Microbial Interactions Influencing the Emergence of Pathogenic Vibrios in Oysters

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Project Type: 
Research
Project Number: 
R/CE-137
Inception Date: 
2008
Completion Date: 
2010

Participants:

Stephen Jones N.H. Sea Grant Collaborator
Cheryl Whistler UNH - Department of Molecular, Cellular & Biomedical Sciences Principal Investigator
Vaughn Cooper UNH - Department of Molecular, Cellular & Biomedical Sciences Co-Principal Investigator
Randi Desy UNH - Department of Molecular, Cellular & Biomedical Sciences Technician

Students Involved:

Crystal Ellis UNH - Department of Molecular, Cellular & Biomedical Sciences
Freddie Ta UNH - Department of Molecular, Cellular & Biomedical Sciences
Matt Gerding UNH - Department of Molecular, Cellular & Biomedical Sciences
Rachel Donner UNH - Department of Molecular, Cellular & Biomedical Sciences
Megan Striplin UNH - Department of Molecular, Cellular & Biomedical Sciences
Jenny Mahoney UNH - Department of Molecular, Cellular & Biomedical Sciences
Brian Schuster UNH - Department of Molecular, Cellular & Biomedical Sciences
Tucker Noyes UNH - Department of Molecular, Cellular & Biomedical Sciences
Anna Tyzick UNH - Department of Biological Sciences
Jong Yu UNH - Department of Molecular, Cellular & Biomedical Sciences
Nicole Lefebvre UNH - Department of Biological Sciences
Abstract: 
Within the last two decades the number of gastroenteritis outbreaks due to Vibrio parahaemolyticus and Vibrio vulnificus has risen steeply worldwide. Consumption of raw or undercooked shellfish represents a common mechanism for infection by these vibrios. Due in part to changing patterns in human land and resource use, and variability in seasonal extremes that may represent larger changes in climate, these emergent pathogens are a public health concern in north temperate regions not previously at risk (McLaughlin et al., 2005; Fitzpatrick, 2002; DePaola et al., 2000; Abbott et al., 1989). Public health and shellfish program managers are now challenged to better understand ecological dynamics that drive shellfish-borne Vibrio illnesses.
 
One major question is whether changing estuarine conditions simply affect total abundance of all bacteria, including potential Vibrio pathogens, or whether certain physical or biological conditions actually may selectively amplify minority pathogenic strains. Because pathogenic biovars are typically rare and are usually not differentiated from total bacterial counts, we require a more nuanced perspective of factors influencing estuarine microbial ecology, including the role and relevance of other microbes residing in shellfish. Furthermore, the ability of bacteria to exchange genes with neighboring populations highlights the potential for spread of virulence determinants among non-pathogens.
 
Thus, this proposal focuses on how physical factors, such as temperature, salinity, and dissolved nutrients, affect vibrios within the context of other resident microbes that may favor or antagonize potential pathogens. This proposal also will delineate in detail the genetic relationships between pathogenic and nonpathogenic Vibrio isolates and quantify the extent of their past and future potential genetic exchange. Both of these objectives will aid development of sensitive and accurate typing methods for pathogenic Vibrios in susceptible ecosystems. More generally, by examining the interplay of physical conditions and microbial community interactions on the emergence of pathogenic biovars, we will be better equipped to predict broader infectious threats and protect human health.
Objectives: 
1. Map the population genetic structure and virulence potential of V. vulnificus and V. parahaemolyticus and determine the extent of recombination between strains using multi-locus sequence typing (MLST).
 
2. Determine the correlated effects of the resident microbial community on the abundance and distribution of Vibrio populations in oysters.
 
Methodology: 

The presence of Vibrio vulnificus and Vibrio parahaemolyticus harboring virulence genes in microbial community DNA isolated from oysters will be determined by multiplex PCR. From cultured isolates we will determine the interspecies population structure using multi-locus seuqence typing (MLST). From the same oyster community DNA, we will determine intraspecies population structure, by amplifying the 16s rDNA variable region (V6) and using massively parallel pyrosequencing (424 sequencing technology).

Rationale: 

It is believed that Northern Vibrio isolates are not pathogenic; however, human infections have been traced to local shellfish. The proposed study will examine the ecology of Vibrios thus, aiding in risk assessment, management, and education. We will determine the proportion of pathgenic biovars and estimate the recombination between strains, as this could lead to a shift in the proportion of pathgenic biovars within the population. Due to the precedent of microbial antagonism and the indication that normal microbiota actively exclude Vibrios from shellfish, we will determine the identity of microbial members in Vibrio infested and Vibrio free oysters.

Accomplishments: 

2013

Vibrio research at UNH leads to formulation of control plans to protect human health and preserve shellfish businesses
N.H. Sea Grant-funded research helped inform Vibrio control plans that increase the awareness of the potential risks from eating raw or undercooked shellfish and the appropriate measures to reduce those risks and protect the New England shellfish industry.

RELEVANCE: An increasing number of New England shellfish beds are being temporarily closed down due to the presence of pathogenic Vibrios — bacteria that may be present in raw and undercooked shellfish and can lead to gastroenteritis in humans. Changing ecological and climatic conditions may influence the abundance and strain distribution of pathogenic Vibrios in New England shellfish such as oysters.

RESPONSE: With funding provided by NHSG, researchers identified components of climate change that alter microbial communities in oysters and potentially impact the abundance and distribution of pathogenic Vibrios.

RESULTS: Researchers presented this information to the public, shellfish growers and regulators at numerous venues in 2013. These information sessions assisted in the formulation of various Vibrio control plans aimed at protecting human health and preserving shellfish businesses in New England.

New microbial typing methods improve identification of pathogens and climate change impacts in N.H. study
N.H. Sea Grant-funded researchers conducted analyses to determine if there are correlations between Vibrio abundance and strain distribution in response to changing ecological and climatic conditions. The unique microbial typing methods developed as part of this study were used in 2013 for analyses of environmental and clinical samples from Maine, New Hampshire, Massachusetts and Connecticut. Methodologies developed from NHSG research will help shellfish managers and scientists to better understand the role of climate change on pathogenic Vibrios and encourage region-wide analyses for improved region-wide decision-making.

Researchers inform Mass. and Maine Departments of Health about emergence of pathogens in shellfish
With funding provided by N.H. Sea Grant, researchers conducted studies to determine if there are correlations between Vibrio abundance and strain distribution in response to changing ecological and climatic conditions. In 2013, researchers provided information about environmental epidemiology to the Mass. and Maine Departments of Public Health related to the emergence of pathogens in each state’s commercial shellfish. This information will help health officials to be more prepared for potential Vibrio outbreaks and allow them to inform the public and shellfish farmers about safe handling and consumption guidelines.

Researchers inform N.H. oyster farmers about safe shellfish handling practices to minimize human illness
The presence of pathogenic Vibrios in shellfish can cause gastroenteritis in humans who consume raw or undercooked shellfish containing the pathogen. With funding provided by N.H. Sea Grant, researchers conducted analyses to determine the role of changing environmental factors in the prevalence and distribution of pathogenic Vibrios in N.H.’s Great Bay. In 2013, researchers held a roundtable discussion to inform N.H. oyster farmers about harvest practices that minimize risks of Vibrio diseases to consumers. This educational event provided oyster farmers with research-based advice, helping them to maintain safe product handling protocols that protect human health while ensuring a successful harvest.

N.H. pathogenic Vibrio research leads to development of monitoring programs and detection methods in neighboring states
An increasing number of shellfish beds are being temporarily closed down due to the presence of pathogenic Vibrios — bacteria that may be present in raw and undercooked shellfish and can lead to gastroenteritis in humans. With funding provided by N.H. Sea Grant, researchers sampled oysters in N.H.’s Great Bay and conducted analyses to determine pathogenic Vibrio abundance and distribution in response to changing ecological conditions. In 2013, researchers helped Conn. and Mass. state shellfish programs to design V. parahaemolyticus monitoring programs and laboratory detection methods for subsequent years based on their NHSG-funded research. These efforts will help to create a more coordinated and widespread response to Vibrios, ensuring adequate steps are taken to protect human health and preserve the economic benefits of shellfish beds.

Technologies developed by N.H. Sea Grant researchers to help understand pathogens
N.H. Sea Grant-funded researchers developed several technologies related to understanding the environmental emergence of Vibrio parahaemolyticus in the New England region, including strain typing and metagenetic sequencing methods. These methodologies will improve pathogen identification, discrimination and sensitive detection to help protect human health.

2012

Populations of One Pathogenic Species of Vibrios Declining in Great Bay Estuary
Within the last two decades the number of gastroenteritis outbreaks due to pathogenic strains of Vibrios in undercooked or raw shellfish has risen steeply worldwide. Improved monitoring of these strains is necessary for a more complete view of their abundance, distribution and potential for human exposure. NHSG-funded researchers conducted studies in Great Bay Estuary to determine abundance of Vibrios. Their research indicates that V. vulnificus populations are in decline compared to historical data. This finding was incorporated into a large dataset that will be used to generate a relational database for analysis and will be made available to the public upon completion. Results from this project will enable comparisons with other study sites and assist in the development of shellfish management policies in the U.S.

Researchers Identify Genetic Correlation between Vibrio parahaemolyticus and Cyanobacteria
Changing patterns of human land and resource use, along with variability in seasonal extremes, have contributed to the northward spread of emergent pathogens including certain strains of Vibrios. NHSG-funded researchers conducted additional analyses of the V. parahaemolyticus strain from the metagenomic library to improve their understanding of the potential microbial interactions that influence this emergence. Their analyses indicate that there is a genetic correlation between V. parahaemolyticus and cyanobacteria. This finding helps researchers understand ecological determinants in the occurrence and survival of V. parahaemolyticus in N.H. estuarine waters.

Genetic Analyses Provide Improved Understanding of Pathogen Origin, Potential for Future Outbreaks
Consumption of raw or undercooked shellfish is a common mechanism by which humans are infected with pathogenic strains of Vibrios that lead to gastroenteritis. With funding provided by NHSG, researchers conducted genetic sequencing analysis of V. parahaemolyticus. Their data indicate that strains from recent outbreaks — including those caused by people eating oysters harvested in Mass. — are similar to strains found in N.H. waters and oysters. This suggests that N.H. strains may evolve into pathogenic strains and that the Mass. clinical strains may also have evolved from local populations rather than being brought in via external sources. Results from this research provide shellfish managers and scientists with an improved understanding of pathogen origin and the potential for future outbreaks.

2011

Researchers Link Water Temperature and Fluctuations in Salinity to Occurrences of Vibrios
Within the last two decades the number of gastroenteritis outbreaks due to “Vibrio parahaemolyticus” and “V. vulnificus” has risen steeply worldwide. Consumption of raw or undercooked shellfish is a common mechanism by which humans are infected. N.H. Sea Grant-funded researchers examined the microbial interactions that influence the seasonal abundance and distribution of pathogenic Vibrios found in oysters in Great Bay. Their research indicates that increasing temperature and major rainfall events that influence the bay’s salinity impact the occurrences of Vibrios, which could in turn help predict the potential for gastroenteritis outbreaks.

Discovery of “Vibrio cholerae” in Great Bay Marks Northern-most Occurrence of Pathogen
While conducting research on bacterial “Vibrio” species found in New Hampshire’s Great Bay Estuary, N.H. Sea Grant-funded researchers found the first documented strains of “Vibrio cholerae,” a pathogenic species that famously causes the disease cholera. Although strains found here are incapable of causing cholera, these strains were associated with oysters and may cause gastroenteritis. The discovery is the northern-most occurrence of this species, and the population will serve as an important model for understanding niche expansion and adaptation, particularly as climate change impacts the local ecology.

Researchers Establish Population Genetic Structure Map for "Vibrio parahaemolyticus"
Using multi-locus sequence typing, NHSG researchers established a population genetic structure map for "Vibrio parahaemolyticus," a microbe found in oysters that can cause gastroenteritis if a pathogenic strain is consumed. This map will help researchers to characterize the evolution and mechanisms of pathogen emergence and could indicate the potential for future outbreaks.

Researchers determine influences for the presence and emergence of pathogenic Vibrios in oysters
RELEVANCE: Consumption of raw or undercooked bivalve shellfish has caused an increasing number of gastroenteritis outbreaks throughout the world in recent years. These outbreaks have often been associated with pathogenic strains of Vibrio vulnificus and V. parahaemolyticus, both of which are present in the Great Bay Estuary. Changing physical conditions, climate change and the microbial community interactions in the estuary may influence the abundance and population structure of pathogenic Vibrio strains.

RESPONSE: Researchers used genetic analyses to map the population structure of Vibrio strains to determine the potential for genetic exchange between pathogenic and nonpathogenic strains. Physico-chemical factors in the estuary were recorded and oyster samples were collected. 

RESULTS: Researchers have detected Vibrio cholera for the first time in the Great Bay Estuary. However, they have yet to determine its particular environmental niche. No pathogenic strains of Vibrio vulnificus were detected in the estuary and the population is lower and more rarely detected compared to historical data, possibly due to increased estuarine salinity. Total populations of V. parahaemolyticus increased with increasing temperature and decreasing dissolved oxygen. There is no evidence that the pathogenic and nonpathogenic strains have undergone genetic exchange with one another.

RECAP: Researchers now have a better understanding of the Vibrio species in the Great Bay Estuary, which will aid in the development of sensitive, accurate molecular typing methods for pathogenic Vibrios species.

Publications

Available from the National Sea Grant Library (use NHU number to search) or NH Sea Grant

Journal Article

  • Mahoney, J., M. Gerding, S. Jones and C. Whistler (2010). Comparison of the pathogenic potentials of environmental and clinical "Vibrio parahaemolyticus" strains indicates a role for temperature regulation in virulence. Applied and Environmental Microbiology 76(22):7459-7465, November 2010.
  • Schuster, B., A. Tyzik, R. Donner, M. Striplin, S. Almagro-Moreno, S. Jones, V. Cooper and C. Whistler (2011). Ecology and genetic structure of a northern temperate "Vibrio cholerae" population related to toxigenic isolates. Applied and Environmental Microbiology 77(21):7568-7575, Nov. 2011.
  • Ellis, C., B. Schuster, M. Striplin, S. Jones, C. Whistler and V. Cooper (2012). Influence of seasonality on the genetic diversity of "Vibrio parahaemolyticus" in New Hampshire shellfish waters as determined by multilocus sequence analysis. Applied and Environmental Microbiology 78(10):3778-3782.

Thesis/Dissertation

  • Schuster, B. (2010). Microbial interactions with oysters from the Great Bay Estuary: characterization of endemic "Vibrio cholerae" and oyster metagenetics. Master's Thesis, University of New Hampshire.
  • Mahoney, J. (2011). Existing regulatory circuitries govern backbone and acquired host association factors in the human pathogen "Vibrio parahaemolyticus." Doctoral dissertation, University of New Hampshire.
  • Yu, J. (2011). Incidence, abundance, postharvest processing and population diversity of pathogenic vibrios in oysters from the Great Bay Estuary. Master's Thesis, University of New Hampshire.

Proceeding

  • Jones, S., M. Striplin, J. Mahoney, V. Cooper and C. Whistler (2009). Incidence and abundance of pathogenic Vibrio species in the Great Bay Estuary, New Hampshire. Proceedings of the Seventh International Conference on Molluscan Shellfish Safety, Lassus, P. (ed.), pp. 127-134, Nantes, France, June 14-19, 2009.