1) Vibrio vulnificus (Vv) is an opportunistic human pathogen found in the Great Bay Estuary (GBE) of New Hampshire. While cases of Vv infection have increased in the Gulf of Maine, there have been no reported cases from GBE strains. 2) The study analyzed 37 Vv isolates from GBE using short read genome sequencing and multilocus sequence typing (MLST). Most isolates contained novel sequence types not in databases, suggesting genetic diversity. 3) Analysis of virulence genes and a phylogenetic marker showed GBE isolates were more similar to environmental strains and different from known pathogenic strains, supporting the hypothesis that GBE strains are non-pathogenic.

1. Molecular Typing by srst2 Analysis of Vibrio vulnificus Isolates
from the New Hampshire Great Bay Estuary
Sarah E. Sanders1, Katherine Kiley1, Brian Moore1, Jordan Ramsdell2, Stephen Jones2 and Loren A. Launen1
Abstract
References
Results
Introduction
Vibrio vulnificus (Vv) is an opportunistic human pathogen that resides in seawater and
brackish areas, including the Great Bay Estuary (GBE) of New Hampshire. Infections with
Vv are caused by wound exposure to seawater or by consumption of raw oysters and,
although rare, can be fatal in immunocompromised persons. Although the level of
vibriosis has been increasing in recent years in the Gulf of Maine region (Urquhart et al.
2016), to our knowledge there has never been a case of Vv infection due to Vv from the
GBE, suggesting local populations are non-pathogenic. Interestingly, the number of Vv
isolates recovered in surveillance samples has increased in recent years (Fig. 1, and Kiley
et al. poster), providing us a considerable collection of strains for analysis.
5 8
28
23
55
78
150
0
20
40
60
80
100
120
140
160
2007 2008 2009 2012 2013 2014 2015
FIGURE 1: NUMBER OF VIBRIO VULNIFICUS ISOLATED
FROM THE GBE IN YEARS SAMPLED
We hypothesize that the Vibrio vulnificus strains in the GBE are non-pathogenic and are
most genetically similar to environmental (non-pathogenic) Vv found in other regions.
1. Bisharat, N., Cohen, D. I., Harding, R. M., Falush, D., Crook, D. W., Peto, T., & Maiden, M. C. (2005). Hybrid Vibrio vulnificus. Emerging Infectious
Diseases, 11(1), 30–35. http://doi.org/10.3201/eid1101.040440
2. Inouye, M., et al(. 2014, November 20). SRST2: Rapid genomic surveillance for public health and hospital microbiology labs. Genome Medicine.
doi:10.1101/006627
3. Kumar S., Stecher G., and Tamura K. (2015). MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and
Evolution (submitted).
4. Reynaud, Y., Pitchford, S., De Decker, S., Wikfors, G. H., & Brown, C. L. (2013). Molecular Typing of Environmental and Clinical Strains of Vibrio
vulnificus Isolated in the Northeastern USA. PLoS ONE, 8(12), e83357. http://doi.org/10.1371/journal.pone.0083357
5. Sanders, S, Jones, S, Launen, L (2015). A Characterization of Potentially Pathogenic Vibrio vulnificus Isolates Found in the New Hampshire Great Bay
Estuary. Poster.
6. Urquhart, Erin A., Stephen H. Jones, Jong W. Yu, Brian M. Schuster, Ashley L. Marcinkiewicz, Cheryl A. Whistler, and Vaughn S. Cooper. PLOS ONE PLoS
ONE 11.5 (2016): e0155018. doi:10.1371/journal.pone.0155018
Hypothesis
Approach & Methods
Gene Designation Description
16s rRNA HK
Phylogenetic marker, A in environmental strains,
B in clinical strains
ARSA V Arylsulfatase A, sulfate metabolism
mtlABC V Mannitol/fructose-specific protein
nanA V Sialic acid metabolism
pilF V Pilus assembly protein, cell movement
vcg V
virulence correlated gene (C / E type for
clinical/environmental)
vvhA V Hemolysin / cytolysin,Vv marker
wza V Capsule production
wzb V Capsule production
wzc V Capsule production
Vibrio vulnificus (Vv) is an opportunistic pathogen deadly to immunocompromised
individuals. Although Vv occurs in the Great Bay Estuary (GBE) of New Hampshire, to
our knowledge none of the cases of vibriosis reported in the Gulf of Maine (Urquhart et
al. 2016), are due to GBE strains of Vv. We hypothesize that the Vv strains in the GBE
are non-pathogenic and genetically similar to environmental (non-clinical) Vv found
elsewhere. Using short read whole genome sequences of isolates from 2009 – 2015, we
compared several house-keeping and virulence-associated genes, with existing
(Bisharat et al. 2005), and custom, MLST strategies, using the read-mapping program
SRST2 (Innouye et al. 2013). Although preliminary, our data suggest that the GBE
isolates are genetically diverse (containing 34 novel MLST sequence types (ST) and only
one known ST), and similar to environmental strains in their 16S rRNA structure, and
genes important in pathogenicity-related functions such as capsule synthesis. The
population structure of Vv in the GBE has changed in the past year at one of our two
sampling locations, indicating that while current populations may not be pathogenic,
changing environmental conditions could favor pathogenic strains in future.
Isolate Tissue Source Site M/D/Y 16s rRNA glp gyrB pilF vcg vvhA wza wzb wzc
1022 Oyster E OR 7/12/09 A + + + E + + + +
2584 Oyster E OR 8/16/12 A + + + E + - - -
2785 Oyster E OR 8/23/12 A + + + E + + + -
3741 Oyster E NI 7/10/13 A + + + E + + + +
3801 Oyster E OR 7/10/13 A + + + E + + + 92%
3914 Oyster E NI 8/1/13 A + + + E + - - -
3919 Oyster E NI 8/1/13 A + + + E + + + 90%
3925 Oyster E NI 8/1/13 A + + + E + + + 94%
3927 Oyster E NI 8/1/13 A + + + E + - - -
3930 Oyster E NI 8/1/13 A + + + E + + + -
3971 Oyster E OR 8/1/13 A + + + E + - - -
3979 Oyster E OR 8/1/13 A + + + E + + + 90%
3984 Oyster E OR 8/1/13 A + + + E + + + -
4888 Oyster E OR 6/20/14 A + + + E + + + 93%
4963 Oyster E NI 6/20/14 A + + + E + + + 92%
5048 Oyster E OR 7/7/14 A + + + E + + + -
5054 Oyster E OR 7/7/14 B + + + E + - - -
5067 Oyster E OR 7/7/14 B + + + C + - - -
5071 Oyster E OR 7/7/14 A + + + C/E + - - -
5523 Oyster E NI 7/30/14 A + + + E + 94% + -
5557 Oyster E SCS 8/1/14 A + + + E + 93% + 94%
5706 Oyster E NI 8/13/14 A + + + E + - - -
5708 Oyster E NI 8/13/14 A + + + E + - - -
5710 Oyster E NI 8/13/14 A + + + E + + + -
6848 Oyster E OR 8/3/15 A + + + E + + - -
6858 Oyster E OR 8/3/15 A + + + E + + + +
7084 Oyster E OR 8/17/15 A + + + E + + + -
7093 Oyster E OR 8/17/15 A + + + E + + - -
7096 Oyster E OR 8/17/15 A + + + E + - - -
7207 Oyster E NI 8/24/15 A + + + E + + + -
7210 Oyster E NI 8/24/15 A + + + E + + + -
7226 Oyster E OR 8/24/15 A + + + E + 94% + +
7242 Oyster E OR 8/24/15 A + + + E + - + -
7245 Oyster E OR 8/24/15 A + + + E + + + -
7246 Oyster E OR 8/24/15 A + + + E + + + -
7341 Oyster E OR 9/1/15 A + + + E + - + -
7427 Oyster E OR 9/15/15 A + + + E + - - -
M06-24 Blood C CA ~1990 B + + + C + + + +
CMCP6 Blood C S.Korea ~2003 B + + + C + + + -
 MLST analysis using the 10 housekeeping gene-based scheme of Bisharat et al (2005)
was conducted on 39 isolates. Only 3 isolates (all from Oyster River, collected in 2015)
matched a known sequence type (ST 59). The remaining 36 isolates contained 33
novel sequence types not currently found in the pubmlst.org database for Vibrio
vulnificus.
 Genetic analysis including combined housekeeping and virulence-associated genes
suggests the population of Vv in the GBE in 2015 differed from that existing in
previous years, possibly indicating the expansion of some strains in response to
environmental changes. This analysis was better able to capture differences in strains
than analysis of the 16S rRNA alone.
 Analysis of the 16S rRNA gene, as well as combinations of housekeeping and
virulence associated genes suggests that GBE isolates of Vv are not similar to well-
studied pathogenic strains, supporting our hypothesis.
 All of our results support the existence of considerable genetic diversity in Vibrio
vulnificus found within oysters in the Great Bay Estuary.
Acknowledgements
A. B.
Figure 5: Phylogenetic trees (neighbor-joining) created using (A) 16S rRNA
sequences and (B) concatenated 16S rRNA-glyp-gyrB-pilF-vvhA sequences
(Reynaud et al. 2013). Parameters were: Kimura-2-parameter, 1000 bootstraps, condensed.
Table 2: Analysis of selected virulence and housekeeping genes. “+” indicates gene
detected and confirmed by BLAST (95%, E value >3.0x10-3), see Table 1 for other symbols.
Conclusions
We used short read sequence typing (SRST2) developed by Katherine Holt’s lab at the
University of Melbourne (Inouye et al. 2014) to analyze the genetic diversity of 37 Vibrio
vulnificus isolates. SRST2 is a read mapping-based tool for fast and accurate detection of
genes, alleles and multi-locus sequence types (MLST) from whole genome sequencing
(WGS) data.
Figure 2: Project Experimental Approach
Table 1: Virulence-related and Marker Genes
Used to Develop Custom MLST Analysis
Figure 4: SRST2 Overview
(Inouye et al. 2014).
Figure 3: MLST Scheme Using 10 House-keeping Genes
(Bisharat et. al 2005).
1Keene State College
2University of New Hampshire
Multi-locus sequence typing (MLST) types bacterial strains
for epidemiological and evolutionary analysis. We used
MLST to detect known and novel sequence types (STs,
Figure 3) and to analyze virulence associated genes and a
phylogenetic marker (Table 1).
SRST2 allows the facile analysis of specific
loci within whole genome data for
molecular typing and evolutionary
analysis. It is sensitive enough to detect
genes and call alleles at >5X coverage.
*7226 (circled) matches ST
59 in the pubmlst.org Vv
database.
Ms. Katie Featherston, Chantale Lacroix, Marianne O’Brien and Andressa Gutierrez from
Keene State College assisted with the technical aspects and administration of this work.
This research was supported by the National Science Foundations Grant to NH EPSCoR
#1330641. Summer support for the author was provided by an Institutional
Development Award (IDeA) from the National Institute of General Medical Sciences of
the National Institutes of Health under grant number P20GM103506.