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  • MICROBIAL INTERACTIONS WITH OYSTERS FROM THE GREAT BAYESTUARY: CHARACTERIZATION OF ENDEMIC Vibrio cholerae AND OYSTER

    METAGENETICS

    BY

    BRIAN MICHAEL SCHUSTER

    B. S., University of New Hampshire, 2008

    THESIS

    Submitted to the University of New Hampshire

    in Partial Fulfillment of

    the Requirements of the Degree of

    Master of Science

    In

    Microbiology

    December, 2010

  • UMI Number: 1489963

    All rights reserved

    INFORMATION TO ALL USERSThe quality of this reproduction is dependent upon the quality of the copy submitted.

    In the unlikely event that the author did not send a complete manuscriptand there are missing pages, these will be noted. Also, if material had to be removed,

    a note will indicate the deletion.

    UMTDissertation Publishing

    UMI 1489963Copyright 2011 by ProQuest LLC.

    All rights reserved. This edition of the work is protected againstunauthorized copying under Title 17, United States Code.

    ProQuest

    ProQuest LLC789 East Eisenhower Parkway

    P.O. Box 1346Ann Arbor, Ml 48106-1346

  • examined and approved.

    U iyCtCt.'- 'CXThesis Director, Cheryl A. Whistler,

    Assistant professor, Microbiology

    ..--^aughn S. Cooper,Associ^profess^r, Microbiology

    / Stephen H. Jones,/Research Associate professor, Natm^Resourc>landJhnvironment

    Y3) SiODate

  • TABLE OF CONTENTS

    LISTOFTABLES iv

    LISTOFFIGURES v

    ABSTRACT vi

    CHAPTER PAGE

    I. INTRODUCTION l

    II PHENOTYPIC AND MULTI-LOCUS SEQUENCE ANALYSIS OF Vibrio choleraeISOLATED FROM THE GREAT BAY ESTUARY OF NEW HAMPSHIREREVEALS GENETIC RECOMBINATION WITHIN THIS DIVERSE ENDEMICPOPULATION 5

    Introduction 5

    Methods

    Results 1379Discussion ^

    III METAGENETIC ANALYSIS REVEALS HOST ASSOCIATED POPULATIONSWITHIN OYSTERS COMPARED TO THE SURROUNDING WATER 24

    Introduction ^

    Methods 29

    Results and Discussion 34

    Future Directions 46

    REFERENCES 47

    iii

  • LIST OF TABLES

    Table 2.1: Summary of V. cholerae collected from the Great Bay Estuary 14Table 2.2: Abundance and environmental factor correlations 15

    Table 3.1: Concentration of V. parahaemolyticus in samples 41

    IV

  • LIST OF FIGURES

    Figure 1.1: Great Bay Estuary Shellfish Classification Map 3Figure 2.1: Consensus neighbor-joining tree of Great Bay Estuary isolates 17Figure 2.2: Splits Tree and Phi Test of Recombination of GBE isolates 19Figure 3.1: Water versus oyster abundances of different phyla per site 36Figure 3.2: Water versus oyster abundances of different Proteobacteria 38Figure 3.3: High versus low V. parahaemolyticus concentration oysters 42Figure 3.4: Distribution of total Vibrio 44

    ?

  • ABSTRACT

    BACTERIAL ECOLOGY OF THE GREAT BAY ESTUARY: THE DISCOVERY OF

    ENDEMIC Vibrio cholerae AND DEVELOPING A METAGENETICS APPROACH

    TO EXPLORE THE OYSTER MICROBIOTA

    By

    Brian Michael Schuster

    University of New Hampshire. December, 2010

    Within the last two decades the number of gastroenteritis seafood-borne outbreaks

    due to Vibrio species has increased. These infections are usually caused by theconsumption of raw or undercooked shellfish. I took two approaches to describe theecology of Vibrios in the Great Bay Estuary, Durham, New Hampshire. First, Iperformed a phenotypic and multi-locus sequence analysis of 31 V cholerae strainsisolated from oysters, water and sediment in 2008 and 2009. 1 concluded that the V.cholerae population is endemic, diverse and highly recombinatory, but none of theisolates contain the virulence markers indicative of clinical strains. Next, I performed a

    metagenetic microbial census of 39 individual oysters and the overlying water from theNannie Island and Oyster River oyster beds. I concluded that the microbial populations inthe oyster are differential from the overlying water. Furthermore, the microbialpopulations structure in V. parahaemolyticus contaminated oysters contain uniquesignatures.

    vi

  • CHAPTERI

    INTRODUCTION

    Over at least the past thirty years, outbreaks of Vibrio infections have increasedsharply, especially in industrialized nations where disease incidence typically stabilizesor diminishes. The consumption of raw shellfish, particularly oysters, is a common vector

    for gastroenteritis infections caused by Vibrios. Changes in dietary habits, changes inland and resource usage, and especially seasonal weather changes which are possiblyindicative of global climate change have raised concerns about emerging pathogens inenvironments atypical for these outbreaks, including Northern coastal United States(DePaola et al, 2000; McLaughlin et al, 2005; Constantin de Magny et al, 2009; Lipp etal, 2003). These new public health trends challenge researchers to explore ecologicaldynamics that are driving these outbreaks of shellfish-borne Vibrio infections.

    Little is known about natural populations of Vibrio parahaemolyticus and Vibrio

    cholerae in cold temperate regions where disease is rare but is recently emerging.Environmental Vibrios do not have equal potential to cause disease, and multiple biovarswith different levels of virulence often coexist (Deepanjali et al, 2005; DePaola et al,

    2000; Louis et al, 2003). In many cases, infections caused by these organisms intemperate regions are simply attributed to travelers contracting the disease elsewhere, butother incidents are not as clear (MacRae et al, 1983; McLaughlin et al, 2005). In cases

    where the pathogens emerge from these temperate environments, it is not known in what

    1

  • context of the natural microbial population these arise. For example, different strains orspecies, either non-pathogenic or pathogenic, could displace one other as environmentalconditions influence changes in the local population structure. Another potential is geneflow between strains with different potentials to cause disease, and different potentials tosurvive in their particular environment, in which new hybrid strains can arise. Fewstudies have explored the dynamics of these pathogens and non-pathogens as they relateto each other and as they relate to the total microbial population in the environment.

    Another question is how changes in environmental conditions affect thepopulation dynamics of Vibrios, especially in northern environments which experienceextremely cold winters and warm summers. Some studies have been performed thatdemonstrate increasing temperature of surface waters and other environmental conditionsincrease the total V. cholerae population (Constantin de Magny et al, 2009; Lipp et al,2002), but have not yet explored how this dynamic influences interactions betweendifferent biovars of Vibrios including recombination. Recombination in naturalpopulations is particularly important to understand because most Vibrio pathogenicityislands and toxins are transferred horizontally (Garg et al, 2003). A particular concern innorthern regions is that non-pathogenic but robust cold adapted strains could exchangegenes with transient pathogenic strains that likely cannot persist in the cold environment.

    The Great Bay Estuary (GBE) in New Hampshire is a unique location to studythese dynamics because changes in environmental conditions will probably approachconditions similar to endemic outbreak regions (New England Regional Assessment,2001; Sokolov et al, 2009). Incidences of these organisms that typically reside in warmerconditions have already been reported (O'Neill et al, 1992), but little is known about

    2

  • how these endemic strains interact with each other, and their surrounding microbial

    inhabitants. Due to extreme changes in environmental conditions over relatively small

    spatial scales, the GBE provides an excellent opportunity to study the dynamics of themicrobial populations residing there. Additionally, different oyster beds in the GBE are

    influenced by different factors. The Oyster River (OR) oyster bed site is classified as

    prohibited for shellfish harvesting because of close proximity to a wastewater treatment

    facility, which causes low dissolved oxygen and high dissolver organic carbon levels (Fig1.1). Comparably, the Nannie Island (NI) oyster bed is more distant from the facility andis thought to be less influenced by waste water effluent. NI is generally accepted as acleaner site, so that recreational oyster harvesting is approved there (Fig. 1.1).

    EW%i:-* Kr.**' Sl**

    Figure 1.1: Great Bay Estuary Shellfish Classification Map. Courtesy of the NHShellfish Program.

    We took two approaches to study microbial populations associated with oysters inthe GBE, one focused on a single human pathogenic species, and another focused on themixed microbial populations associated with Vibrio free and Vibrio infested oysters.First, we will use a newly discovered endemic population of V. cholerae to 1) correlate

    3

  • incidence and abundance with environmental conditions, 2) describe the structure ofthat

    particular population including strain relatedness to each other and with known toxigenicstrains, and 3) explore any evidence of recombination between the local strains with a

    multi-locus sequence analysis (MLSA). In a parallel study to understand microbial

    populations in the GBE as they correlate and interact with a pathogen, V.parahaemolyticus, we will perform a microbial population survey by a 16s rDNAmetagenetic approach. This survey will allow us to explore 1) differentiations betweenpopulations from various environments, including the OR or NI, and 2) identify host andwater specific microbial community members. Understanding the dynamics of thesepopulations, how they interact, how they differ, and how th

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