Reductions of Vibrio parahaemolyticus in oysters after bacteriophage application during depuration
Introduction
Vibrio parahaemolyticus is an enteric pathogen that is widely distributed in coastal areas, and is a causative agent of gastroenteritis in humans after consumption of contaminated seafood (Feldhusen, 2000, Potasman et al., 2002, Sala et al., 2009, Su and Liu, 2007). V. parahaemolyticus are the most prevalent seafood-associated pathogenic bacteria in the world, and are frequently found in the oysters of China (Chen et al., 2010, Drake et al., 2007). Oysters are filter feeders that accumulate food particles and small organisms by circulating large volumes of seawater through their system; consequently, microorganisms, including human pathogens, become absorbed along with nutrients and accumulate in their bodies (Lees, 2000, Meujo et al., 2010). In China, oysters are consumed raw or lightly cooked; therefore, contaminated oysters are potential vectors for pathogenic V. parahaemolyticus (Larsen et al., 2013, Lees, 2000, Ye et al., 2012).
Many methods have been applied to eliminate human pathogens, including Vibrio spp., from oysters. Several post-harvest processes (PHP) have been reported previously: heat/cool pasteurization (Andrews et al., 2000, Cook, 2003), irradiation (Andrews et al., 2003, Mahmoud, 2009, Mahmoud and Burrage, 2009), high hydrostatic pressure (Kural and Chen, 2008, Ma and Su, 2011, Prapaiwong et al., 2009), ultraviolet (UV) exposure (Hamamoto et al., 2007, Phuvasate et al., 2012), and rapid freezing with frozen storage (Liu et al., 2009). Recently, combined PHP techniques were also studied, such as high hydrostatic pressure combined with moderate heating (Ye et al., 2012). However, all of these processes have shown negative effects on the consumers' culinary experience of oysters. Furthermore, several oysters commonly die during these processing techniques. All of these limitations of PHP influence the consumption of oysters by consumers (Chae et al., 2009, Larsen et al., 2013).
Shellfish depuration is a method applied to eliminate human pathogens from live oysters. Depuration is a control process in which shellfishes are reared in seawater treated by chlorine, ozone, or UV light for a few hours in order to reduce pollutants in their bodies by filtering (Croci et al., 2002, Rodrick and Schneider, 2003, Wang et al., 2010). This method was established for reducing fecal contamination in shellfish, such as Salmonella and Escherichia coli, but is generally not effective against Vibrio spp. (Lee et al., 2008, Otwell et al., 1991). However, low temperature depuration was found to be effective in reducing V. parahaemolyticus in oysters by 2–3 log as estimated by most probable number (MPN)/g (Phuvasate et al., 2012); although this process took several days. Additionally, the chemical treatments commonly used for seawater disinfection result in chemical hazards in seafood. The development and evaluation of new strategies, with no adverse effects on the oysters, to reduce V. parahaemolyticus in raw oysters is a prospective research area.
Feeding oysters with bacteriophages that are natural and harmless to humans or animals is a relatively inexpensive method (Sulakvelidze et al., 2001). Because bacteriophages are compatible with food, they can be applied for therapeutic or bio-sanitization purposes (Hanlon, 2007, Skurnik and Strauch, 2006). The potential of bacteriophages to control bacterial diseases has been supported by several studies demonstrating an efficient reduction of pathogen levels in aquaculture species, such as shrimp (Karunasagar et al., 2007, Vinod et al., 2006) and finfish (Nakai and Park, 2002, Park and Nakai, 2003). However, the application of phages in shellfish depuration has not yet been thoroughly studied. This study was conducted to evaluate the effectiveness of a single bacteriophage (VPp1) aimed at V. parahaemolyticus, and to identify the multiplicity of infection (MOI) level along with optimum temperature to achieve a better purification effect in live oysters infected (artificially contaminated) with pathogenic V. parahaemolyticus (ATCC 17802).
Section snippets
Phage preparation
The bacteriophage VPp1, a lytic phage which was previously isolated from sewage samples by our laboratory (Yong et al., 2013), was proved to be effective in lysing V. parahaemolyticus at a much lower MOI (0.0001) and the phage titer was approximately 109 plaque-forming units (PFU)/mL. It was replicated with V. parahaemolyticus in 2216E broth (Qingdao Haibo Technology Co. Ltd.) at 37 °C overnight with rotation (130 rpm). Double-plaque assays were performed, as described by Adams (1959) to determine
Mortality–temperature relationships
The survivals of oysters associated with different levels of V. parahaemolyticus accumulation at different temperatures, ranging from 12 to 22 °C, are shown in Table 1. In the negative control (without V. parahaemolyticus), the highest morality rates observed were 36% at 22 °C and 5% at 20 °C. No deaths occurred at 16 °C or 12 °C after 48 h of V. parahaemolyticus accumulation, both in the negative control and infected groups. The overall mortality rate was higher in infected oysters (> 50%) compared
Discussion
Bacteriophages, as specific pathogen-killers, are effective agents for controlling bacterial infections in aquaculture, and do not affect normal flora (Hawkins et al., 2010, Park and Nakai, 2003). Protective effects of bacteriophages have been reported across different fields by many researchers (Bueno et al., 2012, Lim et al., 2012, Oliveira et al., 2010, Stelios et al., 2011, Steven et al., 2011).
Oysters are always sold live, and depuration is often the preferred method for controlling
Conclusion
To our knowledge, this is the first report of a depuration trial using bacteriophage in the oyster depuration process. In this study, the data provide proof of the principle that the application of the bacteriophage (VPp1) could reduce the population of V. parahaemolyticus in infected oysters.
The depuration process described in the paper requires low initial investment and running costs. Moreover, oysters could be maintained alive during processing. Therefore, the application of bacteriophage
Acknowledgments
This work was supported by the National Natural Science Funding of China (Grant No. 31071540) and the “National Science & Technology Pillar Program” (2012BAD28B05).
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