Development of a Rapid Detection Method for
Waterborne E. coli O157:H7
M. J. Collins, R. M. Hoffman, B. T. Argue, J. H. Standridge
WI State Laboratory of Hygiene, Madison, WI
Poster Q-496
Marty Collins
Wisconsin State Lab of Hygiene
2601 Agriculture Drive
Madison, WI 53718
(608) 224-6260 Phone
(608) 224-6213 Fax
collinmj@mail.slh.wisc.edu
Abstract
Methods
Background: E. coli O157:H7 is a highly virulent pathogen implicated in food and
waterborne outbreaks. Documented outbreaks include over 10,000 cases with several
hundred deaths. Waterborne outbreaks represent 10% of the reported epidemics.
Existing methods for detection of E. coli O157:H7 in water are non-standardized, have
poor sensitivity, or require enrichment steps resulting in assay times exceeding 72
hours. These factors significantly hamper outbreak investigations. The intent of this
study was to develop a rapid and sensitive method to detect E. coli O157:H7 in water.
Methods: Various concentration techniques including diatomaceous earth, and 0.45
micron membrane filtration were evaluated prior to choosing filtration through 142
mm diameter, 0.4 micron polycarbonate track-etched membrane filters. Filters were
placed in 25 mL modified buffered peptone water and incubated at 37C for six hours.
The organisms were concentrated from enrichment broth using immunomagnetic
separation with anti-E.coli O157:H7 Dynabeads®. Bead-cell complexes were stained
with a FITC-conjugated anti-E. coli O157:H7 and screened using flow cytometry.
Samples exhibiting fluorescence were considered presumptive positive, plated onto
cefixime-tellurite-sorbitol-MacConkey agar and CHROMagar O157 plates,
incubated for 18 hours, and typical colonies confirmed with O antisera agglutination.
Results: Multiple spikes into complex lake water samples confirmed the sensitivity
of the assay down to 10 organisms/500 mL. The assay was field tested with 129
samples collected and analyzed during a 10 week swimming season. None of the
samples were positive for E. coli O157:H7.
Conclusions: The assay described above provides a much improved time frame for
detecting E. coli O157:H7 in water samples. In a public health laboratory with flow
cytometry capabilities, presumptive positives can be attained in less than 8 hours, with
confirmed results available even in laboratories without flow cytometers, within 24
hours. The assay also improves sensitivity down to 10 organisms/ 500 mL, an
acceptable level for public health investigations.
•Organisms: Either E. coli O157:H7 ATCC isolate 700927 or a clinical isolate were used for all experiments. Cells were
grown to log phase in modified buffered peptone water at 37ºC. Stock concentrations were determined by flow cytometry
using Flow Count™ Beads (Beckman-Coulter, Fullerton, CA) with methods previously validated in our laboratory. Stock
cultures were serially diluted in sterile saline to achieve concentrations ranging from 10 – 10,000 organisms.
Introduction
E. coli 0157:H7 has been implicated in waterborne disease outbreaks affecting
thousands of individuals throughout North America, Europe and the Far East.
Fatalities from complications of infection have occurred in almost all of these
outbreaks in part due to the absence of effective clinical therapies. A number of these
epidemics have been associated with failures of disinfection systems or compromises
in the integrity of the water distribution systems serving the community and the
incidence of disease is expected to increase over the next twenty years based on the
rising number of epidemics observed in recent years. Current E. coli analytic methods
are incapable of detecting this highly virulent strain and hence may be ineffective for
routine monitoring purposes, particularly in communities relying upon water sources
on or near concentrated farming operations. The intent of this study was to develop a
rapid and sensitive method to detect E. coli O157:H7 in water.
•Field testing: 129 recreational water samples from 3 area lakes were processed using the methods listed below. Positive
controls consisting of natural samples spiked with 103 E. coli O157:H7 and negative reagent water controls were assayed
alongside each set of samples.
Sample concentration
Three filtration techniques were evaluated by filtering 500 mL
of natural water collected from three Madison, WI area lakes
and from a local pond. Techniques included:
• Diatomaceous earth:
• Filta-Max® concentrator tube (adapted from the
FiltaMax system, Idexx Laboratories, Portland, ME);
• 142 mm diameter, 0.4 micron polycarbonate, tracketched (PCTE) Millipore Isopore™ membrane filters
(Millipore, Bedford, MA).
Flow rate and volume limitations determined. PCTE filters
were chosen due to ease of use and cost.
Enrichment and Immunomagnetic separation (IMS)
PCTE filters were transferred into 145 mm petri dishes (bottom left) containing 25 mL of a modified buffered peptone water
enrichment broth that had been further modified to include 10 g casamino acids, 6 g yeast extract, 10 g lactose and 10 mg/L
acriflavin-HCl and incubated at 37° C for 6 hours. A 1 mL aliquot was withdrawn and organisms were concentrated by
immunomagnetic separation (bottom right). with anti-E.coli O157:H7 Dynabeads® (Dynal Biotech, Oslo, Norway).
Flow cytometric detection and confirmation
Bead-cell complexes were stained with a FITC-conjugated anti-E. coli O157:H7 antibody
(KPL, Gaithersburg, MD) and screened with a Beckman-Coulter EPICS XL flow
cytometer (top left). Formation of a fluorescent magnetic bead-organism complex (top
right, positive) indicates the presence of E. coli O157:H7, while the failure to form such a
complex (top right, negative) indicates the sample does not contain E. coli O157:H7.
Samples exhibiting fluorescence (bottom left) were considered presumptive positive and
plated (bottom right) onto cefixime-tellurite-sorbitol-MacConkey agar and
CHROMagar™ O157 plates (CHROMagar, Paris, France) or Rainbow O157 agar
(Biolog, Hayward, CA). Plates were incubated for 18 hours at 37° C and typical colonies
confirmed by O antisera agglutination.
Flow cytometric histograms depicting peak FITC-fluorescence (X-axis) vs. log forward angle light scatter (Yaxis). The presence of fluorescent events indicates the presence of E. coli O157:H7. Panels A-C show results
from distilled water seeded with 0, 10, and 100 E.coli O157:H7 in 500 ml. Panels B-D show results from lake
water seeded with 0, 10, and 100 E.coli O157:H7 in 500 ml.
Negative control
10 organisms/500 ml
100 organisms/500 ml
B
C
E
F
A
Distilled
water
D
Lake
water
Rainbow O157 Agar-Black colonies are E. coli O157
Conclusions
CT-SMAC-E.coli O157
CHROMagar O157-E.coli O157
We have developed an E. coli 0157:H7 method utilizing IMS with flow cytometric detection following
concentration and a 6 hour enrichment. This method is capable of detecting 10 organisms in 500 mL natural
water samples – a significant sensitivity improvement over currently accepted methods which may take up to
72 hours for confirmation. In a public health laboratory with flow cytometry capabilities, presumptive
positives can be attained in less than 8 hours, with confirmed results available even in laboratories without
flow cytometers, within 24 hours.
Results
1. Both the FiltaMax concentrator tube filtration system and the PCTE membrane were
capable of filtering 500 mL natural water samples. Diatomaceous earth filters were not
capable of filtering the entire sample volume. PCTE membranes were chosen due to ease
of use and cost.
2. Both reagent water and natural water samples spiked with 10, 100, and 1000 organisms
were positive using the IMS/flow cytometric method described above.
3. The assay was field tested with 129 samples collected and analyzed during a 10 week
swimming season. None of the samples were positive for E. coli O157:H7. Positive and
negative control samples were acceptable for all samples analyzed.
Future Research
•
•
•
•
Delayed exposure of various antibiotics and use of media additives (e.g., antioxidants) to improve
culturability of viable but non-culturable organisms
PCR confirmation
Determining optimal enrichment temperatures for culturing viable but non-culturable organisms
Further comparison of CT-SMAC, CHROMagar O157, & Rainbow Agar O157
Acknowledgements
This project is being conducted by the Wisconsin State Laboratory of Hygiene, Madison Department of Public
Health, and the United States Geological Survey. This project is being funded by the U.S. EPA, Project #
R82933901-0.