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 1Keene State College 2University of New Hampshire Abstract Figure 3: MLST Scheme Using 10 House-keeping Genes (Bisharat et. al 2005). Figure 4: SRST2 Overview (Inouye et al. 2014). 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. 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. 2013), 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.  All isolates obtained prior to 2015 Table 1: Virulence-related and Marker Genes Used to Develop Custom MLST Analysis Gene Designation Description 16s rRNA HK Phylogenetic marker, A in environmental strains, B in clinical strains ARSA V Arylsulfatase A, sulfate metabolism mtlABC Mannitol/fructose-specific protein nanA Sialic acid metabolism pilF Pilus assembly protein, cell movement vcg virulence correlated gene (C / E type for clinical/environmental) vvhA Hemolysin / cytolysin, Vv marker wza Capsule production wzb wzc Introduction Vibrio vulnificus (Vv) is an opportunistic human pathogen that resides in seawater and brackish areas, inclusing 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. All isolates obtained during 2015 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 for molecular typing and evolutionary analysis. It is sensitive enough to detect genes and call alleles at >5X coverage. B. Well studied pathogenic Vv controls A. *7226 (circled) matches ST 59 in the pubmlst.org Vv database. Conclusions Results 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. 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.. 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 + 2584 8/16/12 - 2785 8/23/12 3741 NI 7/10/13 A  3801 92% 3914 8/1/13 3919 90% 3925 94% 3927 3930 3971 3979 3984 4888 6/20/14 93% 4963 5048 7/7/14 5054 B 5067 C 5071 C/E 5523 7/30/14 5557 SCS 8/1/14 5706 8/13/14 5708 5710 6848 8/3/15 6858 7084 8/17/15 7093 7096 7207 8/24/15 7210 7226 7242 7245 7246 7341 9/1/15 7427 9/15/15 M06-24 Blood CA ~1990 CMCP6 S.Korea ~2003 Hypothesis 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 Acknowledgements Approach & Methods 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 Figure 2: Project Experimental Approach Isolate Vibrio vulnificus Extract gDNA Confirm Identity (vvhA PCR) Sequence gDNA (Illumina, 150bp Paired End Reads Whole Genome Comparison Analysis (ROARY) Gene Specific Analysis (Molecular Typing by MLST) References 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 Inouye, M., et al(. 2014, November 20). SRST2: Rapid genomic surveillance for public health and hospital microbiology labs. Genome Medicine. doi:10.1101/006627 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 Kumar S., Stecher G., and Tamura K. (2015). MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets..Molecular Biology and Evolution (submitted). Sanders, S, Jones, S, Launen, L (2015). A Characterization of Potentially Pathogenic Vibrio vulnificus Isolates Found in the New Hampshire Great Bay Estuary. Poster. We used short read sequence typing (SRST2) developed by Katherine Holt’s lab at the University of Brisbane (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.