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Detecting potentially virulent Vibrio vulnificus strains in raw oysters by quantitative loop-mediated isothermal amplification.

ABSTRACT: Vibrio vulnificus is a leading cause of seafood-related deaths in the United States. Sequence variations in the virulence-correlated gene (vcg) have been used to distinguish between clinical and environmental V. vulnificus strains, with a strong association between clinical ones and the C sequence variant (vcgC). In this study, vcgC was selected as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, sensitive, specific, and quantitative detection of potentially virulent V. vulnificus strains in raw oysters. No false-positive or false-negative results were generated among the 125 bacterial strains used to evaluate assay specificity. The detection limit was 5.4 CFU per reaction for a virulent V. vulnificus strain (ATCC 33815) in pure culture, 100-fold more sensitive than that of PCR. In spiked raw oysters, the assay was capable of detecting 2.5 × 10(3) CFU/g of V. vulnificus ATCC 33815, while showing negative results for a nonvirulent V. vulnificus strain (515-4c2) spiked at 10(7) CFU/g. After 6 h of enrichment, the LAMP assay could detect 1 CFU/g of the virulent V. vulnificus strain ATCC 33815. Standard curves generated in pure culture and spiked oysters suggested a good linear relationship between cell numbers of the virulent V. vulnificus strain and turbidity signals. In conclusion, the LAMP assay developed in this study could quantitatively detect potentially virulent V. vulnificus in raw oysters with high speed, specificity, and sensitivity, which may facilitate better control of V. vulnificus risks associated with raw oyster consumption.

SUBMITTER: Han F

PROVIDER: S-EPMC3126371 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Detecting potentially virulent Vibrio vulnificus strains in raw oysters by quantitative loop-mediated isothermal amplification.

Han Feifei F Wang Fei F Ge Beilei B

Applied and environmental microbiology 20110225 8

Vibrio vulnificus is a leading cause of seafood-related deaths in the United States. Sequence variations in the virulence-correlated gene (vcg) have been used to distinguish between clinical and environmental V. vulnificus strains, with a strong association between clinical ones and the C sequence variant (vcgC). In this study, vcgC was selected as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, sensitive, specific, and quantitative detection of potentia ...[more]

PMID: 21357428

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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).

Dataset Information

Detecting potentially virulent Vibrio vulnificus strains in raw oysters by quantitative loop-mediated isothermal amplification.

Publications

Detecting potentially virulent Vibrio vulnificus strains in raw oysters by quantitative loop-mediated isothermal amplification.

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