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MICROBIAL INTERACTIONS WITH OYSTERS FROM THE GREAT BAYESTUARY: CHARACTERIZATION OF ENDEMIC Vibrio cholerae AND OYSTER
BRIAN MICHAEL SCHUSTER
B. S., University of New Hampshire, 2008
Submitted to the University of New Hampshire
in Partial Fulfillment of
the Requirements of the Degree of
Master of Science
UMI Number: 1489963
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TABLE OF CONTENTS
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
Results 1379Discussion ^
III METAGENETIC ANALYSIS REVEALS HOST ASSOCIATED POPULATIONSWITHIN OYSTERS COMPARED TO THE SURROUNDING WATER 24
Results and Discussion 34
Future Directions 46
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
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
BACTERIAL ECOLOGY OF THE GREAT BAY ESTUARY: THE DISCOVERY OF
ENDEMIC Vibrio cholerae AND DEVELOPING A METAGENETICS APPROACH
TO EXPLORE THE OYSTER MICROBIOTA
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.
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
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
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
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