Analysis of bacterial communities in the eastern oyster (Crassostrea virginica) with emphasis on Vibrio vulnificus dynamics under refrigeration by Raphael Ryan Wood A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama December 12, 2011 Keywords: Eastern oyster (Crassostrea virginica), Vibrio vulnificus, cold shock, refrigeration Copyright 2011 by Raphael Ryan Wood Approved by Covadonga R. Arias, Chair, Associate Professor of Fisheries and Allied Aquacultures Thomas A. McCaskey, Professor of Animal Sciences Omar A.Oyarzabal, Associate Professor of Biological Sciences Alabama State University Craig A. Shoemaker, Affiliate Associate Professor, Fisheries and Allied Aquacultures Abstract In this study we determined the effect of refrigeration on the total bacterial, total Vibrio spp., and V. vulnificus populations present within the eastern oyster (Crassostrea virginica), and the effects of cold shock (35°C to 4°C) on the complete V. vulnificus transcriptome. Oysters from two different locations, the Auburn University Shellfish Laboratory, Dauphin Island, AL, and a commercial processor, were compared during two weeks under refrigeration conditions. During the course of the experiment, total aerobic bacteria counts increased by two logs. Ribosomal Intergenic Sequence Analysis (RISA) and Denaturing Gradient Gel Electrophoresis (DGGE) were used to determine changes within the total population, while DGGE was used to evaluate changes of the Vibrio spp. population over the two week period. RISA-derived data showed that the microbial communities at Day 1 were clearly different from both Day 7 and 14 samples. Within the Day 1 cluster, samples were subdivided based on location. On Day 7 and 14, samples could also be subdivided by date and origin. Vibrio-specific DGGE also allowed for the clustering of samples by location on Day 1, which also clustered away from Days 7 and 14. Bands corresponding to both V. parahaemolyticus and V. vulnificus decreased in prevalence, while those corresponding to V. mimicus increased during the two weeks of refrigeration, particularly on Day 7. Interestingly, V. vulnificus counts determined by colony dot-blot hybridization remained unchanged throughout the experiment. Vibrio vulnificus isolates were recovered from oysters and genotyped as 16S type A, B or AB by ii restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (ALFP) analyses. 16S type B isolates (typically associated with clinical cases) comprised 53% of the isolates. An important note was the identification of 16S type B-specific AFLP bands that differentiated type B from type A and AB. A DNA microarray of V. vulnificus showed that 165 genes out of 4,488 altered their expression profiles by more than twofold. The highest induction observed occurred in two of the five categorized cold shock genes, cspA and cspB, which showed a complementary and persistent expression pattern during cold shock suggesting a homologous role. Other genes showing a significant fold increase included ribosomal genes, protein folding regulators, and membrane genes. iii Acknowledgments First I would like to thank my major professor Dr. Cova Arias for giving me the opportunity to move to her lab to complete my PhD. I would also like to thank my committee members, Dr. Thomas A. McCaskey, Dr. Omar A. Oyarzabal, and Dr. Craig A. Shoemaker for their time and expertise. I thank Óscar Olivares-Fuster for his guidance and friendship throughout my time here and my lab mates, Suttinee Limthammahisornm Matt Lewis, Zhen Tao, Stacey LaFrentz, Haitham Hussien Mohammed, and Wenlong Cai. I appreciate the work of Scott Rikard at the Auburn University Shellfish Laboratory for his help with oysters. I would like to thank the administration staff in the Department of Fisheries and Allied Aquacultures for their help over the years. Thank you to my friends, both old and new, and to my family, Granny, Papa, Mom, Andrew, April, and Wendy. And a special thank you to my beautiful wife Ashley. I could not have made it here without your love and support. iv Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iv List of Tables .................................................................................................................... vii List of Figures .................................................................................................................. viii I. Literature Review.....................................................................................................1 II. Objectives ..............................................................................................................26 III. Microbial community dynamics in the eastern oyster (Crassostrea virginica) under refrigeration .................................................................................................28 Abstract ......................................................................................................29 Introduction ................................................................................................31 Materials and Methods ...............................................................................33 Results ........................................................................................................36 Discussion ..................................................................................................41 IV. Dynamics of Vibrio vulnificus genotypes in oysters (Crassostrea virginica) under refrigeration............................................................................................................54 Abstract ......................................................................................................55 Introduction ................................................................................................56 Materials and Methods ...............................................................................58 Results ........................................................................................................60 Discussion ..................................................................................................63 v V. Evaluation of global gene expression during cold shock in the human pathogen Vibrio vulnificus .....................................................................................................75 Abstract ......................................................................................................76 Introduction ................................................................................................77 Materials and Methods ...............................................................................79 Results ........................................................................................................82 Discussion ..................................................................................................86 Cumulative Bibliography .................................................................................................104 vi List of Tables 1. Total aerobic bacterial counts recovered from raw oysters during this study .......................................................................................................................46 2. Vibiro species identified using Vibiro sp. specific primers ...................................47 3. Total bacteria counts and V. vulnificus Most Probable Number per gram oyster meat .............................................................................................................68 4. Genes used in Real-Time RT-PCR ........................................................................92 5. Genes identified by microarray analysis to be up/down regulated ≥ 2 fold during cold shock ...................................................................................................93 vii List of Figures 1. RISA patterns of bacteria present in oyster samples. The dendrogram was derived by UPGMA cluster analysis of the RISA profiles from 67 individual oysters ...................................................................................................48 2. Multidimensional scaling (MDS) plot showing the relatedness of individual bacterial 16-23S intergenic sequences based on RISA data .................50 3. Dendrogram of DGGE 16S rDNA fingerprints for total bacteria present based... ...................................................................................................................51 4. Dendrogram of Vibrio-specific DGGE fingerprints ..............................................52 5. Multidimensional scaling (MDS) plot showing the relatedness of individual Vibrio-specific DGGE fingerprints.......................................................53 6. Map showing the locations at where the oysters were harvested used in this study ................................................................................................................69 7. Percentage of total V. vulnificus 16S types isolated from each sampling date .........................................................................................................................70 8. Graph showing the total number of V. vulnificus