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.     

Behaviour of Escherichia coli strains during semi-hard and hard raw milk cheese production

The behaviour of two Escherichia coli strains, one with a high and the other with a low thermotolerance phenotype, was investigated during production and ripening of Swiss semi-hard and hard raw milk cheese. In semi-hard cheese, counts of E. coli increased during production, before a log-linear decrease occurred during ripening, with a faster rate of reduction in core than in rind samples, and faster reduction of the more heat-sensitive strain in rind samples. Nevertheless, at the end of semi-hard cheese ripening, E. coli were present at least at 1.3 log₁₀ cfu g⁻¹ in rind samples and remained detectable by enrichment of core samples. During the first day of hard cheese production, both E. coli strains were almost completely inactivated. Detection by enrichment was possible in one of twelve spiked cheeses after 16 weeks, indicating the potential of a thermotolerant E. coli strain to survive until the end of ripening.     

Prevalence and characteristics of Shiga toxin-producing Escherichia coli in Swiss raw milk cheeses collected at producer level

The aim of this study was to describe the prevalence, serotypes, and virulence genes of Shiga toxin-producing Escherichia coli (STEC) isolated from raw milk cheese samples collected at the producer level with the purpose of determining whether raw milk cheeses in Switzer-land represent a potential source of STEC pathogenic for humans. Raw milk cheese samples (soft cheese, n = 52; semihard and hard cheese, n = 744; all produced from Swiss cows’, goats’, and sheep's milk) collected at the producer level throughout Switzerland within the national sampling plan during the period of March 2006 to December 2007 were analyzed. Of the 432 cheese samples obtained in the year 2006 and the 364 samples obtained in the year 2007, 16 (3.7%) and 23 (6.3%), respectively, were found to be stx positive. By colony dot-blot hybridization, non-O157 STEC strains were isolated from 16 samples. Of the 16 strains, 11 were typed into 7 E. coli O groups (O2, O15, O22, O91, O109, O113, O174), whereas 5 strains were nontypeable (ONT). Among the 16 STEC strains analyzed, stx₁ and stx₂ variants were detected in 1 and 15 strains, respectively. Out of the 15 strains with genes encoding for the Stx2 group, 4 strains were positive for stx₂, 6 strains for stx_2d2, 2 strains for stx_2-O118, 1 strain for stx_2-06, 1 strain for stx_2g, 1 strain for stx₂ and stx_2d2, and 1 strain for stx₂ and stx_2g. Furthermore, 3 STEC strains harbored E-hlyA as a further putative virulence factor. None of the strains tested positive for eae (intimin). Results obtained in this work reinforce the suggestion that semihard raw milk cheese may be a potential vehicle for transmission of pathogenic STEC to humans.     

Prevalence of Shiga toxin-producing Escherichia coli in beef

Over the past two decades, many human illness outbreaks were attributed to consumption of undercooked beef products containing Shiga toxin-producing Escherichia coli (STEC). The illnesses included mild or bloody diarrhea, hemorrhagic colitis, and the life-threatening hemolytic uremic syndrome (HUS). Tracing these outbreaks to O157 and an increasing number of non-O157 STEC strains suggests that beef safety concerns will continue to rise and may negatively affect the beef industry. To effectively address these concerns, it is critical to evaluate the role of beef in STEC infections. In this review, published reports on beef contamination were evaluated to assess prevalence rates and health risks of STEC isolates. Global testing of beef showed wide ranges of prevalence rates of O157 (from 0.01% to 54.2%) and non-O157 (from 1.7% to 62.5%) STEC. Of the 155 STEC serotypes found in beef, 31 and 25 are known to cause HUS and/or other illnesses, respectively.     

Mathematical modeling of growth of non-O157 Shiga toxin-producing Escherichia coli in raw ground beef

Abstract: The objective of this study was to investigate the growth of Shiga toxin‐producing Escherichia coli (STEC, including serogroups O45, O103, O111, O121, and O145) in raw ground beef and to develop mathematical models to describe the bacterial growth under different temperature conditions. Three primary growth models were evaluated, including the Baranyi model, the Huang 2008 model, and a new growth model that is based on the communication of messenger signals during bacterial growth. A 5 strain cocktail of freshly prepared STEC was inoculated to raw ground beef samples and incubated at temperatures ranging from 10 to 35 °C at 5 °C increments. Minimum relative growth (<1 log₁₀ cfu/g) was observed at 10 °C, whereas at other temperatures, all 3 phases of growth were observed. Analytical results showed that all 3 models were equally suitable for describing the bacterial growth under constant temperatures. The maximum cell density of STEC in raw ground beef increased exponentially with temperature, but reached a maximum of 8.53 log₁₀ cfu/g of ground beef. The specific growth rates estimated by the 3 primary models were practically identical and can be evaluated by either the Ratkowsky square‐root model or a Bělehrádek‐type model. The temperature dependence of lag phase development for all 3 primary models was also developed. The results of this study can be used to estimate the growth of STEC in raw ground beef at temperatures between 10 and 35 °C. Practical Application: Incidents of foodborne infections caused by non‐O157 Shiga toxin‐producing Escherichia coli (STEC) have increased in recent years. This study reports the growth kinetics and mathematical modeling of STEC in ground beef. The mathematical models can be used in risk assessment of STEC in ground beef.     

可视化环介导等温扩增法检测牛奶中产志贺毒素大肠埃希氏菌

为建立一种快速、简单、灵敏的产志贺毒素大肠埃希氏菌（Shiga toxin-producing Escherichia coli,STEC）检测方法,本研究以stx基因作为检测靶基因和利用羟基萘酚蓝（hydroxy naphthol blue,HNB）指示剂建立了一种STEC的可视化环介导等温扩增（Loop Mediated Isothermal Amplification, LAMP）方法。根据GenBank公布的STEC菌株stx1和stx2基因核苷酸序列为靶序列,分别设计并合成五组特异性LAMP引物,优化反应条件和体系,进行特异性和灵敏度试验。同时,将建立好的LAMP方法与聚合酶链式反应（polymerase chain reaction, PCR）检测方法相比较,并对人工污染STEC的脱脂乳进行检测。结果表明,建立的可视化LAMP方法在64℃反应50 min后可凭肉眼观察（紫罗兰色到天蓝色）判定结果。该方法特异性良好,与大肠埃希菌、沙门菌、金黄色葡萄球菌等均无交叉反应,可以准确区分STEC与非STEC菌。灵敏度的检测结果表明,stx1-LAMP和stx2-LAMP检测方法的检测限分别为3.54×10-4 ng/μL和3.54×10-5 ng/μL,而常规PCR的检测限stx1和stx2分别为3.54×10-3 ng/μL和3.54×10-2 ng/μL,即stx1-LAMP检测方法的灵敏度是stx1-PCR的10倍,stx2-LAMP检测方法的灵敏度是stx2-PCR的1000倍。对人工污染STEC菌牛奶样品进行检测,stx1-LAMP和stx2-LAMP分别可检测到细菌浓度为103 CFU/mL和102 CFU/mL的加标样品。本研究为STEC菌株提供了一种特异性强、灵敏度高、成本低,适用于现场和基层检测的可视化LAMP 检测方法。     

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.     

Role of cellulose in protecting Shiga toxin-producing Escherichia coli against osmotic and chlorine stress

This study was undertaken to determine the role of cellulose in protecting Shiga toxin-producing Escherichia coli (STEC) against osmotic and chlorine treatments. STEC cells producing cellulose (19B and 49B) and their respective cellulose-deficient counterparts (19D or 49D) were subjected to osmotic (1, 2, and 3 M NaCl) or chlorine (25, 50, and 100 μg/ml sodium hypochlorite) treatments. The survival of STEC cells was determined at different treatment intervals. Populations of 19B cells were significantly higher (P < 0.05) than those of 19D cells at all sampling intervals for the chlorine treatments, at 24- to 48-h intervals for the 1 M NaCl treatment, and at 9- to 48-h intervals for the 2 M NaCl treatment. Significant differences in populations of 49B and 49D cells were observed after 9, 36, and 48 h of treatment with 2 M NaCl and after 3, 12, 36, and 48 h of treatment with 3 M NaCl (P < 0.05). Populations of 49B cells were higher than those of 49D cells (P < 0.05) also after 5 to 10 min of treatment with 50 μg/ml sodium hypochlorite and 3 to 10 min of treatment with 100 μg/ml sodium hypochlorite. The protective effects conferred by cellulose may explain the greater survival of cellulose-producing STEC under adverse environmental conditions.     

食源性产志贺毒素大肠杆菌的分离及菌株特征分析

了解不同食品中产志贺毒素大肠杆菌的流行情况、菌株特征及潜在致病性。方法 对我国不同地区采集的355份食品样品进行产志贺毒素大肠杆菌分离鉴定,对菌株进行stx1/stx2基因分型、eae等毒力基因检测,并对菌株进行多位点序列分型(MLST)分析。结果 355份样品中44份stx2基因阳性,共分离出11株非O157 产志贺毒素大肠杆菌,其中3株携带stx2a亚型,3株携带stx2e亚型,1株携带stx2b亚型,4株不能分型;5株携带ehxA、saa毒力基因,2株携带subA基因,1株携带katP基因;MLST将11株菌分为7个不同的ST型,存在与溶血性尿毒综合症患者肠出血性大肠杆菌分离株(HUS-associated enterohemorrhagic E.coli,HUSEC)及主要流行血清群产志贺毒素大肠杆菌亲缘关系较近的ST型别。结论 我国食品中存在一定程度的非O157产志贺毒素大肠杆菌污染,部分菌株具有潜在致病性,应加强对食品中STEC的监测。     

Prevalence of Shiga toxin-producing Escherichia coli in food products of animal origin as determined by molecular methods

In this study we report on the prevalence and distribution of Shiga toxin-producing Escherichia coli (STEC) in food products of animal origin, collected in the Piedmont region of Italy, as determined by a combination of quantitative PCR (qPCR) protocols, applied directly to the samples, and of culture-dependent isolation and subsequent molecular identification and characterization of isolates. The qPCR protocols were developed and optimized in this study and targeted the rpoB gene (as a marker for total E. coli) and the stx?, stx? and eaeA genes (as markers for potentially virulent E.coli). They were then used to test for STEC in 101 food samples, before and after enrichment. A STEC prevalence of 42% (21/50) for dairy products and 70% (36/51) for meat products was obtained. A total of 54 STEC isolates were recovered from dairy and meat samples, resulting in a prevalence of 36% and 27% in dairy and meat products, respectively, by the culture method. A large number of strains carried the stx? gene (39 out of the 54 STEC strains) compared to strains that carried stx? (30 out of 54); only 11 out of 54 strains contained the eaeA gene, while 14 strains contained both stx? and stx?. Eight of the 54 isolates belonged to the O157 serogroup, and none belonged to serogroups O26, O145, O111 or O103. Strains isolated from meat products were diverse, as determined by Enterobacterial repetitive intergenic consensus-PCR (ERIC), while those isolated from dairy products were more similar and grouped together by cluster analysis. The results of the qPCR approach showed a high prevalence of STEC in dairy and meat based products, mainly fermented, indicating a possible safety risk for these types of food commodities.     

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.     

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.     

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.     

伊犁地区肉牛源产志贺毒素大肠杆菌的血清型和ERIC-PCR基因型研究

目的:了解产志贺毒素大肠杆菌在伊犁地区肉牛养殖环境和加工环节中的污染状况及其遗传多样性,为产业链中食源性致病性大肠杆菌的风险监测和控制提供基础数据。方法:采用传统方法和PCR方法对养殖环节的饲草料和粪便及屠宰环节的553份样品进行产志贺毒素大肠杆菌的污染调查,对分离鉴定的产志贺毒素大肠杆菌进行7种常见血清型（O145、O157、O45、O103、O111、O26、O121）的PCR检测和ERIC-PCR的基因分型。结果:检测553份样品中有39株编码志贺毒素基因,产志贺毒素大肠杆菌（STEC）的检测率是7.1%。常见血清型PCR检测中血清型O111有2株菌,检出率是5.1%;O145有5株菌,检出率是12.8%。ERIC-PCR基因分型产志贺毒素大肠杆菌有10种基因亚型,分成3簇,A簇有23株菌,相似性在59%¹00%,表明这些菌株之间的亲缘关系较近。结论:伊犁地区肉牛粪便是产志贺毒素大肠杆菌的污染源,这些菌株的亲缘关系较近。      

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.     

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.     

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.     

Phage types, virulence genes and PFGE profiles of Shiga toxin-producing Escherichia coli O157:H7 isolated from raw beef, soft cheese and vegetables in Lima (Peru)

The present study was conducted in Lima Metropolitana to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in raw beef, raw ground beef, soft cheese and fresh vegetables, sampled at different markets in the city. Between October 2000 and February 2001, 407 food samples were collected from different markets in the 42 districts of Lima Metropolitana. Samples were assayed for E. coli O157 by selective enrichment in modified Tryptic Soy Broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Fifty (12.3%) of 407 food samples resulted positive for E. coli O157 isolation (23 of 102 ground beef; 15 of 102 beef meat; eight of 102 soft cheese and four of 101 fresh vegetables). Thirty-five E. coli O157 isolates were further analysed for the presence of virulence genes. All 35 were positive by PCR for O157 rfbE, fliCh7, eae-gamma1 and ehxA genes. In addition, genes encoding Shiga toxins were detected in 33 of 35 isolates, five isolates (14%) encoded stx(1), stx(2), and 28 (80%) stx2 only. The isolates were of seven different phage types (PT4, PT8, PT14, PT21, PT34, PT54, and PT87) with three phage types accounting for 80% of isolates: PT4 (15 isolates), PT14 (8 isolates), and PT21 (5 isolates). Interestingly, the majority (31 of 35; 89%) of E. coli O157:H7 isolates characterized in this study belonged mainly to the phage types previously found in STEC O157:H7 strains associated with severe human disease in Europe and Canada. Pulsed-field gel electrophoresis (PFGE) of 32 isolates revealed 14 XbaI-PFGE groups (I to XIV) of similarity >85%, with 23 (72%) isolates grouped in five clusters. Some isolates from different districts presented a high clonal relatedness. Thus, PFGE group VIII clustered eleven strains from nine different districts. The broad range of PFGE subtypes found in this study demonstrates the natural occurrence of many genetic variants among STEC O157:H7 spread in Lima.