Shiga-toxigenic Escherichia coli O157:H7 (STEC
O157) is a zoonotic pathogen. Although it is most frequently thought of
as a foodborne pathogen, it can also be transmitted via environmental
reservoirs1 or animal contact, especially contact with cattle
or goats.2,3 In recent years, a number of STEC O157 outbreaks
have been associated with petting zoos and state and county fairs.
Primary isolation of STEC O157 is an important part
of any epidemiological investigation. Standard methods for the isolation
of STEC O157 from humans in clinical settings typically involve
spreading fecal swabs on Sorbital MacConkey (SMAC) media.4
These methods, however, are frequently inadequate in outbreak
investigations for isolation of STEC O157 from environmental sources,
such as dirt or animal feces.2,5 Sick humans commonly shed
more than 105 cfu/g.6 In contrast, STEC O157 is
not a pathogen for ruminants and, when present, is only a minority
component of ruminant feces or of the soil microbiota. So, methods that
are sufficient for culture from clinically ill humans result in
overgrowth of the competing microflora from animal feces or
environmental samples.
Selective enrichment and IMS
The use of a selective enrichment, combined with
immunomagnetic separation and plating on CHROMagar with antibiotics
works well to isolate STEC O157 from a variety of animal and
environmental matrices.2,5 The first step in the procedure is
the selective enrichment for STEC O157. The goal is to enrich for the
E coli, while inhibiting the growth of the background organisms that
may also be present in the sample. One broth that works well is 1.5X (60
g/L) Brilliant Green Bile Broth (BGB). For each gram (g) of sample, 9
milliliters (mL) of 1.5X BGB is added, up to a maximum of 10 g of sample
with 90 mL of 1.5X BGB. After manual homogenization of the sample with
the 1.5X BGB, enrichments should be incubated at 37°C
for six hours.
The second step is the use of immunomagnetic
separation (IMS) using E coli O157 serotype-specific beads. This
step concentrates the small number of STEC O157 cells that are present
in the enrichment broth. For each enrichment, 20 uL of Dynal anti-O157
paramagnetic beads should be combined with 1 mL of enrichment culture
washed twice with PBS (pH 7.4) containing 0.05% Tween 20 detergent and
re-suspended in a final volume of 50 uL of wash buffer.
The third step is plating on CHROMagar O157 (CHROMagar,
Paris, France) containing 18 microliter (uL) per liter of 3.5% potassium
tellurite solution (TCA). After IMS, 60 uL of beads should be spread on
TCA, and incubated at 37°C
for 24 hours. Suspect STEC O157 colonies appear mauve-pink on TCA, while
most competing background flora have blue or white colonies. TCA is
preferred over SMAC for this application because the clearly contrasting
colony morphology of STEC O157 compared to the background flora on TCA
allows for the quick identification of even single suspect colonies in a
near-lawn of non-target colonies, and because the colony morphology
remains stable over time (unlike SMAC, on which the STEC O157 colony
morphology may change over time). The tellurite concentration
suggested here was specifically formulated and evaluated for maximum
recovery of STEC O157 from animal feces and environmental samples, and
differs from the standard concentrations recommended for use by the
manufacturer.
Stock show outbreak
These techniques were used to isolate STEC O157 from
four samples collected as part of an outbreak investigation at a
livestock show. Samples were collected into individual Whirl-Pak bags
and shipped at room temperature overnight to the laboratory for culture.
Samples were logged and weighed upon arrival, and were composed of dirt
and manure weighing between 0.73 g and 10 g each.
Ten colonies were picked from each TCA plate, for a
total of 40 suspect isolates. All suspects were serotyped for the O157
and H7 antigens with an enzyme immunoassay using anti-E coli O157
and anti-E coli H7 monoclonal antibodies and characterized by PCR
for Shiga-toxin and virulence genes.7 Representative
isolates from each of the four samples were characterized by DNA
fingerprinting, and all were exact matches to the DNA fingerprint
patterns of the human cases.
Using these methods can isolate STEC O157 from all
four environmental samples, including samples that weigh less than 1 g.
These methods are robust and can be an effective addition to the STEC
O157 culture techniques currently employed for epidemiological
investigations.
Lisa Durso, PhD,
is a research microbiologist working for the U.S. Department of
Agriculture’s Agricultural Research Service in Lincoln, NE. Hugh F. Maguire, PhD,
is the Microbiology and Molecular Science program manager for the
Colorado Department of Public and Environment, Laboratory Services
Division.
Alicia Cronquist, RN, MPH,
is the Foodborne and Enteric Disease coordinator for the Disease Control
and Environmental Epidemiology Division, Colorado Department of Public
Health and Environment.James L. Bono, PhD,
is a research microbiologist working for the USDA’s Agricultural
Research Service in Clay Center, NE.
References
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coli O157:H7 and Campylobacter jejuni associated with a
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Escherichia coli O157:H7 infections among livestock
exhibitors and visitors at a Texas county fair. Vector Borne
Zoonotic Dis. 2005;5:193-201. - Keen JE, Wittum TE, Dunn JR, Bono JL, Durso LM.
Shiga-toxigenic Escherichia coli O157 in agricultural fair
livestock, United States. Emerg Infect Dis. 2006;12:780-786. - Slutsker L, Ries AA, Greene KD, Wells JG, et al.
Escherichia coli O157:H7 diarrhea in the United States:
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zoo visits, North Carolina State Fair, October-November 2004.
Arch Pediatr Adolesc Med. 2009;163:42-48. - Holland JL, Louie L, Simor AE, Louie M. PCR
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J Clin Microbiol. 2000;38:4108-4113. - Paton AW, Paton JC. Detection and characterization of shiga toxigenic
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