Evaluation of Organisms Isolated Using an Antibody-Based Detection Method for Waterborne Escherichia coli O157:H7 M. J. Collins, R. M. Hoffman--WI State Laboratory of Hygiene, Madison, WI

1
Marty Collins WI State Lab of Hygiene 2601
Agriculture Drive Madison, WI 53718 (608)
224-6260 Phone collinmj_at_mail.slh.wisc.edu
Evaluation of Organisms Isolated Using an
Antibody-Based Detection Method for Waterborne
Escherichia coli O157H7M. J. Collins, R. M.
Hoffman--WI State Laboratory of Hygiene, Madison,
WI
Poster Q-422
Abstract Introduction E. coli O157H7 is a
highly virulent pathogen that causes nearly
73,000 illnesses in the United States each year.
From 1982-2002, the CDC reported 350 E. coli O157
outbreaks, 9 of which were waterborne. Existing
methods for detection of E. coli O157H7 in water
are non-standardized, have poor sensitivity, and
are time intensive. These factors have prompted
the development of several antibody-based
detection methods for detecting E. coli O157H7
in water. However, methods based on antigenicity
may not provide sufficient information of an
organisms virulence and public health risk. This
study summarizes data obtained from water samples
tested by an antibody-based method. Methods
During the 2005 sampling season, 121 surface
water samples from lakes near Madison, WI and
riverine systems and inshore areas of Lake
Michigan near Milwaukee, WI were tested for the
presence of E. coli O157H7. Each sample was
filtered through a 142 mm, 0.4 micron PCTE
membrane filter, enriched in 25 mL modified
buffered peptone water broth, and incubated at
42C for 6 hours. Samples were concentrated by
IMS using anti-E. coli O157H7 Dynabeads,
stained with a FITC-conjugated anti-E. coli
O157H7 antibody and screened using flow
cytometry. Samples exhibiting fluorescence were
considered presumptive positive and plated onto
CHROMagar O157 and Rainbow Agar O157 plates then
incubated at 42C for 18 hours. Isolated colonies
were confirmed by O-antisera agglutination.
Positive isolates were analyzed by PCR for
detection of rfbE, eae, stx1, stx2 genes and
H-typed by RFLP-PCR.  Isolates were also tested
for enterohemolysin production by plating onto
EBA agar and glucoronidase activity as determined
by the MUG reaction. Results Of the121 samples
collected, 30 samples tested presumptively
positive for E. coli O157. Thirteen isolates were
obtained from 11 water samples that tested
positive by O157 antisera agglutination testing. 
Three additional isolates collected in 2004 that
also tested positive were also included for
analysis.  All 16 isolates were PCR positive for
the rfbE gene. Eight isolates lacked all toxin
genes tested, 5 isolates contained eae only, and
1 isolate contained eae and stx2 genes. Overall,
the 13 isolates were found to belong to one of
six serotypes detected and 9 phenotypes
detected. Conclusions These data demonstrate
that methods relying primarily on antigenicity
may not provide sufficient information to
adequately assess an organisms virulence and
public health risk. Detection methods should
include detection of toxin-production genes.
Introduction E. coli O157H7 is a highly virulent
pathogen that causes nearly 73,000 illnesses in
the United States each year. From 1982-2002, the
CDC reported 350 E. coli O157 outbreaks, 9 of
which were waterborne. Existing methods for
detection of E. coli O157H7 in water are
non-standardized, have poor sensitivity, and are
time intensive. These factors have prompted the
development of several antibody-based detection
methods for detecting E. coli O157H7 in water.
However, methods based on antigenicity may not
provide sufficient information of an organisms
virulence and public health risk. This study
summarizes data obtained from water samples
tested by an antibody-based method. 
Results Of 121 samples collected, 30 samples
tested presumptively positive for E. coli O157.
Thirteen isolates were obtained from 11 water
samples that tested positive by O157 antisera
agglutination testing.  Three additional isolates
collected in 2004 that also tested positive for
E. coli O157 were also included in the analysis. 
All 16 isolates tested positive for the rfbE
gene. Eight isolates lacked all toxin genes
tested, 5 isolates contained eae only, and 1
isolate contained eae and stx2 genes. From the 16
identified isolates, 6 serotypes and 9 phenotypes
were observed.  Only one isolate (E. coli
O157H7) tested positive for enterohemolysin.
Isolate Shiga Toxin Intimin Entero-hemolysin Glucoronidase Isolate ID
1 (2004) - - - E. coli O157H12
2 (2004) - - - E. coli O157H16
3 (2004) - - - E. coli O157H12
4 - - - - E. coli O157H-
5 - - - - E. coli O157H-
6 - - - - E. coli O157H-
7 - - - - E. coli O157H-
8 - - - - E. coli O157H-
9 - - - - E. coli O157H-
10 - - - E. coli O157H-
11 - - E. coli O157H-
12 - - E. coli O157H-
13 - - - E. coli O157H12
14 - - E. coli O157H32
15 - - - E. coli O157H6/41
16 Stx1 - Stx2 - E. coli O157H7
Control strain Stx1 Stx2 - E. coli O157H7
Rainbow O157 Agar-Black colonies are E. coli O157
CT-SMAC-E.coli O157
CHROMagar O157-E.coli O157

• Methods
• In 2005, 121 surface water samples were
  collected from lakes near Madison, WI and
  riverine systems and inshore areas of Lake
  Michigan near Milwaukee, WI. These samples were
  tested for the presence of E. coli O157H7 using
  an antibody-based method developed in our
  laboratory.
• Samples were filtered through 142 mm, 0.4 µM
  PCTE membrane filters and enriched in modified
  buffered peptone water broth at 42C for 6 hours.
  
• Samples were concentrated by IMS using anti-E.
  coli O157H7 Dynabeads, stained with a
  FITC-conjugated anti-E. coli O157H7 antibody and
  screened using flow cytometry.
• Samples exhibiting fluorescence were considered
  presumptive positive and plated onto CHROMagar
  O157 and Rainbow Agar O157 plates then incubated
  at 42C for 18-24 hours.
•   Isolated colonies were confirmed by
  O-antisera agglutination. Positive isolates were
  analyzed by PCR for detection of rfbE, eae, stx1,
  stx2 genes and H-typed by RFLP-PCR. Isolates
  were also tested for enterohemolysin production
  by plating onto EBA agar and glucoronidase
  activity as determined by the MUG reaction.

Conclusions The presumptive screening method used
in this study detected several E. coli O157
strains in addition to E. coli O157H7. These
data demonstrate that E. coli O157H7 detection
methods relying primarily on O157 antigenicity
may not provide adequate information to assess an
organisms virulence and public health
risk. Furthermore, the data show that while
antibody screening may prove a useful screening
tool, these methods cannot detect other
enterohaemorrhagic and enterotoxigenic strains
that also pose a public health concern. 
Therefore, detection methods should incorporate
testing for toxin-production genes.