Virulence gene profiling of enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains: a basis for molecular risk assessment of typical and atypical EPEC strains

<p>Abstract</p> <p>Background</p> <p>Enterohaemorrhagic <it>E. coli </it>(EHEC) can cause severe disease such as bloody diarrhoea and haemolytic uraemic syndrome in humans. Besides production of Shiga toxins, the presence of LEE (<it>eae</it>-gene) and non-LEE (<it>nle</it>) encoded effector genes harboured on O-islands OI-122, OI-71 and OI-57 is associated with EHEC virulence and their frequency in outbreaks. Genes encoded by the EHEC-plasmid are putative virulence markers of EHEC. EHEC-plasmids, LEE and non-LEE effector genes have also been detected in some strains of enteropathogenic <it>E. coli </it>(EPEC). The objective of this study was to analyze the relationship between EHEC and EPEC for virulence genes encoded by genomic O-islands and by the EHEC-plasmids.</p> <p>Results</p> <p>Nle genes <it>ent/espL2</it>, <it>nleB </it>and <it>nleE </it>(OI-122), <it>nleA</it>, <it>nleF </it>and <it>nleH1-2 </it>(OI-71), <it>nleG5-2 </it>and <it>nleG6-2 </it>(OI-57), <it>espK </it>(CP-933N) and the EHEC-plasmid encoded genes <it>ehxA</it>, <it>espP</it>, <it>etpD </it>and <it>katP </it>were searched in 73 typical and in 235 atypical enteropathogenic <it>E. coli </it>(EPEC) strains. Typical and atypical EPEC each fall into two clusters. Cluster 1 typical (n = 46) and atypical (n = 129) EPEC strains were characterized by the presence of OI-122 encoded genes and grouped together with 64 investigated EHEC strains. Cluster 2 typical (n = 27) and atypical (n = 106) strains grouped together with 52 LEE-negative, Shiga toxin-producing <it>E. coli </it>(STEC) and with 21 apathogenic <it>E. coli </it>strains. Typical EPEC Cluster 1 strains belonged to serotypes frequently involved in severe illness and outbreaks in children (O111:H2, O114:H2, O55:H6, O127:H6 and O142:H6). Atypical EPEC Cluster 1 strains were characterized by serotypes related to EHEC (O26:H11, O55:H7, O145:H28, O103:H2 and O103:H25).</p> <p>Conclusion</p> <p>The OI-122 encoded <it>nleB </it>gene was found to be most closely associated with Cluster 1 strains and may serve as a diagnostic tool for the identification of virulent EHEC and EPEC seropathotypes. OI-71 encoded genes <it>nleA</it>, <it>nleF </it>and <it>nleH1-2 </it>are less associated with Cluster 1 strains. EHEC-plasmid, OI-57 and CP-933 associated genes showed only weak similarities with virulent Cluster 1 EHEC and EPEC strains.</p

Revisiting the STEC Testing Approach: Using espK and espV to Make Enterohemorrhagic Escherichia coli (EHEC) Detection More Reliable in Beef

Current methods for screening Enterohemorrhagic Escherichia coli (EHEC) O157 and non-O157 in beef enrichments typically rely on the molecular detection of stx, eae, and serogroup-specific wzx or wzy gene fragments. As these genetic markers can also be found in some non-EHEC strains, a number of “false positive” results are obtained. Here, we explore the suitability of five novel molecular markers, espK, espV, ureD, Z2098, and CRISPRO26:H11 as candidates for a more accurate screening of EHEC strains of greater clinical significance in industrialized countries. Of the 1739 beef enrichments tested, 180 were positive for both stx and eae genes. Ninety (50%) of these tested negative for espK, espV, ureD, and Z2098, but 12 out of these negative samples were positive for the CRISPRO26:H11 gene marker specific for a newly emerging virulent EHEC O26:H11 French clone. We show that screening for stx, eae, espK, and espV, in association with the CRISPRO26:H11 marker is a better approach to narrow down the EHEC screening step in beef enrichments. The number of potentially positive samples was reduced by 48.88% by means of this alternative strategy compared to the European and American reference methods, thus substantially improving the discriminatory power of EHEC screening systems. This approach is in line with the EFSA (European Food Safety Authority) opinion on pathogenic STEC published in 2013

Revisiting the STEC Testing Approach: Using espK and espV to Make Enterohemorrhagic Escherichia coli (EHEC) Detection More Reliable in Beef

Current methods for screening Enterohemorrhagic Escherichia coli (EHEC) O157 and non-O157 in beef enrichments typically rely on the molecular detection of stx, eae, and serogroup-specific wzx or wzy gene fragments. As these genetic markers can also be found in some non-EHEC strains, a number of “false positive” results are obtained. Here, we explore the suitability of five novel molecular markers, espK, espV, ureD, Z2098, and CRISPRO26:H11 as candidates for a more accurate screening of EHEC strains of greater clinical significance in industrialized countries. Of the 1739 beef enrichments tested, 180 were positive for both stx and eae genes. Ninety (50%) of these tested negative for espK, espV, ureD, and Z2098, but 12 out of these negative samples were positive for the CRISPRO26:H11 gene marker specific for a newly emerging virulent EHEC O26:H11 French clone. We show that screening for stx, eae, espK, and espV, in association with the CRISPRO26:H11 marker is a better approach to narrow down the EHEC screening step in beef enrichments. The number of potentially positive samples was reduced by 48.88% by means of this alternative strategy compared to the European and American reference methods, thus substantially improving the discriminatory power of EHEC screening systems. This approach is in line with the EFSA (European Food Safety Authority) opinion on pathogenic STEC published in 2013

A multiplex real-time PCR assay targeting virulence and resistance genes in Salmonella enterica serotype Typhimurium

<p>Abstract</p> <p>Background</p> <p>Typhimurium is the main serotype of <it>Salmonella enterica </it>subsp. <it>enterica </it>implicated in food-borne diseases worldwide. This study aimed to detect the prevalence of ten markers combined in a macro-array based on multiplex real-time PCR. We targeted characteristic determinants located on pathogenicity islands (SPI-2 to -5, virulence plasmid <it>pSLT </it>and <it>Salmonella </it>genomic island 1 (SGI1)) as well as a specific 16S-23S rRNA intergenic spacer sequence of definitive type 104 (DT104). To investigate antimicrobial resistance, the study also targeted the presence of genes involved in sulfonamide (<it>sul1</it>) and beta-lactam (<it>bla</it>_TEM) resistance. Finally, the <it>intI1 </it>determinant encoding integrase from class 1 integron was also investigated.</p> <p>Results</p> <p>A total of 538 unrelated <it>S</it>. Typhimurium strains isolated between 1999 and 2009 from various sources, including food animals, food products, human and environmental samples were studied. Based on the combined presence or absence of these markers, we distinguished 34 different genotypes, including three major genotypes encountered in 75% of the studied strains, Although SPI determinants were almost always detected, SGI1, <it>intI1</it>, <it>sul1 </it>and <it>bla</it>_TEMdeterminants were found 47%, 52%, 54% and 12% of the time respectively, varying according to isolation source. Low-marker patterns were most often detected in poultry sources whereas full-marker patterns were observed in pig, cattle and human sources.</p> <p>Conclusion</p> <p>The GeneDisc^®assay developed in this study madeit easier to explore variability within serotype Typhimurium by analyzing ten relevant gene determinants in a large collection of strains. This real-time multiplex method constitutes a valuable tool for strains characterization on epidemiological purposes.</p

The Escherichia coli Serogroup O1 and O2 Lipopolysaccharides Are Encoded by Multiple O-antigen Gene Clusters

Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR- assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains

Combination of whole genome sequencing and supervised machine learning provides unambiguous identification of eae-positive Shiga toxin-producing Escherichia coli

Introduction: The objective of this study was to develop, using a genome wide machine learning approach, an unambiguous model to predict the presence of highly pathogenic STEC in E. coli reads assemblies derived from complex samples containing potentially multiple E. coli strains. Our approach has taken into account the high genomic plasticity of E. coli and utilized the stratification of STEC and E. coli pathogroups classification based on the serotype and virulence factors to identify specific combinations of biomarkers for improved characterization of eae-positive STEC (also named EHEC for enterohemorrhagic E.coli) which are associated with bloody diarrhea and hemolytic uremic syndrome (HUS) in human. Methods: The Machine Learning (ML) approach was used in this study on a large curated dataset composed of 1,493 E. coli genome sequences and 1,178 Coding Sequences (CDS). Feature selection has been performed using eight classification algorithms, resulting in a reduction of the number of CDS to six. From this reduced dataset, the eight ML models were trained with hyper-parameter tuning and cross-validation steps. Results and discussion: It is remarkable that only using these six genes, EHEC can be clearly identified from E. coli read assemblies obtained from in silico mixtures and complex samples such as milk metagenomes. These various combinations of discriminative biomarkers can be implemented as novel marker genes for the unambiguous EHEC characterization from different E. coli strains mixtures as well as from raw milk metagenomesPeer Reviewe

Development and validation of high-resolution melting assays for the detection of potentially virulent strains of \u3ci\u3eEscherichia coli\u3c/i\u3e O103 and O121

Virulent strains of Shiga toxin-producing Escherichia coli (STEC) serogroups O103 and O121 are considered adulterants in beef. Two high-resolution melting (HRM) real-time PCR assays were standardized for the specific detection and discrimination of potentially virulent and avirulent strains of E. coli O103 and O121. The O103 HRM assay offered the possibility to distinguish clearly STEC O103:H2 from STEC O103:H25. The two standardized assays were extensively validated using 215 pure culture strains, laboratory inoculated food samples, and naturally contaminated beef (n = 84) and pork (n = 84) enrichments collected from the red meat surveillance program. Both HRM assays showed 100% inclusivity and exclusivity using pure culture strains and enriched spiked food samples. Data from this study shows the ability of the standardized assays to specifically detect the strains of each target serogroup and, most importantly, to differentiate the strains present into potentially virulent or avirulent groups. The assays standardized in this study can be helpful for food surveillance programs and help mitigate product loss due to the presence of avirulent strains lacking crucial virulence genes (stx and eae)

Genetic Diversity and Pathogenic Potential of Attaching and Effacing Escherichia coli O26:H11 Strains Recovered from Bovine Feces in the United States

Escherichia coli O26 has been identified as the most common non-O157 Shiga toxin-producing E. coli (STEC) serogroup to cause human illnesses in the United States and has been implicated in outbreaks around the world. E. coli has high genomic plasticity, which facilitates the loss or acquisition of virulence genes. Attaching and effacing E. coli (AEEC) O26 strains have frequently been isolated from bovine feces, and there is a need to better characterize the relatedness of these strains to defined molecular pathotypes and to describe the extent of their genetic diversity. High-throughput real-time PCR was used to screen 178 E. coli O26 isolates from a single U.S. cattle feedlot, collected from May to July 2011, for the presence or absence of 25 O26 serogroup-specific and virulence-associated markers. The selected markers were capable of distinguishing these strains into molecularly defined groups (yielding 18 unique marker combinations). Analysis of the clustered regularly interspaced short palindromic repeat 1 (CRISPR1) and CRISPR2a loci further discriminated isolates into 24 CRISPR types. The combination of molecular markers and CRISPR typing provided 20.8% diversity. The recent CRISPR PCR target SP_O26-E, which was previously identified only in stx 2-positive O26:H11 human clinical strains, was identified in 96.4% (161/167 [95% confidence interval, 99.2 to 93.6%]) of the stx-negative AEEC O26:H11 bovine fecal strains. This supports that these stx-negative strains may have previously contained a prophage carrying stx or could acquire this prophage, thus possibly giving them the potential to become pathogenic to humans. These results show that investigation of specific genetic markers may further elucidate our understanding of the genetic diversity of AEEC O26 strains in bovine feces

Genetic Diversity and Virulence Potential of Shiga Toxin-Producing Escherichia coli O113:H21 Strains Isolated from Clinical, Environmental, and Food Sources

Shiga toxin-producing Escherichia coli strains of serotype O113:H21 have caused severe human diseases, but they are unusual in that they do not produce adherence factors coded by the locus of enterocyte effacement. Here, a PCR microarray was used to characterize 65 O113:H21 strains isolated from the environment, food, and clinical infections from various countries. in comparison to the pathogenic strains that were implicated in hemolytic-uremic syndrome in Australia, there were no clear differences between the pathogens and the environmental strains with respect to the 41 genetic markers tested. Furthermore, all of the strains carried only Shiga toxin subtypes associated with human infections, suggesting that the environmental strains have the potential to cause disease. Most of the O113:H21 strains were closely related and belonged in the same clonal group (ST-223), but CRISPR analysis showed a great degree of genetic diversity among the O113:H21 strains.French Joint Ministerial Program of R&D against CBRNE RisksFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Food & Drug Adm, Div Microbiol, College Pk, MD 20740 USAFrench Agcy Food Environm & Occupat Hlth & Safety, Lab Food Safety, Maisons Alfort, FranceFood & Drug Adm, Div Mol Biol, Laurel, MD USAUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilFed Inst Risk Assessment, Natl Reference Lab Escherichia coli, Berlin, GermanyInst Nacl Enfermedades Infecciosas ANLIS Dr Carlo, Serv Fisiopatogenia, Buenos Aires, DF, ArgentinaUniv Melbourne, Peter Doherty Inst Infect & Immun, Dept Microbiol & Immunol, Melbourne, Vic, AustraliaUniv Adelaide, Res Ctr Infect Dis, Sch Mol & Biomed Sci, Adelaide, SA, AustraliaUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilFrench Joint Ministerial Program of R&D against CBRNE Risks: C17609-2Web of Scienc

Phylogenetic relationship and virulence composition of Escherichia coli O26:H11 cattle and human strain collections in Scotland; 2002-2020

O26 is the commonest non-O157 Shiga toxin (stx)-producing Escherichia coli serogroup reported in human infections worldwide. Ruminants, particularly cattle, are the primary reservoir source for human infection. In this study, we compared the whole genomes and virulence profiles of O26:H11 strains (n = 99) isolated from Scottish cattle with strains from human infections (n = 96) held by the Scottish Escherichia coli O157/STEC Reference Laboratory, isolated between 2002 and 2020. Bovine strains were from two national cross-sectional cattle surveys conducted between 2002–2004 and 2014–2015. A maximum likelihood phylogeny was constructed from a core-genome alignment with the O26:H11 strain 11368 reference genome. Genomes were screened against a panel of 2,710 virulence genes using the Virulence Finder Database. All stx-positive bovine O26:H11 strains belonged to the ST21 lineage and were grouped into three main clades. Bovine and human source strains were interspersed, and the stx subtype was relatively clade-specific. Highly pathogenic stx2a-only ST21 strains were identified in two herds sampled in the second cattle survey and in human clinical infections from 2010 onwards. The closest pairwise distance was 9 single-nucleotide polymorphisms (SNPs) between Scottish bovine and human strains and 69 SNPs between the two cattle surveys. Bovine O26:H11 was compared to public EnteroBase ST29 complex genomes and found to have the greatest commonality with O26:H11 strains from the rest of the UK, followed by France, Italy, and Belgium. Virulence profiles of stx-positive bovine and human strains were similar but more conserved for the stx2a subtype. O26:H11 stx-negative ST29 (n = 17) and ST396 strains (n = 5) were isolated from 19 cattle herds; all were eae-positive, and 10 of these herds yielded strains positive for ehxA, espK, and Z2098, gene markers suggestive of enterohaemorrhagic potential. There was a significant association (p &lt; 0.001) between nucleotide sequence percent identity and stx status for the bacteriophage insertion site genes yecE for stx2 and yehV for stx1. Acquired antimicrobial resistance genes were identified in silico in 12.1% of bovine and 17.7% of human O26:H11 strains, with sul2, tet, aph(3″), and aph(6″) being most common. This study describes the diversity among Scottish bovine O26:H11 strains and investigates their relationship to human STEC infections