Cold Storage and Mild Heat Treatment as Processing Aids to Reduce the Numbers of Vibrio vulnificus in Raw Oysters

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...

mentioning

confidence: 99%

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

Wright

Garrido

Debuex

et al. 2007

“…However, with the exception of V. cholerae, V. vulnificus, and V. parahaemolyticus, relatively little is known about the susceptibility of any of the lesser-known vibrios to various food preservation methods. Cook and Ruple (20) reported that decimal reduction time of 78 s at 47°C was useful in decreasing the number of V. vulnificus in raw oysters and that heating oysters for 10 min in water at 50°C was sufficient to reduce V. vulnificus populations to undetectable levels. Thermal D values (times required to reduce the viable population of a given strain by 90%) and Z values (absolute values of the temperature required to reduce 1 log unit scale of D values) for encapsulated V. vulnificus cells were reported by Kim et al (31) to be higher than those for unencapsulated cells.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Vibrio fluvialis -LikeStrains Implicated in Limp LobsterDisease

Tall

Fall

Pereira

et al. 2003

Studies were undertaken to characterize and determine the pathogenic mechanisms involved in a newly described systemic disease in Homarus americanus (American lobster) caused by a Vibrio fluvialis-like microorganism. Nineteen isolates were obtained from eight of nine lobsters sampled. Biochemically, the isolates resembled V. fluvialis, and the isolates grew optimally at 20°C; none could grow at temperatures above 23°C. The type strain (1AMA) displayed a thermal reduction time (D value) of 5.77 min at 37°C. All of the isolates required at least 1% NaCl for growth. Collectively, the data suggest that these isolates may embody a new biotype. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates revealed five closely related subgroups. Some isolates produced a sheep hemagglutinin that was neither an outer membrane protein nor a metalloprotease. Several isolates possessed capsules. The isolates were highly susceptible to a variety of antibiotics tested. However, six isolates were resistant to erythromycin. Seventeen isolates harbored plasmids. Lobster challenge studies revealed that the 50% lethal dose of a plasmid-positive strain was 100-fold lower than that of a plasmid-negative strain, suggesting that the plasmid may enhance the pathogenicity of these microorganisms in lobsters. Microorganisms that were recovered from experimentally infected lobsters exhibited biochemical and PFGE profiles that were indistinguishable from those of the challenge strain. Tissue affinity studies demonstrated that the challenge microorganisms accumulated in heart and midgut tissues as well as in the hemolymph. Culture supernatants and polymyxin B lysates of the strains caused elongation of CHO cells in tissue culture, suggesting the presence of a hitherto unknown enterotoxin. Both plasmid-positive and plasmid-negative strains caused significant dose-related intestinal fluid accumulations in suckling mice. Absence of viable organisms in the intestinal contents of mice suggests that these microorganisms cause diarrhea in mice by intoxication rather than by an infectious process. Further, these results support the thermal reduction data at 37°C and suggest that the mechanism(s) that led to fluid accumulation in mice differs from the disease process observed in lobsters by requiring neither the persistence of viable microorganisms nor the presence of plasmids. In summary, results of lobster studies satisfy Koch's postulates at the organismal and molecular levels; the findings support the hypothesis that these V. fluvialis-like organisms were responsible for the originally described systemic disease, which is now called limp lobster disease.Catastrophic losses of Homarus americanus (American lobster) have been most consistently associated with gaffkemia, a disease caused by Aerococcus viridans subsp. homari (52,59). Recognized more than half a century ago as a cause of heavy mortalities in natural populations and impounded lobsters on the east coast of North America, infection usually results when A. viridans breaches the integu...

“…During the experiments, oysters were kept at 23°C on a bench top so that any reduction in the Vv-GFP level could be attributed to the acidic conditions alone. Samples were plated for recovery on TSAN 2 K with and without sodium pyruvate immediately following exposure (time zero); after 4,8,12,16,20, and 24 h for pH 5.0 (both acetic and citric acids); and after 2, 4, 6, and 8 h for pH 4.0 (acetic acid) or pH 3.5 (citric acid).…”

Section: Methodsmentioning

confidence: 99%

Validation of a Green Fluorescent Protein-Labeled Strain of Vibrio vulnificus for Use in the Evaluation of Postharvest Strategies for Handling of Raw Oysters

Drake

Elhanafi

Bang

et al. 2006

In this paper we describe a biological indicator which can be used to study the behavior of Vibrio vulnificus, an important molluscan shellfish-associated human pathogen. A V. vulnificus ATCC 27562 derivative that expresses green fluorescent protein (GFP) and kanamycin resistance was constructed using conjugation. Strain validation was performed by comparing the GFP-expressing strain (Vv-GFP) and the wild-type strain (Vv-WT) with respect to growth characteristics, heat tolerance (45°C), freeze-thaw tolerance (؊20 o and ؊80°C), acid tolerance (pH 5.0, 4.0, and 3.5), cold storage tolerance (5°C), cold adaptation (15°C), and response to starvation. Levels of recovery were evaluated using nonselective medium (tryptic soy agar containing 2% NaCl) with and without sodium pyruvate. The indicator strain was subsequently used to evaluate the survival of V. vulnificus in oysters exposed to organic acids (citric and acetic acids) and various cooling regimens. In most cases, Vv-GFP was comparable to Vv-WT with respect to growth and survival upon exposure to various biological stressors; when differences between the GFP-expressing and parent strains occurred, they usually disappeared when sodium pyruvate was added to media. When V. vulnificus was inoculated into shellstock oysters, the counts dropped 2 log 10 after 11 to 12 days of refrigerated storage, regardless of the way in which the oysters were initially cooled. Steeper population declines after 12 days of refrigerated storage were observed for both iced and refrigerated products than for slowly cooled product and product held under conservative harvest conditions. By the end of the refrigeration storage study (22 days), the counts of Vv-GFP in iced and refrigerated oysters had reached the limit of detection (10 2 CFU/oyster), but slowly cooled oysters and oysters stored under conservative harvest conditions still contained approximately 10 3 and >10 4 CFU V. vulnificus/ oyster by day 22, respectively. The Vv-GFP levels in the oyster meat remained stable for up to 24 h when the meat was exposed to acidic conditions at various pH values. Ease of detection and comparability to the wild-type parent make Vv-GFP a good candidate for use in studying the behavior of V. vulnificus upon exposure to sublethal stressors that might be encountered during postharvest handling of molluscan shellfish.The consumption of contaminated seafood is a significant cause of food-borne disease in the United States, and raw molluscan shellfish is one of the most important commodities associated with disease transmission (7). Bivalves are filter feeders that concentrate microorganisms in their digestive tracts and can serve as passive carriers of food-borne pathogens, especially since they are often consumed whole and raw. Vibrio vulnificus is an environmentally ubiquitous marine bacterium, and high numbers of this species can be isolated from United States Gulf Coast bivalve molluscan shellfish harvested during warm summer months. This organism is associated with several disease syndromes, the m...