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Applied and Environmental Microbiology, February 2009, p. 862-865, Vol. 75, No. 3
0099-2240/09/$08.00+0     doi:10.1128/AEM.01158-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Shiga Toxins, and the Genes Encoding Them, in Fecal Samples from Native Idaho Ungulates

Jeremy J. Gilbreath, Malcolm S. Shields,^* Rebekah L. Smith, Larry D. Farrell, Peter P. Sheridan, and Kathleen M. Spiegel

Department of Biological Sciences, Idaho State University, Pocatello, Idaho

Received 23 May 2008/ Accepted 22 November 2008

Top ABSTRACT INTRODUCTION REFERENCES

 
Cattle are a known reservoir of Shiga toxin-producing Escherichia coli. The prevalence and stability of Shiga toxin and/or Shiga toxin genes among native wild ungulates in Idaho were investigated. The frequency of both Shiga genes and toxin was similar to that reported for Idaho cattle (19%).

Top ABSTRACT INTRODUCTION REFERENCES

 
First described in 1982, enterohemorrhagic Escherichia coli have emerged as a major cause of human morbidity. Shiga toxin (Stx)-producing E. coli (STEC) are considered zoonotic emerging infectious agents (1). For a comprehensive review of STEC disease and virulence factors, see references 2, 3, 4, 8, 20, 21, and 26.

A recognized relationship exists between STEC infections (especially O157-H7) and the association of cattle and humans (12, 27), notably in Idaho, where STEC occurrence in both humans and cattle is high (R. L. Smith, J. J. Gilbreath, and K. M. Spiegel, unpublished data). STEC are transferred by the fecal-oral route, so wild animals and cattle sharing common areas would likely experience interspecies transfer (23). Although not studied extensively, O157:H7 isolates have been identified in wild deer (7, 9, 10, 23, 24) and have been implicated in at least one human infection (16, 27).

Nonetheless, wild deer are rarely screened for either Shiga toxin or its genes.

Fecal pellets (collected from live mule deer and elk in Idaho from December 2005 to March 2006) cultured in tryptic soy broth (TSB) (Becton Dickinson, Franklin Lakes, NJ) at 37°C were screened for Shiga toxin (Remel ProSpecT Shiga toxin microplate ELISA kit; Remel, Inc., Lexena, KS) and the stx₁, stx₂, eae (intimin), and ehx (enterohemolysin, also known as hemolysin A [hlyA]) genes (25) from total DNA (isolated using the Puregene DNA purification system [Gentra, Minneapolis, MN]) via PCR (Table 1).

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TABLE 1. PCR primersa

Fecal cultures (n = 160) screened in this manner yielded 31/160 (19.4%) positive for Shiga toxin or the stx₁ or stx₂ genes (Table 2). From this pool, 26/31 (83.9%) amplified either stx₁ or stx₂ genes but no toxin was detected, while 5/31 (16.1%) exhibited both stx genes and toxin. The breakdown of the stx gene content was as follows: 15/26 (45.5%), stx₁ only; 10/31 (30.3%), stx₂ only; and 5/31 (15.2%), stx₁ and stx₂.

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TABLE 2. Stx detection in fecal culturesa

Fecal cultures testing positive via enzyme-linked immunosorbent assay (ELISA) and/or PCR were plated on sorbitol MacConkey agar (Becton Dickinson, Franklin Lakes, NJ) 18 to 24 h at 37°C. Both fermenter and nonfermenter colonies were transferred to TSB and incubated overnight at 37°C. These enriched subcultures were rescreened by ELISA and PCR. To ensure axenic cultures, isolates were regrown overnight at 37°C in R2 broth (Remel, Lexena, KS), streaked to R2 agar, and incubated overnight at 37°C. Isolates were then grown similarly in LB broth (Becton Dickinson, Franklin Lakes, NJ) and streaked for isolation onto LB agar, yielding axenic isolates (n = 156).

From each of the 11 amplitypes (described below), 10 to 15 randomly chosen colonies were transferred to grid coordinates on LB agar. Colony-lift hybridization studies were done using radioactive DNA probes for stx₁ and stx₂, constructed as previously described (11), using Stx1a COOH/Stx1a NH (stx_1a), and Stx2-BGR F/R (stx_2ab) primer sets (Table 1).

The instability of both stx genes (as described previously [14, 19]) was observed by DNA:DNA hybridization of subcultures. The instability of these genes has both clinical and epidemiological implications, impacting diagnosis and patient care as well as the ability to track STEC outbreaks. For example, only 3/10 (30%) of the subcultured isolates from fecal culture 6155 remained positive for stx₁. Similarly, only 5/10 (50%) of subcultured isolates from fecal culture 6160 remained positive for stx₂. Similar hybridization results were obtained with other isolates during subculture.

For isolates exhibiting a loss of hybridizable stx genotype, one hybridization-positive and one hybridization-negative representative were compared by repetitive-element PCR (rep-PCR) (6). To ensure these variations were not merely due to multiple bacterial strains with similar colony morphology, rep-PCR fingerprinting of each isolate was done. Of the 156 colonies screened, 11 distinct amplitypes were obtained, and all 11 were identified as E. coli (API 20E [bioMerieux, La Balme les Grottes, France]) (Table 3). All subcultured isolates retained identical rep-PCR fingerprints, regardless of stx₁ or stx₂ hybridization, and thus were (nearly) isogenic with their Shiga toxin-positive progenitors.

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TABLE 3. Toxigenic profiles of E. coli isolates

None of the 11 E. coli amplitypes (Table 3) were serotyped. The cultures were examined for Stx1 or Stx2 toxin by ELISA and for stx₁ and stx₂ genes by hybridization and PCR. Stx1 or Stx2 toxin was detected in 8/11 (72.7%), 2/11 (18.2%) hybridized to both stx₁ and stx₂, 5/11 (45.5%) hybridized to stx₁ only, and 4/11 (36.3%) hybridized to stx₂ only. These results did not agree with the PCR screening, for which 5/11 (45.5%) amplified both stx₁ and stx₂, 1/11 (9.1%) contained stx₁ only, and 5/11 (45.5%) contained stx₂ only. The most likely explanation for this apparent discrepancy is the extremely high sensitivity of PCR in cultures where the target was highly unstable. Detection via colony hybridization requires a substantially larger number of target sequences to produce a positive signal. Therefore, those without hybridization signals but that are PCR positive after serial passages were referred to as genetically unstable for the stx gene targets (Table 3).

Pathogenicity islands, like the locus of enterocyte effacement (26), greatly enhance the pathogenicity of STEC and are associated with more serious disease (15). We identified 5/11 (45.5%) of these stx-bearing strains as also containing the eae gene. The enterohemolysin gene (ehx) was detected in 2/11 (18.1%) of the isolates (these same two isolates also carried the eae gene) (Table 3). By using sorbitol MacConkey agar, 11/11 (100%) of these isolates were determined to be sorbitol fermenters (i.e., presumptive non-O157 serotypes). Although the strains described here are not known pathogens, the presence of the eae, ehx, and stx genes clearly increases their pathogenic potential (21, 25).

Our data indicates wild ruminants are significant reservoirs of STEC containing ancillary virulence factors. The STEC frequency demonstrated here among wild ungulates (19.4% overall [22.3% in elk, 15.3% in deer]) is similar to the highest reported among dairy cattle (13) and is considerably higher than the frequency of O157:H7 reported among U.S. deer (7, 9, 24). Both sorbitol fermentative and nonfermentative E. coli were isolated in this study, and both produced detectable Stx toxin and/or presented detectable stx genes.

The major route of STEC infection in humans and animals is fecal contamination. The presence of large numbers of STEC-colonized cattle in a given area may result in an increased likelihood of other ruminants (including wildlife) being colonized. This is especially important considering that STEC have been shown to survive in fecal material in the environment for more than 20 months (2). Heightened interactions between wild animals and cattle will likely lead to interspecies transfer.

In Idaho, the large numbers of range cattle suggest frequent opportunities for fecal-oral transmission. Neither the rate of transfer of STEC nor the ecology of the genes involved has been established. One hypothesis is that the rate of lateral exchange of these specific toxin genes among E. coli is going to be proportional to the number and frequency of fecal-oral contacts between infected and susceptible animal hosts. Another is that a continued emphasis on strain serology does little to illuminate possible bacterial interactions at the genetic and ecological levels.

A report in 2007 from the Idaho Bureau of Laboratories indicated that more than half of all human STEC infections statewide are due to non-O157 serotypes (18). Our data clearly supports the CDC's 2003 recommendation of using Shiga toxin as the screening criteria for STEC rather than strain-specific serology (5).

 
We gratefully acknowledge the Idaho Department of Fish and Game for ungulate fecal samples and the Molecular Research Core Facility at Idaho State University for the use of their facilities.

This research was partially supported by NIH grant P20 RR016454 from the INBRE program of the National Center for Research Resources. R.L.S. was supported by the American Society for Clinical Laboratory Science Allegiance Healthcare graduate research award.

 
* Corresponding author. Mailing address: Department of Biological Sciences, Idaho State University, 921 South 8th Avenue, Stop 8007, Pocatello, ID 83209-8007. Phone: (208) 282-5719. Fax: (208) 282-4750. E-mail: shiemalc{at}isu.edu

Published ahead of print on 5 December 2008.

Top ABSTRACT INTRODUCTION REFERENCES

 

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Applied and Environmental Microbiology, February 2009, p. 862-865, Vol. 75, No. 3
0099-2240/09/$08.00+0     doi:10.1128/AEM.01158-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Gilbreath, J. J. Articles by Spiegel, K. M. Search for Related Content PubMed PubMed Citation Articles by Gilbreath, J. J. Articles by Spiegel, K. M. Agricola Articles by Gilbreath, J. J. Articles by Spiegel, K. M.