Development and Validation of the MAST ISOPLEX®VTEC Kit for Simultaneous Detection of Shiga Toxin/Verotoxin 1 and 2 (stx1/vt1 and stx2/vt2) with Inhibition Control (IC) in a Rapid Loop-Mediated Isothermal Amplification (LAMP) Multiplex Assay.

Loop-mediated isothermal amplification (LAMP) is a cost-effective, rapid, and highly specific method of replicating nucleic acids. Adding multiple targets into a single LAMP assay to create a multiplex format is highly desirable for clinical applications but has been challenging due to a need to develop specific detection techniques and strict primer design criteria. This study describes the evaluation of a rapid triplex LAMP assay, MAST ISOPLEX®VTEC, for the simultaneous detection of Shiga toxin/verotoxin 1 and 2 (stx1/vt1 and stx2/vt2) genes in verotoxigenic Escherichia coli (E. coli) (VTEC) isolates with inhibition control (IC) synthetic DNA using a single fluorophore-oligonucleotide probe, MAST ISOPLEX®Probes, integrated into the primer set of each target. MAST ISOPLEX®Probes used in the MAST ISOPLEX®VTEC kit produce fluorescent signals as they integrate with reaction products specific to each target, allowing tracking of multiple amplifications in real time using a real-time analyzer. Initial validation on DNA extracts from fecal cultures and synthetic DNA sequences (gBlocks) showed that the MAST ISOPLEX®VTEC kit provides a method for sensitive simultaneous triplex detection in a single assay with a limit of detection (LOD) of less than 100 target copies/assay and 96% and 100% sensitivity and specificity, respectively.

Spatiotemporal Dynamics of Sporadic Shiga Toxin-Producing Escherichia coli Enteritis, Ireland, 2013-2017.

The Republic of Ireland regularly reports the highest annual crude incidence rates of Shiga toxin-producing Escherichia coli (STEC) enteritis in the European Union, ≈10 times the average. We investigated spatiotemporal patterns of STEC enteritis in Ireland using multiple statistical tools. Overall, we georeferenced 2,755 cases of infection during January 2013-December 2017; we found >1 case notified in 2,340 (12.6%) of 18,641 Census Small Areas. We encountered the highest case numbers in children 0-5 years of age (n = 1,101, 39.6%) and associated with serogroups O26 (n = 800, 29%) and O157 (n = 638, 23.2%). Overall, we identified 17 space-time clusters, ranging from 2 (2014) to 5 (2017) clusters of sporadic infection per year; we detected recurrent clustering in 3 distinct geographic regions in the west and mid-west, all of which are primarily rural. Our findings can be used to enable targeted epidemiologic intervention and surveillance.

A high-throughput genomic screen identifies a role for the plasmid-borne type II secretion system of Escherichia coli O157:H7 (Sakai) in plant-microbe interactions.

Shiga-toxigenic Escherichia coli (STEC) is often transmitted into food via fresh produce plants, where it can cause disease. To identify early interaction factors for STEC on spinach, a high-throughput positive-selection system was used. A bacterial artificial chromosome (BAC) clone library for isolate Sakai was screened in four successive rounds of short-term (2 h) interaction with spinach roots, and enriched loci identified by microarray. A Bayesian hierarchical model produced 115 CDS credible candidates, comprising seven contiguous genomic regions. Of the two candidate regions selected for functional assessment, the pO157 plasmid-encoded type two secretion system (T2SS) promoted interactions, while a chaperone-usher fimbrial gene cluster (loc6) did not. The T2SS promoted bacterial binding to spinach and appeared to involve the EtpD secretin protein. Furthermore, the T2SS genes, etpD and etpC, were expressed at a plant-relevant temperature of 18 °C, and etpD was expressed in planta by E. coli Sakai on spinach plants.

The Locus of Heat Resistance Confers Resistance to Chlorine and Other Oxidizing Chemicals in Escherichia coli.

Some chlorine-resistant Escherichia coli isolates harbor the locus of heat resistance (LHR), a genomic island conferring heat resistance. In this study, the protective effect of the LHR for cells challenged by chlorine and oxidative stress was quantified. Cloning of the LHR protected against NaClO (32 mM; 5 min), H2O2 (120 mM; 5 min), and peroxyacetic acid (105 mg/liter; 5 min) but not against 5.8 mM KIO4, 10 mM acrolein, or 75 mg/liter allyl isothiocyanate. The lethality of oxidizing treatments for LHR-negative strains of E. coli was about 2 log10 CFU/ml higher than that for LHR-positive strains of E. coli The oxidation of cytoplasmic proteins and membrane lipids was quantified with the fusion probe roGFP2-Orp1 and the fluorescent probe BODIPY581/591, respectively. The fragment of the LHR coding for heat shock proteins protected cytoplasmic proteins but not membrane lipids against oxidation. The middle fragment of the LHR protected against the oxidation of membrane lipids but not of cytoplasmic proteins. The addition of H2O2, NaClO, and peroxyacetic acid also induced green fluorescent protein (GFP) expression in the oxidation-sensitive reporter strain E. coli O104:H4 Δstx2::gfp::amp Cloning of pLHR reduced phage induction in E. coli O104:H4 Δstx2::gfp::amp after treatment with oxidizing chemicals. Screening of 160 strains of Shiga toxin-producing E. coli (STEC) revealed that none of them harbors the LHR, additionally suggesting that the LHR and Stx prophages are mutually exclusive. Taking our findings together, the contribution of the LHR to resistance to chlorine and oxidative stress is based on the protection of multiple cellular targets by different proteins encoded by the genetic island.IMPORTANCE Chlorine treatments are used in water and wastewater sanitation; the resistance of Escherichia coli to chlorine is thus of concern to public health. We show that a genetic island termed the locus of heat resistance (LHR) protects E. coli not only against heat but also against chlorine and other oxidizing chemicals, adding to our knowledge of the tools used by E. coli to resist stress. Specific detection of the oxidation of different cellular targets in combination with the cloning of fragments of the LHR provided insight into mechanisms of protection and demonstrated that different fragments of the LHR protect different cellular targets. In E. coli, the presence of the LHR virtually always excluded other virulence factors. It is tempting to speculate that the LHR is maintained by strains of E. coli with an environmental lifestyle but is excluded by pathogenic strains that adapted to interact with vertebrate hosts.

Molecular subtyping and clonal relatedness of human and cattle verotoxin-producing Escherichia coli O157:H7 isolates.

Verotoxin-producing Escherichia coli O157:H7 is the dominant serotype isolated from patients with hemolytic-uremic syndrome (HUS) and, Argentina has the highest rate of HUS in the world. However, not all O157:H7 isolates have the same ability to infect and cause disease in humans. It has been postulated that O157:H7 strains integrate subpopulations related to the origin and virulence. In order to study the population structure and genetic diversity of VTEC O157:H7 from Argentina, a combination of molecular subtyping methods such as multiple loci VNTR analysis (MLVA), single nucleotide polymorphisms (SNP) and phylogroups assignment were used. According to MLVA, high genetic diversity was found among strains isolated from cattle, humans and food. On the other hand, 92% of the isolates presented the allele tir 255 T > A T and 95% were assigned to phylogroup E. We did not find a significant association between the isolates origin and the allele T presence (P > 0,05) postulated as significantly overrepresented in human isolates. Our results show that human and cattle VTEC O157:H7 isolates from Argentina are a homogeneous group and, although it presents high genetic diversity in relation to their MLVA and virulence profiles, it is not possible to distinguish divergent populations. The presence in all the strains of a high number of T3SS effectors genes and the no association of genetic subtypes with strain source, is an alert about the potential risk in public health that VTEC O157:H7 cattle strains possess and, at less, a partial explication about the high incidence of HUS in Argentina.

Antibody response to lipopolysaccharides and recombinant proteins of Shiga toxin (STX)-producing Escherichia coli (STEC) in children with haemolytic uraemic syndrome in Poland.

Typical haemolytic uraemic syndrome (STEC-HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), is a serious, life-threating disease that mainly affects children. Bacteriological and genetic tests are commonly used in the routine laboratory diagnosis of STEC-HUS; however, serological methods have emerged as useful and reliable diagnostic tools, especially when bacterial isolation fails. In this study, we present the results of the serological investigation of 72 paediatric patients suspected for HUS, hospitalized during 2011-2019 at the Department of Pediatrics and Nephrology of Children's Hospitals in Poland. During the routine laboratory investigation STEC strains were isolated only from nine stool samples. However, serological investigations confirmed 45 cases of STEC infections in children with HUS. In this study, 22 (48·9%) paediatric patients were infected by E. coli serotype O26, 11 (24·4%) by serotype O145, 9 (20·0%) by serotype O157, and 3 (6·7%) by E. coli serotype O111. In the majority of these patients, in addition to a high level of IgA, IgG and IgM antibodies to lipopolysaccharide of particular E. coli serotypes, antibodies to recombinant proteins Tir, Stx2b and intimin were detected. Our results confirm that serological tests are useful in the diagnosis of STEC-HUS. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that serological analysis greatly complements bacterial isolation and helps in the diagnosis and confirmation of Shiga toxin (verotoxin)-producing Escherichia coli (STEC) infections. Serological tests can be performed to qualify the patient for the typical haemolytic uraemic syndrome (STEC-HUS). In Poland, STEC-HUS in children is mostly caused by the E. coli serotype O26, which indicates that there is an increasing number of non-O157 STEC infections associated with human diseases in Europe.

Identification and molecular characteristics of verotoxin-producing Escherichia coli (VTEC) from bovine and pig carcasses isolated in Poland during 2014-2018.

The presence of verotoxin-producing Escherichia coli (VTEC) on bovine (n = 330) and pig (n = 120) carcasses in Poland was investigated using the ISO/TS 13136 standard. A total of 115 (34.8%) and 37 (30.8%) cattle and pig samples were positive in real-time PCR, respectively. Isolation of the bacteria revealed that from bovine carcasses 37 (32.2%) VTEC were obtained whereas only 5 (13.5%) pig carcasses were positive for the stx gene. The VTEC were characterized using whole genome sequencing (WGS) and bovine isolates were classified into 25 serotypes with the most prevalent O113:H21 (5 strains) whereas pig strains belonged to 5 different serotypes which were not identified among cattle strains. The majority of bovine VTEC (35; 94.6% isolates) were positive for the stx2 gene, either alone or together with the stx1 gene. All strains isolated from pig carcasses resulted positive for the stx2 gene only. Only two isolates of bovine origin contained the eaeA intimin gene, together with the ehxA and lpfA markers. VTEC were highly molecularly diverse as shown by classification into 29 different MLST STs. The obtained results suggest that further studies related to cattle and pig carcasses are needed to assess the role of these sources for human VTEC infections.

Do changes in STEC diagnostics mislead interpretation of disease surveillance data in Switzerland? Time trends in positivity, 2007 to 2016.

BackgroundLaboratory-confirmed cases of Shiga toxin-producing Escherichia coli (STEC) have been notifiable to the National Notification System for Infectious Diseases in Switzerland since 1999. Since 2015, a large increase in case numbers has been observed. Around the same time, syndromic multiplex PCR started to replace other diagnostic methods in standard laboratory practice for gastrointestinal pathogen testing, suggesting that the increase in notified cases is due to a change in test practices and numbers.AimThis study examined the impact of changes in diagnostic methods, in particular the introduction of multiplex PCR panels, on routine STEC surveillance data in Switzerland.MethodsWe analysed routine laboratory data from 11 laboratories, which reported 61.9% of all STEC cases from 2007 to 2016 to calculate the positivity, i.e. the rate of the number of positive STEC tests divided by the total number of tests performed.ResultsThe introduction of multiplex PCR had a strong impact on STEC test frequency and identified cases, with the number of tests performed increasing sevenfold from 2007 to 2016. Still, age- and sex-standardised positivity increased from 0.8% in 2007 to 1.7% in 2016.ConclusionIncreasing positivity suggests that the increase in case notifications cannot be attributed to an increase in test numbers alone. Therefore, we cannot exclude a real epidemiological trend for the observed increase. Modernising the notification system to address current gaps in information availability, e.g. diagnostic methods, and improved triangulation of clinical presentation, diagnostic and serotype information are needed to deal with emerging disease and technological advances.

[Parnaparin sodium - modern therapy options and prevention of venous thromboembolic complications]. / Parnaparin natriya ­ sovremennye vozmozhnosti terapii i profilaktiki venoznykh tromboembolicheskikh oslozhnenii.

Venous thromboembolic complications (VTEC) including pulmonary embolism and acute thrombosis of deep and superficial veins of the lower extremities are often observed in postoperative period. Low-molecular-weight heparin (LMWH) is a common remedy for prevention and treatment of VTEC due to high efficiency, safety, easy use and dosage. According to the modern literature data, LMWH is characterized by different effectiveness in relation to VTEC and risk of bleeding in patients after surgical and traumatological procedures, as well as in ones with severe forms of chronic venous diseases. However, their antithrombin activity varies significantly depending on mean molecular weight. The authors analyze LMWH action mechanism, pharmacokinetic and pharmacodynamic features of LMWH, in particular parnaparin sodium. Efficacy, safety and tolerability of this remedy for various forms of VTEC (superficial and deep vein thrombosis, thrombophlebitis, complicated forms of chronic venous diseases), its advantages for prevention of VTEC after various surgical and orthopedic interventions are considered.

[Prevention of thromboembolic complications in otorhinolaryngological surgery]. / Profilaktika tromboembolicheskikh oslozhnenii pri otorinolaringologicheskikh khirurgicheskikh vmeshatel'stvakh.

The article reviews current clinical guidelines and recent publications on the perioperative prophylaxis of venous thromboembolism (VTE) in ENT-HNS patients. The literature review is descriptive and includes foreign sources and national guidelines on diagnosis, treatment, and prevention of VTE. The average risk of VTE in otorhinolaryngology - head and neck surgery is lower than in the other surgical specialties, not reaching 1% in total. Major oncologic head and neck surgery carries higher risk of VTE. When prescribing anticoagulants after ENT-HNS surgery, one should predict and compare the risk of postoperative bleeding with the risk of VTE.

Evaluation of whole-genome sequencing for outbreak detection of Verotoxigenic Escherichia coli O157:H7 from the Canadian perspective.

BACKGROUND: Rapid and accurate identification of Verotoxigenic Escherichia coli (VTEC) O157:H7 is dependent on well-established, standardized and highly discriminatory typing methods. Currently, conventional subtyping tests for foodborne bacterial pathogen surveillance are rapidly being replaced with whole-genome sequencing (WGS) in public health laboratories. The capacity of WGS to revolutionize global foodborne disease surveillance has positioned this tool to become the new gold standard; however, to ensure evidence standards for public health decision making can still be achieved, the performance of WGS must be thoroughly validated against current gold standard methods prior to implementation. Here we aim to verify the performance of WGS in comparison to pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) for eight retrospective outbreaks of VTEC O157:H7 from the Canadian perspective. Since real-time implementation and routine use of WGS in public health laboratories is highly reliant on standardized data analysis tools, we also provide a comparative analysis of two popular methodologies for WGS analyses; an in-house developed single nucleotide variant phylogenomics (SNVPhyl) pipeline and the BioNumerics whole genome multilocus sequence typing (wgMLST) tool. To provide a useful and consistent starting point for examining laboratory-based surveillance data for VTEC O157:H7 in Canada, we also aim to describe the number of genetic differences observed among outbreak-associated isolates. RESULTS: WGS provided enhanced resolution over traditional subtyping methods, and accurately distinguished outbreak-related isolates from non-outbreak related isolates with high epidemiological concordance. WGS also illuminated potential linkages between sporadic cases of illness and contaminated food, and isolates spanning multiple years. The topologies generated by SNVPhyl and wgMLST were highly congruent with strong statistical support. Few genetic differences were observed among outbreak-related isolates (≤5 SNVs/ < 10 wgMLST alleles) unless the outbreak was suspected to be multi-strain. CONCLUSIONS: This study validates the superiority of WGS and indicates the BioNumerics wgMLST schema is suitable for surveillance and cluster detection of VTEC O157:H7. These findings will provide a useful and consistent starting point for examining WGS data for prospective laboratory-based surveillance of VTEC O157:H7, but however, the data will continue to be interpreted according to context and in combination with epidemiological and food safety evidence to inform public-health decision making in Canada.

A 10-year analysis of VTEC microbiological clearance times, in the under-six population of the Midlands, Ireland.

Verotoxin-producing Escherichia coli (VTEC) is a significant problem in the under-six population in the Midlands, Ireland. VTEC spreads by person-to-person transmission and children attending childcare facilities are excluded until they achieve two consecutive negative stool samples. This report analyses 10 years data on the number of days children under the age of six take to microbiologically clear VTEC. We identified from our data that the median clearance time for VTEC was 39 days, interquartile range (IQR) 27-56 days, maximum clearance time 283 days. At 70 days from onset of infection, 90% of children had cleared the infection. These findings were slightly more prolonged but consistent with international literature on VTEC clearance times for children. Asymptomatic children cleared VTEC infection significantly faster (median time 25 days IQR 13-43 days) than symptomatic children (median time 43 days IQR 31-58 days). Symptomatic children older than 1 year of age cleared VTEC infection significantly faster (median time 42 days IQR 31-57) than symptomatic children year under 1 year (median time 56 days IQR 35-74 days). This report identifies clear data which can be used to more accurately advise parents on time periods required to achieve microbiological clearance from VTEC.

Changing Diagnostic Methods and Increased Detection of Verotoxigenic Escherichia coli, Ireland.

The recent paradigm shift in infectious disease diagnosis from culture-based to molecular-based approaches is exemplified in the findings of a national study assessing the detection of verotoxigenic Escherichia coli infections in Ireland. The methodologic changes have been accompanied by a dramatic increase in detections of non-O157 verotoxigenic E. coli serotypes.

Whole-Genome Characterization and Strain Comparison of VT2f-Producing Escherichia coli Causing Hemolytic Uremic Syndrome.

Verotoxigenic Escherichia coli infections in humans cause disease ranging from uncomplicated intestinal illnesses to bloody diarrhea and systemic sequelae, such as hemolytic uremic syndrome (HUS). Previous research indicated that pigeons may be a reservoir for a population of verotoxigenic E. coli producing the VT2f variant. We used whole-genome sequencing to characterize a set of VT2f-producing E. coli strains from human patients with diarrhea or HUS and from healthy pigeons. We describe a phage conveying the vtx2f genes and provide evidence that the strains causing milder diarrheal disease may be transmitted to humans from pigeons. The strains causing HUS could derive from VT2f phage acquisition by E. coli strains with a virulence genes asset resembling that of typical HUS-associated verotoxigenic E. coli.

A longitudinal study of risk factors for shedding of VTEC O157 by young cattle in herds with known E. coli O157 carriage.

A longitudinal study in England and Wales of two dairy, five beef-fattener and three beef-suckler herds was carried out to identify risk factors for young cattle excreting verocytotoxin-producing Escherichia coli O157 (VTEC O157). A total of 1383 cattle, selected into cohorts at 0-24 months were sampled between March 2000 and February 2001. Mixed-effects logistic regression was employed to identify significant associations between VTEC O157 isolation from rectal faecal samples and explanatory factors (P < 0·001 unless shown). The results revealed a positive association with feeding root crops and a negative association with animals fed silage, milk (P = 0·001) or grain (P = 0·027). Cattle in suckler herds (P = 0·001) and those changing group between sampling visits were identified as negatively associated with VTEC O157 presence. The recovery of VTEC O157 varied throughout the year. However, the winter period from December to February was a risk factor in the multivariable analysis. Cattle in pens were 4·7 times more likely to shed VTEC O157 than those group-housed or at pasture. VTEC O157 detected in pooled environmental faecal pats and biofilm of the water supply within a group's enclosure were positively associated with an animal's VTEC O157 status in the multivariable logistic regression, as was detection of VTEC O157 in the pooled faecal pats at the previous visit.

Evaluation of molecular typing of foodborne pathogens in European reference laboratories from 2012 to 2013.

In 2012, the European Centre for Disease Prevention and Control (ECDC) initiated external quality assessment (EQA) schemes for molecular typing including the National Public Health Reference Laboratories in Europe. The overall aim for these EQA schemes was to enhance the European surveillance of food-borne pathogens by evaluating and improving the quality and comparability of molecular typing. The EQAs were organised by Statens Serum Institut (SSI) and included Salmonella enterica subsp. enterica, verocytotoxin-producing Escherichia coli (VTEC) and Listeria monocytogenes. Inter-laboratory comparable pulsed-field gel electrophoresis (PFGE) images were obtained from 10 of 17 of the participating laboratories for Listeria, 15 of 25 for Salmonella, but only nine of 20 for VTEC. Most problems were related to PFGE running conditions and/or incorrect use of image acquisition. Analysis of the gels was done in good accordance with the provided guidelines. Furthermore, we assessed the multilocus variable-number tandem repeat analysis (MLVA) scheme for S. Typhimurium. Of 15 laboratories, nine submitted correct results for all analysed strains, and four had difficulties with one strain only. In conclusion, both PFGE and MLVA are prone to variation in quality, and there is therefore a continuous need for standardisation and validation of laboratory performance for molecular typing methods of food-borne pathogens in the human public health sector.

Novel sequence types of non-O157 Shiga toxin-producing Escherichia coli isolated from cattle.

The objective of this study was to assess the genetic diversity of non-O157 Shiga toxin-producing Escherichia coli (STEC) isolates from cattle. Multi-locus sequence typing (MLST) was used to identify and compare the sequence types (STs) of 43 non-O157 STEC cattle isolates using the EcMLST database curated by the STEC Center at Michigan State University. For the 43 isolates, 19 STs were identified and 10 of those STs were novel compared to those in EcMLST. For the 43 isolates, 19 different serotypes were identified. STEC O22:H8, O174:H28 and O8:H19 were most common, and STEC O8 isolates were the most diverse, with seven different STs for isolates with that O group. STEC strains with O types identified in this study have been isolated from cattle by other researchers, as well as from cases of human gastroenteritis. Of the 10 novel STs identified, six were found to be closely related to previously identified STs, indicating that populations of non-O157 STEC in cattle are similar to those from other sources, including human clinical cases. Significance and impact of the study: The foodborne pathogen Shiga toxin-producing Escherichia coli (STEC) is a significant public health concern. One of the main reservoirs for STEC are cattle, which can directly or indirectly contribute to STEC in the food supply. The genetic subtype data presented here highlight the diversity of STEC that can be isolated from cattle. These results further our understanding of the ecology of STEC in the primary production environment, which is important for developing effective control measures to reduce this pathogen in the food supply.

Use of lactic acid with electron beam irradiation for control of Escherichia coli O157:H7, non-O157 VTEC E. coli, and Salmonella serovars on fresh and frozen beef.

Lactic acid pre-treatment was examined to enhance the antimicrobial action of electron (e-) beam irradiation of beef trim. Meat samples were inoculated with Escherichia coli O157:H7, non-O157 VTEC E. coli or Salmonella cocktails and treated with 5% lactic acid at 55 °C. Samples were packaged aerobically or vacuum-packed, kept at 4 °C and treated with 1 kGy e-beam energy. Frozen samples were treated with 1, 3 or 7 kGy and stored at -20 °C for ≤ 5 d. Lactic acid enhanced the antimicrobial action of 1 kGy e-beam treatment against Salmonella by causing an additional <1.8 log CFU/g reduction. One kGy treatment of refrigerated samples reduced VTEC E. coli viability by 4.5 log CFU/g, and while lactic acid did not improve the reduction, after freezing additive effects were found. After 3 kGy irradiation, Salmonella was reduced by 2 and 4 log CFU/g in the irradiated and lactic acid plus irradiated samples, respectively. Lactic acid pre-treatment was of limited value with 1 kGy treatment for improving control of toxigenic E. coli in fresh beef trim, however, it would be useful with low dose irradiation for controlling both VTEC E. coli and Salmonella in frozen product.

Comparison of two methods for the detection of verotoxin producing E.coli in human faecal samples during an outbreak of HUS in Apulia, Southern Italy.

This study evaluated the diagnostic performances of an ELISA method and a molecular method for the detection of verotoxin in faecal samples during an outbreak of haemolytic-uraemic syndrome (HUS) occurring in Apulia, Southern Italy. Two of the 16 faecal samples were positive for verotoxin when analysed by ELISA and resulted PCR positive for stx1, stx2, eaeA and serogroup O26. The other 14 faecal samples resulted negative with both tests. The detection of verotoxin in faecal samples by ELISA is a simple, sensitive, specific and rapid method (2 hours) of considerable utility for routine clinical testing laboratories without access to more specialized diagnostic procedures.

Identification of two allelic variants of toxB gene and investigation of their distribution among Verocytotoxin-producing Escherichia coli.

Verocytotoxin-producing Escherichia coli (VTEC) are food borne pathogens causing severe human infections. The virulence genes asset of VTEC is complex and has not been completely defined yet. Nonetheless, all the virulence genes described so far have been described as conveyed by mobile genetic elements. A gene, termed toxB, has been identified in a large virulence plasmid of VTEC O157, later described in similar plasmids carried by VTEC O26 and O145. In this study we identified for the first time an intact copy of toxB gene in a plasmid present in a VTEC O111 strain and observed the existence of two allelic variants of the gene, that we termed toxB1 and toxB2. We investigated the distribution of the two alleles in a panel of VTEC strains belonging to different serogroups and demonstrated that this gene is present only in VTEC serogroups associated with the most severe forms of the infections such as those belonging to the five serogroups O157, O26, O111, O103 and O145 and that the two alleles segregate with the serogroup of the hosting strains. In particular the toxB1 variant was only present in VTEC O157 while the toxB2 allele was present in the remaining four VTEC serogroups.