Detection by multiplex real-time polymerase chain reaction assays and isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 in ground beef

Six Shiga toxin-producing Escherichia coli (STEC) serogroups, which include O26, O45, O103, O111, O121, and O145, are responsible for the majority of non-O157 STEC infections in the United States, representing a growing public health concern. Cattle and other ruminants are reservoirs for these pathogens; thus, food of bovine origin may be a vehicle for infection with non-O157 STEC. Methods for detection of these pathogens in animal reservoirs and in food are needed to determine their prevalence and to develop intervention strategies. This study describes a method for detection of non-O157 STEC in ground beef, consisting of enrichment in modified tryptic soy broth at 42°C, followed by real-time multiplex polymerase chain reaction (PCR) assays targeting stx(1), stx(2), and genes in the O-antigen gene clusters of the six serogroups, [corrected] and then immunomagnetic separation (IMS) followed by plating onto Rainbow? Agar O157 and PCR assays for confirmation of isolates. All ground beef samples artificially inoculated with 1-2 and 10-20 CFU/25?g of ground beef consistently gave positive results for all of the target genes, including the internal amplification control using the multiplex real-time PCR assays after enrichment in modified tryptic soy broth for a total of 24?h (6?h at 37°C and 18?h at 42°C). The detection limit of the real-time multiplex PCR assays was ～50 CFU per PCR. IMS for O26, O103, O111, and O145 was performed with commercially available magnetic beads, and the IMS beads for O45 and O121 were prepared using polyclonal antiserum against these serogroups. A large percentage of the presumptive colonies of each serogroup picked from Rainbow Agar O157 were confirmed as the respective serogroups; however, the percent recovery of STEC O111 was somewhat lower than that of the other serogroups. This work provides a method for detection and isolation in ground beef and potentially other foods of non-O157 STEC of major public health concern.     

Applicability of a multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces

An 11-gene multiplex polymerase chain reaction (mPCR) was developed based on genes that code for serogroup-specific O-antigens and four major virulence factors (intimin, enterohemorrhagic hemolysin, and Shiga toxins [Stx] 1 and 2), to detect O157 and the "top six" non-O157 (O26, O45, O103, O111, O121, and O145) Shiga toxin-producing Escherichia coli (STEC). The assay specificity was validated with pure cultures of seven major STEC (185 strains), 26 other STEC (65 strains), non-STEC (five strains), and 33 strains of other genera and species. Sensitivity of the assay with cattle fecal sample spiked with pooled cultures of seven major STEC was 10(5) colony-forming units (CFU)/g before enrichment and 10(2) CFU/g after enrichment. The applicability of the assay to detect STEC in fecal samples (n=50), before and after enrichment, was evaluated by comparing with culture-based methods for O26, O111, and O157. The mPCR assay of 50 fecal samples showed seven (14%) positive before enrichment and 23 (46%) positive after enrichment for one or more of the seven O-groups. Overall, 17 isolates from 17 fecal samples and 27 isolates (four for O26, three for O45, and 20 for O103) from 19 fecal samples were obtained, by culture-based methods, for O157 and non-O157 serogroups, respectively. None of the 27 non-O157 isolates possessed the stx genes, suggesting that cattle harbor Shiga toxin-negative E. coli belonging to the "top six" non-O157 serogroups. Our data, although based on a limited number of samples, suggest that the sensitivities of the mPCR and culture-based methods in detecting the seven serogroups of STEC in feces differed between O-groups. An obvious limitation of our mPCR is that the concurrent detection of virulence genes and the serogroups in a sample does not necessarily associate the virulence genes with the prevalent serogroups in the same sample. The major application of our 11-gene mPCR assay may be in identifying putative colonies of STEC obtained by culture-based methods.     

Prevalence and characterization of Escherichia coli O26 and O111 in retail minced beef in Ireland

Verocytotoxigenic Escherichia coli (VTEC) O157 are recognized as bacterial pathogens with significant public health impact. However, other serogroups, including O26, O111, O103, and O145, have the potential to cause the same spectrum of illness. In this study, 800 minced (ground) beef samples covering a large geographical region in Ireland were collected and tested for Escherichia coli (E. coli) O26 and E. coli O111 by conventional microbiological protocols. Two minced beef samples (0.25%) tested positive for E. coli O26, indicating fecal contamination. None of these isolates possessed verocytotoxin-encoding genes, (vt1/vt2 also known as stx1/stx2), the hemolysinencoding gene (hlyA), or the E. coli attachment-effacement (eae) gene, as determined by polymerase chain reaction (PCR). None of the beef samples analyzed contained E. coli O111. Although the E. coli O26 isolates were nonvirulent, the presence of these isolates in raw minced beef is an indication of fecal contamination and therefore potentially of public health significance.     

Development and assessment of a rapid method to detect Escherichia coli O26, O111 and O157 in retail minced beef

A molecular-based detection method was developed to detect Escherichia coli O26, O111 and O157 in minced (ground) beef samples. This method consists of an initial overnight enrichment in modified tryptone soya broth (mTSB) and novobiocin prior to DNA extraction and subsequent serogrouping using a triplex PCR. This method has a low limit of detection and results are available within 24 hours of receipt of samples. Once optimized, this rapid method was utilized to determine the prevalence of these E. coli serogroups in six hundred minced beef samples all of which were previously examined by immunomagnetic separation (IMS) and selective plating for E. coli O26 and O111. Using IMS, two E. coli O26 isolates were detected. No E. coli O111 were recovered. The multiplex PCR technique described here did not detect E. coli O111 nor O157 in any of the samples, however six minced beef samples were positive for E. coli O26 using our method, only two of these were previously detected by IMS and culture. Application of molecular methods are useful to support culture-based approaches thereby further contributing to risk reduction along the food chain.     

Shiga Toxin Subtypes Associated with Shiga Toxin-Producing Escherichia coli Strains Isolated from Red Deer, Roe Deer, Chamois, and Ibex

Abstract A total of 52 Shiga toxin-producing Escherichia coli (STEC) strains, isolated from fecal samples of six ibex, 12 chamois, 15 roe deer, and 19 red deer were further characterized by subtyping the stx genes, examining strains for the top nine serogroups and testing for the presence of eae and ehxA. Eleven of the 52 strains belonged to one of the top nine STEC O groups (O26, O45, O91, O103, O111, O113, O121, O145, and O157). Eight STEC strains were of serogroup O145, two strains of serogroup O113, and one strain of serogroup O157. None of the strains harbored stx2a, stx2e, or stx2f. Stx2b (24 strains) and stx1c (21 strains) were the most frequently detected stx subtypes, occurring alone or in combination with another stx subtype. Eight strains harbored stx2g, five strains stx2d, three strains stx1a, two strains stx2c, and one strain stx1d. Stx2g and stx1d were detected in strains not harboring any other stx subtype. The eae and ehxA genes were detected in two and 24 STEC strains, respectively. Considering both, the serogroups and the virulence factors, the majority of the STEC strains isolated from red deer, roe deer, chamois, and ibex do not show the typical patterns of highly pathogenic STEC strains. To assess the potential pathogenicity of STEC for humans, strain isolation and characterization is therefore of central importance.     

Properties of Vero cytotoxin-producing Escherichia coli of human and animal origin belonging to serotypes other than O157:H7

Eight non-O157:H7 Vero cytotoxin (VT)-producing Escherichia coli (VTEC) strains isolated from ill persons and nine bovine and lamb strains of serogroups matching the human strains, were characterized for various properties known to be associated with E. coli virulence. Five different serogroups were represented: O5, O55, O103, O111 and O153. The bovine and lamb strains produced VT1, while 3 human strains produced VT1, 3 produced VT2 and 2 were positive for both VT1 and VT2. The strains were non-haemolytic on horse blood agar, did not produce either heat stable toxin A (STA) or heat labile toxin (LT), and were noninvasive. The CVD419 probe which has been proposed to identify enterohaemorrhagic E. coli (EHEC) hybridized with all of the O5 and O103 strains, none of the O55 and O153 strains, and 3 of the 4 O111 strains. The strains carried several different sized plasmids and hybridization of Southern blots with the CVD419 probe identified plasmids ranging in size from 42 x 10(6) to 90 x 10(6). The strains did not hybridize with the enteroadherence factor (EAF) probe derived from an enteropathogenic strain and associated with the ability to give localized adherence to HEp-2 cells. Nevertheless five of the strains adhered in a localized pattern to HEp-2 cells and Intestine 407 cells. Adhesion to either HEp-2 or Intestine 407 cells did not correlate with hybridization with the CVD419 probe or haemagglutinating properties.     

Laboratory-confirmed non-O157 Shiga toxin-producing Escherichia coli--Connecticut, 2000-2005

Shiga toxin-producing Escherichia coli (STEC) infection causes diarrhea that is often bloody and can result in potentially life-threatening hemolytic uremic syndrome (HUS). Escherichia coli O157:H7 is the most common cause of STEC infection in the United States, producing 73,000 illnesses annually, according to the last estimate in 1999. Unlike O157, however, little is known about the incidence of non-O157 strains. Because STEC other than O157 are not commonly identified, the incidence, trends, and epidemiology of non-O157 STEC are not well understood. To assess trends in Shiga toxin enzyme immunoassay (Stx EIA) testing by local clinical laboratories, the Connecticut Department of Public Health (CTDPH) analyzed results of confirmatory testing conducted in the state laboratory during 2000--2005. The findings indicated that a total of 403 STEC infections were reported by clinical laboratories in Connecticut, including 207 identified as STEC by Stx EIA testing alone, and that the use of Stx EIA increased from 2000 to 2005. Use of Stx EIA without prompt culture confirmation can delay or prevent serotyping and subtyping of isolates and detection of both O157 and non-O157 STEC outbreaks. Public health authorities in all states should ensure that clinical laboratories forward Stx EIA-positive specimens to the state laboratory for isolation and identification of STEC, as recommended by the Association of Public Health Laboratories and CDC.     

Detection of Shiga toxin-producing Escherichia coli O26, O45, O103, O111, O113, O121, O145, and O157 serogroups by multiplex polymerase chain reaction of the wzx gene of the O-antigen gene cluster

O-antigens on the surface of Escherichia coli are important virulence factors that are targets of both the innate and adaptive immune system and play a major role in pathogenicity. O-antigens that are responsible for antigenic specificity of the strain determine the O-serogroup. E. coli O26, O45, O103, O111, O113, O121, O145, and O157 have been the most commonly identified O-serogroups associated with Shiga toxin-producing E. coli (STEC) implicated in outbreaks of human illness all over the world. A multiplex polymerase chain reaction assay was developed to simultaneously detect the eight STEC O-serogroups targeting the wzx (O-antigen-flippase) genes of all O-antigen gene clusters. The sensitivity of the multiplex polymerase chain reaction was found to be 10 colony forming units for each O-group when enriched in broth and 100 colony forming units when enriched in artificially inoculated apple juice diluted with tryptic soy broth for 16?h at 37°C. The method can be used for detecting STEC O-groups simultaneously and may be exploited for improving the safety of food products.     

Virulence properties of Escherichia coli strains belonging to serogroups O26, O55, O111 and O128 isolated in the United Kingdom in 1991 from patients with diarrhoea.

Some strains of Escherichia coli belonging to serogroups O26, O55, O111 or O128 produce Vero cytotoxin (VT). These serogroups are included in the range of enteropathogenic E. coli (EPEC) serogroups for which commercial antisera are available. In an attempt to obtain information on VT-producing strains other than those of serogroup O157, 122 strains belonging to these four serogroups and isolated in 1991 from patients with diarrhoea in the United Kingdom were tested for hybridization with VT probes. Only 18 of the 122 strains were VT-positive and these were O26 or O128. However 90 strains hybridized with the E. coli attaching and effacing (eae) probe (including 14 VT-positive strains) and 17 with the enteroaggregative E. coli (EAggEC) probe. For 78 eae-positive and 9 EAggEC-positive strains, tissue culture tests correlated with the probe results as the strains gave, respectively, either localized adhesion and a positive fluorescent-actin staining test or a characteristic aggregative attachment. A total of 111 of the 122 strains belonging to serogroups O26, O55, O111 or O128 possessed properties that may be associated with the ability to cause human diarrhoeal disease, and similar studies are needed on strains from the other classical EPEC serogroups.     

The prevalence and characterization of verotoxin-producing Escherichia coli isolated from cattle and pigs in an abattoir in Hong Kong

The aim of the study was to define the prevalence of verotoxin-producing Escherichia coli (VTEC) in cattle and pigs in a Hong Kong abattoir. Faecal and carcass samples collected from 986 cattle and 487 pigs from an abattoir were tested for verotoxin (VT) by PCR and cytotoxicity assays. VTEC was isolated from 415 and 1-8% of cattle faecal and carcass samples and from 2.1 and 0.2% of porcine faecal and carcass samples, respectively. Amongst 409 VTEC isolates from cattle, 9 were serotype O157:H7 and eaeA+. The most prevalent vt genotype among bovine VTEC was vtl+vt2 (73.8%) and in porcine VTEC was vt2e+ (30%). None of the porcine VTEC isolates and 9.3% of the bovine VTEC isolates was eaeA+. The non-O157 serogroup VTEC isolates carrying eaeA and EHEC-hlyA belonged to serogroups O172, O15, O84, O91, O110 and O121. The local dietary preference for pork or chicken (rather than beef), the low VTEC carriage in pigs, the rarity of additional virulence factors (caeA) in VTEC isolated from cattle may explain the apparently low incidence of human diarrhoeal disease associated with VTEC in Hong Kong hitherto. However, the presence of non-O157 VTEC strains carrying the eacA virulence marker in cattle highlights the fact that sole reliance on sorbitol-MacConkey agar for screening human VTEC isolates may underestimate the human disease burden. The changing dietary habits of the population in Hong Kong reinforce the need for continued vigilance.     

Antimicrobial resistance of Escherichia coli O26, O103, O111, O128, and O145 from animals and humans

Susceptibilities to fourteen antimicrobial agents important in clinical medicine and agriculture were determined for 752 Escherichia coli isolates of serotypes O26, O103, O111, O128, and O145. Strains of these serotypes may cause urinary tract and enteric infections in humans and have been implicated in infections with Shiga toxin-producing E. coli (STEC). Approximately 50% of the 137 isolates from humans were resistant to ampicillin, sulfamethoxazole, cephalothin, tetracycline, or streptomycin, and approximately 25% were resistant to chloramphenicol, trimethoprim-sulfamethoxazole, or amoxicillin-clavulanic acid. Approximately 50% of the 534 isolates from food animals were resistant to sulfamethoxazole, tetracycline, or streptomycin. Of 195 isolates with STEC-related virulence genes, approximately 40% were resistant to sulfamethoxazole, tetracycline, or streptomycin. Findings from this study suggest antimicrobial resistance is widespread among E. coli O26, O103, O111, O128, and O145 inhabiting humans and food animals.     

Modelling the epidemiology and transmission of Verocytotoxin-producing Escherichia coli serogroups O26 and O103 in two different calf cohorts

Mathematical models are constructed to investigate the population dynamics of Verocytotoxin-producing Escherichia coli (VTEC) serogroups O26 and O103 in two different calf cohorts. We compare the epidemiological characteristics of these two serogroups within the same calf cohort as well as the same serogroups between the two calf cohorts. The sources of infection are quantified for both calf cohort studies. VTEC serogroups O26 and O103 mainly differ in the rate at which calves acquire infection from sources other than infected calves, while infected calves typically remain infectious for less than 1 week regardless of the serogroups. Fewer than 20% of VTEC-positive samples are the result of calf-to-calf transmission. PFGE typing data are available for VTEC-positive samples to further subdivide the serogroup data in one of the two calf cohort studies. For serogroup O26 but not O103, there is evidence for unequal environmental exposure to infection with different PFGE types.     

Human infections with non-O157 Shiga toxin-producing Escherichia coli, Switzerland, 2000-2009

We characterized 97 non-O157 Shiga toxin (stx)-producing Escherichia coli strains isolated from human patients during 2000-2009 from the national reference laboratory in Switzerland. These strains belonged to 40 O:H serotypes; 4 serotypes (O26:H11/H-, O103:H2, O121:H19, and O145:H28/H-) accounted for 46.4% of the strains. Nonbloody diarrhea was reported by 23.2% of the patients, bloody diarrhea by 56.8%. Hemolytic uremic syndrome developed in 40.0% of patients; serotype O26:H11/H- was most often associated with this syndrome. Forty-five (46.4%) strains carried stx2 genes only, 36 strains (37.1%) carried stx1, and 16 (16.5%) strains carried stx1 and stx2. Genes encoding enterohemolysin and intimin were detected in 75.3% and 70.1% of the strains, respectively. Resistance to ≥1 antimicrobial agent was present in 25 isolates. High genetic diversity within strains indicates that non-O157 stx-producing E. coli infections in Switzerland most often occurred as single cases.     

Immunomagnetic separation as a sensitive method for isolating Escherichia coli O157 from food samples.

Minced beef samples inoculated with Escherichia coli O157 were cultured in buffered peptone water supplemented with vancomycin, cefsulodin and cefixime (BPW-VCC) and subcultured to cefixime tellurite sorbitol MacConkey (CT-SMAC) agar both directly and after immunomagnetic separation (IMS) of the organism with magnetic beads coated with an antibody against E. coli O157 (Dynabeads anti-E. coli O157, Dynal, Oslo). E. coli O157 was recovered from initial inocula of 200 organisms/g by direct subculture and 2 organisms/g by IMS. Twelve strains of E. coli O157 of different combinations of phage type, H antigen and toxin genotype were all recovered from initial inocula of two organisms/g by IMS. Non-specific binding of other organisms to the magnetic beads could be reduced by washing of the beads in PBS with Tween-20 0.002-0.005% E. coli O157 was not bound by magnetic coated with an unrelated antibody. During investigation of a dairy herd that was possibly linked to a small outbreak of infection with E. coli O157, the organism was isolated from 2 of 279 forestream milk samples from individual cattle; both isolates were made only by the IMS technique. IMS is rapid, technically simple, and a specific method for isolation of E. coli O157 and will be useful in epidemiological studies.     

Phenotypic and genetic markers for serotype-specific detection of Shiga toxin-producing Escherichia coli O26 strains from North America

Phenotypic and genetic markers of Shiga toxin-producing Escherichia coli (STEC) O26 from North America were used to develop serotype-specific protocols for detection of this pathogen. Carbohydrate fermentation profiles and prevalence of gene sequences associated with STEC O26 (n = 20) were examined. Non-STEC O26 (n = 17), E. coli O157 (n = 20), E. coli O111 (n = 22), and generic E. coli (n = 21) were used as comparison strains. Effects of supplements: cefixime-tellurite, 4-methylumbelliferyl-beta-D-glucuronide (MUG) and chromogenic additives (5-bromo4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-GlcA) and o-nitrophenyl-beta-D-galactopyranoside (ONPG), added to isolation agar media were examined. Tests for presence of gene sequences encoding beta intimin (eae beta), Shiga toxin 1 and 2 (stx1 and stx2), H7 flagella (flicCh7), enterohemolysin (ehlyA), O26 somatic antigen (wzx), and high pathogenicity island genes (irp2 and fyuA) were conducted using multiplex polymerase chain reaction. Pulsed-field gel electrophoresis (PFGE) of XbaI restriction endonuclease genomic DNA digests was used to establish clonality among E. coli O26 strains. Of the 26 carbohydrates tested, only rhamnose had diagnostic value. Rhamnose non-fermenters included STEC O26 (100%), non-STEC O26 (40%), generic E. coli (29%), E. coli O111 (23%), and E. coli O157 (0%). Rhamnose non-fermenting colonies growing on Rhamnose-McConkey agar supplemented with X-GlcA, X-Gal, or ONPG, respectively, were blue, white, or faint yellow, whereas rhamnose-fermenters were red. Blue colonies from X-GlcA-containing media were the most well-defined and easiest to pick for further tests. All STEC O26 were MUG-fluorescent, while STEC O157 (n = 18) were non-fluorescent. E. coli O111 and generic E. coli strains were either MUG-positive or-negative. Serotype-specific detection of STEC O26 was achieved by selecting cefixime-tellurite-resistant, MUG-fluorescent, rhamnose-nonfermenting colonies, which carried stx1, eae beta, irp2, and wzx gene sequences. STEC O26 prevalence in dairy farm environmental samples determined using the developed isolation and genetic detection protocols was 4%. PFGE indicated the presence of one major cluster of E. coli O26 with 72-100% DNA fragment-length digest similarity among test strains. The serotype-specific detection methods described herein have potential for routine application in STEC O26 diagnosis.     

Evaluation of universal pre-enrichment broth for isolation of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes from dairy farm environmental samples

Use of universal pre-enrichment broth (UPB) as a primary enrichment medium for detection of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes from dairy farm environmental samples was evaluated. There were no differences in bacterial growth between UPB and selective primary enrichment broths for each pathogen inoculated individually or in combination at 10(1) and 10(2) colony forming units/mL. In addition, no differences were observed when UPB and selective primary enrichment broths were compared for detection efficiency of pathogens in artificially contaminated raw milk and fecal samples. Listeria enrichment broth (LEB) was compared with UPB to support growth of L. monocytogenes from naturally contaminated environmental samples. Listeria monocytogenes was isolated from seven of 30 samples enriched in UPB and six of 30 samples enriched in LEB. Dairy farm environmental samples were examined for recovery of the three pathogens using UPB. Subsequent isolation was achieved using selective secondary enrichment of each pathogen. Listeria monocytogenes, Salmonella spp., and E. coli O157:H7 were isolated in 13.4% (30 of 224), 8.9% (20 of 224), and 2.2% (five of 224) of samples, respectively. Isolation rates of the three pathogens were somewhat higher than in previous reports. Overall, UPB supported growth of test pathogens to detectable levels within 24 h. Our results demonstrate that UPB has potential for routine use in isolation of foodborne pathogens from diverse environmental samples.     

A one year study of Escherichia coli O157 in raw beef and lamb products

Between April 1996 and March 1997 we examined 5093 samples of raw beef and lamb products for the presence of E. coli O157. Samples were purchased from 81 small butchers' shops in south Yorkshire. In March 1997 we also examined five samples of dried mint for the presence of E. coli O157. Strains of E. coli O157 were isolated by enrichment culture in modified buffered peptone water followed by immunomagnetic separation and culture of magnetic beads onto cefixime tellurite sorbitol MacConkey agar. Strains were characterized by phage typing, toxin genotyping and plasmid analysis. Strains of E. coli O157 were isolated from 72 (1.4%) of 5093 samples; it was isolated from 36 (1.1%) of 3216 samples of beef products and from 29 (2.9%) samples of lamb products. The highest prevalence was found in lamb sausages and lamb burgers where E. coli O157 was isolated from 3 (4.1%) of 73 and 18 (3.7%) of 484 samples respectively. Strains of E. coli O157 were isolated most frequently during early summer. Strains of E. coli O157 were also isolated from 2 of 5 samples of dried mint although we did not determine how the mint had become contaminated. All isolates of E. coli O157 were Verocytotoxin-producing as determined by both Vero cell assay and DNA hybridization for the genes encoding Verocytotoxin and all were positive for the eaeA gene. A combination of phage typing, toxin genotyping and plasmid profile subdivided the 72 strains of E. coli isolated into 20 different subtypes, of which 18 were indistinguishable from strains isolated previously from cattle and sheep; of these 18 strains, 8 were indistinguishable from strains isolated from human cases of infection during the study period.     

Identifying Shiga toxin-producing Escherichia coli: results of a laboratory evaluation

Laboratory procedures for identifying Escherichia coli O157 and other Shiga toxin-producing E. coli (STEC) strains vary considerably, causing concern that these infections may be underdiagnosed. E. coli O157 may be screened for by culture on sorbitol-containing selective media; however, no selective medium is available for isolation of non-O157 STEC. Shiga toxins may be detected using enzyme immunoassay or real-time polymerase chain reaction; however, the organism is not isolated for subsequent characterization. The Centers for Disease Control and Prevention (CDC) recommends using both routine bacterial culture on sorbitol-containing medium and an assay for Shiga toxins to identify STEC. An evaluation of laboratories revealed limited compliance with these recommendations. Enhancing laboratory procedures to comply with the CDC guidelines is essential for public health surveillance.     

Isolation and characterization of Shiga toxin-producing Escherichia coli (STEC) in retail edible beef by-products

The prevalence of Shiga toxin-producing Escherichia coli (STEC) was investigated in 350 edible beef intestinal samples, including omasum (n=110), abomasum (n=120), and large intestines (n=120), collected from traditional beef markets in Seoul, Korea. A total of 23 STEC strains were isolated from 15 samples (four strains from three omasa, 10 from five abomasa, and nine from seven large intestines). The O serotypes and toxin gene types of all STEC isolates were identified, and antimicrobial resistance was assessed using the disk diffusion method. The isolation rates of STEC from edible beef intestines were 2.8% in omasum, 4.2% in abomasums, and 5.9% in large intestines. All STEC isolates harbored either stx1, or both stx1 and stx2 genes simultaneously. Among the 23 isolates, 13 strains were identified as 11 different O serogroups, and 10 strains were untypable. However, enterohemorrhagic Esherichia coli O157, O26, and O111 strains were not isolated. The highest resistance rate observed was against tetracycline (39%), followed by streptomycin (35%) and ampicillin (22%). Of the 23 isolates, 12 isolates (52%) were resistant to at least one antibiotic, nine (39%) isolates were resistant to two or more antibiotics, and one isolate from an abmasum carried resistance against nine antibiotics, including beta-lactam/beta-lactamase inhibitor in combination and cephalosporins. This study shows that edible beef by-products, which are often consumed as raw food in many countries, including Korea, can be potential vehicles for transmission of antimicrobial-resistant pathogenic E. coli to humans.     

Cross reactivity of enterohemorrhagic Escherichia coli O157:H7-specific sera with non-O157 serotypes

Escherichia coli O157:H7 is an important food- and water-borne pathogen of humans, causing Hemorrhagic Colitis and Haemolytic Uremic Syndrome. Colonization of both cattle and human hosts is mediated through the action of effector molecules secreted via a Type III secretion system, a mechanism shared by other enterohemorrhagic E. coli (EHEC). We recently reported that vaccination of cattle with Type III-secreted proteins (TTSPs) resulted in decreased shedding of the organism following both experimental infection as well as under conditions of natural exposure. In order to extend this to non-O157 EHEC serotypes, we examined the serological cross reactivity of TTSPs of serotypes O26:H11, O103:H2, O111:NM and O157:H7. Western blotting experiments with polyclonal antisera directed against serotype O157:H7 TTSPs suggested that there was significant cross reactivity, although there was limited cross reactivity when two Tir- and EspA-specific monoclonal antibodies were used. Groups of cattle were then vaccinated with TTSPs produced from each of the above serotypes and the magnitude and specificity of the responses were measured. All animals responded well with antibodies to TTSPs of the homologous serotype. However, limited cross reactivity was observed against the others. No cross reactivity was observed against Tir and EspA of serotype O157:H7. These results suggest that vaccination of cattle with TTSPs as a means of reducing the risk of EHEC transmission to humans will induce protection that is serotype specific.