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Applied and Environmental Microbiology, November 2006, p. 7205-7211, Vol. 72, No. 11
0099-2240/06/$08.00+0 doi:10.1128/AEM.01091-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Validation of a Green Fluorescent Protein-Labeled Strain of Vibrio vulnificus for Use in the Evaluation of Postharvest Strategies for Handling of Raw Oysters
,
S. L. Drake,
D. Elhanafi,
W. Bang,
M. A. Drake,
D. P. Green, and
L. A. Jaykus*
Food Science Department, North Carolina State University, Raleigh, North Carolina 27695-7624
Received 11 May 2006/ Accepted 4 September 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 (–20o 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 log10 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 (102 CFU/oyster), but slowly cooled oysters and oysters stored under conservative harvest conditions still contained approximately 103 and >104 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.
* Corresponding author. Mailing address: Food Science Department, North Carolina State University, Raleigh, NC 27695-7624. Phone: (919) 513-2074. Fax: (919) 513-0014. E-mail: leeann_jaykus{at}ncsu.edu.
Paper number FSR 06-25 in the Journal Series of the Department of Food Science, North Carolina State University, Raleigh.
Published ahead of print on 15 September 2006.
Applied and Environmental Microbiology, November 2006, p. 7205-7211, Vol. 72, No. 11
0099-2240/06/$08.00+0 doi:10.1128/AEM.01091-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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