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