Dataset Information

Development of enhanced selective media for detection of Vibrio parahaemolyticus in oysters.

ABSTRACT: This study was undertaken to develop enhanced selective media for detection of Vibrio parahaemolyticus in oysters. Primarily, tryptic soy agar (TSA) was supplemented with 4.5-5% NaCl, 0.1-0.5% oxgall, and/or 1-2% sodium citrate, and adjusted to pH 8-9. A total of 21 Vibrio spp., 24 indicators, and 26 food-borne isolates were streaked on the modified media, followed by 24 h of incubation at 37 °C. While all the indicators and isolates failed to grow on TSA containing 5% NaCl, 0.5% oxgall, and 2% sodium citrate (TSA_OSS1; pH 9), V. parahaemolyticus was culturable on this selective medium. Particularly, the ability of TSA_OSS1 to quantify V. parahaemolyticus in oysters was superior to thiosulphate citrate bile salts sucrose (TCBS) agar. V. parahaemolyticus distinctly produced its white-yellowish, round, and edge-pointed colony on TSA_OSS1. TSAO_SS1 with high selectivity potentials over TCBS may be a promising alternative for detection of V. parahaemolyticus in seafoods or natural reservoirs.

Supplementary information

The online version contains supplementary material available at (10.1007/s10068-021-00877-0).

SUBMITTER: Yoon JH

PROVIDER: S-EPMC8017045 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Development of enhanced selective media for detection of Vibrio parahaemolyticus in oysters.

Yoon Jae-Hyun JH Bae Young-Min YM Song Hana H Lee Soyul S Moon Sung-Kwon SK Oh Se-Wook SW Lee Sun-Young SY

Food science and biotechnology 20210206 3

This study was undertaken to develop enhanced selective media for detection of Vibrio parahaemolyticus in oysters. Primarily, tryptic soy agar (TSA) was supplemented with 4.5-5% NaCl, 0.1-0.5% oxgall, and/or 1-2% sodium citrate, and adjusted to pH 8-9. A total of 21 Vibrio spp., 24 indicators, and 26 food-borne isolates were streaked on the modified media, followed by 24 h of incubation at 37 °C. While all the indicators and isolates failed to grow on TSA containing 5% NaCl, 0.5% o ...[more]

PMID: 33868758

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Project description:Vibrio parahaemolyticus and Vibrio vulnificus are naturally occurring estuarine bacteria and are the leading causes of seafood-associated infections and mortality in the United States. Though multiple-antibiotic-resistant V. parahaemolyticus and V. vulnificus strains have been reported, resistance patterns in vibrios are not as well documented as those of other foodborne bacterial pathogens. Salinity relaying (SR) is a postharvest processing (PHP) treatment to reduce the abundances of these pathogens in shellfish harvested during the warmer months. The purpose of this study was to evaluate the antimicrobial susceptibility (AMS), pathogenicity, and genetic profiles of V. parahaemolyticus and V. vulnificus recovered from oysters during an oyster relay study. Isolates (V. parahaemolyticus [n = 296] and V. vulnificus [n = 94]) were recovered from oysters before and during the 21-day relaying study to detect virulence genes (tdh and trh) and genes correlated with virulence (vcgC) using multiplex quantitative PCR (qPCR). AMS to 20 different antibiotics was investigated using microbroth dilution, and pulsed-field gel electrophoresis (PFGE) was used to study the genetic profiles of the isolates. Twenty percent of V. vulnificus isolates were vcgC+, while 1 and 2% of V. parahaemolyticus were tdh+ and trh+, respectively. More than 77% of the V. vulnificus isolates and 30% of the V. parahaemolyticus isolates were resistant to at least one antimicrobial. Forty-eight percent of V. vulnificus and 8% of V. parahaemolyticus isolates were resistant to two or more antimicrobials. All isolates demonstrated a high genetic diversity, even among those isolated from the same site and having a similar AMS profile. No significant effects of the relaying process on AMS, virulence genes, or PFGE profiles of V. vulnificus and V. parahaemolyticus were observed.IMPORTANCE Analysis of the antibiotic resistance profiles of V. vulnificus and V. parahaemolyticus isolated from oysters during this study indicated that more than 48% of V. vulnificus isolates were resistant to two or more antimicrobials, including those recommended by the CDC for treating Vibrio infections. Also, the V. parahaemolyticus isolates showed high MICs for some of the Vibrio infection treatment antibiotics. Monitoring of AMS profiles of this bacterium is important to ensure optimal treatment of infections and improve food safety. Our study showed no significant differences in the AMS profiles of V. vulnificus (P = 0.26) and V. parahaemolyticus (P = 0.23) isolated from the oysters collected before versus after relaying. This suggests that the salinity of the relaying sites did not affect the AMS profiles of the Vibrio isolates, although it did reduce the numbers of these bacteria in oysters (S. Parveen et al., J Food Sci 82:484-491, 2017, https://doi.org/10.1111/1750-3841.13584).

Project description:This study identified Vibrio parahaemolyticus in oyster and seawater samples collected from Delaware Bay from June through October of 2016. Environmental parameters including water temperature, salinity, dissolved oxygen, pH, and chlorophyll a were measured per sampling event. Oysters homogenate and seawater samples were 10-fold serially diluted and directly plated on CHROMagarᵀᴹ Vibrio medium. Presumptive V. parahaemolyticus colonies were counted and at least 20% of these colonies were selected for molecular chracterization. V. parahaemolyticus isolates (n = 165) were screened for the presence of the species-specific thermolabile hemolysin (tlh) gene, the pathogenic thermostable direct hemolysin (tdh)/ thermostable related hemolysin (trh) genes, the regulatory transmembrane DNA-binding gene (toxR), and V. parahaemolyticus metalloprotease (vpm) gene using a conventional PCR. The highest mean levels of the presumptive V. parahaemolyticus were 9.63×103 CFU/g and 1.85×103 CFU/mL in the oyster and seawater samples, respectively, during the month of July. V. parahaemolyticus levels in oyster and seawater samples were significantly positively correlated with water temperature. Of the 165 isolates, 137 (83%), 110 (66.7%), and 108 (65%) were tlh+, vpm+, and toxR+, respectively. Among the V. parahaemolyticus (tlh+) isolates, 7 (5.1%) and 15 (10.9%) were tdh+ and trh+, respectively, and 24 (17.5%), only oyster isolates, were positive for both genes. Potential pathogenic strains that possessed tdh and/or trh were notably higher in oyster (39%) than seawater (15.6%) isolates. The occurrence of total V. parahaemolyticus (tlh+) was not necessarily proportional to the potential pathogenic V. parahaemolyticus. Co-occurrence of the five genetic markers were observed only among oyster isolates. The co-occurrence of the gene markers showed a relatedness potential of tdh occurrence with vpm. We believe exploring the role of V. parahaemolyticus metalloprotease and whether it is involved in the toxic activity of the thermostable direct hemolysin (TDH) protein can be of significance. The outcomes of this study will provide some foundation for future studies regarding pathogenic Vibrio dynamics in relation to environmental quality.

Project description:BackgroundVibrio parahaemolyticus is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are known as major virulence determinants of V. parahaemolyticus. Most V. parahaemolyticus isolates from the environment do not produce TDH or TRH. Total V. parahaemolyticus has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total V. parahaemolyticus using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of Vibrio parahaemolyticus.ResultsThe assay provided markedly more sensitive and rapid detection of V. parahaemolyticus strains than conventional biochemical and PCR assays. The assay correctly identified 143 V. parahaemolyticus strains, but did not detect 33 non-parahaemolyticus Vibrio and 56 non-Vibrio strains. Sensitivity of the LAMP assay for direct detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples was 5.3 x 10(2) CFU per ml/g (2.0 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13-22 min in a single colony on TCBS agar from each of 143 V. parahaemolyticus strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of V. parahaemolyticus required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination.ConclusionThe LAMP assay is a sensitive, rapid and simple tool for the detection of V. parahaemolyticus and will facilitate the surveillance for control of contamination of V. parahaemolyticus in seafood.

Project description:UnlabelledIn the summer of 2010, Vibrio parahaemolyticus caused an outbreak in Maryland linked to the consumption of oysters. Strains isolated from both stool and oyster samples were indistinguishable by pulsed-field gel electrophoresis (PFGE). However, the oysters contained other potentially pathogenic V. parahaemolyticus strains exhibiting different PFGE patterns. In order to assess the identity, genetic makeup, relatedness, and potential pathogenicity of the V. parahaemolyticus strains, we sequenced 11 such strains (2 clinical strains and 9 oyster strains). We analyzed these genomes by in silico multilocus sequence typing (MLST) and determined their phylogeny using a whole-genome MLST (wgMLST) analysis. Our in silico MLST analysis identified six different sequence types (STs) (ST8, ST676, ST810, ST811, ST34, and ST768), with both of the clinical and four of the oyster strains being identified as belonging to ST8. Using wgMLST, we showed that the ST8 strains from clinical and oyster samples were nearly indistinguishable and belonged to the same outbreak, confirming that local oysters were the source of the infections. The remaining oyster strains were genetically diverse, differing in >3,000 loci from the Maryland ST8 strains. eBURST analysis comparing these strains with strains of other STs available at the V. parahaemolyticus MLST website showed that the Maryland ST8 strains belonged to a clonal complex endemic to Asia. This indicates that the ST8 isolates from clinical and oyster sources were likely not endemic to Maryland. Finally, this study demonstrates the utility of whole-genome sequencing (WGS) and associated analyses for source-tracking investigations.ImportanceVibrio parahaemolyticus is an important foodborne pathogen and the leading cause of bacterial infections in the United States associated with the consumption of seafood. In the summer of 2010, Vibrio parahaemolyticus caused an outbreak in Maryland linked to oyster consumption. Strains isolated from stool and oyster samples were indistinguishable by pulsed-field gel electrophoresis (PFGE). The oysters also contained other potentially pathogenic V. parahaemolyticus strains with different PFGE patterns. Since their identity, genetic makeup, relatedness, and potential pathogenicity were unknown, their genomes were determined by using next-generation sequencing. Whole-genome sequencing (WGS) analysis by whole-genome multilocus sequence typing (wgMLST) allowed (i) identification of clinical and oyster strains with matching PFGE profiles as belonging to ST8, (ii) determination of oyster strain diversity, and (iii) identification of the clinical strains as belonging to a clonal complex (CC) described only in Asia. Finally, WGS and associated analyses demonstrated their utility for trace-back investigations.

Dataset Information

Development of enhanced selective media for detection of Vibrio parahaemolyticus in oysters.

Supplementary information

Publications

Development of enhanced selective media for detection of <i>Vibrio parahaemolyticus</i> in oysters.

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