Epidemiological Characteristics of Shiga Toxin-Producing Escherichia coli Responsible for Infections in the Polish Pediatric Population.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in children and the elderly. Stool samples were collected from 180 children hospitalized in five pediatric centers in Poland in 2018-2022. Direct stx1/stx2 gene detection by PCR in feces and E. coli isolates was performed. Antibiotic susceptibility was tested according to EUCAST v.12. Randomly selected isolates were serotyped with O157 antiserum and genotyped by pulsed-field gel electrophoresis (PFGE). A total of 44 E. coli isolates were confirmed as STEC by PCR. Among them, 84.4% were positive for stx2, and equally 6,8% for only stx1 and both stx1 and stx2 genes. The stx1 gene was also found in one Citrobacter freundii isolate. E. coli serotype O157 was present in 97.6% of the isolates. STEC infections most often occurred between June-October with a peak in July and August (51%). The highest, 77.8% of STEC isolates were found in the 1-5 years old group. No extended-spectrum ß-lactamases (ESBL) were found. Resistance only to amoxicillin/clavulanic acid (24.4%), piperacillin/tazobactam (3%), cefotaxime (6%), gentamicin (6%), ciprofloxacin (3%), azithromycin (3%), trimethoprim/sulfamethoxazole (24,2%) was detected. PFGE analysis showed 18 PFGE types with no clonal distribution. Eight isolates with A, B, and C PFGE types showed genetic relatedness in the type with no detection of transmission way of distribution. STEC strains pose a serious threat to human health, therefore demographic and epidemiological characteristics are crucial for their surveillance.

Real-time PCR primers and probes for the detection of Shiga toxin genes, including novel subtypes.

Shiga toxin-producing Escherichia coli (STEC) are foodborne enteric pathogens. STEC are differentiated from other E. coli by detection of Shiga toxin (Stx) or its gene (stx). The established nomenclature of Stx identifies ten subtypes (Stx1a, Stx1c, Stxd, Stx2a to Stx2g). An additional nine subtypes have been reported and described (Stx1e, Stx2h to Stx2o). Many PCR protocols only detect a subset of Stx subtypes which limits their inclusivity. Here we describe a real-time PCR assay inclusive of the DNA sequences of representatives of all currently described Stx subtypes. A multiplex real-time PCR assay for detection of stx was developed using nine primers and four probes. Since the identification of STEC does not require differentiation of stx subtypes, the probes use the same fluorescent reporter to enable detection of multiple possible targets in a single reaction. The PCR mixture includes an internal positive control to detect inhibition of the reaction. Thus, the protocol can be performed on a two-channel real-time PCR platform. To reduce the biosafety risk inherent in the use of STEC cultures as process controls, the protocol also includes the option of a non-pathogenic E. coli transformant carrying a plasmid encoding the targeted fragment of the stx2a sequence. The inclusivity of the PCR was assessed against colonies of 137 STEC strains and one strain of Shigella dysenteriae, including strains carrying single copies of stx representing fourteen subtypes (stx1 a, c, d; stx2 a-j and o). Five additional subtypes (stx1e, 2k, 2l, 2m and 2n) were represented by E. coli transformed with plasmids encoding toxoid (enzymatically inactive A subunit) sequences. The exclusivity panel consisted of 70 bacteria, including 21 stx-negative E. coli. Suitability for food analysis was assessed with artificially inoculated ground beef, spinach, cheese, and apple cider. The real-time PCR generated positive results for all 19 stx subtypes, represented by colonies of STEC, S. dysenteriae and E. coli transformants carrying stx toxoid plasmids. Tests of exclusivity panel colonies were all negative. The real-time PCR detected the presence of stx in all inoculated food enrichments tested, and the presence of STEC was confirmed by isolation.

A challenging STEC strain isolation from patients' stools: an O166:H15 STEC strain with the stx2 gene.

Two patients with acute gastroenteritis tested positive for Shiga toxin-producing Escherichia coli (STEC) by polymerase chain reaction (PCR), and both strains carried the Shiga toxin 2 encoding gene. Since routine culture using CHROMagar STEC failed to recover these isolates, immunomagnetic separation (IMS) targeting the top six non-O157:H7 serotypes was used for isolate recovery. After two subsequent IMS runs, the STEC strains were isolated from trypticase soy broth with and without overnight enrichment for runs 1 and 2, respectively. Serotyping based on whole-genome sequencing revealed that both patients carried the strain O166:H15 STEC with the stx2 gene. Hence, the magnetic beads used in IMS appeared to have cross-reactivity with other E. coli serotypes. When the STEC isolates from both stools were cultured on CHROMagar STEC and sheep blood agar (BAP), two distinct colony sizes were apparent after overnight incubation. The small and large colonies were picked and separately cultured on both media, and colony growth was observed for 2 weeks at room temperature after an initial overnight incubation at 37°C. After 1 week, the colonies showed concentric ring structures with a darker center and a lighter surrounding on CHROMagar STEC and a "fried egg"-resembling structure with a raised circular center and a flat surrounding on BAP. Both colony types remained morphologically different on CHROMagar STEC throughout the 15 days. However, on BAP, their appearance was comparable by day 7. IMPORTANCE: Shiga toxin-producing E. coli (STEC) infections can lead to severe complications such as bloody diarrhea and hemolytic uremic syndrome (HUS), especially in young children and the elderly. Strains that carry the shiga toxin 2 gene (stx2), such as O157:H7, have been mostly linked with severe disease outcomes. In recent years, outbreaks caused by non-O157:H7 strains have increased. E. coli O166:H15 has been previously reported causing a gastroenteritis outbreak in 1996 as a non-STEC strain, however the O166:H15 serotype we recovered carried the stx2 gene. It was particularly challenging to isolate this strain from stools by culture. Consequently, we tested immunomagnetic separation for the STEC recovery, which was a novel approach on clinical stools. Virulence genes were included for the characterization of these isolates.

First Isolation of the Heteropathotype Shiga Toxin-Producing and Extra-Intestinal Pathogenic (STEC-ExPEC) E. coli O80:H2 in French Healthy Cattle: Genomic Characterization and Phylogenetic Position.

The emerging heteropathotype shigatoxigenic (STEC) and extra-intestinal pathogenic Escherichia coli (ExPEC) O80:H2 has been the second leading cause of pediatric HUS in France since the mid-2010s. In contrast with other highly pathogenic STEC serotypes, for which ruminants have clearly been identified as the main human infection source, this heteropathotype's reservoir remains unknown. In this context, we describe for the first time the isolation of seven STEC O80:H2 strains from healthy cattle on a single cattle farm in France. This study aimed at (i) characterizing the genome and (ii) investigating the phylogenetic positions of these O80:H2 STEC strains. The virulomes, resistomes, and phylogenetic positions of the seven bovine isolates were investigated using in silico typing tools, antimicrobial susceptibility testing and cgMLST analysis after short-read whole genome sequencing (WGS). One representative isolate (A13P112V1) was also subjected to long-read sequencing. The seven isolates possessed ExPEC-related virulence genes on a pR444_A-like mosaic plasmid, previously described in strain RDEx444 and known to confer multi-drug resistance. All isolates were clonally related and clustered with human clinical strains from France and Switzerland with a range of locus differences of only one to five. In conclusion, our findings suggest that healthy cattle in France could potentially act as a reservoir of the STEC-ExPEC O80:H2 pathotype.

Comparison of Two Shiga Toxin-producing Escherichia coli (STEC) Isolation Protocols in Raw Cow's Milk Cheese Enrichment Broths: Direct STEC Isolation Versus Techniques Based on Immuno-concentration.

The presence of Shiga toxin-producing Escherichia coli (STEC) in dairy products made with raw milk is a major concern for food safety authorities and industries. Two approaches have been proposed to isolate STEC from food. In the IC-Protocol (immuno-concentration protocol), specific serogroups are identified in the enrichment broth after the detection of the stx and eae genes. An immuno-concentration of the targeted serogroups is performed before isolating them on specific media. In the DI-Protocol (direct isolation protocol), a direct isolation of all STEC present in the enrichment broth is carried out after the detection of stx genes. We compared the ability of these two methods to isolate STEC O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 after artificial inoculation in four different raw milk cheeses. Across all serogroups and cheese types, STEC were isolated in 83.3% of samples when using the IC-Protocol but only 53.3% of samples with the DI-Protocol. For two cheese types, the DI-Protocol failed to isolate STEC O157:H7 strains altogether. Our results suggest that IC-Protocol is a robust methodology to effectively isolate STEC across a range of cheese types.

Characterization of Atypical Shiga Toxin Gene Sequences and Description of Stx2j, a New Subtype.

Shiga toxin (Stx) is the definitive virulence factor of Shiga toxin-producing Escherichia coli (STEC). Stx variants are currently organized into a taxonomic system of three Stx1 (a, c, and d) and seven Stx2 (a, b, c, d, e, f, and g) subtypes. In this study, seven STEC isolates from food and clinical samples possessing stx2 sequences that do not fit current Shiga toxin taxonomy were identified. Genome assemblies of the STEC strains were created from Oxford Nanopore and Illumina sequence data. The presence of atypical stx2 sequences was confirmed by Sanger sequencing, as were Stx2 expression and cytotoxicity. A strain of O157:H7 was found to possess stx1a and a truncated stx2a, which were originally misidentified as an atypical stx2. Two strains possessed unreported variants of Stx2a (O8:H28) and Stx2b (O146:H21). In four of the strains, we found three Stx subtypes that are not included in the current taxonomy. Stx2h (O170:H18) was identified in a Canadian sprout isolate; this subtype has only previously been reported in STEC from Tibetan Marmots. Stx2o (O85:H1) was identified in a clinical isolate. Finally, Stx2j (O158:H23 and O33:H14) was found in lettuce and clinical isolates. The results of this study expand the number of known Stx subtypes, the range of STEC serotypes, and isolation sources in which they may be found. The presence of the Stx2j and Stx2o in clinical isolates of STEC indicates that strains carrying these variants are potential human pathogens.

Large-Scale Phylogenetic Analysis Reveals a New Genetic Clade among Escherichia coli O26 Strains.

Shiga toxin-producing Escherichia coli (STEC) O26 is the predominant non-O157 serogroup causing hemolytic uremic syndrome worldwide. Moreover, the serogroup is highly dynamic and harbors several pathogenic clones. Here, we investigated the phylogenetic relationship of STEC O26 at a global level based on 1,367 strains from 20 countries deposited in NCBI and Enterobase databases. The whole-genome-based analysis identified a new genetic clade, called ST29C4. The new clade was unique in terms of multilocus sequence type (ST29), CRISPR (group Ia), and dominant plasmid gene profile (ehxA+/katP-/espP-/etpD-). Moreover, the combination of multiple typing methods (core genome single nucleotide polymorphism [SNP] typing, CRISPR typing, and virulence genes analysis) demonstrated that this new lineage ST29C4 was in the intermediate phylogenetic position between ST29C3 and other non-ST29C3 strains. Besides, we observed that ST29C4 harbored extraintestinal pathogenic E. coli (ExPEC)-related virulence gene (VG), tsh, and STEC-associated VG, stx2a, suggesting the emergence of a hybrid pathogen. The ST29C4 strains also exhibited high similarity in stx2a-prophage and integrase with the O104:H4 strain, further demonstrating its potential risk to human health. Collectively, the large-scale phylogenetic analysis extends the understanding of the clonal structure of O26 strains and provides new insights for O26 strain microevolution. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) O26 is the second prevalent STEC serogroup only to O157, which can cause a series of diseases ranging from mild diarrhea to life-threatening hemolytic uremic syndrome (HUS). The serogroup is highly diverse and multiple clones are characterized, including ST29C1-C3 and ST21C1-C2. However, the phylogenetic relationship of these clones remains fully unclear. In this study, we revealed a new genetic clade among O26 strains, ST29C4, which was unique in terms of CRISPR, multilocus sequence type (MLST), and plasmid gene profile (PGP). Moreover, the combination of multiple typing methods demonstrated that this new clone was located in the intermediate phylogenetic position between ST29C3 and other non-ST29C3 strains (i.e., ST29C1-C2 and ST21C1-C2). Overall, the large-scale phylogenetic analysis extends our current understanding of O26 microevolution.

Prevalence of genotypic antimicrobial resistance in clinical Shiga toxin-producing Escherichia coli in Norway, 2018 to 2020.

Introduction. Shiga toxin-producing Escherichia coli (STEC) can cause severe to fatal disease in humans. Antimicrobial treatment is sometimes necessary, but contraindicated due to undesirable clinical outcome. However, recent studies have shown promising outcomes following antimicrobial treatment. Before the establishment of a possible antimicrobial treatment strategy for STEC infections, the prevalence of antimicrobial resistance in STEC needs to be determined.Gap Statement. The resistance status of Norwegian clinical STEC is not known and should be assessed.Aim. We aim to characterize genotypic antimicrobial resistance determinants in clinical STEC in Norway, and determine the prevalence of genotypic resistance in order to inform possible antimicrobial treatment options for STEC infections.Methodology. We included all clinical STEC submitted to the Norwegian Reference Laboratory from March 2018 to April 2020. All samples were whole-genome sequenced and screened for genotypic antimicrobial resistance,virulence determinants and plasmid incompatibility groups. We performed phylogenetic clustering of STEC by core-genome multi-locus sequence typing, and statistical association analyses between isolate characteristics and genotypic resistance.Results. A total of 459 STEC were analysed. For 385 (83.9 %) STEC we did not identify any antimicrobial resistance determinants. Seventy-four STEC (16.1 %) harboured antimicrobial resistance determinants against one or more antimicrobial classes. The most frequent genotypic resistance was identified against aminoglycosides (10.5 %). Thirty-nine STEC (8.5 %) had a multi-drug resistance (MDR) genotype. Genotypic resistance was more prevalent in non-O157 than O157 STEC (P=0.02). A positive association was seen between genotypic resistance and the low-virulent STEC O117:H7 phylogenetic cluster (no. 14) (P<0.001). Genotypic resistance was not significantly associated to high-virulent STEC. STEC O146:H28 and isolates harbouring the plasmid replicon type IncQ1 were positively associated with MDR.Conclusion. The overall prevalence of genotypic resistance in clinical STEC in Norway is low (16.1 %). Genotypic resistance is more prevalent in non-O157 strains compared to O157 strains, and not significantly associated to high-virulent STEC. Resistance to antimicrobials suggested for treatment, especially azithromycin is low and may present an empiric treatment alternative for severe STEC infections.

The global population structure and evolutionary history of the acquisition of major virulence factor-encoding genetic elements in Shiga toxin-producing Escherichia coli O121:H19.

Shiga toxin (Stx)-producing Escherichia coli (STEC) are foodborne pathogens causing serious diseases, such as haemorrhagic colitis and haemolytic uraemic syndrome. Although O157:H7 STEC strains have been the most prevalent, incidences of STEC infections by several other serotypes have recently increased. O121:H19 STEC is one of these major non-O157 STECs, but systematic whole genome sequence (WGS) analyses have not yet been conducted on this STEC. Here, we performed a global WGS analysis of 638 O121:H19 strains, including 143 sequenced in this study, and a detailed comparison of 11 complete genomes, including four obtained in this study. By serotype-wide WGS analysis, we found that O121:H19 strains were divided into four lineages, including major and second major lineages (named L1 and L3, respectively), and that the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS) was acquired by the common ancestor of O121:H19. Analyses of 11 complete genomes belonging to L1 or L3 revealed remarkable interlineage differences in the prophage pool and prophage-encoded T3SS effector repertoire, independent acquisition of virulence plasmids by the two lineages, and high conservation in the prophage repertoire, including that for Stx2a phages in lineage L1. Further sequence determination of complete Stx2a phage genomes of 49 strains confirmed that Stx2a phages in lineage L1 are highly conserved short-tailed phages, while those in lineage L3 are long-tailed lambda-like phages with notable genomic diversity, suggesting that an Stx2a phage was acquired by the common ancestor of L1 and has been stably maintained. Consistent with these genomic features of Stx2a phages, most lineage L1 strains produced much higher levels of Stx2a than lineage L3 strains. Altogether, this study provides a global phylogenetic overview of O121:H19 STEC and shows the interlineage genomic differences and the highly conserved genomic features of the major lineage within this serotype of STEC.

Emergence of New ST301 Shiga Toxin-Producing Escherichia coli Clones Harboring Extra-Intestinal Virulence Traits in Europe.

O80:H2 enterohemorrhagic Escherichia coli (EHEC) of sequence type ST301 is one of the main serotypes causing European hemolytic and uremic syndrome, but also invasive infections, due to extra-intestinal virulence factors (VFs). Here, we determined whether other such heteropathotypes exist among ST301. EnteroBase was screened for ST301 strains that were included in a general SNP-phylogeny. French strains belonging to a new heteropathotype clone were sequenced. ST, hierarchical clusters (HC), serotype, resistome, and virulome were determined using EnteroBase, the CGE website, and local BLAST. The ST301 general phylogeny shows two groups. Group A (n = 25) is mainly composed of enteropathogenic E. coli, whereas group B (n = 55) includes mostly EHEC. Three serotypes, O186:H2, O45:H2 and O55:H9, share the same virulome as one of the O80:H2 sub-clones from which they derive subsequent O-antigen switches. The O55:H9 clone, mainly present in France (n = 29), as well as in the UK (n = 5) and Germany (n = 1), has a low background of genetic diversity (four HC20), although it has three Stx subtypes, an H-antigen switch, and genes encoding the major extra-intestinal VF yersiniabactin, and extended-spectrum beta-lactamases. Diverse heteropathotype clones genetically close to the O80:H2 clone are present among the ST301, requiring close European monitoring, especially the virulent O55:H9 clone.

Genetic and antimicrobial resistance profiles of non-O157 Shiga toxin-producing Escherichia coli from different sources in Egypt.

BACKGROUND: The Shiga toxin-producing Escherichia coli (STEC) represented a great risk to public health. In this study, 60 STEC strains recovered from broiler and duck fecal samples, cow's milk, cattle beef, human urine, and ear discharge were screened for 12 virulence genes, phenotypic and genotypic antimicrobial resistance, and multiple-locus variable-number tandem-repeat analysis (MLVA). RESULTS: The majority of strains harbored Shiga toxin 1 (stx1) and stx1d, stx2 and stx2e, and ehxA genes, while a minority harbored stx2c subtype and eaeA. We identified 10 stx gene combinations; most of strains 31/60 (51.7%) exhibited four copies of stx genes, namely the stx1, stx1d, stx2, and stx2e, and the strains exhibited a high range of multiple antimicrobial resistance indices. The resistance genes blaCTX-M-1 and blaTEM were detected. For the oxytetracycline resistance genes, most of strains contained tetA, tetB, tetE, and tetG while the tetC was present at low frequency. MLVA genotyping resolved 26 unique genotypes; genotype 21 was highly prevalent. The six highly discriminatory loci DI = 0.9138 are suitable for the preliminary genotyping of STEC from animals and humans. CONCLUSIONS: The STEC isolated from animals are virulent, resistant to antimicrobials, and genetically diverse, thus demands greater attention for the potential risk to human.

Molecular surveillance of shiga toxigenic Escherichia coli in selected beef abattoirs in Osun State Nigeria.

Shiga toxigenic strains of E. coli (STEC) known to be etiological agents for diarrhea were screened for their incidence/occurrence in selected abattoirs sources in Osogbo metropolis of Osun State, Nigeria using a randomized block design. Samples were plated directly on selective and differential media and E. coli isolates. Multiplex PCR analysis was used to screen for the presence of specific virulence factors. These were confirmed serologically as non-O157 STEC using latex agglutination serotyping kit. Sequence analysis of PCR products was performed on a representative isolate showing the highest combination of virulence genes using the 16S gene for identification purposes only. Results showed that the average cfu/cm2 was significantly lower in the samples collected at Sekona-2 slaughter slab compared with those collected at Al-maleek batch abattoir and Sekona-1 slaughter slab in ascending order at P = 0.03. Moreover, the average cfu/cm2 E. coli in samples collected from butchering knife was significantly lower when compared with that of the workers' hand (P = 0.047) and slaughtering floor (P = 0.047) but not with the slaughter table (P = 0.98) and effluent water from the abattoir house (P = 0.39). These data suggest that the abattoir type may not be as important in the prevalence and spread of STEC as the hygiene practices of the workers. Sequence analysis of a representative isolate showed 100% coverage and 96.46% percentage identity with Escherichia coli O113:H21 (GenBank Accession number: CP031892.1) strain from Canada. This sequence was subsequently submitted to GenBank with accession number MW463885. From evolutionary analyses, the strain from Nigeria, sequenced in this study, is evolutionarily distant when compared with the publicly available sequences from Nigeria. Although no case of E. coli O157 was found within the study area, percent occurrence of non-O157 STEC as high as 46.3% at some of the sampled sites is worrisome and requires regulatory interventions in ensuring hygienic practices at the abattoirs within the study area.

Detection of novel sequence types and zoonotic transmission potentiality among strains of Shiga toxigenic Escherichia coli (STEC) from dairy calves, animal handlers and associated environments.

Shiga toxigenic Escherichia coli (STEC) is one of the most important food-borne zoonotic bacterial pathogens responsible for causing gastrointestinal infections, haemorrhagic colitis and haemolytic uremic syndrome. The present study was aimed to isolate and characterize STEC from neonatal dairy calves, animal handlers and their surrounding environment and to establish the genetic relationship among isolates by multilocus sequence typing (MLST). A total number of 115 samples were collected and processed for the isolation of E. coli. The occurrence rate of E. coli was 92.2% (106/115), of which, 18 were typed as STEC. Antibacterial susceptibility analysis revealed 11 (61.1%) strains as multiple drug-resistant (MDR). MLST analysis has delineated 16 sequence types (STs) including nine novel STs. Among STs, ST58 dominated with three strains and was recovered from the environment and neonatal calves. Strains from neonatal calves and humans showed genetic relatedness with significant bootstrap support values indicative of zoonotic transmission potentiality. Analysis of 211 global isolates belonging to 61 STs indicated predominant STs (ST 21, ST 33 and ST 3416) that can be either host-specific (ST 33 and ST 3416) or can be shared among human and bovine hosts (ST 21). The MLST analysis indicates genetic relatedness among isolates and the results predispose inter-host transmission and zoonotic spread.

The emerging importance of Shiga toxin-producing Escherichia coli other than serogroup O157 in England.

Introduction. Shiga toxin-producing Escherichia coli (STEC) can cause severe disease and large outbreaks. In England, the incidence and clinical significance of STEC serogroups other than O157 (non-O157) is unknown due to a testing bias for detection of STEC O157. Since 2013, the implementation of PCR to detect all STEC serogroups by an increasing number of diagnostic laboratories has led to an increase in the detection of non-O157 STEC.Hypothesis/Gap statement. Due to a bias in testing methodologies to select for STEC serogroup O157 in frontline diagnostic laboratories in most countries, very little surveillance data have been previously generated on non-O157 STEC.Aim. Five years (2014-2018) of STEC national surveillance data were extracted and descriptive analysis undertaken to assess disease severity of non-O157 STEC strains.Methods. Data from 1 January 2014 to 31 December 2018 were extracted from the National Enhanced Surveillance System for STEC and analysed.Results. The implementation of Gastrointestinal Polymerase Chain Reaction (GI-PCR) has resulted in a four-fold increase in the detection of non-O157 STEC cases between 2014 and 2018. There were 2579 cases infected with 97 different non-O157 serogroups. The gender distribution was similar amongst STEC O157 and non-O157 STEC cases with 57 and 56 % of cases being female respectively, but a significantly higher proportion of cases (P <0.001) under 5 years of age was observed among STEC O157 (22 %) cases compared to non-O157 STEC (14 %). The most common non-O157 serogroups were O26 (16 %), O146 (11 %), O91 (10 %), O128 (7 %), O103 (5 %) and O117 (3 %). Overall, rates of bloody diarrhoea were highest in O26 (44 %) and O103 (48 %) cases and lowest in STEC O117 cases (17 %). Strains harbouring Shiga toxin stx1a caused the highest proportion of diarrhoea (93 %) and caused the same level of bloody diarrhoea as stx2a (39 %). However, stx2a caused the highest proportion of vomiting (46 %), hospitalisation (49 %) and considerably more HUS (29 %) than other stx profiles.Conclusion. The implementation of PCR targeting stx at diagnostic laboratories has shown that non-O157 STEC, most notably STEC O26, are an emerging risk to public health.

Pathogenic potential assessment of the Shiga toxin-producing Escherichia coli by a source attribution-considered machine learning model.

Instead of conventional serotyping and virulence gene combination methods, methods have been developed to evaluate the pathogenic potential of newly emerging pathogens. Among them, the machine learning (ML)-based method using whole-genome sequencing (WGS) data are getting attention because of the recent advances in ML algorithms and sequencing technologies. Here, we developed various ML models to predict the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) isolates using their WGS data. The input dataset for the ML models was generated using distinct gene repertoires from positive (pathogenic) and negative (nonpathogenic) control groups in which each STEC isolate was designated based on the source attribution, the relative risk potential of the isolation sources. Among the various ML models examined, a model using the support vector machine (SVM) algorithm, the SVM model, discriminated between the two control groups most accurately. The SVM model successfully predicted the pathogenicity of the isolates from the major sources of STEC outbreaks, the isolates with the history of outbreaks, and the isolates that cannot be assessed by conventional methods. Furthermore, the SVM model effectively differentiated the pathogenic potentials of the isolates at a finer resolution. Permutation importance analyses of the input dataset further revealed the genes important for the estimation, proposing the genes potentially essential for the pathogenicity of STEC. Altogether, these results suggest that the SVM model is a more reliable and broadly applicable method to evaluate the pathogenic potential of STEC isolates compared with conventional methods.

Characterization of antimicrobial susceptibility, extended-spectrum ß-lactamase genes and phylogenetic groups of Shigatoxin producing Escherichia coli isolated from patients with diarrhea in Iran.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) are among common foodborne bacterial pathogens and healthy livestock are the main source of this bacterium. Severe diseases attribute to two types of cytotoxin Stx1 and Stx2, which are also called Shiga toxin (Stx). Infection of humans with STEC may result in Acute diarrhea with or without bleeding, hemorrhagic colitis (HC) and the hemolytic uremic syndrome (HUS). As antibiotic resistance is increasingly being reported among STEC isolates obtained from livestock and patients worldwide, in this study the pattern of antibiotic resistance in clinical isolates was determined. METHODS: Stool samples were collected from patients with diarrhea. All samples were cultured and identified by biochemical and molecular tests. Antimicrobial susceptibility test and assessment of extended-spectrum ß-lactamase (ESBL)-related genes were conducted. Moreover, phylogenetic groups were analyzed using quadruplex PCR, and DNA analysis assessed multi-locus sequence types (MLST). RESULTS: Out of 340 E. coli samples, 174 were identified as STEC by PCR. Antimicrobial susceptibility test results showed that, 99.4%, 96% and 93.1% of isolates were susceptible to imipenem/ertapenem, piperacillin-tazobactam and amikacin, respectively. The highest resistance was towards ampicillin (68.4%), followed by trimethoprim-sulfamethoxazole (59.8%), and tetracycline (57.5%). A total of 106 (60.9%) isolates were multidrug resistance (MDR) and 40.8% of isolates were determined to be extended spectrum ß-lactamase producers. In 94.4% of isolates, genes responsible for ESBL production could be detected, and blaTEM was the most prevalent, followed by blaCTX-M9. Furthermore, phylogenetic grouping revealed that majority of STEC strains belonged to Group C, followed by Groups E, B2 and A. MLST unveiled diverse ST types. CONCLUSION: A periodical surveillance studies and thorough understanding of antibiotic resistant profiles in STEC isolates could help select effective antibiotic treatment for patients and develop strategies to effectively manage food contamination and human infections.

Prevalence, characterization and antibiotic resistance of Shiga toxigenic Escherichia coli serogroups isolated from fresh beef and locally processed ready-to-eat meat products in Lagos, Nigeria.

Fresh beef and meat products have been implicated in outbreaks of Shiga toxin-producing Escherichia coli (STEC) worldwide. This study investigated the prevalence of E. coli O157: H7 and non-O157 STEC serogroups in fresh beef in the open market and street vended meat products (n = 180) in Lagos metropolis, Nigeria. A combination of culture media and immunomagnetic separation followed by typing for associated virulence factors and serotypes was performed. Antimicrobial susceptibility testing was performed on the isolated STEC serotypes using the disk diffusion method. A total of 72 STEC serogroup isolates were detected from 61 out of 180 samples. The O157 STEC serotypes were detected in fresh beef, suya, minced meat and tsire with prevalence of 20.8% while non-O157 STEC serogroups were detected in all the samples. Molecular typing revealed 25% (n = 18) of the STEC serogroups showed presence of all the stx1, stx2, eaeA, fliCH7 and rfbEO157 virulence factors while 54.2% (n = 39) possessed a combination of two virulence genes. Multidrug resistance was discovered in 23.6% (n = 17) of the total STEC serogroups. Locally processed ready-to-eat meat products in Lagos metropolis, Nigeria harbour potentially pathogenic multi-drug resistant STEC serogroups that can constitute public health hazard.

Epidemiological investigation of recurrent outbreaks of haemolytic uraemic syndrome caused by Shiga toxin-producing Escherichia coli serotype O55:H7 in England, 2014-2018.

Recurrent outbreaks of haemolytic uraemic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli (STEC) serotype O55:H7 occurred in England between 2014 and 2018. We reviewed the epidemiological evidence to identify potential source(s) and transmission routes of the pathogen, and to assess the on-going risk to public health. Over the 5-year period, there were 43 confirmed and three probable cases of STEC O55:H7. The median age of cases was 4 years old (range 6 months to 69 years old) and over half of all cases were female (28/46, 61%). There were 36/46 (78.3%) symptomatic cases, and over half of all cases developed HUS (25/46, 54%), including two fatal cases. No common food or environmental exposures were identified, although the majority of cases lived in rural or semi-rural environments and reported contact with both wild and domestic animals. This investigation informed policy on the clinical and public health management of HUS caused by STEC other than serotype O157:H7 (non-O157 STEC) in England, including comprehensive testing of all household contacts and household pets and more widespread use of polymerase chain reaction assays for the rapid diagnosis of STEC-HUS.

Validation strategy of a bioinformatics whole genome sequencing workflow for Shiga toxin-producing Escherichia coli using a reference collection extensively characterized with conventional methods.

Whole genome sequencing (WGS) enables complete characterization of bacterial pathogenic isolates at single nucleotide resolution, making it the ultimate tool for routine surveillance and outbreak investigation. The lack of standardization, and the variation regarding bioinformatics workflows and parameters, however, complicates interoperability among (inter)national laboratories. We present a validation strategy applied to a bioinformatics workflow for Illumina data that performs complete characterization of Shiga toxin-producing Escherichia coli (STEC) isolates including antimicrobial resistance prediction, virulence gene detection, serotype prediction, plasmid replicon detection and sequence typing. The workflow supports three commonly used bioinformatics approaches for the detection of genes and alleles: alignment with blast+, kmer-based read mapping with KMA, and direct read mapping with SRST2. A collection of 131 STEC isolates collected from food and human sources, extensively characterized with conventional molecular methods, was used as a validation dataset. Using a validation strategy specifically adopted to WGS, we demonstrated high performance with repeatability, reproducibility, accuracy, precision, sensitivity and specificity above 95 % for the majority of all assays. The WGS workflow is publicly available as a 'push-button' pipeline at https://galaxy.sciensano.be. Our validation strategy and accompanying reference dataset consisting of both conventional and WGS data can be used for characterizing the performance of various bioinformatics workflows and assays, facilitating interoperability between laboratories with different WGS and bioinformatics set-ups.