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Microbial Community and Vibrio Parahaemolyticus Population Dynamics in Relayed Oysters

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Microbial Community and Vibrio Parahaemolyticus Population Dynamics in Relayed Oysters University of New Hampshire University of New Hampshire Scholars' Repository Doctoral Dissertations Student Scholarship Fall 2017 MICROBIAL COMMUNITY AND VIBRIO PARAHAEMOLYTICUS POPULATION DYNAMICS IN RELAYED OYSTERS Michael Anthony Taylor University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/dissertation Recommended Citation Taylor, Michael Anthony, "MICROBIAL COMMUNITY AND VIBRIO PARAHAEMOLYTICUS POPULATION DYNAMICS IN RELAYED OYSTERS" (2017). Doctoral Dissertations. 2288. https://scholars.unh.edu/dissertation/2288 This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. MICROBIAL COMMUNITY AND VIBRIO PARAHAEMOLYTICUS POPULATION DYNAMICS IN RELAYED OYSTERS BY MICHAEL ANTHONY TAYLOR BS, University of New Hampshire, 2002 Master’s Degree, University of New Hampshire, 2005 DISSERTATION Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Microbiology September, 2017 This dissertation has been examined and approved in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology by: Dissertation Director, Stephen H. Jones Research Associate Professor, Natural Resources and the Environment Cheryl A. Whistler, Associate Professor, Molecular, Cellular & Biomedical Sciences Vaughn S. Cooper, Associate Professor, Microbiology & Molecular Genetics, University of Pittsburg School of Medicine Kirk Broders, Assistant Professor, Plant Pathology, Colorado State University College of Agricultural Sciences Thomas Howell, President / Owner, Spinney Creek Shellfish, Inc., Eliot, Maine On March 24, 2017 Original approval signatures are on file with the University of New Hampshire Graduate School. ii ACKNOWLEDGEMENTS I would like to thank the following individuals for helping me with this endeavor: Dr. Steve Jones: Steve, thank you for taking me on as a graduate student six years ago and providing me the flexibility to be an unorthodox student with other priorities as important as school. Thank you for all of the advice, and the time you have dedicated to helping me. Graduate Committee: Thank you to all 5 of my committee members. Each one of you helped me grow professionally. Thank you for your time and willingness to provide guidance and support me as a student. Tom and Laurie Howell: Special thanks to the Howell’s and Spinney Creek Shellfish, Inc. for their generosity with their time and their facility which made this research novel. Co-workers in the lab: Thank you to all of my fellow students and the students and technicians in the Jones and Whistler labs who assisted in this project. Bob Mooney: Bob, thank you for showing me how to be a Microbiologist. Dr. Robert Zsigray: I want to thank the late Bob Zsigray for teaching me about integrity in research and life. Dr. Stephen Torosian: Thank you Steve for my start as a microbiologist and showing me into the lab. Jong Yu: Thank you Jong for teaching me how to process oysters and all of your hard work and support during this project. Mike Hall Mike thank you for the computer science support. Cristiane Taylor: To my wife – I didn’t realize how tough getting a PhD is until I was at the point of no return. Thank you for making sure I did not give up and thank you for being a mother, friend, wife and an unwavering partner through the last 6 years. Funding Sources: NH Agricultural Experiment Station NH Sea Grant College Program NH EPSCoR Program-Track 2 project iii TABLE OF CONTENTS ACKNOWLEDGEMENTS............................................................................................ iii LIST OF TABLES.......................................................................................................... v LIST OF FIGURES ....................................................................................................... vi ABSTRACT .................................................................................................................. vii CHAPTER PAGE I. INTRODUCTION........................................................................................ 1 II. VARYING SUCCESS OF RELAYING TO REDUCE VIBRIO PARAHAEMOLYTICUS LEVELS IN OYSTERS (CRASSOSTREA VIRGINICA) …………………………………………… 12 INTRODUCTION……………………………………………………. 13 RESULTS…………………………………………………………….. 17 DISCUSSION………………………………………………………… 26 MATERIALS AND METHODS…………………………………….. 30 III. OYSTER MICROBIAL COMMUNITY CHANGES ASSOCIATED WITH VIBRIO PARAHAEMOLYTICUS CONCENTRATIONS IN RELAYED OYSTERS………………………………………………………………… 34 INTRODUCTION……………………………………………………. 35 RESULTS…………………………………………………………….. 37 DISCUSSION………………………………………………………… 60 MATERIALS AND METHODS……………………………………... 64 REFERENCES………………………………………………………………………... 71 APPENDICES………………………………………………………………………… 88 APPENDIX A CHAPTER II SUPPLEMENTAL MATERIAL……………………… 89 APPENDIX B CHAPTER III SUPPLEMENTAL MATERIAL……………………... 96 iv LIST OF TABLES CHAPTER II Table 1 Statistical analyses of V. parahaemolyticus (Vp) levels (MPN/g or mL) in oyster and water samples analyzed during relay experiments from 2011 to 2014……………………………………………………. 22 CHAPTER III Table 1 V. parahaemolyticus concentrations and 16S sequence abundance for Vibrio spp. in paired Day 0 and 14 oyster samples from the four most effective relay experiments: 2011-12……………………… 41 Table 2 Identified genera that increased from Day 0 to Day 14 in oysters from the most effective relay experiments…………………………… 44 Table 3 Non-parametric Multi Response Permutation Procedure (MRPP) analysis of Taxa Differences within Sample Matrices and Years – 2011-2013 Relay Experiments……………………………………….. 49 Table 4 The diversity and relative abundance of taxa in oysters, water and both water and oysters in each study year (2011, 2012 and 2013)…... 58 Table 5 Vibrio spp. used in the competition assay with environmental strain G3654 of Vibrio parahaemolyticus from Spinney Creek……… 60 APPENDIX Table A.1 2011-2014 Oyster and Water Sample MPN Metadata………………… 91 Table B.1 Oyster and water sample V. parahaemolyticus MPN/g concentrations and 16S count metadata for samples submitted for 16S rRNA gene sequencing………………………………………………………… 97 Table B.2 CASAVA Quality Data for Oyster and Water Samples……………….. 98 v LIST OF FIGURES CHAPTER II Fig. 1 The Great Bay estuary of Maine and New Hampshire, USA, with harvest site (PR) in the Piscataqua River and the relay site (SC) in Spinney Creek ………………………………………………… 16 Fig. 2 Water temperature (°C) and salinity (PPT-parts per thousand) at the relay (SC) and harvest (PR) sites………………………………… 19 Fig. 3 V. parahaemolyticus levels (MPN/g) on Day 0 of relay experiments in oysters from PR and water samples from PR and SC……………… 21 Fig. 4 V. parahaemolyticus levels (MPN/g) in oyster samples analyzed during each sampling day of relay experiments from 2011 to 2014….. 24 CHAPTER III Fig. 1 Alpha rarefaction curves for sequences within the observed OTUs….. 39 Fig. 2 Linear regression analysis between V. parahaemolyticus MPN concentrations and Vibrio spp. rRNA gene abundances in all samples………………………………………………………… 42 Fig. 3 The relationship between V. parahaemolyticus concentrations and genera abundance in relayed oyster samples…………………….. 46 Fig. 4 Principal coordinates analysis of all oyster and water samples based on pair-wise sample Bray-Curtis dissimilarity measures……… 48 Fig. 5 The relative abundance and relatedness between OTUs associated with fresh and relayed oyster and water samples during 2011-13…….. 55 Fig. 6 Venn diagram of the number of identified taxa found in 100% of the oyster and water samples by year and Day 14 of relay…. 57 APPENDIX Fig. A.1 Lab procedure for processing oysters after field collection..…………. 90 vi ABSTRACT MICROBIAL COMMUNITY AND VIBRIO PARAHAEMOLYTICUS POPULATION DYNAMICS IN RELAYED OYSTERS by Michael Anthony Taylor University of New Hampshire, September, 2017 The CDC estimates that 45,000 people are sickened each year by foodborne Vibrio parahaemolyticus in the United States. Filter-feeding bivalve shellfish, such as oysters, are routinely inhabited by human pathogenic V. parahaemolyticus and there currently is not a contaminant management process that effectively reduces concentrations of V. parahaemolyticus in oysters. The transplanting of V. parahaemolyticus -laden oysters to an area with low concentrations or no V. parahaemolyticus, called oyster relay, is one reduction strategy that holds promise for treating live oysters. A key consideration for effective strategies to reduce Vibrio spp. in shellfish is the influence of microbiota in natural seawater. Our aim for this study was to identify taxa shifts in the microbial composition of oyster and water samples during relay that correlated with the reduction of V. parahaemolyticus. We hypothesize that changes in bacterial taxa within the oyster microbiome occur during relay and influence the reduction of V. parahaemolyticus in relayed oysters. Oysters with varying concentrations of V. parahaemolyticus were evident from relay experiments carried out in a local body of water with elevated salinity and low concentrations of V. parahaemolyticus over 4 consecutive years. Overall, V. parahaemolyticus levels were reduced during 14-day relay in 9 of the 14 monthly trials to target reduction levels (<100 V. parahaemolyticus/g).
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