identifying likely sources of fecal contamination in little lagoon, alabama
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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. Project Background. - PowerPoint PPT PresentationTRANSCRIPT
Identifying likely sources of fecal contamination in Little
Lagoon, Alabama
Dr. Alice OrtmannUniversity of South
AlabamaDauphin Island Sea Lab
Image: AL.com, Mobile Press Register
Project Background• Water quality sampling suggested an issue with
fecal coliforms (FCB) in Little Lagoon• Not correlated with any measured variables
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
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.
Sample Sites within Little Lagoon
Gulf of Mexico
Bon Secour Bay
Pass5 4
3
21
Sample each site from March 2011-February 2012Collect 40 ‘random’ samples throughout the year for E. coli analysis
Google Earth Image
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
FCB were often above the regulatory limit during the study
March
AprilMay
JuneJuly
August
September
October
November
December
January
February
100
1000
10000
100000
Site1 Site2 Site3 Site4 Site5Feca
l Col
iform
Bac
teria
(CFU
/L)
E. coli communities through out the lagoon are simple and well mixed
Standard
mdh communitiesfrom Little Lagoon
Lagoon communities are significantly different than inflow communities
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
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
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
Bacteroidales genes were detected at all sites throughout the year
March
AprilMay
JuneJuly
August
September
October
November
December
January
February
0E+00
1E+06
2E+06
3E+06
4E+06 Site1 Site2 Site3 Site4 Site5
BacU
ni G
enes
(cop
ies/
L)
**8 samples missing from analysis
The abundance of BacUni did not correlate with the abundance of FCB
0E+00 1E+05 2E+05 3E+05 4E+05 5E+05 6E+05 7E+05 8E+05 9E+05 1E+060E+00
1E+04
2E+04
3E+04
BacUni Genes (copies/L)
Feca
l Col
iform
Bac
teria
(CFU
/L)
Bacteroidales from cows and dogs was much lower than BacUni estimates
March
AprilMay
JuneJuly
August
September
October
November
December
January
February
0E+00
1E+04
2E+04
3E+04Site1 Site2 Site3 Site4 Site5
BacC
ow+B
acDo
g (c
opie
s/L)
***BacDog, extremely high:Site1 January and Site5 February 1.6107 2.3106
Together, they represent a small fraction of the total Bacteroidales
March AprilMay
JuneJuly
August
Septem
ber
October
November
December
January
February
0
100
200
300
400
500
600
BacC
ow+B
acDo
g (%
of B
acUn
i)
March AprilMay
JuneJuly
August
September
October
November
December
January
February
0
10
20
30
40
50
60 Site1 Site2 Site3 Site4 Site5
BacC
ow+B
acDo
g (%
of B
acU
ni)
And they do not correlate well with FCBs
0E+00 1E+04 2E+04 3E+040E+00
1E+04
2E+04
3E+04
BacCow+BacDog genes (copies/L)
Feca
l Col
iform
Bac
teria
(CFU
/L)
BacHum was always detected in Little Lagoon
March
AprilMay
JuneJuly
August
September
October
November
December
January
February
0E+00
1E+04
2E+04
3E+04
4E+04
5E+04
6E+04Site1 Site2 Site3 Site4 Site5
BacH
um G
enes
(cop
ies/
L)
BacHum was also not correlated with the abundance of FCB
1E+03 1E+04 2E+04 3E+04 4E+04 5E+04 6E+041E+02
1E+04
2E+04
3E+04
BacHum Genes (copies/L)
Feca
l Col
iform
Bac
teria
(CFU
/L)
Most of the time, a small number of the BacUni could be accounted for by human
Bacteroidales
March
AprilMay
JuneJuly
August
September
October
November
December
January
February
0
10
20
30
40
50
60
70
80
90
100Site1 Site2 Site3 Site4 Site5
BacH
um G
enes
(% o
f Bac
Uni
)
A significant amount of Bacteroidales genes are not accounted for by humans, dogs or cows
March
AprilMay
JuneJuly
August
September
October
November
December
January
February
1E+00
1E+01
1E+02
1E+03
1E+04
1E+05
1E+06
1E+07
Site1 Site2 Site3 Site4 Site5
"Mis
sing
" Ba
cter
oida
les (
copi
es/L
)
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
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
Image from: http://www.mygulfshoresrentalhouse.com