Enumeration of Vibrio parahaemolyticus in oyster tissues following artificial contamination and depuration

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1 Letters in Applied Microbiology ISSN ORIGINAL ARTICLE Enumeration of Vibrio parahaemolyticus in oyster tissues following artificial contamination and depuration D. Wang, S. Yu, W. Chen, D. Zhang and X. Shi Department of Food Science & Technology and Bor Luh Food Safety Center, School of Agriculture and Biology, Shanghai JiaoTong University, Shanghai, China Keywords dynamic, enumeration, oyster tissues, Vibrio parahaemolyticus. Correspondence Xianming Shi, Department of Food Science and Technology, Shanghai JiaoTong University, 800 Dongchuan Rd., Shanghai , China : received 15 March 2010, revised 25 April 2010 and accepted 26 April 2010 doi: /j x x Abstract Aims: To evaluate enumeration of Vibrio parahaemolyticus in oyster tissues following artificial contamination and depuration. Methods and Results: After inoculating with V. parahaemolyticus (ATCC 17802) and incubating for 24 h, the contaminated oysters were depurated with artificial seawater for 14 days. At each step, the tissue homogenate supernatants of oysters were spread-plated onto thiosulfate-citrate-bile salt-sucrose agar, followed by colony confirmation by the polymerase chain reaction. The pathogen was detected in the gills, digestive glands (including stomach, digestive ducts and digestive diverticula), adductor muscle and mantle cilia. After a 48-h depuration period at C, the retention rate of V. parahaemolyticus in the gills (28Æ1%) and digestive glands (13Æ5%) was higher than that in adductor muscle and mantle cilia (1Æ4 and 2Æ4%, respectively). Conclusions: The population of V. parahaemolyticus in the digestive glands was the highest among all tissues tested, followed by the gills. The data indicate that digestive glands and gills are good sample candidates for direct monitoring of V. parahaemolyticus contamination in oysters. Significance and Impact of the Study: This is the first report on the dynamics of V. parahaemolyticus in various oyster tissues following artificial contamination and depuration. This study provides information to help in monitoring for V. parahaemolyticus in commercial oysters. Introduction Vibrio parahaemolyticus, a halophilic bacterium, is one of the major causative agents of acute gastroenteritis (Potasman et al. 2002; Sala et al. 2009). A thermostable direct haemolysin (TDH) and a TDH-related haemolysin are the major virulence factors (Drake et al. 2007). Many outbreaks of V. parahaemolyticus that have occurred throughout the world (Fukushima et al. 2007; Sala et al. 2009) have been associated with consumption of raw or under-cooked oysters (Feldhusen 2000; Potasman et al. 2002; DePaola et al. 2003; Mahmoud and Burrage 2009; Sala et al. 2009). In China (Wang et al. 2007), the United States and Japan (Su and Liu 2007), V. parahaemolyticus is currently a major cause of bacterial diarrhoea associated with seafood consumption. Oysters can bioaccumulate V. parahaemolyticus (Potasman et al. 2002; Drake et al. 2007) and are important vehicles for the transmission of the pathogen (Potasman et al. 2002; Su and Liu 2007). For detection of V. parahaemolyticus in oysters, however, several hours of enrichment are necessary even when sensitive molecular detection methods are used (Nordstrom et al. 2007; Yamazaki et al. 2008). Thus, prolonged testing time extends the time for release of oysters to market, which can have adverse effects on the quality and market value of this highly perishable food. To reduce testing time and improve the accuracy of results, efforts need to be made to identify the target tissues that contain a high concentration of the pathogen. Therefore, the abundance of V. parahaemolyticus in different oyster tissues needs to be investigated. 104 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010)

2 D. Wang et al. Abundance of V. parahaemolyticus in oyster tissues Green fluorescent protein (GFP) tracing has been utilized to indirectly locate V. parahaemolyticus in the gills and other tissues (Cabello et al. 2005). Although V. parahaemolyticus has been detected in the gills (Sarkar et al. 1985; Cabello et al. 2005; Luan et al. 2008) and in individual oysters (Parveen et al. 2008), the actual levels of the pathogen in specific oyster tissues are not yet clear. Therefore, this study investigated the abundance of V. parahaemolyticus (ATCC 17802) in various oyster tissues following artificial contamination and depuration, to determine which tissues may be suitable for direct monitoring of this pathogen. Materials and methods Bacterium and culture conditions TDH-producing V. parahaemolyticus (ATCC 17802), which was a factor causing food poison, was cultured in Luria Bertani medium with 2% NaCl (NLB, OXOID, Ltd, England) for 10 h at 37 C. The growth curve was plotted based on the OD 600 values, and enumeration was performed as previously described (Wang et al. 2010). Artificial seawater preparation and oyster cultivation and testing The artificial seawater with a salinity of 1Æ8 to 2Æ0% was prepared as follows: One kilogram of Formula Grade A Reef salt (Qingdao Tianyili International Trading Co. Ltd, China) was dissolved in l of tap water purified with an Elix-5 system (0Æ22 lm; Millipore, Billerica, MA). One hundred fresh oysters (Crassostrea gigas), collected randomly from the Jiangyang market in Shanghai, China, between December 2008 and January 2009, were evenly distributed in 400 l of artificial seawater (in six tanks) as described previously (Wang et al. 2010) and acclimatized for 6 days prior to contamination with V. parahaemolyticus. In each tank, there were 16 oysters with ca. 67 l of artificial seawater. The artificial seawater was continuously circulated using pumps at 800 l per h and maintained at C in an air-conditioned room. The seawater was replaced every 12 h, except during the artificial contamination period. Any dead oysters were removed from the tanks before replacing the artificial seawater. The oysters were fed with green alga (Chlorella protothecoides CS-41), which was cultured in our laboratory under the culture conditions described previously (Qu et al. 2008). After acclimatization for 4 days, five oysters (150 g) were randomly collected, dissected and homogenized with a grinder (Wang et al. 2008b) in 300 ml of sterile physiological saline. The homogenates were centrifuged (Eppendorf, Borsdorf, Leipzig, Germany) at 500 g for 3 min at 4 C, and the supernatants were 10-fold serially diluted in triplicate and were spread-plated onto thiosulfate-citrate-bile salt-sucrose (TCBS) agar with disposable spreading rods. The plates were incubated at 37 C for 16 h. To determine whether the oysters were contaminated with V. parahaemolyticus, blue-green colonies, the typical colour of V. parahaemolyticus on TCBS, were enumerated and identified by PCR as described previously (Wang et al. 2006). Artificial contamination of oysters After testing for the presence of V. parahaemolyticus, the oysters were randomly divided into two groups, and 20 oysters were placed in each tank with artificial seawater as described earlier. The treatment group (n = 60) was inoculated with V. parahaemolyticus at a final concentration of 10 6 CFU ml )1 and incubated for 24 h (Kelly and Dinuzzo 1985; Schwab et al. 1998; Wang et al. 2010). Another group (n = 40) was incubated in noncontaminated seawater as a negative control. Evaluation of oyster tissues and seawater during bioaccumulation and depuration During the 24-h contamination period in the artificial seawater, four oysters were randomly collected and dissected after 0, 12 and 24 h of bioaccumulation, and various tissues were analysed for V. parahaemolyticus, including the gills, digestive glands (stomach, digestive ducts and digestive diverticula), adductor muscle and the mantle cilia, as previously reported (Wang et al. 2008b). The artificial seawater was replaced after the 24-h bioaccumulation period, and in the following 14 days, the artificial seawater was replaced every 12 h. After the 24-h contamination period, oyster tissues were evaluated for V. parahaemolyticus after 12, 24, 36, 48, 72, 96, 168 and 336 h of depuration. To avoid cross contamination, the oysters were aseptically shucked, and the different tissues were dissected and homogenized as described earlier. Oyster tissues from the negative control group were also processed in the same manner. At the same time, the artificial seawater was also sampled and analysed for V. parahaemolyticus. Seawater (1Æ0 ml) from the culture tanks was centrifuged (Eppendorf) at 8000 g (Deepanjali et al. 2005) for 3 min at 4 C. The pellet was resuspended in 1Æ0 ml of sterile physiological saline, and 10-fold serial dilutions were made and plated (100 ll) onto TCBS plates. After 16 h incubation at 37 C, blue-green colonies on the TCBS plates were counted. Tissue samples from four oysters were combined, and three replicates of the homogenates were evaluated for Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010)

3 Abundance of V. parahaemolyticus in oyster tissues D. Wang et al. V. parahaemolyticus. The retention rate of V. parahaemolyticus in different tissues was determined during 48 h depuration. The retention rate was calculated as follows: retention rate = (bacterial density) (bacterial density after 24 h inoculation) 100%. DNA extraction Five blue-green colonies from each tissue were selected randomly, resuspended in 1Æ0 ml of sterile water, and heated in boiling water for 10 min. The tubes were chilled on ice for 2 min, and then centrifuged at g for 5 min at 4 C. The supernatant (containing bacterial DNA) was stored at )80 C until analysis. PCR assay for confirmation of V. parahaemolyticus in oyster tissues Genomic DNA was amplified by the PCR as previously described (Wang et al. 2006), and the PCR product, a 476-bp fragment of the toxr gene, was sequenced. Negative (without DNA template) and positive (V. parahaemolyticus genomic DNA as template) controls were run in each PCR assay under the same conditions. All PCR assays were performed in triplicate. Statistical analyses The numbers of V. parahaemolyticus in different oyster tissues and in artificial seawater were converted to base 10 logarithms before being analysed by Origin 7.5 (Originlab, Northampton, MA). Statistical analyses were performed using the spss 13.0 software (Chicago, IL, USA) for the Tukey s HSD test. Results Estimation of V. parahaemolyticus in oysters before artificial contamination No or very few blue-green colonies were obtained on TCBS plates from the oysters used for artificial contamination (Fig. 1; negative control) after acclimatization in seawater. PCR assays performed using DNA from the blue-green colonies gave negative results for V. parahaemolyticus. Abundance of V. parahaemolyticus in oyster tissues during bioaccumulation As shown in Fig. 1, V. parahaemolyticus was detected in all of the oyster tissues. After 24 h of incubation, the population of V. parahaemolyticus had increased to 4Æ45 log CFU g )1 in the digestive glands followed by 3Æ95 log CFU g )1 in the gills (Fig. 1). At the same time, the bacterial concentration in the artificial seawater had declined from 6 log CFU ml )1 to 3Æ67 log CFU ml )1 (Fig. 1). The bacterial density in the adductor muscle and mantle cilia was lower than that in other tissues (Fig. 1). Abundance of V. parahaemolyticus in oyster tissues during depuration The level of V. parahaemolyticus in all of the tissues declined during 72 h of depuration (Fig. 1). The retention rate of the pathogen in different tissues was evaluated during 48 h of depuration, and there was a less decline of the pathogen in the gills compared to the other tissues (Table. 1). After a 14-day depuration, the level of Log 10 (CFU g 1 ) Bioaccumulation Depuration Hours 336 Figure 1 Evaluation of V. parahaemolyticus (ATCC 17802) populations in oyster tissues and in artificial seawater during the bioaccumulation and depuration periods. The numbers of V. parahaemolyticus were converted to base 10 logarithms. (h) Digestive glands, (d) Muscle, (4) Gills, ( ) Cilia of mantle, (s) Artificial seawater, ( ) Negative control. Bars represent standard error. 106 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010)

4 D. Wang et al. Abundance of V. parahaemolyticus in oyster tissues Table 1 The retention rate of V. parahaemolyticus in oyster tissues calculated every 12 h for 2 days Time (h) Gills Digestive glands Adductor muscle Mantle cilia Artificial seawater Æ7 ±0Æ067 a 48Æ2 ±0Æ089 c 59Æ0 ±0Æ125 b,c 9Æ97 ± 0Æ012 d 77Æ3 ±0Æ086 a,b Æ0 ±0Æ074 a 41Æ2 ±0Æ020 b 27Æ9 ±0Æ018 c 9Æ5 ±0Æ006 d 17Æ4 ±0Æ015 d 36 81Æ1 ±0Æ068 a 37Æ8 ±0Æ015 b 25Æ0 ±0Æ046 c 7Æ5 ±0Æ008 d 4Æ2 ±0Æ004 d 48 28Æ1 ±0Æ050 a 13Æ5 ±0Æ038 b 1Æ4 ±0Æ004 c,d 2Æ4 ±0Æ022 c,d 0Æ3 ±0Æ004 d Means (n = 3) in the column with different superscripts are significantly different (P < 0Æ05) with the retention rate of V. parahaemolyticus in oyster tissues according to Tukey s HSD test. V. parahaemolyticus in the different tissues was approximately 0Æ5 1Æ0 log CFU g )1. (Fig. 1). PCR detection of V. parahaemolyticus Although PCR results of the blue-green colonies from the TCBS plates from oyster tissues before artificial contamination were negative, all of the blue-green colonies from artificially contaminated samples yielded PCR positive results. The results of the sequence (tox R gene) analysis using BLAST on the NCBI database confirmed that the colonies were V. parahaemolyticus (ATCC17802). The tox R gene accession number is: GQ Discussion The distribution of V. parahaemolyticus in oyster tissues has been indirectly traced using GFP, and fluorescence was detected in the gills, visceral mass and adductor muscle (Cabello et al. 2005). The current study focused on the enumeration of V. parahaemolyticus in specific tissues of oysters and confirmed that V. parahaemolyticus can be accumulated efficiently in different tissues, presenting a significant health threat to oyster consumers. Because studies have demonstrated that there is an apparent positive correlation between water temperature and the prevalence of V. parahaemolyticus (DePaola et al. 2003; Deepanjali et al. 2005), the oysters used for the artificial contamination were collected in late winter, when they were less likely to be contaminated with V. parahaemolyticus. The PCR results confirmed that the oysters were clear of V. parahaemolyticus contamination. In this study, 13Æ5% of V. parahaemolyticus remained in the digestive glands after 48 h of depuration. V. parahaemolyticus could be detected in these organs even after 14 days of depuration. The results confirmed that efficient depuration of V. parahaemolyticus from oyster tissues does not occur using fresh seawater (Nappier et al. 2009). It has been suggested that depuration should be carried out at lower temperatures (Chae et al. 2009) with added measures such as ozone (Croci et al. 2002), ultraviolet light (Hamamoto et al. 2007) and disinfectant treatments (Wang et al. 2010) in conjunction with depuration. The results of this study indicated that the digestive glands and the gills can accumulate V. parahaemolyticus efficiently. These organs have been used as target tissues for molecular detection of food-borne viruses (Le Guyader et al. 2000; Kou et al. 2008; Nappier et al. 2009). In addition, the gills contain substantially less PCR inhibitors (Wang et al. 2008a). The current evidence suggests that the digestive glands and the gills could be promising target tissues for direct detection of V. parahaemolyticus in oysters by molecular methods, possibly without sample enrichment. Future studies will examine the ability to detect low levels of V. parahaemolyticus in artificially contaminated oysters and in naturally contaminated oysters using the gills and digestive glands as the target tissues. Acknowledgements This work was jointly supported by the grants no. 2009BAK43B31 from the Ministry of Science and Technology of China, no from the China Postdoctoral Science Foundation and no. 09DZ from Science & Technology Commission of Shanghai Municipality. We also thank Dr. Pina Fratamico from the USDA Eastern Regional Research Center for helpful revision of this manuscript. References Cabello, A., Espejo, R. and Romero, J. (2005) Tracing Vibrio parahaemolyticus in oysters (Tiostrea chilensis) using a Green Fluorescent Protein tag. J Exp Mar Biol Ecol 327, Chae, M., Cheney, D. and Su, Y. (2009) Temperature effects on the depuration of Vibrio parahaemolyticus and Vibrio vulnificus from the American oyster (Crassostrea virginica). J Food Sci 74, M62 M66. Croci, L., Suffredini, E., Cozzi, L. and Toti, L. (2002) Effects of depuration of molluscs experimentally contaminated with Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010)

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