1. 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

2. 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]

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]

4. 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]

5. 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]

6. 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

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

8. 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

9. 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

10. 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.

11. 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.

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

13. 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

14. 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

15. References
Vibrio gram stain: accessed 1/19/ :14 a.m.
Vibrio fischeri flagella: accessed 2/27/16 3:05 p.m.
Oyster gif: accessed 2/27/16 3:08 p.m.
V. parahaemolyticus cartoon: accessed 1/19/ :07 a.m.
V. vulnificus cartoon: accessed 1/19/ :09 a.m.
Vv Oysters: accessed 2/27/16 3:04 p.m.
Vv hand: accessed 2/27/16 3:24 p.m.
Healed hand: accessed 2/27/16 3:25 p.m.
Oyster Vp: accessed 2/9/16 2:09 p.m.
CHROMagar: accessed 3/3/2016 3:52 p.m.
TCBS: accessed 3/3/2016 3:51 p.m.
Neuse River color image: 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:
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
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: