Seasonal variation in abundance of total and pathogenic Vibrio parahaemolyticus bacteria in oysters along the southwest coast of India

The seasonal abundance of Vibrio parahaemolyticus in oysters from two estuaries along the southwest coast of India was studied by colony hybridization using nonradioactive labeled oligonucleotide probes. The density of total V. parahaemolyticus bacteria was determined using a probe binding to the tlh (thermolabile hemolysin) gene, and the density of pathogenic V. parahaemolyticus bacteria was determined by using a probe binding to the tdh (thermostable direct hemolysin) gene. Furthermore, the prevalence of V. parahaemolyticus was studied by PCR amplification of the toxR, tdh, and trh genes. PCR was performed directly with oyster homogenates and also following enrichment in alkaline peptone water for 6 and 18 h. V. parahaemolyticus was detected in 93.87% of the samples, and the densities ranged from <10 to 10(4) organisms per g. Pathogenic V. parahaemolyticus could be detected in 5 of 49 samples (10.2%) by colony hybridization using the tdh probe and in 3 of 49 samples (6.1%) by PCR. Isolates from one of the samples belonged to the pandemic serotype O3:K6. Twenty-nine of the 49 samples analyzed (59.3%) were positive as determined by PCR for the presence of the trh gene in the enrichment broth media. trh-positive V. parahaemolyticus was frequently found in oysters from India.     

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.     

Improved recovery of pathogenic Vibrio parahaemolyticus from oysters using colony hybridization following enrichment

The traditional streak plating and alternative spread-plating methods were compared for detection of pathogenic Vibrio parahaemolyticus (Vp) in oyster enrichments. We found the alternative method to be more efficient: it was quicker (2d vs. 3d) and had a significantly (p < 0.05) greater detection rate than streak plating.     

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 >10(4) 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.     

Incidence of Vibrio parahaemolyticus in U.S. coastal waters and oysters

Oyster and seawater samples were collected seasonally from May 1984 through April 1985 from shellfish-growing areas in Washington, California, Texas, Louisiana, Alabama, Florida, South Carolina, Virginia, and Rhode Island which had been designated as approved or prohibited by the National Shellfish Sanitation Program. Fecal coliforms counts, aerobic plate counts, and Vibrio parahaemolyticus densities were determined for the samples. Mean V. parahaemolyticus density was more than 100 times greater in oysters than in water, whereas density of fecal coliforms was approximately 10 times higher in oysters. Seasonal and geographical distributions of V. parahaemolyticus were related to water temperature, with highest densities in samples collected in the spring and the summer along the Gulf coast. The synthetic DNA probe for thermostable direct hemolysin hybridized with 2 of 50 isolates, 1 of which was positive by the Kanagawa test.     

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 > or = 16 microg/ml). Additionally, V. parahaemolyticus displayed significantly higher MICs for cefotaxime, ciprofloxacin, and tetracycline than V. vulnificus.     

Incidence of Vibrio parahaemolyticus in U.S. coastal waters and oysters.

Oyster and seawater samples were collected seasonally from May 1984 through April 1985 from shellfish-growing areas in Washington, California, Texas, Louisiana, Alabama, Florida, South Carolina, Virginia, and Rhode Island which had been designated as approved or prohibited by the National Shellfish Sanitation Program. Fecal coliforms counts, aerobic plate counts, and Vibrio parahaemolyticus densities were determined for the samples. Mean V. parahaemolyticus density was more than 100 times greater in oysters than in water, whereas density of fecal coliforms was approximately 10 times higher in oysters. Seasonal and geographical distributions of V. parahaemolyticus were related to water temperature, with highest densities in samples collected in the spring and the summer along the Gulf coast. The synthetic DNA probe for thermostable direct hemolysin hybridized with 2 of 50 isolates, 1 of which was positive by the Kanagawa test.     

Effect of essential oils on pathogenic and biofilm-forming Vibrio parahaemolyticus strains

Abstract

In this study, the effect of three essential oils (EOs) – clove oil (CO), thyme oil (TO), and garlic oil (GO), which are generally recognized as safe – on the planktonic growth, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), motility, biofilm formation, and quorum sensing (QS) of Vibrio parahaemolyticus was investigated. All three EOs showed bacteriostatic activity, with MICs in the range 0.02%–0.09% (v/v). CO and TO completely controlled planktonic growth at 0.28% and 0.08% (v/v), which is four times their MIC (4?×?MIC), after 10?min, whereas GO completely controlled growth at 0.36% (v/v) (4?×?MIC) after treatment for 20?min. V. parahaemolyticus motility was significantly reduced by all three EOs at 4?×?MIC (0.28% for CO, 0.08% for TO, and 0.36% for GO), whereas QS was controlled and biofilm formation reduced by all three EOs at 8?×?MIC (0.56% for CO, 0.16% for TO, and 0.72% for GO) after 30?min of treatment. These results suggest that CO, TO, and GO have a significant inhibitory effect on V. parahaemolyticus cells in biofilm sand thus represent a promising strategy for improving food safety. These results provide the evidence required to encourage further research into the practical use of the proposed EOs in food preparation processes.     

Use of an oligonucleotide probe to detect Vibrio parahaemolyticus in artificially contaminated oysters.

A 26-mer oligonucleotide specific to Vibrio parahaemolyticus was synthesized from a 1,275-bp thermostable direct hemolysin (tdh) gene. This oligonucleotide probe specifically reacted with DNA from 89 of 95 V. parahaemolyticus isolates but not with DNA from other vibrios or other enteric and nonenteric organisms (n = 48). The probe hybridized with Southern blots of 0.5-kb HindIII-restricted chromosomal DNA fragments from all but five V. parahaemolyticus test isolates. The probe could be used to directly identify V. parahaemolyticus in artificially contaminated food without an isolation step.     

Use of an oligonucleotide probe to detect Vibrio parahaemolyticus in artificially contaminated oysters.

A 26-mer oligonucleotide specific to Vibrio parahaemolyticus was synthesized from a 1,275-bp thermostable direct hemolysin (tdh) gene. This oligonucleotide probe specifically reacted with DNA from 89 of 95 V. parahaemolyticus isolates but not with DNA from other vibrios or other enteric and nonenteric organisms (n = 48). The probe hybridized with Southern blots of 0.5-kb HindIII-restricted chromosomal DNA fragments from all but five V. parahaemolyticus test isolates. The probe could be used to directly identify V. parahaemolyticus in artificially contaminated food without an isolation step.     

A comparison between farmed oysters using floating cages and oysters grown on-bottom reveals more potentially human pathogenic Vibrio in the on-bottom oysters

Eating raw oysters can come with serious health risks, as oysters can potentially contain bacteria of the Vibrio genus that cause food-borne infections. Vibrio bacteria are concentrated by oysters and, when consumed, infections can result with severe symptoms such as diarrhoea, lesions on the extremities, or even death. Vibrio spp. concentrations are strongly affected by season, location, and other factors such as temperature and salinity. Previous research in North Carolina oysters has been conducted on wild and farmed oysters but not at the same time. Farmed, or aquaculture raised, oysters are considerably different from wild oysters and could possibly pose different health risks. Farmed oysters are handled, raised from seed, and often grown using suspended grow-out systems called ‘floating cages’. Therefore, farmed oysters can be grown at the surface of the estuary, while wild oysters typically grow at the bottom of the water column. This project compared the concentrations of Vibrio spp. in suspended, farm-grown oysters and wild oysters at three sites, using a paired approach with farmed and wild oysters sampled in proximity. An important part of this comparison was identifying pathogenicity of the bacteria isolated from the samples. Distinction was made between off- and on-bottom farming. Interestingly, on-bottom oysters had more pathogenic V. vulnificus than off-bottom oysters.     

Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR

A real time polymerase chain reaction (PCR) assay was developed and evaluated to detect the presence of the thermostable direct hemolysin gene (tdh), a current marker of pathogenicity in Vibrio parahaemolyticus. The real time PCR fluorogenic probe and primer set was tested against a panel of numerous strains from 13 different bacterial species. Only V. parahaemolyticus strains possessing the tdh gene generated a fluorescent signal, and no cross-reaction was observed with tdh negative Vibrio or non-Vibrio spp. The assay detected a single colony forming unit (CFU) per reaction of a pure culture template. This sensitivity was achieved when the same template amount per reaction was tested in the presence of 2.5 microl of a tdh negative oyster:APW enrichment (oyster homogenate enriched in alkaline peptone water overnight at 35 degrees C). This real time technique was used to test 131 oyster:APW enrichments from an environmental survey of Alabama oysters collected between March 1999 and September 2000. The results were compared to those previously obtained using a streak plate procedure for culture isolation from the oyster:APW enrichment combined with use of a non-radioactive DNA probe for detection of the tdh gene. Real time PCR detected tdh in 61 samples, whereas the streak plate/probe method detected tdh in 15 samples. Only 24 h was required for detection of pathogenic V. parahaemolyticus in oyster:APW enrichments by real time PCR, whereas the streak plate/probe method required 3 days and was more resource intensive. This study demonstrated that real time PCR is a rapid and reliable technique for detecting V. parahaemolyticus possessing the tdh gene in pure cultures and in oyster enrichments.     

Evaluation of two direct plating methods using nonradioactive probes for enumeration of Vibrio parahaemolyticus in oysters

Oysters (Crassostrea virginica) were collected monthly from May 1998 to April 1999 from Mobile Bay, Ala., and analyzed to determine Vibrio parahaemolyticus densities at zero time and after 5, 10, and 24 h of postharvest storage at 26 degrees C. After 24 h of storage at 26 degrees C, oysters were transferred to a refrigerator at 3 degrees C and then analyzed 14 to 17 days later. The V. parahaemolyticus numbers were determined by the most-probable-number procedure using alkaline phosphatase-labeled DNA probe VPAP, which targets the species-specific thermolabile hemolysin gene (tlh), to identify suspect isolates (MPN-VPAP procedure). Two direct plating methods, one using a VPAP probe (Direct-VPAP) and one using a digoxigenin-labeled probe (Direct-VPDig) to identify suspect colonies, were compared to the MPN-VPAP procedure. The results of the Direct-VPAP and Direct-VPDig techniques were highly correlated (r = 0.91), as were the results of the Direct-VPAP and MPN-VPAP procedures (r = 0.91). The correlation between the Direct-VPDig and MPN-VPAP results was 0.85. The two direct plating methods in which nonradioactive DNA probes were used were equivalent to the MPN-VPAP procedure for identification of total V. parahaemolyticus, and they were more rapid and less labor-intensive.     

四重PCR检测副溶血性弧菌及其毒力基因方法的建立

【目的】建立同时检测副溶血性弧菌tox R、tdh、trh、tlh基因的四重PCR快速检测方法。【方法】分别以副溶血性弧菌的tox R、tdh、trh、tlh 4个基因为靶基因,设计4对特异性引物,对4对引物浓度和退火温度进行优化,获得最佳引物比例和扩增条件,建立快速检测致病性副溶血性弧菌的四重PCR体系。通过特异性验证、灵敏度验证以及模拟样品检测进行方法确认。【结果】四重PCR体系扩增条带与预期相符,即115 bp(tox R)、244 bp(tdh)、418 bp(trh)、759 bp(tlh)4个目的条带;用74株副溶血性弧菌和37株非目标菌的测试结果表明,所建立的方法有良好的特异性。该方法对模板DNA的检测灵敏度为50μg/L,纯培养物的检测灵敏度为6.7×103 CFU/m L;副溶血性弧菌含量为1.36 CFU/g的人工模拟样品增菌6 h后,tox R、tlh、tdh、trh 4个基因可同时被检出。【结论】该方法可实现同时检测携带tox R、tdh、trh、tlh 4种基因的副溶血性弧菌,对开展致病性副溶血性弧菌的检测研究具有一定现实意义。     

Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea, Japan

Seawater and organic material (live and/or dead matter deposited on any substratum submersed in seawater) were collected during the cool weather season from a coast of the Seto-Inland Sea, Japan, and analyzed to determine Vibrio parahaemolyticus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by the polymerase chain reaction (PCR), using isolated DNA from enrichment culture of the samples. About 95% of the samples were positive for V. parahaemolyticus (with densities of 3 to >1400 cells per 100 ml water or 10 g organic samples) by the most-probable-number (MPN)-PCR technique with species-specific toxR primers, but only 40% were positive by the conventional MPN-culture technique (with densities ranging from 3 to 240 cells per 100 ml water or 10 g organics). Furthermore, the tdh and trh genes were positive in 55% and 20% of samples, respectively, by the MPN-PCR technique. No tdh and trh gene-positive strains were isolated by the conventional MPN-culture procedure. The difference in detection between the MPN-culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detection sensitivities and other factors.     

Predictive models for the effect of storage temperature on Vibrio parahaemolyticus viability and counts of total viable bacteria in Pacific oysters (Crassostrea gigas)

Vibrio parahaemolyticus is an indigenous bacterium of marine environments. It accumulates in oysters and may reach levels that cause human illness when postharvest temperatures are not properly controlled and oysters are consumed raw or undercooked. Predictive models were produced by injecting Pacific oysters (Crassostrea gigas) with a cocktail of V. parahaemolyticus strains, measuring viability rates at storage temperatures from 3.6 to 30.4°C, and fitting the data to a model to obtain parameter estimates. The models were evaluated with Pacific and Sydney Rock oysters (Saccostrea glomerata) containing natural populations of V. parahaemolyticus. V. parahaemolyticus viability was measured by direct plating samples on thiosulfate-citrate-bile salts-sucrose (TCBS) agar for injected oysters and by most probable number (MPN)-PCR for oysters containing natural populations. In parallel, total viable bacterial counts (TVC) were measured by direct plating on marine agar. Growth/inactivation rates for V. parahaemolyticus were −0.006, −0.004, −0.005, −0.003, 0.030, 0.075, 0.095, and 0.282 log₁₀ CFU/h at 3.6, 6.2, 9.6, 12.6, 18.4, 20.0, 25.7, and 30.4°C, respectively. The growth rates for TVC were 0.015, 0.023, 0.016, 0.048, 0.055, 0.071, 0.133, and 0.135 log₁₀ CFU/h at 3.6, 6.2, 9.3, 14.9, 18.4, 20.0, 25.7, and 30.4°C, respectively. Square root and Arrhenius-type secondary models were generated for V. parahaemolyticus growth and inactivation kinetic data, respectively. A square root model was produced for TVC growth. Evaluation studies showed that predictive growth for V. parahaemolyticus and TVC were “fail safe.” The models can assist oyster companies and regulators in implementing management strategies to minimize V. parahaemolyticus risk and enhancing product quality in supply chains.     

Recovery of chill-stressed Vibrio parahaemolyticus from oysters with enrichment broths supplemented with magnesium and iron salts.

The effects of magnesium and iron salts on the recovery and growth of chill-stressed cells of Vibrio parahaemolyticus were studied. Supplementation of glucose salt Teepol (GST) broth with 20 to 100 mM of Mg2+ significantly (P less than or equal to 0.05) increased the number of cells recovered from oyster homogenate stored at 3 degrees C. Populations detected with supplemented GST were comparable to those obtained with Horie arabinose ethyl violet (HAE) broth, with or without Mg2+. Recovery of V. parahaemolyticus from homogenates stored at -18 degrees C was also improved when enrichment broths supplemented with Mg2+ were used. Ferric iron (added as FeCl3) at 240 microM in GST and 240 or 960 microM in HAE significantly enhanced the extent of recovery of chilled cells. Ferrous iron was generally less effective. Teepol did not influence the growth of nonchilled cells, but significantly reduced the viable population in suspensions of chilled cells when used at a level of 0.4% in GST. The relatively high pH (9.0) of HAE caused a significant reduction in the number of viable, chill-stressed cells of V. parahaemolyticus. The overall results indicated that HAE broth is superior to GST for recovering V. parahaemolyticus from refrigerated and frozen oyster homogenates.