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.

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.

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.

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.

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.

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.

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.

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.

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.

Choice of library preparation affects sequence quality, genome assembly, and precise in silico prediction of virulence genes in shiga toxin-producing Escherichia coli.

Whole genome sequencing (WGS) provides essential public health information and is used worldwide for pathogen surveillance, epidemiology, and source tracking. Foodborne pathogens are often sequenced using rapid library preparation chemistries based on transposon technology; however, this method may miss random segments of genomes that can be important for accurate downstream analyses. As new technologies become available, it may become possible to achieve better overall coverage. Here we compare the sequence quality obtained using libraries prepared from the Nextera XT and Nextera DNA Prep (Illumina, San Diego, CA) chemistries for 31 Shiga toxin-producing Escherichia coli (STEC) O121:H19 strains, which had been isolated from flour during a 2016 outbreak. The Nextera DNA Prep gave superior performance metrics including sequence quality, assembly quality, uniformity of genome coverage, and virulence gene identification, among other metrics. Comprehensive detection of virulence genes is essential for making educated assessments of STECs virulence potential. The phylogenetic SNP analysis did not show any differences in the variants detected by either library preparation method which allows isolates prepared from either library method to be analysed together. Our comprehensive comparison of these chemistries should assist researchers wishing to improve their sequencing workflow for STECs and other genomic risk assessments.

Integrated Approach for the Diagnosis of Shiga Toxin-Producing Escherichia coli Infections in Humans.

Shiga toxin-producing Escherichia coli (STEC) are human pathogens causing severe diseases, such as hemorrhagic colitis and the hemolytic uremic syndrome. The prompt diagnosis of STEC infection is of primary importance to drive the most appropriate patient's management procedures. The methods to diagnose STEC infections include both direct isolation of the STEC from stool samples and the identification of indirect evidences based on molecular, phenotypic, and serological applications. Here, the procedures in use at the Italian Reference Laboratory for E. coli infections are described.

Predicting Host Association for Shiga Toxin-Producing E. coli Serogroups by Machine Learning.

Escherichia coli is a species of bacteria that can be present in a wide variety of mammalian hosts and potentially soil environments. E. coli has an open genome and can show considerable diversity in gene content between isolates. It is a reasonable assumption that gene content reflects evolution of strains in particular host environments and therefore can be used to predict the host most likely to be the source of an isolate. An extrapolation of this argument is that strains may also have gene content that favors success in multiple hosts and so the possibility of successful transmission from one host to another, for example, from cattle to human, can also be predicted based on gene content. In this methods chapter, we consider the issue of Shiga toxin (Stx)-producing E. coli (STEC) strains that are present in ruminants as the main host reservoir and for which we know that a subset causes life-threatening infections in humans. We show how the genome sequences of E. coli isolated from both cattle and humans can be used to build a classifier to predict human and cattle host association and how this can be applied to score key STEC serotypes known to be associated with human infection. With the example dataset used, serogroups O157, O26, and O111 show the highest, and O103 and O145 the lowest, predictions for human association. The long-term ambition is to combine such machine learning predictions with phylogeny to predict the zoonotic threat of an isolate based on its whole genome sequence (WGS).

Functional Analysis of Shiga Toxin-Producing Escherichia coli Biofilm Components in Plant Leaves.

Plants represent alternative or secondary hosts for Shiga toxin-producing Escherichia coli (STEC), enabling transmission of the pathogens through the food chain on horticultural crops. This becomes a public health concern for plants that are eaten raw or minimally processed, such as leafy salad and fruits. STEC actively interact with plants as hosts, and so to determine the mechanistic basis to the interaction, it is necessary to assess STEC gene function in planta. Here, we describe analysis of an STEC biofilm component, curli, that plays a role in STEC colony formation in plant leaves. It also serves as a suitable example of the approaches required for qualitative and quantitative assessment of functional host colonization traits.

Shiga Toxin-Producing E. coli in Animals: Detection, Characterization, and Virulence Assessment.

Cattle and other ruminants are primary reservoirs for Shiga toxin-producing Escherichia coli (STEC) strains which have a highly variable, but unpredictable, pathogenic potential for humans. Domestic swine can carry and shed STEC, but only STEC strains producing the Shiga toxin (Stx) 2e variant and causing edema disease in piglets are considered pathogens of veterinary medical interest. In this chapter, we present general diagnostic workflows for sampling livestock animals to assess STEC prevalence, magnitude, and duration of host colonization. This is followed by detailed method protocols for STEC detection and typing at genetic and phenotypic levels to assess the relative virulence exerted by the strains.