1. Analysis of Vibrio dynamics in the Neuse River Estuary, NC using next generation sequencing amplicon data
Kelsey Jesser, PhD Candidate
UNC Chapel Hill, Institute of Marine Sciences
May 16, 2017

2. Vibrio Vibrio ecology Diverse and naturally occurring genus
Over 100 species, includes several human pathogens
V. parahaemolyticus
V. vulnificus
Warm water
Exist along a salinity gradient
Response environmental parameters not well understood, vary across species and ecosystems
Freshwater
Brackish Water
Coastal Water
Pelagic Water
V. anguillarum
V. cholerae
V. fluvialis
V. navarrensis
V. parahaemolyticus
V. vulnificus
V. logei
V. alginolyticus
Salinity
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
[figure adapted from Urakawa and Rivera 2006]

3. Vibrio detection Easily culturable Molecular methods (ex: QPCR)
Selective and differential, but not effective
Molecular methods (ex: QPCR)
Thinking about NGS approaches
16S rDNA and hsp60 amplicon sequencing
Want species-level resolution
Applications for microbial ecology and monitoring
Change out one figure for a DNA figure
[1, 2]

4. 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 biweekly 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
[3]

5. Sampling methods
Samples collected biweekly May through early September, in coordination with Modmon
Modmon stations 30, 70, and 120
Surface and bottom water
Enhanced sampling after Tropical Storm Collin in early June, 2016
Large rainfall event
100 mL filtered onto 0.4 µm polycarbonate filters
Genomic DNA extracted with MoBio Powersoil kit
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
Collin
[4]

6. Vibrio community hsp60
No sample
No sample

7. Vibrio community 16S
No sample
No sample

8. Vibrio heatmap
De novo otu picking– reads are clustered without any external reference. Clusters all reads based on sequence similarity

9. The x-axis is highly correlated (Spearman’s rho>0. 5, p<0
The x-axis is highly correlated (Spearman’s rho>0.5, p<0.01) with salinity, DO, DOC, DIC, DON, TDN and chlorophyll-a.
NMDS Month
De novo otu picking– reads are clustered without any external reference. Clusters all reads based on sequence similarity

10. The x-axis is highly correlated (Spearman’s rho>0. 5, p<0
The x-axis is highly correlated (Spearman’s rho>0.5, p<0.01) with salinity, DO, DOC, DIC, DON, TDN and chlorophyll-a.
NMDS Storm sampling
De novo otu picking– reads are clustered without any external reference. Clusters all reads based on sequence similarity

11. The x-axis is highly correlated (Spearman’s rho>0. 5, p<0
The x-axis is highly correlated (Spearman’s rho>0.5, p<0.01) with salinity, DO, DOC, DIC, DON, TDN and chlorophyll-a.
NMDS Site and depth
De novo otu picking– reads are clustered without any external reference. Clusters all reads based on sequence similarity

12. Network analysis correlation networks
Positive Spearman rank correlations
R2 > 0.3
De novo otu picking– reads are clustered without any external reference. Clusters all reads based on sequence similarity

13. Network analysis correlation networks
Negative Spearman rank correlations
R2 > 0.3
De novo otu picking– reads are clustered without any external reference. Clusters all reads based on sequence similarity

14. Conclusions
The hsp60 universal target had much higher taxonomic resolution for Vibrio species than 16S
The Vibrio communities in the NRE over the summer were very similar to one another
Vibrio communities sampled in response to Tropical Storm Collin were very similar to others taken during June
Network analyses can identify interactions between important pathogenic species (V. vulnificus and V. parahaemolyticus), total Vibrio, and environmental gradients
Potential applications for monitoring/modeling
Excellent tool for studying microbial ecology
De novo otu picking– reads are clustered without any external reference. Clusters all reads based on sequence similarity

15. Acknowledgements Rachel Noble Noble lab
Committee members Hans Paerl, Jill Stewart, Alecia Septer, and Adrian Marchetti
Modmon monitoring project and the Paerl lab
UNC ROI
Kelsey Jesser
PhD Candidate, Marine Sciences
UNC Chapel Hill
Institute of Marine Sciences

16. References Other figures
Centers for Disease Control and Prevention (2016). Foodborne Diseases Active Surveillance Network (FoodNet): FoodNet Surveillance Report for 2016 (Draft report).
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:
Other figures
CHROMagar: accessed 3/3/2016 3:52 p.m.
DNA: id ?s=170667a; accessed 5/15/2017 3:51 p.m.
Neuse River color image: accessed 5/11/16 11:01 a.m.
Tropical Storm Collin: accessed 5/9/17 2:19 p.m.