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Characterization of Pathogenic and Non-Pathogenic Vibrio

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Characterization of Pathogenic and Non-Pathogenic Vibrio AN ABSTRACT OF THE THESIS OF Jingyi Du for the degree of Master of Science in Food Science and Technology presented on August 31, 2017. Title: Characterization of Pathogenic and Non-pathogenic Vibrio parahaemolyticus Strains and the Antimicrobial Activity of Fruit Juice and Fruit Extracts against Seafood Pathogens Abstract approved: ______________________________________________________ Christina Ann Mireles DeWitt Vibrio parahaemolyticus is frequently isolated or detected from raw seafoods, especially shellfish. Also, Listeria monocytogenes, Salmonella spp. are pathogens that are frequently found in ready-to-eat (RTE) seafood, such as smoked fish and shellfish, seafood salad, cooked shrimp and crabmeat, and seafood consumed raw. Two studies to improve safety in RTE seafoods were conducted. FDA’s regulatory limit, 10,000 cells/g, for V. parahaemolyticus in RTE fish products encompasses both pathogenic and non-pathogenic V. parahaemolyticus. However, limited studies exist on the factors that influence growth and die-off rate of pathogenic and non-pathogenic V. parahaemolyticus strains. This study investigated the influence of temperature (5-30 °C) on the growth (positive µmax) and die-off rates (negative µmax) of pathogenic and non-pathogenic V. parahaemolyticus strains. There was a significant effect of strain type (non-pathogenic vs pathogenic, P < 0.001), temperature (P < 0.001) and a strain type x temperature interaction (P = 0.026). At 5 °C, µmax was negative for both strain types indicating die-off. At 10 °C, µmax was negative for two pathogenic and one non-pathogenic strain. From 15-30 °C, µmax was positive for all strains. When evaluating differences between strain type at each temperature, significant difference (P < 0.001) between strain type only occurred at 30 °C. When evaluating the effect of temperature on µmax within either non-pathogenic or pathogenic strain type, there was no significant difference between 5-15 °C. The mean µmax for non-pathogenic and pathogenic strains was 0.448 and 0.340, respectively. The faster rate of growth of non-pathogenic strains suggests an increased likelihood of positives from environmental sampling, especially as environmental temperature increases. This study revealed differences between growth and die-off rates of pathogenic and non-pathogenic V. parahaemolyticus strains at various temperatures. This information is useful to the risk assessment of raw and RTE seafood consumption. Future studies need to be conducted to investigate the specific mechanism responsible for different responses to temperature change. Foodborne pathogens Listeria monocytogenes, Salmonella spp. and Vibrio parahaemolyticus are especially problematic in RTE foods. Consumer demand for healthy, minimally processed RTE food suggests natural methods are needed to control these pathogens. The aim of this study was to investigate antimicrobial activity of seven fruit extracts against five L. monocytogenes, four Salmonella strains and five V. parahaemolyticus strains from human clinical samples, seafood, raw milk, produce, and meat sources. Pomegranate peel (PPE) exhibited the highest antimicrobial activity and it was found to be similar in efficacy when compared to cranberry juice (CJ), a well-known source of antimicrobial activity. Limited inhibition was observed for blueberry, strawberry, plum meat, whole plum and pomegranate seed. The minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) of PPE for L. monocytogenes was 22.5% and 37.5%, respectively. MBC of PPE against Salmonella was 37.5%. MBC of PPE against V. parahaemolyticus strains was 8.1%. The MIC of PPE against L. monocytogenes was 22.5% while the MIC of PPE against Salmonella was >22.5% and < 37.5%. The MIC of PPE against V. parahaemolyticus strains was >4.86% and <8.1%, which was much lower that against L. monocytogenes and Salmonella. The MBC of CJ against V. parahaemolyticus strains was 12.5%. The MBC of CJ against L. monocytogenes and Salmonella was 25%, whereas the MIC against L. monocytogenes was 12.5%, but this concentration did not fully inhibit the growth of Salmonella strains. The MIC of CJ against Salmonella was >12.5% and < 25% and that against V. parahaemolyticus was>6.25% and < 12.5%. Concentration (P ˂ 0.0001), pathogen type (P ˂ 0.0001) and treatment type (PPE vs CJ; P ˂ 0.0001) have an effect on log reductions. In addition, there was a significant three-way interaction between all main effects (P ˂ 0.0001). This study demonstrated the potential of PPE, a natural food by-product, to be used as an antimicrobial in RTE seafood. More research is needed to optimize concentrations that exhibit effective antimicrobial activity, but have minimal impact on sensory qualities and shelf-life of seafood products. ©Copyright by Jingyi Du August 31, 2017 All Rights Reserved Characterization of Pathogenic and Non-pathogenic Vibrio parahaemolyticus Strains and the Antimicrobial Activity of Fruit Juice and Fruit Extracts against Seafood Pathogens by Jingyi Du A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented August 31, 2017 Commencement June 2018 Master of Science thesis of Jingyi Du presented on August 31, 2017 APPROVED: Major Professor, representing Food Science and Technology Head of the Department of Food Science and Technology Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Jingyi Du, Author ACKNOWLEDGEMENTS I would like to express my deeply appreciation to Dr. Christina Ann DeWitt, my major professor and Dr. Jovana Kovacevic, my co-major professor, for their support help and guidance to my graduation. Their encouragement and knowledge especially helped me through the hard times. I would also like to dedicate the dissertation to my previous major professor, Dr. Yi-Cheng Su, with all the memory we had and his support for my time as a graduate student. Miss you, Dr. Su. I would like to thank my committee members: Dr. Jae Park and Dr. Claudia Häse for their advice and participation in my M.S. program. Thanks to people from OSU and Food Science Department for their knowledge. Many thanks to the Astoria Seafood Laboratory: Dr. Kwon, Dr. Guo, Sue, Craig, and my friends: Clara, Kaitline, Ziwen, Robin, Ning, Nut and many others. Thank them so much for your help and all the time that we spent together. Finally, I am truly thankful to my parents. Without their support and understanding, I could not achieve what I have and become the person that I want to be. I cherish every moment in my Master life. TABLE OF CONTENTS Page Chapter 1 General Introduction ..................................................................................... 1 1.1 Overview .............................................................................................................. 1 1.1.1 Seafood consumption ................................................................................... 1 1.1.2 Seafood hazard ............................................................................................. 1 1.2 Prevalence of L. monocytogenes, Salmonella and V. parahaemolyticus in ready-to eat (RTE) seafood ...................................................................................................... 2 1.2.1 Taxonomy and characteristics of L. monocytogenes, Salmonella and V. parahaemolyticus .................................................................................................. 2 1.2.2 Detection of L. monocytogenes, Salmonella and V. parahaemolyticus ....... 8 1.2.3 Incidence of L. monocytogenes, Salmonella and V. parahaemolyticus in ready-to-eat (RTE) seafood ................................................................................. 14 1.2.4 Outbreaks of L. monocytogenes, Salmonella and V. parahaemolyticus infection in ready-to-eat (RTE) seafood .............................................................. 16 1.3 Regulation of L. monocytogenes, Salmonella and V. parahaemolyticus in ready-to- eat (RTE) seafood .................................................................................................... 18 1.4 Control and prevention of L. monocytogenes, Salmonella and V. parahaemolyticus infection by using plant extracts .............................................................................. 20 1.4.1 Berry ........................................................................................................... 20 1.4.2 Pomegranate ............................................................................................... 26 1.4.3 Plum ........................................................................................................... 29 1.5 Objective ............................................................................................................ 30 Chapter 2 Comparison of Growth and Die-off of Pathogenic and Non-pathogenic Vibrio parahaemolyticus at Various Temperatures ................................................................. 37 2.1 Abstract .............................................................................................................. 38 2.2 Introduction ........................................................................................................ 39 2.3 Material and methods ......................................................................................... 41 2.3.1 Bacteria culture preparation
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