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

Abstract

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...

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