Prevalence of Shiga toxin-producing Escherichia coli in beef cattle

A large number of Shiga toxin-producing Escherichia coli (STEC) strains have caused major outbreaks and sporadic cases of human illnesses, including mild diarrhea, bloody diarrhea, hemorrhagic colitis, and the life-threatening hemolytic uremic syndrome. These illnesses have been traced to both O157 and non-O157 STEC. In a large number of STEC-associated outbreaks, the infections were attributed to consumption of ground beef or other beef products contaminated with cattle feces. Thus, beef cattle are considered reservoirs of STEC and can pose significant health risks to humans. The global nature of the human food supply suggests that safety concerns with beef will continue and the challenges facing the beef industry will increase at the production and processing levels. To be prepared to address these concerns and challenges, it is critical to assess the role of beef cattle in human STEC infections. In this review, published reports on STEC in beef cattle were evaluated to achieve the following specific objectives: (i) assess the prevalence of STEC in beef cattle, and (ii) determine the potential health risks of STEC strains from beef cattle. The latter objective is critically important because many beef STEC isolates are highly virulent. Global testing of beef cattle feces revealed wide ranges of prevalence rates for O157 STEC (i.e., 0.2 to 27.8%) and non-O157 STEC (i.e., 2.1 to 70.1%). Of the 261 STEC serotypes found in beef cattle, 44 cause hemolytic uremic syndrome and 37 cause other illnesses.     

Shiga toxin-producing Escherichia coli in beef heifers grazing an irrigated pasture

Shiga toxin-producing Escherichia coli (STEC) produce toxins that have been associated with several human illnesses. E. coli O157:H7 is the most well-studied STEC and was first associated with consumption of improperly cooked ground beef in 1982. E. coli O157:H7 is not the only foodborne STEC because other STEC serotypes are also associated with human illnesses. The objective of this study was to assess prevalence of STEC in 23 yearling beef (Angus) heifers grazing an irrigated grass pasture in spring (April), summer (July), fall (October), and winter (December) of 1999. A total of 86 fecal samples were rectally collected and were subjected to microbiological testing for the presence of STEC. Nine E. coli isolates from five heifers (one in spring and fall and three in winter) were toxic to Vero cells. Of these isolates, four were E. coli O157:H7, two belonged to the serogroup O6, one O39:NM, one O113:H-, and the final isolate was untypable. The STEC prevalence rate in our herd ranged from 4% (spring) to 15% (winter). Based on detecting both O157:H7 and non-O157:H7 STEC in our heifers, it is clear that screening fecal samples should not be limited to E. coli O157:H7. Identification of STEC-positive cattle prior to slaughter should help in reducing the risk of beef contamination with such foodborne pathogens if pre- and/or postharvest control measures are applied to such animals.     

Thermal resistance parameters for Shiga toxin-producing Escherichia coli in apple juice

The purpose of the present study was to determine the heat resistance of six non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes in comparison to E. coli O157:H7 in single-strength apple juice without pulp. The thermal parameters for stationary-phase and acid-adapted cells of E. coli strains from serogroups O26, O45, O103, O111, O121, O145, and O157:H7 were determined by using an immersed coil apparatus. The most heat-sensitive serotype in the present study was O26. Stationary-phase cells for serotypes O145, O121, and O45 had the highest D(56°C)-value among the six non-O157 serotypes studied, although all were significantly lower (P < 0.05) than that of E. coli O157:H7. At 60°C E. coli O157:H7 and O103 demonstrated the highest D-values (1.37 ± 0.23 and 1.07 ± 0.03 min, respectively). The D(62°C) for the most heat-resistant strain belonging to the serotype O145 was similar (P > 0.05) to that for the most resistant O157:H7 strain (0.61 ± 0.17 and 0.60 ± 0.09 min, respectively). The heat resistance for stationary-phase cells was generally equal to or higher than that of acid-adapted counterparts. Although E. coli O157:H7 revealed D-values similar to or higher than the individual six non-O157 STEC serotypes in apple juice, the z-values for most non-O157 STEC tested strains were greater than those of E. coli O157:H7. When data were used to calculate heat resistance parameters at a temperature recommended in U.S. Food and Drug Administration guidance to industry, the D(71.1°C) for E. coli O157:H7 and non-O157 STEC serotypes were not significantly different (P > 0.05).     

Validation of pepperoni process for control of Shiga toxin-producing Escherichia coli

The objective of this study was to compare the survival of non-O157 Shiga toxin-producing Escherichia coli (STEC) with E. coli O157:H7 during pepperoni production. Pepperoni batter was inoculated with 7 log CFU/g of a seven-strain STEC mixture, including strains of serotypes O26, O45, O103, O111, O121, O145, and O157. Sausages were fermented to pH ≤4.8, heated at 53.3°C for 1 h, and dried for up to 20 days. STEC strains were enumerated at designated intervals on sorbitol MacConkey (SMAC) and Rainbow (RA) agars; enrichments were completed in modified EC (mEC) broth and nonselective tryptic soy broth (TSB). When plated on SMAC, total E. coli populations decreased 2.6 to 3.5 log after the 1-h heating step at 53.3°C, and a 4.9- to 5-log reduction was observed after 7 days of drying. RA was more sensitive in recovering survivors; log reductions on it were 1.9 to 2.6, 3.8 to 4.2, and 4.6 to 5.3 at the end of cook, and at day 7 and day 14 of drying, respectively. When numbers were less than the limit of detection by direct plating on days 14 and 20 of drying (representing a 5-log kill), no more than one of three samples in each experiment was positive by enrichment with mEC broth; however, STEC strains were recovered in TSB enrichment. Freezing the 7-day dried sausage for 2 to 3 weeks generated an additional 1- to 1.5-log kill. Confirmation by PCR revealed that O103 and O157 had the greatest survival during pepperoni productions, but all serotypes except O111 and O121 were occasionally recovered during drying. This study suggests that non-O157 STEC s trains have comparable or less ability than E. coli O157 to survive the processing steps involved in the manufacture of pepperoni. Processes suitable for control of E. coli O157 will similarly inactivate the other STEC strains tested in this study.     

Food as a vehicle for transmission of Shiga toxin-producing Escherichia coli

Contaminated food continues to be the principal vehicle for transmission of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) to humans. A large number of foods, including those associated with outbreaks (alfalfa sprouts, fresh produce, beef, and unpasteurized juices), have been the focus of intensive research studies in the past few years (2003 to 2006) to assess the prevalence and identify effective intervention and inactivation treatments for these pathogens. Recent analyses of retail foods in the United States revealed E. coli O157:H7 was present in 1.5% of alfalfa sprouts and 0.17% of ground beef but not in some other foods examined. Differences in virulence patterns (presence of both stx1 and stx2 genes versus one stx gene) have been observed among isolates from beef samples obtained at the processing plant compared with retail outlets. Research has continued to examine survival and growth of STEC in foods, with several models being developed to predict the behavior of the pathogen under a wide range of environmental conditions. In an effort to develop effective strategies to minimize contamination, several influential factors are being addressed, including elucidating the underlying mechanism for attachment and penetration of STEC into foods and determining the role of handling practices and processing operations on cross-contamination between foods. Reports of some alternative nonthermal processing treatments (high pressure, pulsed-electric field, ionizing radiation, UV radiation, and ultrasound) indicate potential for inactivating STEC with minimal alteration to sensory and nutrient characteristics. Antimicrobials (e.g., organic acids, oxidizing agents, cetylpyridinium chloride, bacteriocins, acidified sodium chlorite, natural extracts) have varying degrees of efficacy as preservatives or sanitizing agents on produce, meat, and unpasteurized juices. Multiple-hurdle or sequential intervention treatments have the greatest potential to minimize transmission of STEC in foods.     

Host-dependent activation of IS1203v excision in Shiga toxin-producing Escherichia coli

IS1203v is an insertion sequence (IS) which is identical to the most abundant IS elements in the genome of Escherichia coli O157:H7. However, there is no sequence homologous to IS1203v in the genome of E. coli K-12. We constructed a system to analyze the excision frequency of IS1203v, and demonstrated that the frequency in E. coli O157:H7 was approximately 10(5) times higher than that in E. coli K-12. We also investigated the excision frequencies of IS1203v in various E. coli isolates, and showed that the excision frequencies of IS1203v-possessing strains were approximately 10(3) times higher than those of IS1203v-nonpossessing strains. The results suggest that the IS1203v-possessing strains use a common system to enhance IS1203v excision.     

An overview of molecular stress response mechanisms in Escherichia coli contributing to survival of Shiga toxin-producing Escherichia coli during raw milk cheese production

The ability of foodborne pathogens to survive in certain foods mainly depends on stress response mechanisms. Insight into molecular properties enabling pathogenic bacteria to survive in food is valuable for improvement of the control of pathogens during food processing. Raw milk cheeses are a potential source for human infections with Shiga toxin-producing Escherichia coli (STEC). In this review, we focused on the stress response mechanisms important for allowing STEC to survive raw milk cheese production processes. The major components and regulation pathways for general, acid, osmotic, and heat shock stress responses in E. coli and the implications of these responses for the survival of STEC in raw milk cheeses are discussed.     

Rapid and reliable detection of Shiga toxin-producing Escherichia coli by real-time multiplex PCR

Escherichia coli O26, O45, O103, O111, O121, O145, and O157 are the predominant Shiga toxin-producing E. coli (STEC) serogroups implicated in outbreaks of human foodborne illness worldwide. The increasing prevalence of these pathogens has important public health implications. Beef products have been considered a main source of foodborne human STEC infections. Robust and sensitive methods for the detection and characterization of these pathogens are needed to determine prevalence and incidence of STEC in beef processing facilities and to improve food safety interventions aimed at eliminating STEC from the food supply. This study was conducted to develop Taqman real-time multiplex PCR assays for the screening and rapid detection of the predominant STEC serogroups associated with human illness. Three serogroup-specific assays targeted the O-antigen gene clusters of E. coli O26 (wzy), O103 (wzx), and O145 (wzx) in assay 1, O45 (wzy), O111 (manC), and O121 (wzx) in assay 2, and O157 (rfbE) in assay 3. The uidA gene also was included in the serogroup-specific assays as an E. coli internal amplification control. A fourth assay was developed to target selected virulence genes for Shiga toxin (stx(1) and stx(2)), intimin (eae), and enterohemolysin (ehxA). The specificity of the serogroup and virulence gene assays was assessed by testing 100 and 62 E. coli strains and non-E. coli control strains, respectively. The assays correctly detected the genes in all strains examined, and no cross-reactions were observed, representing 100 % specificity. The detection limits of the assays were 10(3) or 10(4) CFU/ml for pure cultures and artificially contaminated fecal samples, and after a 6-h enrichment period, the detection limit of the assays was 10(0) CFU/ml. These results indicate that the four real-time multiplex PCR assays are robust and effective for the rapid and reliable detection of the seven predominant STEC serogroups of major public health concern and the detection of their virulence genes.     

Occurrence of Shiga toxin-producing Escherichia coli in a Spanish raw ewe's milk cheese

The aim of the present study was to investigate the occurrence of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in 'Castellano' cheese, a non-cooked and hard or semi-hard Spanish cheese made from ewe's milk. A total of 83 raw milk cheese samples with different ripening times (2.5, 6 and 12 months) were taken at 30 cheese factories. Samples were examined for the presence of STEC using in the first stage the Association of Official Analytical Chemists (AOAC) official method number 997.11, and then, in the second stage, isolates were tested for virulence genes using genotypic (PCR) methods. Three STEC strains were detected in two samples (2.4%) of 'Castellano' cheese, one with 2.5 and the other one with 12 month-ripening period. From those STEC isolates, two were identified as E. coli O14 and the third presented an O-specific polysaccharide not-groupable serologically (ONG). PCR showed that all isolates were characterized by harbouring the Shiga toxin (stx) stx1 gene and by the absence of the genes for stx2, eaeA, and ehxA virulence factors. This study revealed the potential of STEC to survive in long-ripened-hard cheeses.     

Inactivation of Shiga toxin-producing Escherichia coli in single-strength lemon and lime juices containing preservatives

The objective of this study was to determine the inactivation of non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes in comparison with O157 STEC in commercially produced, shelf-stable lemon and lime juices. The present validation tests confirmed that storage of the juices containing preservatives at room temperatures (22°C) for 3 days (72 h) ensures a >6-log reduction of O26, O45, O103, O111, O121, O145, and O157 STEC. These results demonstrate that non-O157 STEC had survival abilities comparable to those of E. coli O157:H7 strains in acidic food products such as lemon and lime juices (pH 2.5 ± 0.1); therefore, the storage conditions deemed to inactivate E. coli O157:H7 similarly inactivate the non-O157 serotypes.     

Serotypes and virulence genes of ovine non-O157 Shiga toxin-producing Escherichia coli in Switzerland

Sixty ovine STEC strains were examined with the aim (i) to serotype the strains, (ii) to characterize virulence factors, and (iii) to discuss possible associations between these factors and to assess the potential pathogenicity of these strains for humans. The 60 sorbitol-positive, non-O157 STEC strains belonged to 19 O:H serotypes, whereas 68% were of five serotypes (O87:H16, O91:H-, O103:H2, O128:H2, O176:H4). 52% belonged to serotypes reported in association with HUS. Five serotypes were not previously reported in sheep strains. Of the 47 strains encoding for stx1 variants, 57% were stx1c- and of the 45 encoding for stx2 variants, 80% were stx2d-positive. Eighty-two percent of the strains showed further putative virulence factors: 13% were eae-, 60% ehxA- and 67% saa-positive. The associations between harboring (i) eae and stx1, stx2, ehxA or no saa and (ii) saa and stx1c or stx2d were significant (P<0.05). The strains belonged to 27 seropathotypes (association between serotypes and virulence factors), but 57% belonged to only six and O91:H-stx1 stx2d saa and O128:H2 stx1c stx2d ehxA saa were the most common. Seven of the eight intimin-positive strains harbored eae. Four strains of serotype O103:H2 and O121:H10 harboring stx2, eae and ehxA showed virulence factors typical for strains associated with severe human disease. However, according to the virulence factors, the majority of the ovine non-O157 STEC strains are assumed low-virulence variants. Nevertheless, as long as the contribution and interaction of these factors in milder disease remains unclear P, a certain risk for humans cannot be excluded.     

Inactivation of Shiga toxin-producing O157:H7 and non-O157:H7 Shiga toxin-producing Escherichia coli in brine-injected, gas-grilled steaks

We quantified translocation of Escherichia coli O157:H7 (ECOH) and non-O157:H7 verocytotoxigenic E. coli (STEC) into beef subprimals after brine injection and subsequently monitored their viability after cooking steaks cut therefrom. Beef subprimals were inoculated on the lean side with ca. 6.0 log CFU/g of a five-strain cocktail of rifampin-resistant ECOH or kanamycin-resistant STEC, and then passed once through an automatic brine-injector tenderizer, with the lean side facing upward. Brine solutions (9.9% ± 0.3% over fresh weight) consisted of 3.3% (wt/vol) of sodium tripolyphosphate and 3.3% (wt/vol) of sodium chloride, prepared both with (Lac(+), pH = 6.76) and without (Lac(-), pH = 8.02) a 25% (vol/vol) solution of a 60% potassium lactate-sodium diacetate syrup. For all samples injected with Lac(-) or Lac(+) brine, levels of ECOH or STEC recovered from the topmost 1 cm (i.e., segment 1) of a core sample obtained from tenderized subprimals ranged from ca. 4.7 to 6.3 log CFU/g; however, it was possible to recover ECOH or STEC from all six segments of all cores tested. Next, brine-injected steaks from tenderized subprimals were cooked on a commercial open-flame gas grill to internal endpoint temperatures of either 37.8 °C (100 °F), 48.8 °C (120 °F), 60 °C (140 °F), or 71.1 °C (160 °F). Regardless of brine formulation or temperature, cooking achieved reductions (expressed as log CFU per gram) of 0.3 to 4.1 of ECOH and 0.5 to 3.6 of STEC. However, fortuitous survivors were recovered even at 71.1 °C (160 °F) for ECOH and for STEC. Thus, ECOH and STEC behaved similarly, relative to translocation and thermal destruction: Tenderization via brine injection transferred both pathogens throughout subprimals and cooking highly contaminated, brine-injected steaks on a commercial gas grill at 71.1 °C (160 °F) did not kill all cells due, primarily, to nonuniform heating (i.e., cold spots) within the meat.     

Genotypic characterization of non-O157 Shiga toxin-producing Escherichia coli in beef abattoirs of Argentina

The non-O157 Shiga toxin-producing Escherichia coli (STEC) contamination in carcasses and feces of 811 bovines in nine beef abattoirs from Argentina was analyzed during a period of 17 months. The feces of 181 (22.3%) bovines were positive for non-O157 STEC, while 73 (9.0%) of the carcasses showed non-O157 STEC contamination. Non-O157 STEC strains isolated from feces (227) and carcasses (80) were characterized. The main serotypes identified were O178:H19, O8:H19, O130:H11, and O113:H21, all of which have produced sporadic cases of hemolytic-uremic syndrome in Argentina and worldwide. Twenty-two (7.2%) strains carried a fully virulent stx/eae/ehxA genotype. Among them, strains of serotypes O103:[H2], O145:NM, and O111:NM represented 4.8% of the isolates. Xba I pulsed-field gel electrophoresis pattern analysis showed 234 different patterns, with 76 strains grouped in 30 clusters. Nine of the clusters grouped strains isolated from feces and from carcasses of the same or different bovines in a lot, while three clusters were comprised of strains distributed in more than one abattoir. Patterns AREXSX01.0157, AREXBX01.0015, and AREXPX01.0013 were identified as 100% compatible with the patterns of one strain isolated from a hemolytic-uremic syndrome case and two strains previously isolated from beef medallions, included in the Argentine PulseNet Database. In this survey, 4.8% (39 of 811) of the bovine carcasses appeared to be contaminated with nonO157 STEC strains potentially capable of producing sporadic human disease, and a lower proportion (0.25%) with strains able to produce outbreaks of severe disease.     

Detection of viable Shiga toxin-producing Escherichia coli by quantitative competitive polymerase chain reaction

With the use of Escherichia coli O157:H7 as a model, a procedure for the quantitative detection of viable Shiga toxin-producing E. coli (STEC) in broth and cooked ground beef enrichments with multiple-time point quantitative competitive polymerase chain reaction (QC-PCR) was developed. The A subunit (a 401-bp fragment) of the stx2 gene was chosen as a target sequence. Immunomagnetic separation (IMS) was used to isolate and concentrate cells from ground beef enrichments. Cell viability was confirmed on the basis of the quantitative increase in the signal of target bands from QC-PCR across multiple time points. The application of IMS increased detection limits relative to those for QC-PCR without IMS. E. coli O157:H7 inoculated at 0.20 CFU/g of cooked ground beef (25 g of ground beef plus 225 ml of Bacto modified EC medium plus novobiocin) was detected and confirmed to be viable in <15 h. A DNA-based molecular approach can be used to determine cell viability.     

Escherichia coli O157 and non-O157 Shiga toxin-producing Escherichia coli in fecal samples of finished pigs at slaughter in Switzerland

Fecal samples from 630 slaughtered finisher pigs were examined by PCR to assess the shedding of Escherichia coli O157 (rfbE) and Shiga toxin-producing E. coli (STEC, stx). The proportion of positive samples was 7.5% for rfbE and 22% for stx. By colony hybridization, 31 E. coli O157 and 45 STEC strains were isolated, and these strains were further characterized by phenotypic and genotypic traits. Among E. coli O157 strains, 30 were sorbitol positive, 30 had an H type other than H7, and none harbored stx genes. Intimin (eae), enterohemolysin (ehxA), EAST1 (astA), and porcine A/E-associated protein (paa) were present in 10, 3, 26, and 6% of strains. Among them, one eae-gamma1-positive O157:H7 strain testing positive for ehxA and astA and two eae-alpha1-positive O157:H45 strains were classified as enteropathogenic E. coli (EPEC). The O157:H45 EPEC harbored the EAF plasmid and the bfpA gene, factors characteristic for typical EPEC. The isolated STEC strains (43 sorbitol positive) belonged to 11 O:H serotypes, including three previously reported in human STEC causing hemolytic uremic syndrome (O9:H-, O26:H-, and O103:H2). All but one strain harbored stx2e. The eae and ehxA genes, which are strongly correlated with human disease, were present in only one O103:H2 strain positive for stx1 and paa, whereas the astA gene was found more frequently (14 strains). High prevalence of STEC was found among finisher pigs, but according to the virulence factors the majority of these strains seem to be of low virulence.     

Diversity of virulence profiles of Shiga toxin-producing Escherichia coli serotypes in food-producing animals in Brazil

The prevalence, serotypes and virulence profiles of Shiga toxin-producing Escherichia coli (STEC) were investigated in 205 healthy beef and dairy cattle, and 106 goats reared in the southeastern region of Minas Gerais State, Brazil. The prevalence of STEC was 57.5% (61/106) in goats, 39.2%, (40/102) in beef cattle and 17.5% (18/103) in dairy cattle. Among the 514 STEC isolates, 40 different serotypes were found and some of them were identified in a specific host. STEC isolates harboring stx(1) corresponded to 15.6% (28/180), 26.7% (16/60) and 24.1% (66/274) in beef cattle, dairy cattle and goats, respectively. stx(2) was found in 30% (54/180), 53.3% (32/60) and 34.7% (95/274) of beef and dairy cattle, and goats. stx(1) plus stx(2) sequences were harbored by 54.4% (98/180), 20% (12/60) and 41.2% (113/274) of beef cattle, dairy cattle and goats, respectively. The eae sequence was found in 15% (9/60) and 0.6% (1/180) of STEC isolates from dairy and beef cattle, respectively, and the toxB gene was found only in one O157:H7 strain isolated from beef cattle. Strains with the genetic profiles stx(2) ehxA iha saa and stx(1) stx(2) ehxA iha saa were the most prevalent among STEC isolates from cattle. Profiles stx(1) stx(2) ehxA iha, stx(2), and stx(1) iha accounted for 75.5% (207 /274) of the STEC isolates from goats. While STEC strains carrying either stx(2) alone or associated with stx(1) were found more frequently in cattle, those harboring sequences stx(1c) and stx(2d) alone or associated with stx(1c) predominated in goats. Our data show a diversity of STEC strains in food-producing animals, most of them carrying genes linked to severe forms of human diseases.     

Growth and enrichment medium for detection and isolation of Shiga toxin-producing Escherichia coli in cattle feces

Detection methods of Shiga toxin-producing Escherichia coli (STEC) in cattle feces varied in using enrichment media containing different antibiotic combinations. To examine efficacy of a new detection method for STEC, three O157:H7 (ATCC 43889, 43890, and 43895) and 41 non-O157:H7 (members of the O1, O15, O26, O86, O103, O111, O125, O127, O128, O136, O146, O153, O158, O165, O166, and O169 serogroups) isolates were tested. These isolates were grown in tryptic soy broth for 6 h, and their concentrations were determined before inoculation of tubes containing 1 g of cattle feces (sterile [experiment 1; evaluating growth] and fresh [experiment 2; evaluating enrichment]) to simulate the high and low levels of STEC shedding by cattle (10(5) versus 102 CFU/g feces, respectively). Eight STEC isolates (the three O157:H7 and five non-O157:H7 selected at random) were tested at a very low level (10 CFU/g feces). The feces were incubated in 50 ml of brain heart infusion broth containing potassium tellurite, novobiocin, and vancomycin (2.5, 20, and 40 mg/liter, respectively) and cefixime (50 microg/liter) at 37 degrees C for 12 h and tested for STEC (VTEC [verotoxin-producing E. coli]-Screen assay [agglutination immunoassay]). Potential STEC isolates were recovered, characterized biochemically, serotyped, and tested for toxin production using Vero (African green monkey kidney) cell toxicity assay and agglutination immunoassay. In both experiments, all the STEC isolates used for fecal inoculation were recovered at the concentrations tested. Our medium supported growth of and enrichment for a wide range of STEC isolates.     

Shiga toxin-producing Escherichia coli in ground beef and lamb cuts: results of a one-year study

Shiga toxin-producing Escherichia coli (STEC) have been associated with a broad spectrum of diarrhoeal syndromes. Some of these cases have been attributed to foods of bovine origin or other foods cross-contaminated by beef products or cow manure. The purpose of this study was to determine the pattern of STEC distribution in selected red meats over time. Samples of ground beef and lamb cuts were collected over a 52-week period from 31 different outlets and 25 g portions were assayed for STEC. STEC were isolated from 46/285 (16%) ground beef and 111/275 (40%) lamb samples using an stx PCR screen followed by colony hybridisation. All isolates were tested by PCR for additional STEC virulence markers with 95% of ground beef isolates shown to possess stx(2) and 80% of lamb cutlet isolates shown to possess stx(1) and stx(2). The enterohaemolysin gene (ehxA) was detected in 65% and 53% of ground beef and lamb isolates respectively. Putative enterohaemorrhagic E. coli (EHEC), i.e. STEC possessing the E. coli attaching and effacing gene (eae) were not isolated. The STEC isolates comprised 18 and 15 different serotypes from ground beef and lamb respectively. STEC of serotypes O157, O111 and O26 (common enterohaemorrhagic E. coli serotypes) were not isolated. Serotypes O174 and O91 were the most common serotypes isolated from ground beef samples and O128 and O91 the most common from lamb cutlet samples. The presence of STEC in retail red meats highlights the need for a clearer understanding of STEC in food and human illness to interpret the public health significance of these findings.     

Shiga toxin-producing Escherichia coli in healthy young beef steers from Argentina: prevalence and virulence properties

Between July 1999 and December 2000, the prevalence of Shiga toxin-producing Escherichia coli (STEC) was established in 200 Argentine healthy young beef steers (14-16 months old) grown under local production systems with a feed grain period of 3-4 months, and the STEC strains isolated were examined in regard to their phenotypic and genotypic characteristics. Stool samples (n = 70) and rectal swabs (n = 130) were taken at the slaughterhouse level. By polymerase chain reaction (PCR), Shiga toxin (stx) gene sequences were detected in 69% of the samples. Eighty-six STEC strains were isolated from 39% of the animals. Serogroups identified, in order of frequency, were: O8 (16 strains), O113 (14), O103 (5), O91 (4), O171 (3), O174 (3), O25 (2), O112 (2), O145 (2), O2, O11, O104, O121, O128, O143, O146, O157. The most frequent serotype isolated was O8:H19 (12.9%). A total of 17 serotypes, including E. coli O157:H7 found in one animal (0.5%), have been previously associated with hemolytic uremic syndrome (HUS), bloody and non-bloody diarrhea in different countries, including Argentina. The prevalent genotype isolated was stx2 (51 of 86, 59.3%). Subtyping of stx2 variants showed the prevalence of stx2vh-b (25.6%) and stx2vh-a types (24.4%), and revealed the presence of an atypical stx2-v. Only 7.0% of STEC strains carried eae, and 33.7% harbored EHEC-hlyA gene. The full virulent genotype (stx/eae/EHEC-hlyA) was found to be present in 4 of the 86 (4.7%) STEC strains isolated. This research indicates that young steers from the main beef-producing area of Argentina are an important reservoir of STEC strains; however, its importance as agents of human diseases in our country has still to be established.     

Comparison of a new enrichment procedure for Shiga toxin-producing Escherichia coli with five standard methods

A new procedure for enrichment of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli was compared to five standard methods: the British Public Health Laboratory Service, International Standard Method, U.S. Department of Agriculture, Canadian Health Products and Food Branch, and U.S. Food and Drug Administration. The new procedure was comparable to the standard methods in its ability to detect target cells inoculated into foods at approximately 1 CFU g(-1). Comparisons were also made of the ability of the six enrichment procedures to detect E. coli O157:H7 against a large background of competitor microorganisms. In these experiments the new procedure yielded more target cells than the other five enrichments by two to three orders of magnitude as determined by enumeration on sorbitol MacConkey agar with tellurite and cefixime and Rainbow agar with tellurite and novobiocin and by verification of presumptive colonies by real-time PCR. For example, the population of enterohemorrhagic E. coli strain 6341 recovered on sorbitol MacConkey agar with tellurite and cefixime after enrichment with the experimental method was 2.42 x 10(8) CFU ml(-1) and 1.80 x 10(6) CFU ml(-1) after enrichment with the Canadian Health Products and Food Branch method, the second most effective in this experiment. In addition, broth cultures resulting from each of the six enrichment procedures were used to prepare templates for real-time PCR detection of stx1/stx2. Resulting threshold cycle (Ct) values after the experimental enrichment were similar to positive control values, whereas the five standard methods produced delayed Ct values or were not detected.