissues of fish exposed to more than 8 mg/kg tPAH50. All fish at these concentrations showed hepatic intravascular congestion, macrovesicular hepatic vacoulation, telangiectasia of secondary lamellae, and lamellar epithelial proliferation in gill tissues. Dose-dependent upregulation of Cyp1a expression in liver tissues was observed. Taxonomic analysis of gill and intestinal commensal bacterial assemblages showed that exposure to oiled sediments led to distinct shifts in commensal bacterial population structures. These data show that chronic exposure to environmentally-relevant concentrations of oiled sediments produces adverse effects in flounder at multiple biological levels

Crude oil impairs immune function and increases susceptibility to pathogenic bacteria in southern flounder - Fig 1

<p><b>(A) Evidence of bloody lesions seen in flounder exposed to oil.</b> Red arrows indicate observed lesions. Fish were not tagged; therefore, lesions were not enumerated. <b>(B) Survival rates in exposure treatments on Day 2 post-exposure to pathogenic bacteria <i>Vibrio anguillarum</i></b>. Bars represent average survival for three tanks per treatment and error bars represent standard error.</p

Nontoxigenic \u3ci\u3eVibrio cholerae\u3c/i\u3e Non-O1/O139 Isolate From a Case of Human Gastroenteritis In the U.S. Gulf Coast

An occurrence of Vibrio cholerae non-O1/O139 gastroenteritis in the U.S. Gulf Coast is reported here. Genomic analysis revealed that the isolate lacked known virulence factors associated with the clinical outcome of a V. cholerae infection but did contain putative genomic islands and other accessory virulence factors. Many of these factors are widespread among environmental strains of V. cholerae, suggesting that there might be additional virulence factors in non-O1/O139 V. cholerae yet to be determined. Phylogenetic analysis revealed that the isolate belonged to a phyletic lineage of environmental V. cholerae isolates associated with sporadic cases of gastroenteritis in the Western Hemisphere, suggesting a need to monitor non-O1/O139 V. cholerae in the interest of public health