Evaluation of Postharvest-Processed Oysters by Using PCR-Based Most-Probable-Number Enumeration of Vibrio vulnificus Bacteria

Abstract

Postharvest processing (PHP) is used to reduce levels of Vibrio vulnificus in oysters, but process validation is labor-intensive and expensive. Therefore, quantitative PCR was evaluated as a rapid confirmation method for most-probable-number enumeration (QPCR-MPN) of V. vulnificus bacteria in PHP oysters. QPCR-MPN showed excellent correlation (R 2 ‫؍‬ 0.97) with standard MPN and increased assay sensitivity and efficiency.Vibrio vulnificus can cause life-threatening, systemic disease (2, 9, 14) that is associated with the consumption of raw oysters. The bacterium is distributed throughout temperate estuaries worldwide (6,7,17,24,25), and environmental conditions of warmer water temperature and lower salinity favor its growth in molluscan shellfish (5,13,16,21,23,25). Warning labels on oyster products and educational programs have not been effective in reducing disease mortality rates for at-risk individuals with underlying diseases, such as cirrhosis, hemochromatosis (iron overload), diabetes, or immune system dysfunction (8). Therefore, the FDA and the Interstate Shellfish Sanitation Conference (ISSC) have mandated postharvest processing (PHP) of oysters harvested from Gulf Coast states in order to reduce V. vulnificus infections (11). Application of PHP methodology requires validation and verification in order to ensure that the process will substantially reduce numbers of V. vulnificus bacteria to levels that are below the predicted threshold for disease. Validation trials for PHP are laborintensive and cost-prohibitive (10, 12), and improved protocols for industry compliance and for risk assessment of V. vulnificus in PHP oysters are urgently needed.The standard method for validating PHP requires that three independent lots of oysters meet the specification of Ͻ30 most probable numbers (MPN)/g of V. vulnificus by using the geometric mean of 10 samples/lot. Levels of V. vulnificus bacteria in oysters are enumerated by MPN endpoint titration of replicate samples in enrichment broth cultures (12), and speciesspecific growth is determined by isolating typical V. vulnificus colonies on selective medium, with subsequent confirmation by DNA probe (26). The present study evaluated a real-time quantitative PCR (QPCR) assay for detection of V. vulnificus growth in MPN enrichment cultures (QPCR-MPN). QPCR increases assay throughput by using automated species-specific confirmation, which is not available with standard PCR. Also, the limit of detection for direct QPCR enumeration of V. vulnificus bacteria in oysters without enrichment is generally 100 CFU/g, but QPCR-MPN assays generally increase the sensitivity of detection to 1 bacterium/g after enrichment (1,18,19,20) and permit detection of V. vulnificus at levels (30 CFU/g) required for validation protocols.Field trials were conducted to assess application of QPCR-MPN to oyster PHP validation, as application of QPCR-MPN to enumerate V. vulnificus bacteria in PHP oysters has not been examined previously and merits further scrutiny. For example, large numbers of dead b...

Comparison of surficial CO2 efflux to other measures of subsurface crude oil degradation

“…Microbial concentrations were measured using quantitative Polymerase Chain Reaction (qPCR), a method to determine microbial concentrations using function-specific DNA fragments (Heid et al, 1996) that has been shown to correlate with the Most Probable Number (MPN) growth method (Lavender and Kinzelman, 2009;Wright et al, 2007). qPCR has been shown to be less labor-intensive than the MPN method, but perhaps more importantly, qPCR data have been shown in some cases to correlate with reaction rates at other sites (Braker and Conrad, 2011;Wang et al, 2011).…”

Vancomycin-Resistant Enterococci and Bacterial Community Structure following a Sewage Spill into an Aquatic Environment

“…V. vulnificus is an autochthonous marine bacterium that does not grow in freshwater environments. Reaction mixtures contained 4 l of DNA sample and 1 l of V. vulnificus DNA (20,000 copies) and were compared to a control reaction mixture containing 4 l of nuclease-free water and 1 l of V. vulnificus DNA (20,000 copies) using previously published cycling conditions and primers (76). Accession number(s).…”

Rapid MPN-Qpcr Screening for Pathogens in Air, Soil, Water, and Agricultural Produce

“…Such steps could be immunomagnetic separation (Warren et al 2007), preamplification of the target DNA (Ishii et al 2013), or nonspecific enrichment (Edel and Kampelmacher 1973), aiming to increase the likelihood of detecting (but not directly enumerating) viable pathogens (Krämer et al 2011;Malorny et al 2004). The combination of nonspecific enrichment followed by DNA extraction and qPCR analysis coupled with most probable number (MPN) estimation was found to be a useful tool to increase the likelihood of pathogen detection when initial concentrations are low (Krämer et al 2011;Russo et al 2014;Wright et al 2007). However, several shortcomings precluded the application of the proposed method to more challenging environmental matrices such as soil or aerosols.…”

“…However, several shortcomings precluded the application of the proposed method to more challenging environmental matrices such as soil or aerosols. The processing schemes presented were directed at specific food matrices, i.e., meat (Krämer et al 2011), oysters (Wright et al 2007), or vegetables freshly cut and eaten raw (Russo et al 2014). These food matrices naturally lack the overwhelming bacterial diversity found in soil or wastewater; thus, pathogen enrichment and detection should be simpler.…”

“…These food matrices naturally lack the overwhelming bacterial diversity found in soil or wastewater; thus, pathogen enrichment and detection should be simpler. In addition, the previously presented methods applied enrichment schemes that were aimed at specific pathogens (Krämer et al 2011;Russo et al 2014;Wright et al 2007). In fact, different enrichments were employed if more than one pathogen was targeted (Russo et al 2014).…”