Detection of E. coli 0157:H7 in the Rio Grande Using PCR
E.
1
Penner ,
A.
1
Vazquez ,
El Paso Community
E.
2
Holguin ,
1
College, El
C.
2
Baeza ,
P. De Los
2
Santos ,
J.
1
Mendoza ,
Paso ,Texas, Transmountain Early College High School, El Paso , Texas
Methods and Materials
The proximity of large populations of livestock around the
Rio Grande River in El Paso, TX and Juarez, Mexico place
the river at risk for contamination with E. coli 0157:H7. This
toxin-producing strain of E.coli has been found in the
intestine of cattle and has the potential to cause severe
disease in humans. The Rio Grande River is an international
boundary between the U.S. and Mexico and is used as a
source of drinking water and irrigation, as well as for
recreation. Runoff from dairy farms has the potential to
contaminate the river and food products grown in this area in
personal and commercial gardens. User-friendly, quick and
effective ways of monitoring contamination of the river are
very important. The MicroScan autoSCAN-4 System by
Siemens has been used in our laboratory to identify E.coli
isolates from the Rio Grande River at a 99.99% probability.
The system also provides the minimal inhibitory
concentration (MIC) for a variety of antibiotics. The E.coli
0157:H7 detection kit from Norgen Biotek Corp. is used in
the food industry and has a high sensitivity and specificity for
E.coli 0157:H7. It can be used with agarose PCR gels or
Real-Time PCR. The objective of this study is to determine if
E. coli 0157:H7 can be isolated from the Rio Grande River.
Water samples were collected from the Rio Grande close to
the mouth of the Montoya drain in the town of Sunland Park
NM. This site is impacted by agriculture runoff, dairy farms,
and municipal streams. EPA guide lines in SOP# 2013
procedure were used to collect samples. Thirty five E.coli
isolates were identified at a 99.99% probability of correct
identification using the MicroScan system. Twenty isolates
with the highest MICs were selected. Among the highest
MICs were Cefotaxime (30µg/ml), Ampicillin (15 µg/ml), and
Amoxicillin / K Clavulanate (15 µg/ml). The isolates were
then processed with the Norgen Biotek Corp. Kit. Agarose
gels were used for the detection of the antigens. Out of the
twenty isolates, three tested positive upon visual inspection
using the STX2 348 Bp band DNA marker positive control in
the kit. Our results show the potential for using these two
systems for quick and effective detection of E.coli O157:H7
in river water.
Step 1
Step 2
Results
Step 3
Step 4
Gram Staining
Sample collected
Magenta colonies
Modified M-tec agar.
Membrane filtration was
done through a 0.47 μm
cellulose membrane
filter.
• Thirty five colonies were isolated using
differential and selective media from the Rio
Grande samples. After testing positive for
E.coli on Modified M-tec agar by Difco, the
isolates were analyzed using the MicroScan
autoSCAN-4 system.
• The 35 isolates were identified at a 99.99%
probability of correct Identification as E.coli
using the MircroScan auto SCAN- 4 bacterial
identification system.
• Upon further inspection, 20 isolates with the
highest MICs were selected to be analyzed for
O157:H7 antigens with the Norgen Biotek
• Three of these isolates were positive for the
O157:H7 strain of E.coli using the Norgen
Biotek kit .
Conclusion
Out of twenty E.coli isolates, three isolates tested tentatively
positive for O157:H7. Further Melt Curve Real Time PCR
analysis are needed to verify positive results.
Isolate potential colonies
of E.coli.
Inoculate and scan to
confirm
E. coli isolates
PCR
amplification of
gene sequence
DNA extraction of pure
E.coli
Results
Figure 5.2
Figure 5.1
1 2
3
4
5
6
7
8
Acknowledgements
• This work was supported in part by the US International
Boundary and Water Commission (IBWC). RISE Student
Elizabeth Penner and research reported in this publication
was supported in part by the National Institute of General
Medical Sciences of the National Institutes of Health under
RISE Award Number R25GM060424. The content is solely
the responsibility of the authors and does not necessarily
represent the official views of the National Institutes of
Health. Carina Baeza, Eduardo Holguin, and Priscilla De Los
Santos, were supported in part by DHS SLA Award #2011-ST062000043
E.coli O157:H7
(STX2) 348 Bp
Works Cited
Introduction
The Rio Grande River is used as a source of drinking
water and irrigation, as well as for recreation along the
region of El Paso, TX and Juarez. Runoff from dairy farms
has the potential to contaminate the river water and food
products grown in this area in personal and commercial
gardens with the strain O157:H7. This toxin-producing
strain of E.coli has been found in the intestine of cattle and
has the potential to cause severe disease in humans
including hemorrhagic diarrhea and hemolytic uremic
syndrome. User-friendly, quick and effective ways of
monitoring contamination of the river is very important .The
objective of this study is to determine if E. coli 0157:H7 can
be isolated from the Rio Grande River.
M. E.
1
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Figure 5.4
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Column 1: DNA Marker
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Column 7: Positive O157:H7 strain
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