Serotypes, virulence genes and intimin types of Shiga toxin (verocytotoxin)-producing Escherichia coli isolates from minced beef in Lugo (Spain) from 1995 through 2003.
ABSTRACT: BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) have emerged as pathogens that can cause food-borne infections and severe and potentially fatal illnesses in humans, such as haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). In Spain, like in many other countries, STEC strains have been frequently isolated from ruminants, and represent a significant cause of sporadic cases of human infection. In view of the lack of data on STEC isolated from food in Spain, the objectives of this study were to determine the level of microbiological contamination and the prevalence of STEC O157:H7 and non-O157 in a large sampling of minced beef collected from 30 local stores in Lugo city between 1995 and 2003. Also to establish if those STEC isolated from food possessed the same virulence profiles as STEC strains causing human infections. RESULTS: STEC were detected in 95 (12%) of the 785 minced beef samples tested. STEC O157:H7 was isolated from eight (1.0%) samples and non-O157 STEC from 90 (11%) samples. Ninety-six STEC isolates were further characterized by PCR and serotyping. PCR showed that 28 (29%) isolates carried stx1 genes, 49 (51%) possessed stx2 genes, and 19 (20%) both stx1 and stx2. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 43 (45%) and in 25 (26%) of the isolates, respectively. Typing of the eae variants detected four types: gamma1 (nine isolates), beta1 (eight isolates), epsilon1 (three isolates), and theta (two isolates). The majority (68%) of STEC isolates belonged to serotypes previously detected in human STEC and 38% to serotypes associated with STEC isolated from patients with HUS. Ten new serotypes not previously described in raw beef products were also detected. The highly virulent seropathotypes O26:H11 stx1 eae-beta1, O157:H7 stx1stx2 eae-gamma1 and O157:H7 stx2eae-gamma1, which are the most frequently observed among STEC causing human infections in Spain, were detected in 10 of the 96 STEC isolates. Furthermore, phage typing of STEC O157:H7 isolates showed that the majority (seven of eight isolates) belonged to the main phage types previously detected in STEC O157:H7 strains associated with severe human illnesses. CONCLUSION: The results of this study do not differ greatly from those reported in other countries with regard to prevalence of O157 and non-O157 STEC in minced beef. As we suspected, serotypes different from O157:H7 also play an important role in food contamination in Spain, including the highly virulent seropathotype O26:H11 stx1 eae-beta1. Thus, our data confirm minced beef in the city of Lugo as vehicles of highly pathogenic STEC. This requires that control measures to be introduced and implemented to increase the safety of minced beef.
Project description:We have analyzed the prevalence of Shiga toxin-producing Escherichia coli (STEC) in stool specimens of patients with diarrhea or other gastrointestinal alterations from the Xeral-Calde Hospital of Lugo City (Spain). STEC strains were detected in 126 (2.5%) of 5,054 cases investigated, with a progressive increase in the incidence from 0% in 1992 to 4.4% in 1999. STEC O157:H7 was isolated in 24 cases (0.5%), whereas non-O157 STEC strains were isolated from 87 patients (1.7%). STEC strains were (after Salmonella and Campylobacter strains) the third most frequently recovered enteropathogenic bacteria. A total of 126 human STEC isolates were characterized in this study. PCR showed that 43 (34%) isolates carried stx(1) genes, 45 (36%) possessed stx(2) genes and 38 (30%) carried both stx(1) and stx(2). A total of 88 (70%) isolates carried an ehxA enterohemolysin gene, and 70 (56%) isolates possessed an eae intimin gene (27 isolates with type gamma1, 20 with type beta1, 8 with type zeta, 5 with type gamma2, and 3 with type epsilon). STEC isolates belonged to 41 O serogroups and 66 O:H serotypes, including 21 serotypes associated with hemolytic uremic syndrome and 30 new serotypes not previously reported among human STEC strains in other studies. Although the 126 STEC isolates belonged to 81 different seropathotypes (associations between serotypes and virulence genes), only four accounted for 31% of isolates. Seropathotype O157:H7 stx(1) stx(2) eae-gamma1 ehxA was the most common (13 isolates) followed by O157:H7 stx(2) eae-gamma1 ehxA (11 isolates), O26:H11 stx(1) eae-beta1 ehxA (11 isolates), and O111:H- stx(1) stx(2) eae-gamma2 ehxA (4 isolates). Our results suggest that STEC strains are a significant cause of human infections in Spain and confirm that in continental Europe, infections caused by STEC non-O157 strains are more common than those caused by O157:H7 isolates. The high prevalence of STEC strains (both O157:H7 and non-O157 strains) in human patients, and their association with serious complications, strongly supports the utilization of protocols for detection of all serotypes of STEC in Spanish clinical microbiology laboratories.
Project description:A total of 514 Shiga toxin-producing Escherichia coli (STEC) isolates from diarrheic and healthy cattle in Spain were characterized in this study. PCR showed that 101 (20%) isolates carried stx(1) genes, 278 (54%) possessed stx(2) genes, and 135 (26%) possessed both stx(1) and stx(2). Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 326 (63%) and in 151 (29%) of the isolates, respectively. STEC isolates belonged to 66 O serogroups and 113 O:H serotypes (including 23 new serotypes). However, 67% were of one of these 15 serogroups (O2, O4, O8, O20, O22, O26, O77, O91, O105, O113, O116, O157, O171, O174, and OX177) and 52% of the isolates belonged to only 10 serotypes (O4:H4, O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and ONT:H19). Although the 514 STEC isolates belonged to 164 different seropathotypes (associations between serotypes and virulence genes), only 12 accounted for 43% of isolates. Seropathotype O157:H7 stx(2) eae-gamma1 ehxA (46 isolates) was the most common, followed by O157:H7 stx(1) stx(2) eae-gamma1 ehxA (34 isolates), O113:H21 stx(2) (25 isolates), O22:H8 stx(1) stx(2) ehxA (15 isolates), O26:H11 stx(1) eae-beta1 ehxA (14 isolates), and O77:H41 stx(2) ehxA (14 isolates). Forty-one (22 of serotype O26:H11) isolates had intimin beta1, 82 O157:H7 isolates possessed intimin gamma1, three O111:H- isolates had intimin type gamma2, one O49:H- strain showed intimin type delta, 13 (six of serotype O103:H2) isolates had intimin type epsilon and eight (four of serotype O156:H-) isolates had intimin zeta. We have identified a new variant of the eae intimin gene designated xi (xi) in two isolates of serotype O80:H-. The majority (85%) of bovine STEC isolates belonged to serotypes previously found for human STEC organisms and 54% to serotypes associated with STEC organisms isolated from patients with hemolytic uremic syndrome. Thus, this study confirms that cattle are a major reservoir of STEC strains pathogenic for humans.
Project description:BACKGROUND: Enteropathogenic Escherichia coli (EPEC) and Shigatoxin-producing Escherichia coli (STEC) share the ability to introduce attaching-and-effacing (A/E) lesions on intestinal cells. The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that also contains the genes encoding intimin (eae). This study reports information on the occurrence of eae positive E. coli carried by healthy cattle at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated EPEC and STEC strains. RESULTS: Of 51 eae positive bovine E. coli strains, 59% were classified as EPEC and 41% as STEC. EPEC strains belonged to 18 O:H serotypes, six strains to typical EPEC serogroups. EPEC strains harbored a variety of intimin variants with eae-beta1 being most frequently found. Moreover, nine EPEC strains harbored astA (EAST1), seven bfpA (bundlin), and only one strain was positive for the EAF plasmid. We have identified a new intimin gene (eta2) in three bovine bfpA and astA-positive EPEC strains of serotype ONT:H45. STEC strains belonged to seven O:H serotypes with one serotype (O103:H2) accounting for 48% of the strains. The majority of bovine STEC strains (90%) belonged to five serotypes previously reported in association with hemolytic uremic syndrom (HUS), including one O157:H7 STEC strain. STEC strains harbored four intimin variants with eae-epsilon1 and eae-gamma1 being most frequently found. Moreover, the majority of STEC strains carried only stx1 genes (13 strains), and was positive for ehxA (18 strains) encoding for Enterohemolysin. Four STEC strains showed a virulence pattern characteristic of highly virulent human strains (stx2 and eae positive). CONCLUSION: Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-harboring E. coli strains. Moreover, cattle have not only to be considered as important asymptomatic carriers of O157 STEC but can also be a reservoir of EPEC and eae positive non-O157 STEC, which are described in association with human diseases.
Project description:The prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) was determined by evaluating its presence in faecal samples from 155 heifers, and 254 dairy cows in 21 farms at North of Portugal sampled between December 2017 and June 2019. The prevalence of STEC in heifers (45%) was significantly higher than in lactating cows (16%) (p<0.05, Fisher exact test statistic value is <0.00001). A total of 133 STEC were isolated, 24 (13.8%) carried Shiga-toxin 1 (stx1) genes, 69 (39.7%) carried Shiga-toxin 2 (stx2) genes, and 40 (23%) carried both stx1 and stx2. Intimin (eae) virulence gene was detected in 29 (21.8%) of the isolates. STEC isolates belonged to 72 different O:H serotypes, comprising 40 O serogroups and 23 H types. The most frequent serotypes were O29:H12 (15%) and O113:H21 (5.2%), found in a large number of farms. Two isolates belonged to the highly virulent serotypes associated with human disease O157:H7 and O26:H11. Many other bovine STEC serotypes founded in this work belonged to serotypes previously described as pathogenic to humans. Thus, this study highlights the need for control strategies that can reduce STEC prevalence at the farm level and, thus, prevent food and environmental contamination.
Project description: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-?1 (4.8%; 19 samples), stx-wzx(O103)-eae-? (1.3%; five samples), stx-ihp1(O145)-eae-?1 (0.8%; three samples), and stx-rfbE(O157)-eae-?1 (0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seven eae?1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Three stx-negative and eae?1-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.
Project description:In this study, 140 cattle STEC isolates belonging to serogroups O157, O26, O145, O121, O103 and O45 were characterized for 38 virulence-associated genes, antimicrobial resistance profiles and genotyped by PFGE. The majority of isolates carried both stx1 and stx2 concurrently, stx2c, and stx2d; plasmid-encoded genes ehxA, espP, subA and saa but lacked katP and etpD and eaeA. Possession of eaeA was significantly associated with the presence of nle genes, katP, etpD, ureC and terC. However, saa and subA, stx1c and stx1d were only detected in eaeA negative isolates. A complete OI-122 and most non-LEE effector genes were detected in only two eaeA positive serotypes, including STEC O157:H7 and O103:H2. The eaeA gene was detected in STEC serotypes that are commonly implicated in severe humans disease and outbreaks including STEC O157:H7, STEC O145:H28 and O103:H2. PFGE revealed that the isolates were highly diverse with very low rates of antimicrobial resistance. In conclusion, only a small number of cattle STEC serotypes that possessed eaeA, had the highest number of virulence-associated genes, indicative of their high virulence. Further characterization of STEC O157:H7, STEC O145:H28 and O103:H2 using whole genome sequencing will be needed to fully understand their virulence potential for humans.
Project description:We have investigated 677 Shiga toxin-producing Escherichia coli (STEC) strains from humans to determine their serotypes, virulence genes, and clinical signs in patients. Six different Shiga toxin types (1, 1c, 2, 2c, 2d, and 2e) were distributed in the STEC strains. Intimin (eae) genes were present in 62.6% of the strains and subtyped into intimins alpha1, beta1, gamma1, epsilon, theta, and eta. Shiga toxin types 1c and 2d were present only in eae-negative STEC strains, and type 2 was significantly (P < 0.001) more frequent in eae-positive STEC strains. Enterohemorrhagic E. coli hemolysin was associated with 96.2% of the eae-positive strains and with 65.2% of the eae-negative strains. Clinical signs in the patients were abdominal pain (8.7%), nonbloody diarrhea (59.2%), bloody diarrhea (14.3%), and hemolytic-uremic syndrome (HUS) (3.5%), and 14.3% of the patients had no signs of gastrointestinal disease or HUS. Infections with eae-positive STEC were significantly (P < 0.001) more frequent in children under 6 years of age than in other age groups, whereas eae-negative STEC infections dominated in adults. The STEC strains were grouped into 74 O:H types by serotyping and by PCR typing of the flagellar (fliC) genes in 221 nonmotile STEC strains. Eleven serotypes (O157:[H7], O26:[H11], O103:H2, O91:[H14], O111:[H8], O145:[H28], O128:H2, O113:[H4], O146:H21, O118:H16, and O76:[H19]) accounted for 69% of all STEC strains. We identified 41 STEC strains belonging to 31 serotypes which had not previously been described as human STEC. Twenty-six of these were positive for intimins alpha1 (one serotype), beta1 (eight serotypes), epsilon (two serotypes), and eta (three serotypes). Our study indicates that different types of STEC strains predominate in infant and adult patients and that new types of STEC strains are present among human isolates.
Project description:Aim:Shiga toxin-producing Escherichia coli (STEC) represent a severe public health issue worldwide, causing life-threatening diseases in the human gastrointestinal tract. This study aimed to determine the occurrence of virulent and antibiotic-resistant STEC in retail meat and milk products and human stool samples and to characterize the genes encoding for virulence and antibiotic resistance among the identified STEC isolates. Materials and Methods:A total of 260 food samples were randomly collected from retail markets in different localities of El Giza Governorate, Egypt. 50 stool specimens were obtained from children that had diarrhea at Embaba Fever Hospital. All collected samples were initially subjected to bacteriological examination and serotyping, and then subsequently, the isolates were exposed to polymerase chain reaction application and sequencing for the identification of the virulence-related genes. Finally, the virulent STEC isolates were tested for antibiotic susceptibility. Results:Serotyping of the 76 biochemically identified isolates showed that 18 were STEC with a predominance of non-O157 (16) while 2 O157:K-serotype was detected only in one food and one human isolate. Molecular identification of the virulence genes illustrated that the minced meat showed the highest prevalence of STEC (8%) as compared to the other food products. In the humans, the O157 was the only serotype that expresses the Shiga toxin-associated gene (eaeA). Antibiotic susceptibility test displayed that 13 of the 17 food and human isolates (76.47%) were resistant to cephalothin (KF30). 9 of the 13 cephalothin-resistant isolates harbor the ? lactamase (blaTEM )-resistant gene. All isolates were sensitive to chloramphenicol, ciprofloxacin, amikacin, and gentamicin. DNA sequencing and phylogenetic analysis of the stx2-positive minced meat isolate revealed a high genetic relatedness with beef minced meat from the USA and Australia. Conclusion:This study showed the predominance of non-O157 among the identified isolates. Minced meat showed the highest prevalence of STEC as compared to the other food products, and this work illustrates the necessity to consider the food products as a potential source of the non-O157 STEC serotypes. DNA sequencing and phylogenetic analysis revealed a high genetic relatedness with beef minced meat from the USA and Australia. This highlights the high probability of worldwide spread of such serotypes, signifying the importance of the one world concept.
Project description:Gradually, conventional methods for foodborne pathogen typing are replaced by whole genome sequencing (WGS). Despite studies describing the overall benefits, National Reference Laboratories of smaller countries often show slower uptake of WGS, mainly because of significant investments required to generate and analyze data of a limited amount of samples. To facilitate this process and incite policy makers to support its implementation, a Shiga toxin-producing Escherichia coli (STEC) O157:H7 (stx1+, stx2+, eae+) outbreak (2012) and a STEC O157:H7 (stx2+, eae+) outbreak (2013) were retrospectively analyzed using WGS and compared with their conventional investigations. The corresponding results were obtained, with WGS delivering even more information, e.g., on virulence and antimicrobial resistance genotypes. Besides a universal, all-in-one workflow with less hands-on-time (five versus seven actual working days for WGS versus conventional), WGS-based cgMLST-typing demonstrated increased resolution. This enabled an accurate cluster definition, which remained unsolved for the 2013 outbreak, partly due to scarce epidemiological linking with the suspect source. Moreover, it allowed detecting two and one earlier circulating STEC O157:H7 (stx1+, stx2+, eae+) and STEC O157:H7 (stx2+, eae+) strains as closely related to the 2012 and 2013 outbreaks, respectively, which might have further directed epidemiological investigation initially. Although some bottlenecks concerning centralized data-sharing, sampling strategies, and perceived costs should be considered, we delivered a proof-of-concept that even in smaller countries, WGS offers benefits for outbreak investigation, if a sufficient budget is available to ensure its implementation in surveillance. Indeed, applying a database with background isolates is critical in interpreting isolate relationships to outbreaks, and leveraging the true benefit of WGS in outbreak investigation and/or prevention.
Project description:Out of 174 bovine Shiga toxin-producing Escherichia coli (STEC) strains isolated from diarrheic calves in Germany and Belgium, 122 strains (70.1%) were selected because of their reactivity with the eae (E. coli attaching and effacing gene) probe ECW1-ECW2. One hundred seven of these eae-positive strains (87.7%) harbored stx1 genes, 13 strains (10.7%) had stx2 genes, and 2 strains (1.6%) had both stx genes. The strains displayed 17 different O types, the majority (97 strains) [79.5%]) belonging to O5 (5 strains), O26 (21 strains), O111 (13 strains) O118 (36 strains), O145 (9 strains), and O157 (13 strains). In the HEp-2 cell adhesion assay, 99 strains (81.1%) showed a localized adhesion, and 80 strains (65.6%) stimulated actin accumulation, as determined in the fluorescence actin staining test. None of the strains harbored genes coding for bundle-forming pili (bfpA), clearly differentiating them from enteropathogenic. E. cole. espB gene sequences were only detectable in 23 (18.9%) of the eae-positive bovine STEC strains. Three different PCRs were established, differentiating between eae sequences of enteropathogenic E. coli strain E2348/69 (O127:H6) and STEC strain EDL933 (O157: H7). Primers matching in the more heterologous downstream eae sequences gave amplicons in only 8 of the 17 O types (O84:H-, O103:H2, O111:H-, O111:H2, O119:H25, O128:H-, O145:H28, and O157:H-). Only 15 STEC strains, belonging to serotypes O111H:-, O111H:2, O145:H28, and O157:H-, gave amplicons in all three eae-specific PCRs. These data demonstrate that bovine STEC strains are a heterogeneous group of pathogenic bacteria, a lot of which share virulence markers with STEC strains causing infections in humans. However, in contrast to human STEC strains, bovine eae-positive STEC strains are mainly restricted to the stx1 genotype. The observation that espB sequences are not highly conserved might have consequences for the serological recognition of the ESPB protein in patients. Like in human STEC strains, eae-related sequences are closely associated with certain E. coli O groups; however, they are not serotype specific.