Isolation of Escherichia coli O157:H7 from Intact Colon Fecal Samples of Swine

Escherichia coli O157:H7 was recovered from colon fecal samples of pigs. Polymerase chain reaction confirmed two genotypes: isolates harboring the eaeA, stx1, and stx2 genes and isolates harboring the eaeA, stx1, and hly933 genes. We demonstrate that swine in the United States can harbor potentially pathogenic E. coli O157:H7.


The Study
Colon samples were collected at a cooperating swine slaughter facility from 305 swine carcasses during evisceration. Two to three inches of distal colon that contained feces at the first point proximal to the rectum was resected and maintained on ice for approximately 2 hours before processing (Figure). Ten grams of feces from each colon was transferred to filterlined sterile plastic bags. One hundred milliliters of brilliant green bile broth (Difco Laboratories, Detroit, MI), prewarmed to 37°C, was added to each filter stomacher bag containing feces and incubated at 37°C for 6 h with shaking (150 rpm) (7). After enrichment, 1.0-mL aliquots were processed by using Dynabeads anti-E. coli O157 (Dynal Biotech, Oslo, Norway), according to manufacturer's instructions with modification. Bead/sample suspensions were incubated at room temperature for 30 min with continuous mixing on a Bellco roller drum (Bellco Glass, Inc., Vineland, NJ) before plating onto sorbitol MacConkey (SMAC; Difco Laboratories), cefixime/tellurite (CT; cefixime-tellurite supplement, Dynal Biotech)-SMAC agars, and rainbow agar O157 (Biolog, Inc., Hayward, CA). Black colonies from rainbow agar O157 and sorbitol-negative colonies from CT-SMAC and SMAC agars were tested for the absence of β-glucuronidase and the ability to ferment lactose by using E. coli broth containing 4-methylumbelliferyl-β-Dglucuronide (MUG) (EC medium with MUG; Difco Laboratories) and MacConkey broth (Difco Laboratories), respectively. Lactose-positive/MUG-negative isolates were serotyped by using the RIM E. coli O157:H7 Latex Test (Remel, Lenexa, KS). Up to 10 E. coli O157 latex agglutination-positive isolates per colon fecal sample were tested for the presence of the rfb O157 gene by using polymerase chain reaction (PCR) (8). Isolates positive for the rfb O157 gene were further characterized for the presence of genes encoding the H7 flagellar protein (fliC H7 ), Shiga toxin 1 (stx 1 ), Shiga toxin 2 (stx 2 ), intimin protein (eaeA), and hemolysin (hly 933 ) (9). We conducted further analysis using antimicrobial resistance patterns, pulsed-field gel electrophoresis (PFGE), and ribotyping on all E. coli O157 PCR-positive isolates containing fliC H7 , stx 1 , stx 2 , eae A , or hly 933 . However, for tabulation purposes, each sample ultimately contributed one isolate. When fliC H7 , stx 1 , stx 2 , eaeA, or hly 933 was not detected in PCR-confirmed E. coli O157 isolates, further analysis was performed on only one E. coli O157 isolate per colon sample.
For PFGE, DNA was digested with 50 U XbaI (Invitrogen Corp., Carlsbad, CA) for 4 h at 37°C. PFGE was performed by using a CHEF Mapper XA system (Bio-Rad, Hercules, CA) at 14°C with pulses ramping from 2.16 s to 63.8 s over 18 h. PFGE patterns were evaluated visually, and isolates were assigned to the same pulsotype when exhibiting a difference of <3 bands from the index isolate. Ribotyping of the E. coli O157 isolates was done by using a RiboPrinter (Qualicon, Inc., Wilmington, DE) as described in the user's manual. Restriction digests were performed on E. coli O157 isolates by using the EcoRI enzyme (Qualicon, Inc.).
A total of 305 colon samples were randomly collected on 8 different days over a 6-month period as follows: collection  (Table). Eighteen (5.9%) of the 305 colon samples had isolates positive for rfb O157 . Isolates from 6 of these 18 colon samples also contained fliC H7 . Two gene combinations based on the presence or absence of stx 1 , stx 2 , eae, and hly 933 were detected in these E. coli O157:H7 PCR-confirmed isolates. The stx 1 , eaeA, and hly 933 virulence pattern was detected in two isolates (isolates 1 and 2) from two of the five colon samples collected on February 16, 2001, and the stx 1 , stx 2 , and eaeA virulence pattern was detected in 22 isolates (isolates 6-27) from 4 of the 50 colon samples collected on May 4, 2001. None of the E. coli O157:H7 isolates recovered contained all four of the virulence genes (stx 1 , stx 2 , eaeA, and hly 933 ). None of the E. coli O157:non-H7 isolates (isolates 3-5, 28-36) in the present study contained stx 1 , stx 2 , eaeA, or hly genes. Non-Shiga toxin-producing E. coli O157:non-H7 isolates have been previously isolated from the feces of pigs (11,12). For slaughterhouse visits on March 8, March 22, June 20, and July 10, 2001, E. coli O157 and E. coli O157:H7 were not recovered from any of the colons sampled.
All E. coli O157:H7 isolates recovered in this study were sensitive to the antimicrobial agents tested, with the exception of one isolate (isolate 15) that was resistant to streptomycin. This isolate was recovered from a colon from which a pan-sen- sitive E. coli O157:H7 was also recovered. The antimicrobial sensitivity pattern of the E. coli O157:non-H7 isolates was more varied than that of the E. coli O157:H7 isolates with five different susceptibility patterns noted. Only one of the E. coli O157:non-H7 isolates was pan-sensitive. These data are similar to previous reports in which antimicrobial resistance among E. coli O157 non-Shiga toxin-producing isolates was higher than that of Shiga toxin-producing E. coli O157 isolates (11).
As previously shown, ribotyping did not discriminate among isolates within the E. coli O157:H7 serotype (13

Conclusions
Results from this study demonstrate that pigs in the United States can harbor E. coli O157:H7. The recovery rate of E. coli O157:H7 from colon fecal samples of pigs reported in this study was 2.0% (6/305). Previous attempts to isolate E. coli O157:H7 from swine feces in the United States have been unsuccessful (12,14). Use of more appropriate methods for sampling, processing, and culturing swine feces may have accounted for the ability to recover and isolate E. coli O157:H7 from swine feces in our study. For example, samples were obtained from the colon, transported on ice, and processed within 2 h of collection. The absence of antibiotics in our enrichment step may have also facilitated the recovery of E. coli O157:H7 from swine feces. Furthermore, although direct comparisons cannot be made between cattle studies, the recovery rate of Shiga toxin-producing E. coli O157 from cattle feces has improved over the past 10 years. This is most likely due to more conducive sampling procedures, culture practices, and detection methods than an increase in true carriers. The detection of E. coli O157 in swine feces has previously been based on the isolation techniques used for the recovery of E. coli O157 from cattle feces. The difficulty in detecting E. coli O157 from swine feces may in part be attributable to differences in the physiologic environment between swine and cattle feces. More appropriate isolation techniques may still be discovered for detecting E. coli O157 in swine.
Although our recovery rates of E. coli O157:H7 from swine are similar to recovery rates in Japan (4), we recovered a genotype in addition to the stx 1 , stx 1 , and eaeA genotype: the stx 1 , eaeA, and hly 933 genotype. In Norway, the recovery rate (0.1%) of E. coli O157:H7 from pig feces was much lower (5). Isolates recovered from Norway possessed the stx 2 and eaeA genes only; however, the presence of the hly 933 gene was not determined (5).
The ability to produce one or more Shiga toxins is an important virulence characteristic of E. coli O157:H7 (1).
However, production of Shiga toxins alone may not be sufficient for E. coli O157:H7 to be pathogenic (1). Other virulence factors such as the intimin protein (involved in the attachment of the E. coli O157 to enterocytes), the presence of a plasmidencoded hemolysin, or both, are important in the pathophysiology of hemorrhagic disease (1). E. coli O157:H7 isolates recovered in this study possessed either two virulence factors, eaeA and hly 933 , in addition to stx 1 or one virulence factor, eaeA, in addition to stx 1 and stx 2 . These isolates can potentially cause disease and should be considered pathogenic to humans. Since human E. coli O157:H7 clinical isolates contain the stx 1 , stx 2 , eaeA, and hly 933 genes, the human pathogenicity of E. coli O157:H7 isolates from pigs that lack the hly gene requires further study.
The clonal nature of the isolates that were isolated on a particular day suggests transmission of E. coli O157 between pigs. Unfortunately, we did not have access to information concerning the source of the pigs from which the samples were collected, the number of pigs slaughtered from a given farm, or the holding facilities and grouping of the pigs before slaughter. Therefore, we do not know whether E. coli O157 transmission between pigs occurred on the farm, in transit, or while the pigs were in a holding pen at the slaughterhouse.
This study did not permit inferences of E. coli O157:H7 isolation rates with respect to the season, nor can inferences of E. coli O157:H7 isolation rates be made with respect to swine or herd prevalence. The relatively low recovery rate of E. coli O157:H7 from swine feces compared to cattle feces warrants further study to determine the significance and prevalence of E. coli O157:H7 in swine and if different enrichment and isolation methods would have an impact on the recovery of E. coli O157:H7 from swine feces. In addition, future studies should be conducted to determine the occurrence of E. coli O157 on swine hides, in swine mouths, and in swine stomachs.