Metagenomic characterization of Vibrio in the Neuse River Estuary, NC Kelsey Jesser, Brett Froelich, Rachel Noble UNC Chapel Hill Institute of Marine Sciences Morehead City, NC

Vibrio Diverse and naturally occurring genus of bacteria Includes several human pathogens Concentrated in oysters and other filter feeders Most abundant in summer months Many species have a moderate salinity requirement Response to other environmental parameters not well understood, vary across species and ecosystems Oysters aren’t prominent in the NRE, but they coinhabit estuarine ecosystems (shallow, eutrophic, lots of phytoplankton); Both oysters and Vibrio are prominent in estuaries Facultative fermentative- can use oxygen to undergo aerobic respiration, but can switch to fermentative metabolism if oxygen is not present Chemoorganotrophic- utilize chemical energy from breaking bonds in organic (not inorganic) molecules Graph shows seasonality of various clinically-relevant Vibrio in the Black Sea, off the coast of Georgia (Kovashil et al. 2015) Photos: Vibrio fisheri (flagella), Vp gram stain [1-3]

Vibriosis in the United States Vibriosis infections have increased ~100-fold since 2000, became a nationally notifiable disease in 2007 [CDC, Trends in Foodborne Illness in the United States 2014, 4, 5]

Vibrio detection Easily culturable Selective and differential, but not effective Culture methods are not sufficiently discriminatory Can’t differentiate clinical v. environmental strains Viable but not culturable state (VNBC) Molecular methods (ex: QPCR) Virulence-gene focused not discriminatory PCR inhibitors Thinking about metagenomic approaches Preliminary work Use figures they use to sell media [10, 11]

Study site: the Neuse River Estuary (NRE) Estuary in eastern North Carolina Minimal tidal influence Flow dominated by river inflow and wind forcing Stratified Freshwater on top of saltwater Monitored by the ModMon monitoring program Shallow, bar-built estuary Minimal tidal influence, flow usually dominated by river inflow and wind forcing Stratified, fresh water on top of saltwater Eutrophic, anthropogenic as well as environmental gradients [12, map from Froelich et al. 2013]

Methods Samples collected 7/22/13 in coordination with Modmon Modmon stations 30, 70, 120 Surface and bottom water 200 mL filtered onto 0.4 µm polycarbonate filters Illumina sequenced extracted DNA 2 MiSeq lanes/sample Paired-end 150 bp reads Annotated with MG-RAST Extracted normalized Vibrio reads (~5% of annotated taxa) Extreme climatic events- tropical storms, hurricanes, drought We’re especially interested in storms as precipitation and wind-driven resuspension events 15 km, 28 km, and 42 km

Total Vibrio Linear regressions– Vp significant negative relationship (p=0.2); Vv no significance (p=0.2)

Vibrio species abundances Multivariate analyses: nMDS Vibrio species abundances nMDS2 nMDS1 NMDS: Based on a similarity/dissimilarity matrix; ranks distances between “objects” based on similarity matrix and plots them in order to preserve those distances; runs through several ordinations/iterations to find best fit or least “stress”—lower stress is better Proximity corresponds to similarity, not the original distance between objects

Vibrio species abundances Multivariate analyses: nMDS Vibrio species abundances nMDS2 NMDS: Based on a similarity/dissimilarity matrix; ranks distances between “objects” based on similarity matrix and plots them in order to preserve those distances; runs through several ordinations/iterations to find best fit or least “stress”—lower stress is better Proximity corresponds to similarity, not the original distance between objects nMDS1

Indirect gradient analysis Spearman’s rank correlation (p<0.05) nMDS1 Salinity DOC DON Salinity (ppt) nMDS2 nMDS1 Spearman’s correlation– based on RANKS, assumes monotonic relationship between variables Significance just means that you can reject the null hypothesis that the two variables are not associated; doesn’t tell you anything about the STRENGTH of the relationship (need to do a regression, calculate R2 to do that) Dissolved organic nitrogen (DON) was calculated by subtracting dissolved inorganic nitrogen (DIN) from total dissolved nitrogen (TDN). If the DIN value used in the calculation was below the detection limit, it was take to be zero for this calculation. At one point DON was determined by high temperature oxidation using the Antek 7000N or Antek 7000V analyzer. Dissolved organic carbon (DOC) concentration was measured using a Shimadzu TOC-5000A Analyzer: Water samples were vacuum filtered (less than 25 kilopascal) using pre-combusted Whatman glass microfibre filters (GF/F). The filtrate was stored in pre-combusted glass scintillation vials with Teflon closures and frozen at -20 degrees Celsius until analysis. The Shimadzu TOC-5000A Analyzer uses high temperature catalytic oxidation followed by non-dispersive infrared analysis of the CO2 produced. Samples were acidified to a pH less than 2 and sparged with air before they were analyzed for non-volatile organic carbon. DOC values in 1996 were run from previously run nutrient samples.

Indirect gradient analysis Spearman’s rank correlation (p<0.05) nMDS1 Salinity DOC DON DOC (μM) nMDS2 nMDS1 DON (µg/L) nMDS1 nMDS2 Spearman’s correlation– based on RANKS, assumes monotonic relationship between variables Significance just means that you can reject the null hypothesis that the two variables are not associated; doesn’t tell you anything about the STRENGTH of the relationship (need to do a regression, calculate R2 to do that) Y-axis (nMDS2) Temperature Turbidity POC PN Chl-a Dissolved organic nitrogen (DON) was calculated by subtracting dissolved inorganic nitrogen (DIN) from total dissolved nitrogen (TDN). If the DIN value used in the calculation was below the detection limit, it was take to be zero for this calculation. At one point DON was determined by high temperature oxidation using the Antek 7000N or Antek 7000V analyzer. Dissolved organic carbon (DOC) concentration was measured using a Shimadzu TOC-5000A Analyzer: Water samples were vacuum filtered (less than 25 kilopascal) using pre-combusted Whatman glass microfibre filters (GF/F). The filtrate was stored in pre-combusted glass scintillation vials with Teflon closures and frozen at -20 degrees Celsius until analysis. The Shimadzu TOC-5000A Analyzer uses high temperature catalytic oxidation followed by non-dispersive infrared analysis of the CO2 produced. Samples were acidified to a pH less than 2 and sparged with air before they were analyzed for non-volatile organic carbon. DOC values in 1996 were run from previously run nutrient samples.

V. vulnificus and V. parahaemolyticus 30S 30B 70S 70B 120S 120B LOG TRANSFORM regressions– Vp significant negative relationship, Vv no significance

Ongoing work Illumina amplicon sequencing in the Neuse River Estuary Sampling every ~two weeks for 1 year, storm sampling 16S rRNA and hsp60 genes Vibrio response to environmental gradients Vibrio relationship to other organisms in the bacterioplankton Combined power of sequencing, QPCR, and culture Identifying environmental drivers of Vibrio communities in time and space Identifying associated bacterial communities

Acknowledgements Rachel Noble, Brett Froelich Noble lab Modmon monitoring project and the Paerl lab Orion Integrated Biosciences UNC ROI Kelsey Jesser PhD Candidate, Marine Sciences UNC Chapel Hill Institute of Marine Sciences kjesser@live.unc.edu 208-308-5653

References Vibrio gram stain: http://synapse.koreamed.org/DOIx.php?id=10.5021/ad.2011.23.S1.S25&vmode=PUBREADER, accessed 1/19/2016 11:14 a.m. Vibrio fischeri flagella: http://en.citizendium.org/wiki/Vibrio_fischerid accessed 2/27/16 3:05 p.m. Oyster gif: http://sploid.gizmodo.com/video-oysters-are-fantastic-at-filtering-dirty-water-1652540034 accessed 2/27/16 3:08 p.m. V. parahaemolyticus cartoon: http://adoptamicrobe.blogspot.com/2006/06/vibrio-parahaemolyticus.html; accessed 1/19/2016 11:07 a.m. V. vulnificus cartoon: http://adoptamicrobe.blogspot.com/2006/06/vibrio-vulnificus.html; accessed 1/19/2016 11:09 a.m. Vv Oysters: http://www.washingtoncitypaper.com/blogs/youngandhungry/2012/04/20/is-an-all-you-can-eat-oyster-fest-really-the-best-way-to-promote-sustainability/; accessed 2/27/16 3:04 p.m. Vv hand: https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcQ9BULvRTuUXA8gGKbdK5Bo8HebU93y7NCpiF4upYj_A-TCz65S; accessed 2/27/16 3:24 p.m. Healed hand: http://www.loyno.edu/lucec/natural-history-writings/flesh-eating-bacteria-coastal-scourge-vibrio-vulnificus-lurking-estuaries; accessed 2/27/16 3:25 p.m. Oyster Vp: http://www.countynewscenter.com/news/its-spring-do-you-know-where-your-oysters-are/; accessed 2/9/16 2:09 p.m. CHROMagar: http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/gram4.htm; accessed 3/3/2016 3:52 p.m. TCBS: http://www.microbiologyinfo.com/thiosulfate-citrate-bile-salts-sucrose-tcbs-agar-composition-principle-uses-preparation-and-colony-morphology/; accessed 3/3/2016 3:51 p.m. Neuse River color image: http://ims.unc.edu/home/research/; accessed 5/11/16 11:01 a.m. Centers for Disease Control and Prevention (2014). Foodborne Diseases Active Surveillance Network (FoodNet): FoodNet Surveillance Report for 2014 (Final Report). Froelich, B.A, J. Bowen, R. Gonzalez, A. Snedeker, R.T. Noble (2013). Mechanistic and statistical models of total Vibrio abundance in the Neuse River Estuary. Water Research 47: 5783- 5793. Urakawa, H., I.N.G. Rivera. (2006). Ch. 12. Aquatic environment. In: F.L. Thompson, B. Austin and J. Swings (eds). The Biology of the Vibrios. ASM Press. Washington, D.C. pg. 175-189. Hsieh, J.L., J.F. Fries, R.T. Noble (2008). Dynamics and predictive modelling of Vibrio spp. in the Neuse River Estuary, North Carolina, USA. Environmental Microbiology 10: 57-64.