1. Dr. Alice Ortmann University of South Alabama Dauphin Island Sea Lab
Identifying likely sources of fecal contamination in Little Lagoon, Alabama
Dr. Alice Ortmann
University of South Alabama
Dauphin Island Sea Lab
Image: AL.com, Mobile Press Register

2. Project Background
Water quality sampling suggested an issue with fecal coliforms (FCB) in Little Lagoon
Not correlated with any measured variables

3. Sea Grant Funded Project
Identify the source of the fecal contamination so an appropriate management plan can be developed
Contamination from humans
higher health risk
can be managed
Contamination from wildlife
low health risk
much harder to manage

4. Use two DNA-based approaches to identify sources
Comparison of E. coli communities within the lagoon to communities in inflowing water.
Quantify total Bacteroidales in the lagoon and determine what percentage came from humans, dogs and cows.

5. Sample Sites within Little Lagoon
Gulf of Mexico
Bon Secour Bay
Pass 5 4 3 2 1
Sample each site from March 2011-February 2012
Collect 40 ‘random’ samples throughout the year for E. coli analysis
Google Earth Image

6. Approach Collect ~20 L of water Quantify FCB (LLPS)
Remove large particles and concentrate cells to 1 ml
PCR amplify 2 genes from E. coli
Generate fingerprints with DGGE
Compare fingerprints from different locations
Quantify total Bacteroidales (BacUni)
Quantify host specific genes
Determine which host contributes the most genes
Approach

7. FCB were often above the regulatory limit during the study

8. E. coli communities through out the lagoon are simple and well mixed
Standard
mdh communities
from Little Lagoon

9. Lagoon communities are significantly different than inflow communities

10. E. coli communities analysis does not indicate a specific source of contamination
Inflow sites included:
small pond
sediment
streams
drainage
freshwater vs. saltwater
E. coli and close relatives can survive in aquatic environments, so these may represent naturally occurring organisms

11. Use qPCR to identify the host of the Bacteroidales
They are a family (multiple species) of Bacteria that live in animal guts
obligate anaerobes
do not grow in aquatic environments
high abundances in feces
quantitative PCR (qPCR)
sensitive with good detection
very specific, using DNA sequences

12. We used four different assays to determine the host of Bacteroidales
BacUni → detect all members of the Bacteroidales regardless of their host
BacHum → detect only Bacteroidales from human hosts
BacDog → detect only Bacteroidales from dog (maybe cat) hosts
BacCow → detect only Bacteroidales from cow (maybe horse) hosts

13. Bacteroidales genes were detected at all sites throughout the year
**8 samples missing from analysis

14. The abundance of BacUni did not correlate with the abundance of FCB

15. Bacteroidales from cows and dogs was much lower than BacUni estimates
***BacDog, extremely high:
Site1 January and Site5 February
1.6 106

16. Together, they represent a small fraction of the total Bacteroidales

17. And they do not correlate well with FCBs

18. BacHum was always detected in Little Lagoon

19. BacHum was also not correlated with the abundance of FCB

20. Most of the time, a small number of the BacUni could be accounted for by human Bacteroidales

21. A significant amount of Bacteroidales genes are not accounted for by humans, dogs or cows

22. Bacteroidales analysis suggests human fecal contamination at low levels
Like reflects the fact there are humans using the lagoon
Maximum estimate: 0.08 g of fecal material/L
Local maximums: reflect recent use
Humans not likely the source of the large increases in FCB
Estimates do not correlate with FCB
Naturally occurring strains/relatives of E.coli

23. Acknowledgements Daniel Presley-E. coli analysis and qPCR
Chris Lee-new BacUni analysis
Justin Liefer-sample collection
LLPS-sample collection analysis
Lei Wang and Natalie Ortell-working with Chris
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