A novel multiplex and glycoprotein-based immunochromatographic serologic IgM test for the rapid diagnosis of Escherichia coli O157 and O145 causing bloody diarrhea and hemolytic uremic syndrome.

Shiga toxin-producing Escherichia coli (STEC) are the main etiological agents of hemolytic uremic syndrome (HUS). Good clinical management of STEC infections and HUS depends on early, rapid, and accurate diagnosis. Here, we have developed and evaluated the first multiplex and glycoprotein-based immunochromatographic test for the detection of IgM antibodies against the O-polysaccharide of the lipopolysaccharide of E. coli O157 and O145 in human serum samples. A retrospective study was carried out resulting in a diagnostic sensitivity of the E. coli O157/O145 LFIA (lateral flow immunoassay) of 97.1% and 98.9% for O157 and O145, respectively, and 97.9% for both serogroups. The diagnostic specificity was 98.7% for O157 and O145, and the overall specificity 97.4%. In samples obtained before 3 days after the onset of diarrhea, the detection percentage was 83%, increasing to 100% from 3 days onward. Finally, the association of bloody diarrhea (BD) or HUS cases to an STEC infection increased from 22.8% to 77.2% when stool culture and stx/Stx detection were combined with serology by LFIA. Our results demonstrate that the E. coli O157/O145 LFIA is a highly accurate and serospecific test for the early and rapid diagnosis of E. coli O157 and O145 infections in BD or HUS cases. This test allows the detection of specific IgM antibodies very early in the course of the infection, making it an ideal diagnostic tool to be implemented in pediatric emergencies and, thus, avoid delays in the application of the correct supportive or specific treatment and prevent complications associated with HUS.

Etiological diagnosis of post-diarrheal hemolytic uremic syndrome (HUS): humoral response contribution.

BACKGROUND: Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolysis, thrombocytopenia, and thrombus formation leading to tissue injury. HUS is classified according to its etiology as post-diarrheal or atypical HUS. Differential diagnosis of both entities continues to be a challenge for pediatric physicians. METHODS: The aim was to improve the rapid etiological diagnosis of post-diarrheal HUS cases based on the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection by screening of stx1/stx2 and rfbO157 in cultured stools by multiplex PCR, and the additional detection of anti-lipopolysaccharide (anti-LPS) O157, O145, and O121 antibodies by Glyco-iELISA test. In addition, we studied patients' relatives to detect circulating pathogenic strains that could contribute to HUS diagnosis and/or lead to the implementation of measures to prevent dissemination of familial outbreaks. This study describes the diagnosis of 31 HUS patients admitted to Hospital Municipal de Niños Prof Dr Ramón Exeni during the 2017-2020 period. RESULTS: Stool PCR confirmed the diagnosis of STEC associated with HUS in 38.7% of patients (12/31), while anti-LPS serology did in 88.9% (24/27). In those patients in which both methods were carried out (n = 27), a strong association between the results obtained was found. We found that 30.4% of HUS patients had at least one relative positive for STEC. CONCLUSIONS: We could identify 96.3% (26/27) of HUS cases as secondary to STEC infections when both methods (genotyping and serology) were used. The results demonstrated a high circulation of STEC in HUS families and the prevalence of the STEC O157 serotype (83%) in our pediatric cohort. A higher-resolution version of the Graphical abstract is available as Supplementary information.

First molecular characterization of Escherichia coli O157:H7 isolates from clinical samples in Paraguay using whole-genome sequencing.

Escherichia coli O157:H7 is a foodborne pathogen implicated in numerous outbreaks worldwide that has the ability to cause extra-intestinal complications in humans. The Enteropathogens Division of the Central Public Health Laboratory (CPHL) in Paraguay is working to improve the genomic characterization of Shiga toxin-producing E. coli (STEC) to enhance laboratory-based surveillance and investigation of foodborne disease outbreaks. Whole genome sequencing (WGS) is proposed worldwide to be used in the routine laboratory as a high-resolution tool that allows to have all the results in a single workflow. This study aimed to carry out for the first time, the genomic characterization by WGS of nine STEC O157:H7 strains isolated from human samples in Paraguay. We were able to identify virulence and resistance mechanisms, MLST subtype, and even establish the phylogenetic relationships between isolates. Furthermore, we detected the presence of strains belonging to hypervirulent clade 8 in most of the isolates studied.

Surveillance of Shiga toxin-producing Escherichia coli associated bloody diarrhea in Argentina.

In Argentina, hemolytic uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli (STEC-HUS) infection is endemic, and reliable data about prevalence and risk factors have been available since 2000. However, information about STEC-associated bloody diarrhea (BD) is limited. A prospective study was performed during the period October 2018-June 2019 in seven tertiary-hospitals and 18 referral units from different regions, aiming to determine (i) the frequency of STEC-positive BD cases in 714 children aged 1-9 years of age and (ii) the rate of progression of bloody diarrhea to HUS. The number and regional distribution of STEC-HUS cases in the same hospitals and during the same period were also assessed. Twenty-nine (4.1%) of the BD patients were STEC-positive, as determined by the Shiga Toxin Quik Chek (STQC) test and/or the multiplex polymerase chain reaction (mPCR) assay. The highest frequencies were found in the Southern region (Neuquén, 8.7%; Bahía Blanca, 7.9%), in children between 12 and 23 month of age (8.8%), during summertime. Four (13.8%) cases progressed to HUS, three to nine days after diarrhea onset. Twenty-seven STEC-HUS in children under 5 years of age (77.8%) were enrolled, 51.9% were female; 44% were Stx-positive by STQC and all by mPCR. The most common serotypes were O157:H7 and O145:H28 and the prevalent genotypes, both among BD and HUS cases, were stx2a-only or -associated. Considering the endemic behavior of HUS and its high incidence, these data show that the rate of STEC-positive cases is low among BD patients. However, the early recognition of STEC-positive cases is important for patient monitoring and initiation of supportive treatment.

PulseNet International Survey on the Implementation of Whole Genome Sequencing in Low and Middle-Income Countries for Foodborne Disease Surveillance.

PulseNet International (PNI) is a global network of 88 countries who work together through their regional and national public health laboratories to track foodborne disease around the world. The vision of PNI is to implement globally standardized surveillance using whole genome sequencing (WGS) for real-time identification and subtyping of foodborne pathogens to strengthen preparedness and response and lower the burden of disease. Several countries in North America and Europe have experienced significant benefits in disease mitigation after implementing WGS. To broaden the routine use of WGS around the world, challenges and barriers must be overcome. We conducted this study to determine the challenges and barriers countries are encountering in their attempts to implement WGS and to identify how PNI can provide support to improve and become a better integrated system overall. A survey was designed with a set of qualitative questions to capture the status, challenges, barriers, and successes of countries in the implementation of WGS and was administered to laboratories in Africa, Asia-Pacific, Latin America and the Caribbean, and Middle East. One-third of respondents do not use WGS, and only 8% reported using WGS for routine, real-time surveillance. The main barriers for implementation of WGS were lack of funding, gaps in expertise, and training, especially for data analysis and interpretation. Features of an ideal system to facilitate implementation and global surveillance were identified as an all-in-one software that is free, accessible, standardized and validated. This survey highlights the minimal use of WGS for foodborne disease surveillance outside the United States, Canada, and Europe to date. Although funding remains a major barrier to WGS-based surveillance, critical gaps in expertise and availability of tools must be overcome. Opportunities to seek sustainable funding, provide training, and identify solutions for a globally standardized surveillance platform will accelerate implementation of WGS worldwide.

Economic evaluation of whole genome sequencing for pathogen identification and surveillance - results of case studies in Europe and the Americas 2016 to 2019.

BackgroundWhole genome sequencing (WGS) is increasingly used for pathogen identification and surveillance.AimWe evaluated costs and benefits of routine WGS through case studies at eight reference laboratories in Europe and the Americas which conduct pathogen surveillance for avian influenza (two laboratories), human influenza (one laboratory) and food-borne pathogens (five laboratories).MethodsThe evaluation focused on the institutional perspective, i.e. the 'investment case' for implementing WGS compared with conventional methods, based on costs and benefits during a defined reference period, mostly covering at least part of 2017. A break-even analysis estimated the number of cases of illness (for the example of Salmonella surveillance) that would need to be avoided through WGS in order to 'break even' on costs.ResultsOn a per-sample basis, WGS was between 1.2 and 4.3 times more expensive than routine conventional methods. However, WGS brought major benefits for pathogen identification and surveillance, substantially changing laboratory workflows, analytical processes and outbreaks detection and control. Between 0.2% and 1.1% (on average 0.7%) of reported salmonellosis cases would need to be prevented to break even with respect to the additional costs of WGS.ConclusionsEven at cost levels documented here, WGS provides a level of additional information that more than balances the additional costs if used effectively. The substantial cost differences for WGS between reference laboratories were due to economies of scale, degree of automation, sequencing technology used and institutional discounts for equipment and consumables, as well as the extent to which sequencers are used at full capacity.

Development and Evaluation of a Novel VHH-Based Immunocapture Assay for High-Sensitivity Detection of Shiga Toxin Type 2 (Stx2) in Stool Samples.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is the main cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening clinical complication characterized by hemolytic anemia, thrombocytopenia, and acute renal failure that mainly affects children. A relevant feature of STEC strains is the production of Stx, and all of them express Stx1 and/or Stx2 regardless of the strain serotype. Therefore, Stx detection assays are considered the most suitable methods for the early detection of STEC infections. Single-domain antibodies from camelids (VHHs) exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis. In this work, we have exploited VHH technology for the development of an immunocapture assay for Stx2 detection. Thirteen anti-Stx2 VHHs previously obtained from a variable-domain repertoire library were selected and evaluated in 130 capture-detection pair combinations for Stx detection. Based on this analysis, two VHHs were selected and a double VHH-based biotin-streptavidin capture enzyme-linked immunosorbent assay (ELISA) with spectrophotometric detection was developed and optimized for Stx2 detection. This assay showed an excellent analytical and clinical sensitivity in both STEC culture supernatants and stool samples even higher than the sensitivity of a commercial ELISA. Furthermore, based on the analysis of stool samples, the VHH-based ELISA showed high correlation with stx2 detection by PCR and a commercial rapid membrane-based immunoassay. The intrinsic properties of VHHs (high target affinity and specificity, stability, and ease of expression at high yields in recombinant bacteria) and their optimal performance for Stx detection make them attractive tools for the diagnosis of HUS related to STEC (STEC-HUS).

An Stx-EAEC O59:NM[H19] strain isolated from a hemolytic uremic syndrome case in Argentina.

Shiga toxin-producing Escherichia coli (STEC) are a heterogeneous group of foodborne pathogens causing a broad spectrum of human disease, from uncomplicated diarrhea to hemolytic uremic syndrome (HUS). In this study, we report an HUS case associated with an O59:NM[H19] strain, harboring stx2a, iha, lpfAO26, lpfAO113 genes associated with STEC, and aatA, aap, pic, sigA, agg4A genes associated with enteroaggregative E. coli (EAEC), named Stx-EAEC. The strain showed low toxicity on Vero cells, and was resistant to streptomycin and trimethoprim/sulfonamides. The child carried the bacteria for more than 100 days. Since the large outbreak associated with Stx-EAEC O104:H4, many strains with similar profiles have been described. In Germany, an O59:NM[H19] strain, with comparable characteristics to the Argentine strain, was isolated from a bloody diarrhea case. In Argentina, this is the first report of an HUS case associated with a Stx-EAEC infection, and represents a new challenge for the surveillance system.

Phylogeographic Analysis Reveals Multiple International transmission Events Have Driven the Global Emergence of Escherichia coli O157:H7.

BACKGROUND: Shiga toxin-producing Escherchia coli (STEC) O157:H7 is a zoonotic pathogen that causes numerous food and waterborne disease outbreaks. It is globally distributed, but its origin and the temporal sequence of its geographical spread are unknown. METHODS: We analyzed whole-genome sequencing data of 757 isolates from 4 continents, and performed a pan-genome analysis to identify the core genome and, from this, extracted single-nucleotide polymorphisms. A timed phylogeographic analysis was performed on a subset of the isolates to investigate its worldwide spread. RESULTS: The common ancestor of this set of isolates occurred around 1890 (1845-1925) and originated from the Netherlands. Phylogeographic analysis identified 34 major transmission events. The earliest were predominantly intercontinental, moving from Europe to Australia around 1937 (1909-1958), to the United States in 1941 (1921-1962), to Canada in 1960 (1943-1979), and from Australia to New Zealand in 1966 (1943-1982). This pre-dates the first reported human case of E. coli O157:H7, which was in 1975 from the United States. CONCLUSIONS: Inter- and intra-continental transmission events have resulted in the current international distribution of E. coli O157:H7, and it is likely that these events were facilitated by animal movements (eg, Holstein Friesian cattle). These findings will inform policy on action that is crucial to reduce the further spread of E. coli O157:H7 and other (emerging) STEC strains globally.

PulseNet Latin America and the Caribbean Network: Present and Future.

PulseNet Latin America and Caribbean (PNLAC) was established in 2003 and is one of seven Regional networks within PulseNet International. The main objectives of the network are to strengthen national and regional laboratory-based foodborne disease surveillance for early detection and investigation of outbreaks to setup control and prevention strategies in contribution to Public Health. Participants perform standardized pulsed-field gel electrophoresis (PFGE) protocols and analysis. For functioning, it is important for the network, the development of national and regional databases (RDBs) and the communication between countries, regionally and internationally. Metadata from over 8600 cases/outbreaks are profiled and isolated by PFGE and are incorporated into the RDB, hosted by the Pan American Health Organization. Currently PNLAC is moving toward whole-genome sequencing to use as a complementary strategy for surveillance. The aim of this article was to describe the experience of the construction of PNLAC, and its contribution to the surveillance of the foodborne diseases at the country and regional levels.

Frequency, characterization and genotypic analysis of Shiga toxin-producing Escherichia coli in beef slaughterhouses of Argentina.

The objectives of this study were: (1) to estimate STEC frequency in hide and carcass samples taken from beef slaughterhouses supplying the domestic market in Argentina, (2) to establish the pheno-genotypic characteristics of STEC and non-toxigenic Escherichia coli of serogroups O26, O45, O103, O121, O111, O145 or O157 isolated from the analyzed samples and, (3) to study their clonal relatedness. Sixty hides and 60 carcasses were analyzed. At the screening step, 48% of hide and 80% of carcass samples tested positive for the stx gene by endpoint PCR. The STEC isolation rate was 5% for hides and 8% for carcasses. The isolation rate of STEC-positive for O26, O45, O103, O111, O145 or O157 serogroups was 0% for hides and 2% for carcasses. With the purpose of studying the clonal relatedness of isolates, macrorestriction fragment analysis by pulsed-field gel electrophoresis was performed. The results indicated cross-contamination between hides and between carcasses of animals in the same lot and, that the origin of carcass contamination was their own hide, or the hides of other animals in the same lot. The high detection rate at the screening step, especially in carcasses, and the evidence of cross-contamination show the need to apply additional in-plant intervention strategies aimed at preventing carcass contamination.

PulseNet International: Vision for the implementation of whole genome sequencing (WGS) for global food-borne disease surveillance.

PulseNet International is a global network dedicated to laboratory-based surveillance for food-borne diseases. The network comprises the national and regional laboratory networks of Africa, Asia Pacific, Canada, Europe, Latin America and the Caribbean, the Middle East, and the United States. The PulseNet International vision is the standardised use of whole genome sequencing (WGS) to identify and subtype food-borne bacterial pathogens worldwide, replacing traditional methods to strengthen preparedness and response, reduce global social and economic disease burden, and save lives. To meet the needs of real-time surveillance, the PulseNet International network will standardise subtyping via WGS using whole genome multilocus sequence typing (wgMLST), which delivers sufficiently high resolution and epidemiological concordance, plus unambiguous nomenclature for the purposes of surveillance. Standardised protocols, validation studies, quality control programmes, database and nomenclature development, and training should support the implementation and decentralisation of WGS. Ideally, WGS data collected for surveillance purposes should be publicly available, in real time where possible, respecting data protection policies. WGS data are suitable for surveillance and outbreak purposes and for answering scientific questions pertaining to source attribution, antimicrobial resistance, transmission patterns, and virulence, which will further enable the protection and improvement of public health with respect to food-borne disease.

[Hygienic-sanitary quality in abattoirs from Tucuman province, Argentina. Detection, isolation and characterization of Shiga toxin-producing Escherichia coli]. / Calidad higiénico-sanitaria en plantas de faena de la provincia de Tucumán. Detección, aislamiento y caracterización de Escherichia coli productor de toxina Shiga.

Cattle are the main reservoir of Shiga toxin-producing Escherichia coli (STEC), and the strategies to prevent the transmission of these microorganisms are concentrated in the slaughtering plant. The aim of this study was to evaluate the hygienic-sanitary quality and the frequency of detection of STEC in beef carcasses in abattoirs from Tucuman province. Two hundred and seventy four beef carcass sponges were processed; the count of generic E. coli was marginal in 9 (3,3%) of them. Escherichia coli O157 was isolated in 4 (1,4%) samples; 2 of which were characterized as stx2c(vh-a)/eae/ehxA whereas the other 2 were non-toxigenic strains. Non-O157 E. coli ONT:H49, stx2a/ehxA/saa was isolated from 1 sample (0,4%). In this work the quality of the analyzed product indicates that the good practices of manufacture are fulfilled in slaughtering facilities in Tucumán province.

Serogroup-specific bacterial engineered glycoproteins as novel antigenic targets for diagnosis of shiga toxin-producing-escherichia coli-associated hemolytic-uremic syndrome.

Human infection with Shiga toxin-producing Escherichia coli (STEC) is a major cause of postdiarrheal hemolytic-uremic syndrome (HUS), a life-threatening condition characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. E. coli O157:H7 is the dominant STEC serotype associated with HUS worldwide, although non-O157 STEC serogroups can cause a similar disease. The detection of anti-O157 E. coli lipopolysaccharide (LPS) antibodies in combination with stool culture and detection of free fecal Shiga toxin considerably improves the diagnosis of STEC infections. In the present study, we exploited a bacterial glycoengineering technology to develop recombinant glycoproteins consisting of the O157, O145, or O121 polysaccharide attached to a carrier protein as serogroup-specific antigens for the serological diagnosis of STEC-associated HUS. Our results demonstrate that using these antigens in indirect ELISAs (glyco-iELISAs), it is possible to clearly discriminate between STEC O157-, O145-, and O121-infected patients and healthy children, as well as to confirm the diagnosis in HUS patients for whom the classical diagnostic procedures failed. Interestingly, a specific IgM response was detected in almost all the analyzed samples, indicating that it is possible to detect the infection in the early stages of the disease. Additionally, in all the culture-positive HUS patients, the serotype identified by glyco-iELISAs was in accordance with the serotype of the isolated strain, indicating that these antigens are valuable not only for diagnosing HUS caused by the O157, O145, and O121 serogroups but also for serotyping and guiding the subsequent steps to confirm diagnosis.

[Isolation and characterization of Escherichia coli O157 in bovine meat products and cattle in the province of Tucuman]. / Aislamiento y caracterización de Escherichia coli O157 en productos cárnicos bovinos y medias reses en la provincia de Tucumán.

Escherichia coli O157 is an emergent pathogen associated with diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. Meat products constitute an important transmission source of this microorganism. The aims of this study were to characterize E. coli O157 isolated from cattle and meat products collected from abattoirs and retail stores, to establish the clonal relatedness among regional isolates and to compare them with those in the national database. Between 2004 and 2013, 169 minced meat, 35 sausage and 216 carcass samples were analyzed. Thirteen E. coli O157 isolates were identified; 6 of which were O157:H7 and characterized as stx2c(vh-a)/eae/ehxA (n = 5) and stx2/eae/ehxA (n = 1). The 7 remaining isolates were non-toxigenic E. coli strains, and serotyped as O157:NT (n = 4), O157:NM (n = 1), O157:ND (n = 1) and O157:H16 (n = 1). The strains yielded different XbaI-PFGE patterns. Compared to the E. coli O157 isolates in the National Database, none of these patterns have been previously detected in strains of different origin in Argentina.

Genotypic characterization of Escherichia coli O157:H7 strains that cause diarrhea and hemolytic uremic syndrome in Neuquén, Argentina.

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens associated with cases of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). E. coli O157:H7 is the dominant serotype in Argentina and also in Neuquén Province, in which HUS incidence is above the national average, with a maximum of 28.6 cases per 100,000 children less than 5 years old reported in 1998. The aim of this study was to characterize a collection of 70 STEC O157 strains isolated from patients with diarrhea and HUS treated in the province of Neuquén, Argentina, between 1998 and 2011. All strains harbored eae, ehxA, rfbO157, and fliCH7 genes, and stx2a/stx2c (78.7%) was the predominant genotype. A total of 64 (91.4%) STEC O157 strains belonged to the hypervirulent clade 8 tested using both 4 and 32 SNP typing schemes. The strains showed the highest values reported in the literature for 6 of the 7 virulence determinants described in the TW14359 O157 strain associated with the raw spinach outbreak in the U.S. in 2006. Clade 8 strains were strongly associated with two of them: ECSP_3286, factor encoding an outer membrane protein that facilitates the transport of the heme complex (P=0.001), and in particular extracellular factor ECSP_2870/2872, coding proteins related to adaptation to plant hosts (P=0.000004). The q933 allele, which has been related to high toxin production, was present in 97.1% of the strains studied for the anti-terminator Q gene. In summary, this study describes, for the first time in Argentina, the almost exclusive circulation of strains belonging to the hypervirulent clade 8, and also the presence of putative virulence factors in higher frequencies than those reported worldwide. These data may help to understand the causes of the particular epidemiological situation related to HUS in Neuquén Province.

Rapid and accurate detection of Shiga toxin-producing Escherichia coli (STEC) serotype O157 : H7 by mass spectrometry directly from the isolate, using 10 potential biomarker peaks and machine learning predictive models.

Introduction. The different pathotypes of Escherichia coli can produce a large number of human diseases. Surveillance is complex since their differentiation is not easy. In particular, the detection of Shiga toxin-producing Escherichia coli (STEC) serotype O157 : H7 consists of stool culture of a diarrhoeal sample on enriched and/or selective media and identification of presumptive colonies and confirmation, which require a certain level of training and are time-consuming and expensive.Hypothesis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a quick and easy way to obtain the protein spectrum of a microorganism, identify the genus and species, and detect potential biomarker peaks of certain characteristics.Aim. To verify the usefulness of MALDI-TOF MS to rapidly identify and differentiate STEC O157 : H7 from other E. coli pathotypes.Methodology. The direct method was employed, and the information obtained using Microflex LT platform-based analysis from 60 clinical isolates (training set) was used to detect differences between the peptide fingerprints of STEC O157 : H7 and other E. coli strains. The protein profiles detected laid the foundations for the development and evaluation of machine learning predictive models in this study.Results. The detection of potential biomarkers in combination with machine learning predictive models in a new set of 142 samples, called 'test set', achieved 99.3 % (141/142) correct classification, allowing us to distinguish between the isolates of STEC O157 : H7 and the other E. coli group. Great similarity was also observed with respect to this last group and the Shigella species when applying the potential biomarkers algorithm, allowing differentiation from STEC O157 : H7Conclusion. Given that STEC O157 : H7 is the main causal agent of haemolytic uremic syndrome, and based on the performance values obtained in the present study (sensitivity=98.5 % and specificity=100.0 %), the implementation of this technique provides a proof of principle for MALDI-TOF MS and machine learning to identify biomarkers to rapidly screen or confirm STEC O157 : H7 versus other diarrhoeagenic E. coli in the future.

Phylogenetically related Argentinean and Australian Escherichia coli O157 isolates are distinguished by virulence clades and alternative Shiga toxin 1 and 2 prophages.

Shiga toxigenic Escherichia coli O157 is the leading cause of hemolytic uremic syndrome (HUS) worldwide. The frequencies of stx genotypes and the incidences of O157-related illness and HUS vary significantly between Argentina and Australia. Locus-specific polymorphism analysis revealed that lineage I/II (LI/II) E. coli O157 isolates were most prevalent in Argentina (90%) and Australia (88%). Argentinean LI/II isolates were shown to belong to clades 4 (28%) and 8 (72%), while Australian LI/II isolates were identified as clades 6 (15%), 7 (83%), and 8 (2%). Clade 8 was significantly associated with Shiga toxin bacteriophage insertion (SBI) type stx(2) (locus of insertion, argW) in Argentinean isolates (P < 0.0001). In Argentinean LI/II strains, stx(2) is carried by a prophage inserted at argW, whereas in Australian LI/II strains the argW locus is occupied by the novel stx(1) prophage. In both Argentinean and Australian LI/II strains, stx(2c) is almost exclusively carried by a prophage inserted at sbcB. However, alternative q(933)- or q(21)-related alleles were identified in the Australian stx(2c) prophage. Argentinean LI/II isolates were also distinguished from Australian isolates by the presence of the putative virulence determinant ECSP_3286 and the predominance of motile O157:H7 strains. Characteristics common to both Argentinean and Australian LI/II O157 strains included the presence of putative virulence determinants (ECSP_3620, ECSP_0242, ECSP_2687, ECSP_2870, and ECSP_2872) and the predominance of the tir255T allele. These data support further understanding of O157 phylogeny and may foster greater insight into the differential virulence of O157 lineages.

Subtyping of Escherichia coli O157:H7 strains isolated from human infections and healthy cattle in Argentina.

Shiga toxin-producing Escherichia coli (STEC) cause nonbloody (NBD) and bloody diarrhea (BD), and hemolytic uremic syndrome (HUS). Cattle have been described as their main reservoir. STEC O157:H7 is recognized as the predominant serotype in clinical infections, but much less is known about the dominant subtypes in humans and animals or their genetic relatedness. The aims of this study were to compare the STEC O157 subtypes found in sporadic human infections with those in the bovine reservoir using stx-genotyping, phage typing, and XbaI-pulsed-field gel electrophoresis (PFGE), and correlate the subtypes with the severity of clinical manifestations. The 280 STEC O157:H7 strains collected included in this study were isolated from HUS (n=122), BD (n=69), and NBD (n=30) cases, and healthy carriers (n=5), and from bovines (n=54) in the abattoirs. The stx-genotyping showed that stx2/stx(2c(vh-a)) was predominant in human (76.1%) and in bovine strains (55.5%), whereas the second more important genotype was stx2 (20.8%) in human and stx(2c(vh-a)) (16.7%) in cattle strains. In human strains, PT4 (37.6%), PT49 (24.3%), and PT2 (18.6%) were the most frequent PTs (80.5%). In bovine isolates, PT2 (26%), PT39 (16.7%), and PT4 and PT49 (11.1% each) were predominant. By XbaI-PFGE, all 280 strains yielded 148 patterns with 75% similarity, and 169 strains were grouped in 37 clusters. Identical PT-PFGE-stx profile combinations were detected in strains of both origins: PT4-AREXH01.0011-stx2/stx(2c(vh-a)) (12 humans and one bovine), PT4-AREXH01.0543-stx2/stx(2c(vh-a)) (one human and four bovines), PT2-AREXH01.0076-stx2/stx(2c(vh-a)) (one human and four bovines), PT49-AREXH01.0175-stx2/stx(2c(vh-a)) (seven humans and one bovine), and PT49-AREXH01.0022-stx2/stx(2c(vh-a)) (seven humans and one bovine). No correlation was found among the stx-genotypes, the phage type, and the clinical symptoms.