Detection of Shiga toxin-producing Escherichia coli serotypes O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 in raw-milk cheeses by using multiplex real-time PCR

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive for stx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28 fliC alleles were highly prevalent and could not be used as reliable targets for screening. Combinations of stx, eae variants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and included stx-wzx(O26)-eae-β1 (4.8%; 19 samples), stx-wzx(O103)-eae-ε (1.3%; five samples), stx-ihp1(O145)-eae-γ1 (0.8%; three samples), and stx-rfbE(O157)-eae-γ1 (0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seven eaeβ1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Three stx-negative and eaeβ1-positive E. coli O26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC from stx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagic E. coli (EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA, katP, and espP, as well as genomic O islands 71 and 122. Except for one strain, they all contained the stx1 variant only, which was reported to be less frequently associated with human cases than stx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.     

Attachment and biofilm formation on stainless steel by Escherichia coli O157:H7 as affected by curli production

AIMS: The aim of this study was to determine the role of curli in attachment and biofilm formation by Escherichia coli O157:H7 on stainless steel. METHODS AND RESULTS: Three curli-deficient strains (43895-, 43894- and E0018-) and three curli over-producing strains (43895+, 43894+ and E0018+) of E. coli O157:H7 were studied. Stainless steel coupons (SSC) were immersed in cell suspensions of each strain for 24 h at 4 degrees C. The number of cells attached to SSC was determined. To determine the ability of attached cells to form biofilm, SSC were immersed in 10% of tryptic soya broth up to 6 days at 22 degrees C. Curli-deficient and curli-producing strains did not differ in their ability to attach to SSC, but only curli-producing strains formed biofilms. CONCLUSIONS: Curli production by E. coli O157:H7 does not affect attachment of cells on stainless steel but curli-producing strains are better able to form biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Curli production by E. coli O157:H7 enhances its ability to form biofilm on stainless steel, thereby potentially resulting in increased difficulty in removing or killing cells by routine cleaning and sanitizing procedures used in food-processing plants.     

Assessment of resistance to colicinogenic Escherichia coli by E. coli O157:H7 strains

AIMS: To assess a collection of 96 Escherichia coli O157:H7 strains for their resistance potential against a set of colicinogenic E. coli developed as a probiotic for use in cattle. METHODS AND RESULTS: Escherichia coli O157:H7 strains were screened for colicin production, types of colicins produced, presence of colicin resistance and potential for resistance development. Thirteen of 14 previously characterized colicinogenic E. coli strains were able to inhibit 74 serotype O157:H7 strains. Thirteen E. coli O157:H7 strains were found to be colicinogenic and 11 had colicin D genes. PCR products for colicins B, E-type, Ia/Ib and M were also detected. During in vitro experiments, the ability to develop colicin resistance against single-colicin producing E. coli strains was observed, but rarely against multiple-colicinogenic strains. The ability of serotype O157:H7 strains to acquire colicin plasmids or resistance was not observed during a cattle experiment. CONCLUSIONS: Escherichia coli O157:H7 has the potential to develop single-colicin resistance, but simultaneous resistance against multiple colicins appears to be unlikely. Colicin D is the predominant colicin produced by colicinogenic E. coli O157:H7 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for resistance development against colicin-based strategies for E. coli O157:H7 control may be very limited if more than one colicin type is used.     

Reliability of O157:H7 ID agar (O157 H7 ID-F) for the detection and isolation of verocytotoxigenic strains of Escherichia coli belonging to serogroup O157

AIMS: The reliability of the O157:H7 ID agar (O157 H7 ID-F) to detect verocytotoxigenic strains of Escherichia coli (VTEC) of serogroup O157 was investigated. METHODS AND RESULTS: This medium, designed to detect strains belonging to the clone of VTEC O157:H7/H-, contains carbohydrates and two chromogenic substrates to detect beta-d-galactosidase and beta-d-glucuronidase and sodium desoxycholate to increase selectivity for Gram-negative rods. A total of 347 strains of E. coli including a variety of serotypes, verocytotoxigenicity of human and animal sources were tested. The green VTEC O157 colonies were easy to detect among the other dark purple to black E. coli colonies. Of 63 O157:H7/H- strains, 59 (93.7%) gave the characteristic green colour. Three of the failed four strains of O157:H- were not verocytotoxigenic, missing only one VTEC O157. Three non-O157 strains gave the characteristic green colour on the medium and were VTEC OR:H- (2) and Ont:H- (1), possibly being degraded variants of the O157 enterohaemorrhagic E. coli clone. CONCLUSIONS: The O157:H7 ID agar (O157 H7 ID-F) was largely successful in isolating VTEC belonging to the O157:H7/H- clone. SIGNIFICANCE AND IMPACT OF THE STUDY: A medium, suitable for isolating strains of VTEC O157 was successfully evaluated and should be useful for the isolation of these pathogens.     

Restriction-site-specific PCR as a rapid test to detect enterohemorrhagic Escherichia coli O157:H7 strains in environmental samples

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in industrialized countries. We developed a rapid and simple test for detecting E. coli O157:H7 using a method based on restriction site polymorphisms. Restriction-site-specific PCR (RSS-PCR) involves the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences, without the use of endonucleases, to generate a set of amplicons that yield "fingerprint" patterns when resolved electrophoretically on an agarose gel. The method was evaluated in a blinded study of E. coli isolates obtained from environmental samples collected at beef cattle feedyards. The 54 isolates were all initially identified by a commonly used polyclonal antibody test as belonging to O157:H7 serotype. They were retested by anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent assay (ELISA). The RSS-PCR method identified all 28 isolates that were shown to be E. coli O157:H7 by the monoclonal antibody ELISA as belonging to the O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains as non-O157:H7. The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than with the polyclonal antibody latex agglutination tests. The RSS-PCR method may be a useful test to distinguish E. coli O157:H7 from a large number of E. coli isolates from environmental samples.     

Evaluation of colicins for inhibitory activity against diarrheagenic Escherichia coli strains, including serotype O157:H7.

Twenty-four Escherichia coli strains producing standard colicins were evaluated for inhibitory activity against 27 diarrheagenic E. coli strains of serotypes O15:H-, O26:(H11, H-), and O111:(H8, H11, H-), including O157:H7, representing diarrheagenic E. coli clones, 3, 4, 8, 9, and 10. Overlay techniques were used to assess inhibition on Luria agar and Luria agar supplemented with 0.25 micrograms of mitomycin C per ml to induce colicin production. As a group, the A colicins (Col) E1 to E8, K, and N inhibited 23 to 25 (85.2 to 92.6%) of the 27 diarrheagenic strains on mitomycin C-containing agar, whereas the most active group B colicins, Col D and Ia, inhibited 9 and 12 (33.3 and 44.4%), of the diarrheagenic strains, respectively. Col G and H and Mcc B17 inhibited 22 to 27 (81.5 to 100%) of the diarrheagenic strains on Luria agar but were suppressed on mitomycin C-containing agar medium. All O157:H7 strains evaluated were sensitive to Col E1 to E8, K, and N on mitomycin C-containing agar and to Col G and H and Mcc B17 on Luria agar. Sensitivity to colicins of the selected set of diarrheagenic strains was in the order diarrheagenic E. coli clone 9 > 4 > 3 > 10 > 8 and was not restricted to strains of a single clone or serotype. Strain 8C from clone 8 was resistant to most test colicins. There is potential for using colicins in foods and agriculture to inhibit sensitive diarrheagenic E. coli strains, including serotype O157:H7.     

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.     

Inactivation of Escherichia coli O157:H7 in simulated human gastric fluid

Human disease caused by Escherichia coli O157:H7 is a function of the number of cells that are present at potential sites of infection and host susceptibility. Such infectious doses are a result, in part, of the quantity of cells that are ingested and that survive human host defenses, such as the low-pH environment of the stomach. To more fully understand the kinetics of E. coli O157:H7 survival in gastric fluid, individual E. coli O157:H7 strains were suspended in various media (i.e., saline, cooked ground beef [CGB], and CGB containing a commercial antacid product [CGB+A]), mixed at various proportions with simulated human gastric fluid (SGF), and then incubated at 37 degrees C for up to 4 h. The highest inactivation rate among nine E. coli O157:H7 strains was observed in saline. Specifically, the average survival rates in 100:1 and 10:1 proportions of SGF-saline were -1.344 +/- 0.564 and -0.997 +/- 0.388 log(10) CFU/h, respectively. In contrast, the average inactivation rate for 10 E. coli O157:H7 strains suspended in 10:1 SGF-CGB was -0.081 +/- 0.068, a rate that was 12-fold lower than that observed for SGF-saline. In comparison, the average inactivation rate for Shigella flexneri strain 5348 in 100:1 and 10:1 SGF-saline was -8.784 and -17.310, respectively. These latter inactivation rates were 7- to 17-fold higher than those for E. coli O157:H7 strains in SGF-saline and were 4-fold higher than those for E. coli O157:H7 strains in SGF-CGB. The survival rate of E. coli O157:H7 strain GFP80EC increased as the dose of antacid increased from one-half to twice the prescribed dose. A similar trend was observed for the matrix pH over the range of pH 1.6 to 5.7, indicating that pH is a primary factor affecting E. coli O157:H7 survival in SGF-CGB+A. These results can be used in risk assessment to define dose-response relationships for E. coli O157:H7 and to evaluate potential surrogate organisms.     

Differences in growth of Salmonella enterica and Escherichia coli O157:H7 on alfalfa sprouts

Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log(10) on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log(10). The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.     

Competition for proline between indigenous Escherichia coli and E. coli O157:H7 in gnotobiotic mice associated with infant intestinal microbiota and its contribution to the colonization resistance against E. coli O157:H7

Previously, we produced two groups of gnotobiotic mice, GB-3 and GB-4, which showed different responses to Escherichia coli O157:H7 challenge. E. coli O157:H7 was eliminated from GB-3, whereas GB-4 mice became carriers. It has been reported that the lag time of E. coli O157:H7 growth in 50% GB-3 caecal suspension was extended when compared to GB-4 caecal suspension. In this study, competition for nutrients between intestinal microbiota of GB-3 and GB-4 mice and E. coli O157:H7 was examined. Amino acid concentrations in the caecal contents of GB-3 and GB-4 differed, especially the concentration of proline. The supplementation of proline into GB-3 caecal suspension decreased the lag time of E. coli O157:H7 growth in vitro. When E. coli O157:H7 was cultured with each of the strains used to produce GB-3 mice in vitro, 2 strains of E. coli (proline consumers) out of 5 enterobacteriaceae strains strongly suppressed E. coli O157:H7 growth and the suppression was attenuated by the addition of proline into the medium. These results indicate that competition for proline with indigenous E. coli affected the growth of E. coli O157:H7 in vivo and may contribute to E. coli O157:H7 elimination from the intestine.     

Evolutionary silence of the acid chaperone protein HdeB in enterohemorrhagic Escherichia coli O157:H7

The periplasmic chaperones HdeA and HdeB are known to be important for cell survival at low pH (pH < 3) in Escherichia coli and Shigella spp. Here we investigated the roles of HdeA and HdeB in the survival of various enterohemorrhagic E. coli (EHEC) following exposure to pH 2.0. Similar to K-12 strains, the acid protections conferred by HdeA and HdeB in EHEC O145 were significant: loss of HdeA and HdeB led to over 100- to 1,000-fold reductions in acid survival, depending on the growth condition of prechallenge cells. However, this protection was much less in E. coli O157:H7 strains. Deletion of hdeB did not affect the acid survival of cells, and deletion of hdeA led to less than a 5-fold decrease in survival. Sequence analysis of the hdeAB operon revealed a point mutation at the putative start codon of the hdeB gene in all 26 E. coli O157:H7 strains analyzed, which shifted the ATG start codon to ATA. This mutation correlated with the lack of HdeB in E. coli O157:H7; however, the plasmid-borne O157-hdeB was able to restore partially the acid resistance in an E. coli O145ΔhdeAB mutant, suggesting the potential function of O157-HdeB as an acid chaperone. We conclude that E. coli O157:H7 strains have evolved acid survival strategies independent of the HdeA/B chaperones and are more acid resistant than nonpathogenic K-12 for cells grown under nonfavorable culturing conditions such as in Luria-Bertani no-salt broth at 28°C. These results suggest a divergent evolution of acid resistance mechanisms within E. coli.     

Rapid procedure for detecting enterohemorrhagic Escherichia coli O157:H7 in food.

A sensitive, specific procedure was developed for detecting Escherichia coli O157:H7 in food in less than 20 h. The procedure involves enrichment of 25 g of food in 225 ml of a selective enrichment medium for 16 to 18 h at 37 degrees C with agitation (150 rpm). The enrichment culture is applied to a sandwich enzyme-linked immunosorbent assay (ELISA) with a polyclonal antibody specific for E. coli O157 antigen as the capture antibody and a monoclonal antibody specific for enterohemorrhagic E. coli of serotypes O157:H7 and O26:H11 as the detection antibody. The ELISA can be completed within 3 h. The sensitivity of the procedure, determined by using E. coli O157:H7-inoculated ground beef and dairy products, including different varieties of cheese, was 0.2 to 0.9 cell per g of food. A survey of retail fresh ground beef and farm raw milk samples with this procedure revealed that 3 (2.8%) of 107 ground beef samples and 11 (10%) of 115 raw milk samples were positive for E. coli O157:H7. Most-probable-number determinations revealed E. coli O157:H7 populations of 0.4 to 1.5 cells per g in the three ground beef samples. In addition to being highly specific, sensitive, and rapid, this procedure is easy to perform and is amenable to use by laboratories performing routine microbiological testing.     

Rapid procedure for detecting enterohemorrhagic Escherichia coli O157:H7 in food.

A sensitive, specific procedure was developed for detecting Escherichia coli O157:H7 in food in less than 20 h. The procedure involves enrichment of 25 g of food in 225 ml of a selective enrichment medium for 16 to 18 h at 37 degrees C with agitation (150 rpm). The enrichment culture is applied to a sandwich enzyme-linked immunosorbent assay (ELISA) with a polyclonal antibody specific for E. coli O157 antigen as the capture antibody and a monoclonal antibody specific for enterohemorrhagic E. coli of serotypes O157:H7 and O26:H11 as the detection antibody. The ELISA can be completed within 3 h. The sensitivity of the procedure, determined by using E. coli O157:H7-inoculated ground beef and dairy products, including different varieties of cheese, was 0.2 to 0.9 cell per g of food. A survey of retail fresh ground beef and farm raw milk samples with this procedure revealed that 3 (2.8%) of 107 ground beef samples and 11 (10%) of 115 raw milk samples were positive for E. coli O157:H7. Most-probable-number determinations revealed E. coli O157:H7 populations of 0.4 to 1.5 cells per g in the three ground beef samples. In addition to being highly specific, sensitive, and rapid, this procedure is easy to perform and is amenable to use by laboratories performing routine microbiological testing.     

The influence of melatonin on growth of E. coli O157:H7 in pure culture and exogenous melatonin on faecal shedding of E. coli O157:H7 in experimentally infected wethers

AIMS: To determine if exogenous melatonin (MEL) influences growth of Escherichia coli O157:H7 in pure culture and if MEL affects faecal shedding patterns of E. coli O157:H7 or total leucocyte counts in sheep. METHODS AND RESULTS: Two strains of E. coli O157:H7 were cultured in the presence of varying concentrations of MEL. Maximal specific growth rates of E.coli O157:H7 strains were not affected by MEL addition in pure culture. Wethers (n = 16) received either 0 (CONT) or 25 mg MEL hd(-1) day(-1) for 21 days. Daily shedding patterns of E. coli O157:H7 were not different (P > 0.10) between groups with faecal populations of E. coli O157:H7 decreasing daily (P < 0.01) in both groups. However, shedding tended to differ between the control and treated group by the end of the experiment. Total WBC and differential leucocyte counts were not affected by treatment. CONCLUSIONS: Melatonin had no affect on specific growth rates in pure culture nor did the administration of exogenous MEL alter bacterial shedding patterns or immune response indicators in experimentally infected wethers exposed to a long photoperiod. SIGNIFICANCE AND IMPACT OF THE STUDY: Although MEL did not affect shedding patterns or gastrointestinal populations of E. coli O157:H7, the tendency for MEL-treated sheep to shed less E. coli O157:H7 towards the end of the experiment warrants further research. Providing MEL for a longer period of time, or at greater concentrations, may elucidate a potential role that MEL plays in the seasonal shedding patterns of E. coli O157:H7 in livestock.     

Presence of Shiga toxin-producing Escherichia coli O157:H7 in living layer hens

AIMS: To evaluate the presence of Shiga toxin-producing strains of Escherichia coli (STEC) of the O157:H7 serotype in living layer hens so as to analyse the role of this avian species as potential reservoir. METHODS AND RESULTS: Cloacal swabs were collected between November 2004 and November 2005 from four intensive management layer hen farms and analysed for STEC O157:H7 by immunomagnetic separation methods and multiplex polymerase chain reaction for stx1 and/or stx2, the E. coli attaching and effacing (eae) and hly genes. STEC was detected in 26 of the 720 samples. CONCLUSIONS: The layer hens analysed were shown to carry STEC O157:H7. The presence of this bacterium in living layer hen farms investigated did not result in any detectable increase in gastrointestinal disease in this species. SIGNIFICANCE AND IMPACT OF THE STUDY: Living layer hens are a novel potential reservoir of E. coli O157:H7.     

Characterization of unexpected growth of Escherichia coli O157:H7 by modeling

Modeling of batch kinetics in minimal synthetic medium was used to characterize Escherichia coli O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for E. coli K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other E. coli strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures, E. coli K-12 had a positive effect on E. coli O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for E. coli K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of E. coli K-12 on E. coli O157:H7 appeared to be similar to the effect of methionine.     

Evaluation of AFLP, a high-resolution DNA fingerprinting method, as a tool for molecular subtyping of enterohemorrhagic Escherichia coli O157:H7 isolates.

The amplified fragment-length polymorphism (AFLPTM) technique is based on the selective PCR amplification of restriction fragments. We investigated the utility of AFLP in the molecular subtyping of enterohemorrhagic Escherichia coli serotype O157:H7 isolates. We analyzed a total of 46 isolates of E. coli O157:H7 along with other serotypes, O26:H11, 0114:H19 and 0119:NT. Isolates of E. coli O157:H7 derived from the same outbreak showed an identical AFLP-banding pattern and were subtyped into the same group, giving results almost consistent with those of a pulsed-field gel electrophoresis (PFGE) study, while other serotypes showed clearly different patterns from those of E. coli O157:H7. These results suggest that the AFLP technique has potential as an alternative tool for the molecular epidemiology of E. coli O157:H7.     

Survey of retail cheeses, dairy processing environments and raw milk for Escherichia coli O157 : H7

Escherichia coli was isolated from 58% (11/19) of retail soft and semi-soft cheeses tested, but E. coli O157 : H7 was not detected. The presence of E. coli in retail cheeses and the lack of baseline data on the prevalence of serotype O157 : H7 strains prompted us to survey ingredients and the environment in 15 cheese and dairy plants. Escherichia coli O157 : H7 was not detected in any of the 1104 samples tested, including 42 raw milk samples. These results suggest that serotype O157 : H7 is not prevalent within dairy product ingredients and processing environments.     

Experimental and field studies of Escherichia coli O157:H7 in white-tailed deer

Studies were conducted to evaluate fecal shedding of Escherichia coli O157:H7 in a small group of inoculated deer, determine the prevalence of the bacterium in free-ranging white-tailed deer, and elucidate relationships between E. coli O157:H7 in wild deer and domestic cattle at the same site. Six young, white-tailed deer were orally administered 10(8) CFU of E. coli O157:H7. Inoculated deer were shedding E. coli O157:H7 by 1 day postinoculation (DPI) and continued to shed decreasing numbers of the bacteria throughout the 26-day trial. Horizontal transmission to an uninoculated deer was demonstrated. Although E. coli O157:H7 bacteria were recovered from the gastrointestinal tracts of deer necropsied from 4 to 26 DPI, attaching and effacing lesions were not apparent in any deer. Results are similar to those of inoculation studies in calves and sheep. In field studies, E. coli O157 was not detected in 310 fresh deer fecal samples collected from the ground. It was detected in feces, but not in meat, from 3 of 469 free-ranging deer in 1997. In 1998, E. coli O157 was not detected in 140 deer at the single positive site found in 1997; however, it was recovered from 13 of 305 dairy and beef cattle at the same location. Isolates of E. coli O157:H7 from deer and cattle at this site differed with respect to pulsed-field gel electrophoresis patterns and genes encoding Shiga toxins. The low overall prevalence of E. coli O157:H7 and the identification of only one site with positive deer suggest that wild deer are not a major reservoir of E. coli O157:H7 in the southeastern United States. However, there may be individual locations where deer sporadically harbor the bacterium, and venison should be handled with the same precautions recommended for beef, pork, and poultry.     

Escherichia coli O157:H7 Shiga toxin-encoding bacteriophages: integrations,excisions, truncations,and evolutionary implications

As it descended from Escherichia coli O55:H7, Shiga toxin (Stx)-producing E. coli (STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1. Between these events, sorbitol-fermenting E. coli O157:H(-) presumably diverged from this clade. We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx. Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy yehV in almost all E. coli O157:H7 strains that lack stx(1) (stx(1)-negative strains). Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region. A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer. An stx(2) bacteriophage usually occupies wrbA in stx(1)(+)/stx(2)(+) E. coli O157:H7, but wrbA is unexpectedly unoccupied in most stx(1)-negative/stx(2)(+) E. coli O157:H7 strains, the presumed progenitors of stx(1)(+)/stx(2)(+) E. coli O157:H7. Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated stx bacteriophages from the E. coli O157:H7 strains tested; bile salts usually attenuate excision. These data demonstrate the unexpected diversity of the chromosomal architecture of E. coli O157:H7 (with novel truncated bacteriophages and multiple stx(2) bacteriophage insertion sites), suggest that stx(1) acquisition might be a multistep process, and compel the consideration of multiple exogenous factors, including antibiotics and bile, when chromosome stability is examined.