Transcript Fingerprinting E. coli communities in Little Lagoon, AL to understand

Fingerprinting E. coli communities in Little Lagoon, AL to
understand their potential sources
Alice C.
1University
1,2
Ortmann ,
James D.
Little Lagoon Sample Sites
Since 2007 the number of fecal coliform bacteria (FCB) in the water column of
Little Lagoon, AL has been monitored in a collaboration between the Little
Lagoon Preservation Society (LLPS) and researchers at the Dauphin Island
Sea Lab (DISL) and Dalhousie University (DU). Over this time period, the
concentrations of FCB at five sites within the lagoon have varied, with some
samples showing concentrations well above the regulatory threshold of 200
CFU l-1. The source of the FCB remains unknown, with no correlation between
the concentration of FCB and any other measured parameter, including
temperature, salinity, nutrients or microalgal community composition (see poster
by MacIntyre et al). FCB may represent a threat to human health or the heath
of other organisms within the lagoon: however until the source of the FCB is
determined, it is impossible to develop a long-term management plan (see
poster by Hatfield et al).
Because FCB represents a wide range of different types of bacteria, including
some that occur naturally within the environment, it is necessary to further
identify the organisms present in the water column of Little Lagoon and link their
identity to potential sources. One method of identifying bacteria is through the
use of DNA fingerprinting. A combination PCR and denaturing gradient gel
electrophoresis (DGGE) has been used to generate fingerprints which represent
the specific E. coli community present in each sample. Samples were collected
during a period when Little Lagoon Pass was closed, when high levels of FCB
were detected, and after the pass was re-opened. (The pass was closed in
response to oil from the Deepwater Horizon spill.) Two different genes in E. coli
have been used for fingerprinting, giving us a clear measure of the diversity of
the E. coli present within Little Lagoon. These fingerprints will be compared to
fingerprints generated from potential sources of FCB contamination to identify
which are the most likely sources of FCB in Little Lagoon.
3
Hatfield ,
Hugh L.
Bon Secour Bay
Results: Salinity and CFU Detection
5
4
Pass
Gulf of Mexico
Location of the sample sites in Little Lagoon, AL. Site 5 is located in Gator Lake
and not within the lagoon itself. Varying levels of development are found around
the lagoon, with little development and a wildlife refuge at the west end. Image
from Google Earth.
Results: Physical conditions
Plot of CFU/ml of coliforms and E. coli vs salinity. As the salinity decreases below
~10 ppt, the number of both coliforms and E. coli detected using Coliscan Easygel
plates (Micrology Laboratories) increases.
Table showing Spearman’s
Rho values for physical
parameters. Bold indicates
significant correlations.
T
S
For total coliforms, >100 colonies on a plate are represented by 10 000 CFS/100 ml.
Results: Amplification of mdh and phoE
DO
S
-0.0475
DO
-0.3289
Day
-0.6478 -0.0140 0.2262
0.3668
Aug. 18
1
For each gene, 2 independent
PCR reactions containing 10
ng of DNA were carried out.
The products were pooled and
concentrated. This gel shows
3 μl of pooled product run on
an agarose gel. mdh is in the
top lanes while phoE is shown
in the bottom lanes.
2 3
Sept. 2
4
5 1 2
3
4 5
Sept. 28
1 2
3
4
Different intensities of the
bands suggest that each
sample contained different
numbers of E. coli cells.
Results: CFU Detection
Filter to remove large
organisms
Results: E. coli community richness
Concentrate
prokaryote cells
Fingerprint communities
with DGGE
Amplify genes
with PCR
TEMPLATE DESIGN © 2008
www.PosterPresentations.com
M 8/18 8/18 8/18 M
1 3 5
9/2 9/2 9/28 9/28 M M
2
5
1 2
M
M 9/28 9/28 9/28 9/28 M
4
3 2
1
9/2 9/2 8/18 M
5 1
5
Day
Temperature
Salinity
DO
Coliforms
E. coli
E. coli
-0.3384
0.0006
-0.3709
-0.3217
0.5048
Other CFU
-0.1357
0.1925
-0.0286
-0.3089
0.2074
-0.2197
1. Temperature and salinity are both correlated with dissolved oxygen (DO).
Temperature decreased over the sample period, but did not differ between
sites. Salinity was always lower in Sites 1 and 5. Increases in salinity near
the end of the sampling correspond to an opening of the pass to the Gulf of
Mexico.
2. The abundance of coliforms and E. coli were correlated with each other,
suggesting a common source. Both were negatively correlated with the day of
sampling, salinity and DO. Coliforms are facultatively anaerobic and may
benefit from lower DO conditions.
3. The E. coli communities based on two different genes are low complexity.
For mdh, two different sequences were detected, with one detected in 13 of
the 14 samples. For phoE, three difference sequences were detected with
one sequences detected in 13 of 14 samples. For each gene there was one
sample for which no fingerprint was generated. The high similarity between E.
coli communities detected in different samples suggests a common source.
4. There appears to be no correlation between E. coli community
composition and the detection of E. coli or coliforms on the plates. Genes
from E. coli could be detected even when no colonies were detected on the
plates. This is likely due to the larger volume of water analyzed using
molecular methods.
References
Table showing Spearman’s Rho values for physical parameters and CFUs detected.
The sample day, salinity and DO were correlated with both total coliforms and E.
coli. Bold indicates significant correlations.
Coliforms
-0.3519
0.2068
-0.7545
-0.5556
UPGMA analysis using Dice similarity index shows two outlying samples and three
main groups. Group 1 (Red) represents communities with 3 mdh sequences and 1
or 2 phoE sequences. Group 2 (Green) contains samples with 2 mdh sequences
and 2 phoE. Group 3 (Blue) communities have only 1 mdh and 1 phoE sequences.
There is no apparent relationship between the clusters of E. coli communities and
other parameters measured.
Conclusions
Measure water
parameters
Extract DNA
from cells
using the
UltraClean
Soil Kit
(MoBio Labs)
9/02 Site3
9/02 Site2
8/18 Site3
9/02 Site1
9/02 Site5
9/28 Site1
9/28 Site2
8/18 Site5
9/28 Site3
8/18 Site1
8/18 Site4
9/02 Site4
9/28 Site4
8/18 Site2
3
http://www.envcoglobal.com/files/MO-YSI-85.jpg
Centrifuge cells
to concentrate
further
Results: Cluster analysis of fingerprints
1
2
Methods
Quantify coliforms
and E. coli
4
MacIntyre
of South Alabama, 2Dauphin Island Sea Lab, 3Little Lagoon Preservation Society, 4Dalhousie University.
Abstract
http://www.micrologylabs.com/:/Pictures/Dish%20B.jpg
Justin D.
1,2
Liefer ,
Esseili MA, Kassem II and Von Sigler LP (2008) Optimization of DGGE community fingerprinting for
characterizing Escherichia coli communities associated with fecal pollution. Water Research 42:44674476.
Lasalde C Rodriguez R and Toranzos GA (2005). Statistical analyses: Possible reasons for
unreliability of source tracking efforts. Applied and Environmental Microbiology 71(8): 4690-4695.
Von Sigler LP (2006). Evaluation of denaturing gradient gel electrophoresis to differentiate
Escherichia coli populations in secondary environments. Environmental Microbiology 8(10): 17031711.
Acknowledgements
Examples of DGGE images. mdh DGGE shows 1 or 2 bands in most samples,
phoE has 1, 2 or 3 bands. One sample was negative for each gene (9/2 Site3, mdh
and 8/18 Site2, phoE).
Special thanks to the members of the Little Lagoon Preservation Society, Courtney
Metzger, Bart Christiaen, Andrea Hale and Natalie Ortell for their assistance. This
work was supported by a grant from the Mississippi-Alabama Sea Grant Consortium
to H. MacIntyre, A. Ortmann and K. Park.