Phylogenomic framework and virulence gene boundaries of emerging Shiga toxin-producing Escherichia coli O118 informed by the comprehensive profiling of 359 O118 genomes

Phylogenomic framework and virulence gene boundaries of emerging Shiga toxin-producing Escherichia coli O118 informed by the comprehensive profiling of 359 O118 genomes | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Phylogenomic framework and virulence gene boundaries of emerging Shiga toxin-producing Escherichia coli O118 informed by the comprehensive profiling of 359 O118 genomes Irvin Rivera , Sara S.K. Konig , Armando L. Rodriguez , Joseph M. Bosilevac , Mark Eppinger doi: https://doi.org/10.1101/2025.04.29.651274 Irvin Rivera 1 Department of Molecular Microbiology & Immunology, University of Texas at San Antonio , San Antonio, TX, United States 2 South Texas Center for Emerging Infectious Diseases (STCEID) , San Antonio, TX, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sara S.K. Konig 1 Department of Molecular Microbiology & Immunology, University of Texas at San Antonio , San Antonio, TX, United States 2 South Texas Center for Emerging Infectious Diseases (STCEID) , San Antonio, TX, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site Armando L. Rodriguez 3 University of Texas at San Antonio, Research Computing Support Group , San Antonio, TX, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Joseph M. Bosilevac 4 U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS), U.S. Meat Animal Research Center, Clay Center , NE, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mark Eppinger 1 Department of Molecular Microbiology & Immunology, University of Texas at San Antonio , San Antonio, TX, United States 2 South Texas Center for Emerging Infectious Diseases (STCEID) , San Antonio, TX, United States Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: mark.eppinger{at}utsa.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Non-O157 Shiga toxin-producing Escherichia coli (STEC), particularly the O118 serogroup, are emerging pathogens linked to severe foodborne illnesses, including hemolytic uremic syndrome. The hallmark of STEC virulence is the production of a potent phage-borne cytotoxin, often accompanied by the locus of enterocyte effacement (LEE). This study explores the genomic landscape, virulence factors, and resistance traits of O118 STEC. We analyzed 357 publicly available O118 genomes across ten H-antigens and included two clinically significant O118:H16 STEC strains sequenced to closure. Pangenome assessment and core genome multilocus sequence typing (MLST) based on 4,160 shared genes revealed phylogenetic clustering by H-type and delineated distinct STEC-phylogroups, alongside relationships to non-STEC pathovars such as uropathogenic E. coli (UPEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC). Identified STEC phylogroups encompassed H6, H12, H16, and H2 strains with diverse Shiga toxin ( stx ) profiles ( stx 1a , stx 2a , stx 2b , stx 2c , stx 2f ). A subset of H2-STEC lacked stx , suggesting potential secondary phage loss. Most STEC groups harbored the locus of enterocyte effacement (LEE). Further, a strong correlation was observed between H-antigens and eae subtypes, with specific pairings such as H6/ eae-ι , H16/ eae-β , and H2/ eae-ε . Horizontally acquired pathogenicity islands—including O-island 122 in H16 strains and a novel pathogenicity-associated island carrying antibiotic resistance—along with other loci related to colonization and interbacterial competition, further enhance these strains’ virulence potential. Our findings underscore the genetic diversity and virulence potential of O118 STEC. Understanding phylogroup-specific traits and resistance markers is crucial for effective surveillance and public health interventions. Importance Shiga toxin-producing Escherichia coli (STEC) are a major public health concern, responsible for illnesses ranging from diarrhea to life-threatening kidney damage. Among non-O157 STEC, serogroup O118 is increasingly recognized as an emerging STEC lineage. This study provides the most comprehensive genomic analysis to date of O118 STEC, offering critical insights into their pathogenome, virulence traits, and antimicrobial resistance inventories. Notably, we identified a novel 74 kb pathogenicity island in clinical strain H16-12089 that encodes a combination of multidrug resistance genes, virulence factors, and interbacterial competition systems. Understanding the genetic makeup and virulence potential of these pathogens is essential for improving surveillance, risk assessment, and public health interventions. 1. Introduction Escherichia coli O118 is an emerging foodborne Shiga toxin-producing E. coli (STEC) serotype with a bovine zoonotic reservoir ( 1 ). Infections can cause severe human gastrointestinal disease and may progress to life-threatening complications, such as hemorrhagic colitis and hemolytic uremic syndrome (HUS) ( 2 , 3 ). Vulnerable populations, such as children and the elderly, are particularly affected ( 4 – 6 ). This underscores the importance of monitoring this emerging serotype in both veterinary and public health contexts and assessing its virulence potential and phylogenetic boundaries ( 4 , 7 ). E. coli are historically classified by their somatic O– and flagellar H-antigens ( 8 – 10 ). Serotype O157:H7 is the dominant causative agent of STEC disease in the U.S. ( 11 – 15 ). However, the incidence of non-O157 infections has steadily increased ( 16 – 19 ). Emerging serogroups O26, O45, O103, O111, O121, and O145 account for most clinical non-O157 STEC infections in the US and are colloquially referred to as the “Big Six” ( 16 , 20 , 21 ). These accounted for 83% of non-O157 STEC infections in the United States from 2000 to 2010 ( 22 – 25 ). As diagnostic testing increased, non-O157 STEC, namely the “Big Six”, surpassed the previously dominant O157:H7 lineage in 2014 ( 21 , 26 ). Between 2021 and 2023, non-O157 infections accounted for 168k annual cases, compared to 96k O157 cases ( 22 , 27 ). The first reported human infection with stx 1 -positive O118:H2 STEC occurred in 1996 during a school outbreak in Japan ( 28 ) linked to salad ( 29 ). Since then, strains of O118 (including its related O151 subgroup) ( 30 , 31 ) have been recognized as an emerging and human pathogenic STEC lineage, many of which possess Shiga toxins, intimin ( eae ), and hemolysins ( 29 , 32 , 33 ). The carried ΦStx prophages feature different combinations of stx -suballeles ( 12 , 34 ) that can also form hybrid toxins ( 35 ). Both stx 1 and stx 2 are potent translation inhibitors ( 29 , 34 , 36 – 41 ) and their alleles have been documented in O118 ( 6 , 32 , 42 ). Stx 2 , particularly stx 2a, and stx 2d, have been associated with elevated cytotoxicity ( 43 – 46 ) compared to the morbidity caused by stx 1a + STEC ( 8 , 17 , 47 , 48 ). However, Stx 1 has been shown to exhibit increased cytotoxicity in Vero cells compared to Stx 2a ( 44 , 49 , 50 ). Adding further complexity, Stx 1 has been reported to reduce the overall toxicity of Stx 2a , potentially through competitive binding or interference at the receptor level ( 44 ). Besides the pathovar-defining Shiga toxin, another notable virulence factor include the locus of enterocyte effacement (LEE). This pathogenicity island encodes a type III secretion system (T3SS) and effectors, the outer membrane adhesin intimin ( eae ), and its translocated receptor ( tir ) ( 9 , 29 , 51 , 52 ). The latter is also carried by enteropathogenic E. coli (EPEC) ( 53 ). Antimicrobial (AR) and multidrug-resistant (MDR) isolates are described for O118 STEC ( 29 , 32 , 54 , 55 ). STEC also carry serogroup-specific virulence plasmids and an array of other plasmids with diverse functions ( 56 – 58 ). Serogroup O118 also contains EPEC, encoding intimin adhesins forming characteristic attaching and effacing lesions ( 4 , 28 ); Enteroaggregative E. coli (EAEC) which form biofilm-like aggregates ( 4 , 16 , 59 – 61 ); and extraintestinal pathogenic E. coli (ExPEC) that are capable of causing severe infections outside the gastrointestinal tract, such as urinary tract infections (UTIs) and bloodstream infections ( 28 , 62 ). In this study, we provide a comprehensive framework for O118 E. coli with a focus on emerging STEC O118. The gathered information on virulence gene content and genome makeup provide a foundation to delineate evolutionary and pathovar boundaries of these emerging and human pathogenic non-O157 lineages. 2. Materials and Methods Bacterial strains analyzed in this study For this study, we analyzed 359 O118 genomes, including three closed genomes, 357 publicly available genomes retrieved from the NCBI Pathogen Detection Isolate Browser ( https://www.ncbi.nlm.nih.gov/pathogens/isolates/ ) (July 2024), and two clinical O118 genomes from our STEC collection that were sequenced to closure. Strain-associated metadata and genome statistics can be found in Table S1 . Genome sequencing, assembly, and annotation Strains were cultured overnight at 37°C with shaking at 220 rpm in lysogeny broth (LB) (Thermo Fisher Scientific, Asheville, NC, USA). To maximize total genomic DNA (gDNA) yields, bacterial overnight cultures were diluted to OD 600 of 0.03 in fresh LB medium and grown at 37°C with shaking at 220 rpm to mid-log phase (OD 600 ∼0.5). Total gDNA was extracted using the Monarch HMW DNA Extraction Kit (New England Biolabs, Ipswich, MA, USA). Genomes were sequenced to closure using Nanopore long-read technology (Oxford Nanopore, UK). Sequencing libraries were prepared with Rapid Barcoding Kit (RBK-114) according to the manufacturer’s instructions and sequenced on the PromethION platform on R10.4.1.Flow Cell (FLO-MIN114). Reads in the fastq format were imported into Galaxy v.22.05 ( 63 ). Default parameters were used for all software unless specified otherwise. Fastq reads were QC-ed using FastQC (v.0.74+Galaxy0) ( http://www.bioinformatics.babraham.ac.uk/projects/fastqc ) and assembled with Flye (v.2.9.3) ( 64 ). The resulting contigs were evaluated with QUAST (v.5.2.0 + Galaxy1). The chromosomal dnaA and plasmid repA genes were designated as the zero point of the closed molecules prior to annotation using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) ( 65 ). Genome assemblies were profiled with SeqKit v2.8.2 to obtain genome statistics ( 66 , 67 ). Phylogenomic analyses and MLST-schemas The O118 genomes, along with the sequence of K-12 substrain MG1655 ( 68 , 69 ), were imported into Ridom SeqSphere+ (v.8.3) (Ridom GmbH, Münster, Germany) for core genome (cg) and targeted Multilocus Sequence Typing (MLST) ( 70 – 72 ). The Sequence Type (ST) was determined according to the EnteroBase schema ( 73 ). Allele sequences for the Achtman scheme, targeting seven housekeeping genes ( adk , fumC , gyrB , icd , mdh , purA , and recA ), were accessed on the EnteroBase website ( https://enterobase.warwick.ac.uk/species/ecoli/download_7_gene ). A cgMLST schema was developed using the closed chromosome of K-12 substrain MG1655 ( 69 ) as seed as previously described ( 74 ). Core and accessory MLST targets were identified according to the inclusion/exclusion criteria of the SeqSphere+ Target Definer. The allele information from the targeted seven-gene schema and the defined core genome gene of the panel strains were used to establish phylogenetic hypotheses using the minimum-spanning method with default settings ( 75 , 76 ). Pathogenome makeup and visualization Chromosomes were compared with progressive Mauve ( 77 ) and visualized in pyGenomeViz-pgv-mauve ( 78 ) ( https://github.com/moshi4/pyGenomeViz ) using seaborne ( 79 ). Chromosomes and carried plasmids were compared and visualized in Blast Ring Image Generator BRIG (v.0.95) ( 80 ). Serotypes were determined in silico using E. coli Typer (ECTyper) ( https://github.com/phac-nml/ecoli_serotyping ) (Galaxy 1.0.0) in Galaxy v.22.05. If the wzy gene was reported absent by ECTyper, BLASTn was used to reevaluate its presence and fragmentation status. The ECTyper database was downloaded as a JSON file from GitHub ( https://github.com/phac-nml/ecoli_serotyping ) and converted into a blast database. The Average Nucleotide Identity (ANI) between all-vs-all 359 genomes was computed with skani ( 81 ), the distance matrix clustered by the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method and visualized using ANIclustermap ( 78 ) ( https://github.com/moshi4/ANIclustermap ). Virulence and antibiotic resistance genes (ARGs) were cataloged with Virulence Factors of Pathogenic Bacteria (VFDB) ( 82 ) and ResFinder ( https://cge.cbs.dtu.dk/services/ResFinder/ ) ( 83 ), respectively. Pathovars were inferred in silico through BLASTp query ( 84 ) against a curated VFDB database of E. coli pathovar-defining virulence genes ( 82 , 85 – 92 ). Ribosomal RNAs and CRISPR/Cas systems were detected with Basic Rapid Ribosomal RNA Predictor (Barrnap) ( 93 ) (v.0.7) and CRISPRCasFinder ( 94 ) (v4.2.20) in Proksee ( 95 ) (v.1.1.0). Anti-phage systems were detected using DefenseFinder ( 96 ) (Galaxy v.1.3.0+galaxy0). Prophages were compared and visualized in Easyfig (v.2.2.2) ( 97 ). Boundaries and locations of intact, partial, or remnant prophages were identified using PHASTEST ( 98 ), followed by manual curation of the ΦStx-prophage and core genome borders by Mauve alignment of the genome to E. coli K-12 substrain MG1655 (GenBank accession U00096 ) ( 68 ) in Geneious Prime (v.2024.0.5). Direct repeats caused by phage integration were identified by BLASTn self-alignment and the Repeat Finder plugin (v1.0.1) in Geneious Prime. Replicase subtyping was performed to characterize the ΦStx 1a -encoding prophages, following the typing scheme introduced by ( 99 ). Replicases were typed with EHEC phage replication unit ( eru ) schema ( 100 , 101 ). The subtypes of phage-borne stx were determined by BLASTn against a curated stx -suballele database, as previously described ( 58 , 102 – 104 ). A hierarchical classification approach was implemented to categorize phage-associated genes from the PHASTEST JSON output. All present phage-associated genes were further curated processing type and name descriptors, along with reported Gene Ontology (GO), for more in-depth functional classification. Genomic islands (GI) were detected with IslandViewer4 ( 105 – 107 ). LEE islands and boundaries were curated, guided by LEE1-LEE4 operon genes espG and espF, respectively, and visualized in EasyFig (v.2.2.2) ( 97 ). Intimin subtypes were determined by BLASTn against a curated database of published eae -subtype sequences, as reported previously ( 58 ). Transposable elements and Insertion Sequence (IS) elements, along with inverted repeats, were cataloged through BLASTn ( 108 ) against the Transposon Central (TnCentral) database ( 109 ), comprised of TnCentral+Integrall+ISFinder entries ( 110 , 111 ) and ISEScan (v.1.7.2.3+Galaxy0) ( 112 ). Plasmid makeup and visualization Plasmids were visualized and decorated in Blast Ring Image Generator BRIG (v.0.95) ( 80 ). Plasmid mobility and incompatibility groups were recorded with Mobilome Typer (MOB-Typer) (v.3.0.3+Galaxy0) ( 113 ) and ABRicate (Galaxy v.1.0.1; ( https://github.com/tseemann/ABRicate ) with options ––minid 95 ––mincov 90 using the PlasmidFinder database ( 108 , 114 ). Using a mash search strategy, phylogenetically related plasmids were identified with the Plasmid DataBase (PLSDB) tool (v.2024.05.31.v2) ( 115 ) ( 116 ). Serotypes of the plasmid-associated chromosomes molecule were determined in silico using ECTyper ( https://github.com/phac-nml/ecoli_serotyping ) (Galaxy v1.0.0) in Galaxy v.22.05. Virulence genes (ARGs) were cataloged with VFDB ( 82 ). Bacteriocins, ribosomally synthesized and post-translationally modified peptides (RiPPs), were recorded with BAGEL4 ( http://bagel4.molgenrug.nl/ ) ( 117 ). Genomic islands were detected with IslandViewer4 ( 105 – 107 ). Mobile Genetic Elements (MGEs) were identified with the mobile orthologous groups database (mobileOG-db) tool ( 118 ) in Proksee ( 95 ). Transposable elements and IS elements were identified with ISEScan (v.1.7.2.3+Galaxy0) ( 112 ) and through BLASTn query ( 108 ) against the TnCentral database, comprised of TnCentral+Integrall+ISFinder entries ( 109 – 111 ). Pan-Genome computation and visualization The global gene reservoir of serogroup O118 was computed by re-annotating all genomes with Prokka (v.1.14.6+galaxy1) ( 119 ). The generated gff3 files were then subjected to pan-genome analysis using Roary (v.3.13.0+galaxy2) ( 120 ). Classification of full-length genes into core, accessory, and shell genes provided information on shared and unique genes according to Roary’s inclusion/exclusion criteria ( 121 ), and visualized Phandango (v.1.2.0) ( 122 ). Results and Discussion Genomes of closed STEC O118 genomes At the time of data collection, three closed serogroup O118 genomes were publicly available, all of which are STEC: H6-strains 2013C-4538 and 2014C-3050 from human stool ( 123 ) and H2-strain EC20017429 isolated in 2017 from a gastroenteritis patient ( 124 ). To support comprehensive analyses of O118 STEC, we sequenced two clinical H16-isolates, strains 12089 and 12867, to closure using long-read sequencing ( 20 , 125 , 126 ). This approach yielded high-quality closed chromosomes of 5,804,173 and 5,685,925 bp and recovered carried plasmids ( Fig. 1 , Fig. 2 ). Genome statistics and strain-associated metadata are provided in Table S1 . The average nucleotide identity processing closed and draft O118 genomes was 99.34%, indicative of the highly conserved chromosomal E. coli backbone ( 127 , 128 ) ( Fig. S1 ). The STEC strains contained about 1 Mb of additional sequence information compared to nonpathogenic E. coli strain K-12 substrain MG1655 ( 68 ), included in this comparison. Mobile genome elements are major drivers of pathogenome diversification ( 38 , 129 – 133 ). The identified prophages, GIs, and IS elements contributed significantly (24.11 to 28.51%) to the genome content of the closed O118 STEC, comparable to observations in other STEC-lineages ( 133 , 134 ) ( Table S2 ). These included pathovar-defining ΦStx prophages ( 29 , 34 , 36 – 41 ) and other virulence hallmarks, such as the LEE Pathogenicity Island ( 29 , 52 , 135 ). IS elements have been utilized to delineate distinct phylogenetic lineages in STEC ( 20 , 136 – 138 ). Download figure Open in new tab Download figure Open in new tab FIG 1 Comparison of O118 STEC chromosomes. BRIG comparison of representative genomes for all identified STEC-phylogroup, considering H-antigen and sequence type, referenced to O118:H16 strains A) 12867 and B) 12089. E. coli K-12 is also included to highlight STEC-specific genome content. Query genomes are color-coded, and their order reflects the inferred phylogenomic relationships. Coding sequences are shown as arrows on the + and – strands, and functional annotations for virulence genes and other loci of interest are highlighted. Download figure Open in new tab Download figure Open in new tab FIG 2 Plasmid inventory of the two sequenced O118 STEC strains. A) The small 6,673 bp ColD-colicin plasmid in H2-strain 12867, B) The 88,842 bp multifunctional ColM-colicin and virulence plasmid of H16-strain 12809 features an array of virulence and conjugation loci, C) , and D) Phylogenetically related plasmids were detected in diverse STEC serogroups. We recorded IS element prevalence and types in the five closed genomes ( Fig. S2 ), showing both shared (IS3_168) but also H-antigen-specific IS elements (e.g., IS3_255, IS4_463). Mauve comparison of closed O118 chromosomes revealed largely genome-wide chromosome synteny but identified several inversions, inversion/transposition, and transposition events associated with adjacent or flanking mobility or ribosomal RNA loci ( Fig. S3 ) ( 139 – 142 ). The sequenced H16-strain 12867 and H16-strain 12089 are both colicinogenic ( Fig. 2 ). Colicins are produced and toxic to E. coli, and support the ability of E. coli to compete for a shared niche ( 143 ). Plasmid p12867-ColD ( 143 ), showed the typical organization into D-lysis, D-immunity, and Colicin D ( 144 ), along with a mobilizable DNA-relaxase mbeA ( 145 ). Additionally, the plasmid-borne SOS inhibition gene psiB was detected ( 146 ). A MASH-based survey identified related plasmids in STEC O157:H7, the Big Six serotypes, and other STEC-associated serotypes ( Table S2-7 ). The larger 88 kb plasmid p12089-ColM encodes Colicin M and its associated M-immunity protein ( 147 ). However, its other gene content identified it as a virulence plasmid. Notable were hemolysin ( hlyCABD) ( 148 ), serine protease ( espP) ( 149 ), fimbrial regulator fimB, and the STEC adherence factor toxB ( 150 ). Additionally, modulators of lipopolysaccharide and exopolysaccharide biosynthesis ( lpxM , icaB ) ( 151 , 152 ) along with SOS-inhibition gene psiB ( 146 ), were also present. Approximately 43% of the plasmid sequence are mobility and conjugation loci ( 153 , 154 ). A MASH-based plasmid survey identified related plasmids among H16 and H2, Big Six and other STEC serogroups ( 155 – 157 ) ( Table S2-7 ). Comparison of ΦStx prophages in closed O118 STEC Comparative genomics provided insights into the shared and specific virulence traits in the identified STEC-phylogroups. Bacteriophages target conserved chromosomal loci and undergo evolutionary acquisition, loss, microevolution, collective shaping, gene content, and pathogenicity traits ( 132 , 158 , 159 ). A comparison of the complete ΦStx 1 prophages extracted from the closed genomes identified two insertion sites: torS and an intergenic insertion upstream of dinI with the phage boundaries marked by flanking direct repeats ( Fig. 3 , Table S2-2 ). The torSTR locus was one of four identified ΦStx 1a integration sites in O26:H11 ST21 ( 160 ), an intergenic attachment site common in E. coli ( 161 ). The ΦStx 1a prophages show an overall high-degree of sequence identity and syntenic organization. The torS ΦStx 1a phage of the H2-strain EC20017429 was distinguished by the presence of an IS3-like element and cluster of three hypothetical genes and a kinase, absent in the other H16 strains. The larger ΦStx 1a of H16-strain 12867 carried a DUF550 protein, type III effectors EspK and EspN, and a hypothetical protein. Download figure Open in new tab FIG 3 Comparison of ΦStx-prophages in closed O118 STEC. BLASTn-based comparison of the ΦStx 1a prophage architectures and gene content. The comparison shows that ΦStx1a phages show considerable genomic plasticity and are inserted at torS or intergenically close to the dinI locus. Locus of Enterocyte Effacement Pathogenicity Island in closed O118 STEC The Locus of Enterocyte Effacement (LEE) pathogenicity island is carried by diverse STEC serogroups, forming characteristic attaching and effacing (A/E) lesions ( 52 , 162 – 166 ). The comparison of LEE islands extracted from the closed O118 STEC genomes ( Fig. 4 ) showed they were highly conserved and organized in five polycistronic operons (LEE1 to 5). These encoded T3SS regulators and effectors ( 167 ). Download figure Open in new tab FIG 4 LEE islands of closed O118 STEC. BLASTn-based comparison of LEE islands inserted into the pheU . The organization of the LEE1 to LEE5 operons and O-island 122, disrupting the LEE island in H2-strain EC20017429, is indicated. The order of strains reflects their inferred phylogenomic position. All were integrated at pheU tRNA, a known LEE integration site in E. coli ( 168 – 172 ). The H2 strain was distinguished by an adjacent O-island 122 within the LEE island boundaries marked by 17 bp direct repeats ( Table S2-4 ). This island encodes non-LEE-encoded (Nle) effectors and transport and regulatory elements that enhance E. coli pathogenicity by promoting intracellular survival, adhesion, immune modulation, and enterotoxin production ( 173 , 174 ). Its carriage has been associated with highly virulent STEC, causing HUS ( 174 , 175 ). The H16 phylogroup, represented by strains 2014C-3050, 2013C-4538, 12867, and 12089, features the β-intimin subtype, whereas the ε-intimin subtype is present in the H2 strain EC20017429 ( Fig. 4 ). Pathogenicity associated multi-drug resistance, virulence, and competition island In strain H16-12089, a novel 74 kb Resistance, Virulence, and Competition (MDRVC) Island with a complex modular architecture was discovered inserted into tRNA-Ser ( 153 ) ( Fig. 5 ). This island featured a tyrosine-type integrase ( 176 ), with borders marked by direct repeats ( 177 ) ( Table S2-4 ). A core component of this island was the MDR-element AMR-SSuT, which has been previously described in STEC O157, though the genomic context was unclear ( 178 ). This island conferred resistance to tetracycline, streptomycin, and sulfonamide. Embedded within this element was streptomycin resistance transposon Tn5393.2 ( 179 ), itself disrupted by an ISVsa5-flanked Tn10 ( 180 ), introducing tetracycline ( 181 , 182 ). The sul2 locus, however, is located outside this Tn5393.2-Tn10 composite, proximate to IS66-like elements, previously linked to antimicrobial resistance in E. coli ( 183 ). Prominently featured were genes that aid interbacterial competition, such as a cdiA -mediated contact-dependent inhibition ( 184 ) associated with VENN motif pre-toxin ( 185 ) and the cbtA-cbeA toxin-antitoxin system ( 186 ). A broad array of virulence loci was catalogued; among these was the hemagglutinin adhesin ( 184 ) and the hemolysins secretion gene ( fhaC ), which likely interacts with the hemolysin on p12089-ColM ( Fig. 2 ). Further diverse regulator genes were associated with stress-response. This included lysR ( 187 ), a DEAD/DEAH-box helicase ( 188 ), alpA ( 189 ), and a bacteriophage defense gene gp2 ( 190 ), along with membrane modeling genes, patatin-like phospholipases and dynamin-like protein rdcB ( 191 , 192 ). DNA-modification enzymes support genomic integrity through repair ( radC ) or endonuclease-mediated defense ( 193 , 194 ). Altogether, the carriage of this multifunctional GI by strain 12809 suggests increased pathogenic potential and fitness, promoting virulence, antimicrobial resistance, stress adaptation, and also genomic stability. Download figure Open in new tab FIG 5 Modular architecture of the MDRVC Island. This newly discovered 74kb multifunctional island, inserted into the tRNA-Ser of H16-strain 12089, features a complex modular architecture. It integrates an antimicrobial multi-drug resistance cassette with gene loci, promoting virulence, interbacterial competition, stress resilience, and overall fitness. Phylogenomic framework and pathovar boundaries of serogroup O118/O151 STEC To support broad-scale analysis, we included all publicly available serogroups O118 and O151 genomes retrieved from the NCBI Pathogen Detection Isolate Browser (July 2024), which were all in the draft stage. These were differentiated by subtle genetic and antigenic changes in their O-antigen sequences and linkages ( 195 – 197 ). Among the 359 analyzed strains, 358 possessed the O151- wzy variant, while a single strain carried O118- wzy ( Table S1-2 ). Strain-associated metadata and genome statistics of this expanded strain panel can be found in Table S1 . This panel of 359 O118 genomes features ten H-antigens, as determined by in silico flagellin ( fliC ) subtyping ( 198 , 199 ). The shared gene inventory was determined at 4,160 genes, comprised of 2,437 core and 1,723 accessory loci, indicative of the genome plasticity found in heterogenous O118 serogroup E. coli ( Fig. 7 , Fig. S4 , Table S4 ). The ANI of 96.32% was computed through an all-vs-all comparison of the 359 O118 genomes ( 128 ). The UPGMA-clustering of ANI values recovered the different H-phylogroups ( Fig. S1 ). The majority belonged to H2 (n=242), followed by H16 (n=93) and H11 (n=11), with relatively few available representative genomes of H10 and H12 (n=3), H5 and H28 (n=2), and a single strain for H6, H8, and H51 ( Table S1-2 ). To investigate the phylogenomic and virulence boundaries of emerging O118 STEC among the heterogenous serogroup O118/O151 strains, we established a phylogenomic framework informed by targeted and core genome MLST ( Fig. 6 , Fig. 7 ). The tree topology partitions the isolates by H-antigen and ST. Pathovars were inferred in silico from their VFDB virulence gene profiles. The STEC phylogroups among serogroup O118 were comprised of H2, H6, H12, and H16 strains. Non-STEC pathovars belonged to EPEC (H5, H11), ETEC (H10, H51), UPEC (H28, H10, H12, H11), and a single strain containing virulence genes associated with both ETEC and EPEC ( Fig. 7 , Table S1-1 , Table S3 ). Notably, H10 antigen belonged to EPEC and UPEC, which formed distinct clusters, also evident in the ANI heatmap ( Fig. S1 ). Download figure Open in new tab FIG 6 MLST-inferred phylogeny of serogroup O118. The relatedness of O118 strains was determined using MLST in Ridom SeqSphere+ through both targeted seven-gene MLST accessed in the EnteroBase website. Numbers on connecting branches indicate the number of genes with differing allele status. The shared gene inventory of 4,160 genes comprises 2,437 core and 1,723 accessory loci according to the inclusion/exclusion criteria of the SeqSphere+ Target Definer. Circle size corresponds to the number of isolates with identical ST. Download figure Open in new tab FIG 7 Phylogeny and distribution of virulence genes. The relatedness of analyzed O118/O151 strains was determined using cgMLST in Ridom SeqSphere+ using the closed chromosome of E. coli strain K12 substrain MG1655 as seed, complemented by the 357 publicly available strains and two closed genomes strains 12089 and 12867. The shared gene inventory was determined at 4,160 genes. These comprise 2,437 core and 1,723 accessory loci according to the inclusion/exclusion criteria of the SeqSphere+ Target Definer. The topology and clusters indicate a strong association between H-antigen, eae -subtype, and ST in LEE+ isolates. A total of 199 distinct virulence genes were cataloged, of which 16 are shared. The STEC-pathogroups are comprised of H-antigens 6, 12, 16, and 2 and carry the stx 1a,2a,2b,2c,2f suballeles. Correlation between Stx status, H-antigens, and intimin subtypes For STEC, the following stx/eae -genotype profiles were recorded: H6 ( stx 2f , eae-ι ), H12 ( stx 2b , eae (-)), H16 ( stx 1a , eae - β ) and H2 ( stx 1a , eae - ε ; stx 2a , eae - ε ; stx 1a , stx 2c , eae - ε ; stx 1a , stx 2a , eae - ε and stx 1a , stx 2c , eae - ε ) ( 102 ). The best-represented STEC groups in the analyzed strain panel were H16 (n=93) and H2 (n=242). H16 STEC were genetically uniform ( stx 1a , eae - β ) compared to H2 STECs with significant toxicity profile heterogeneity. The dominant Stx-genotype was stx 1a + (93%), followed by isolates with combinations of stx 2a ( stx 1a +stx 2c +, n=11) or stx2c ( stx 1a +stx 2a +, n=5), and stx 2a + only (n=2). Within the dominant H2 STEC, we further identified a distinct stx (-) sublineage. Further analyses of the H2 ΦStx 1a phage insertion site at torS ( Fig. 3 ) detected phage remnants ( Fig. S5 ). We thus speculate that this lineage evolved from ST-17 , stx 1a + ancestors and represents Lost Shiga Toxin (LST) isolates ( 125 ). Their closest relatives were stx 2a + strains that similarly lack stx 1a + . However, the draft status of these genomes hindered further investigation into Φthe Stx insertion locus at torS to evaluate the ancestral or evolved ΦStx-status ( Fig. S5 ). We thus speculate that this STEC lineage may have secondarily acquired stx 2a , essentially transitioning between stx (+/−) states ( 125 , 200 , 201 ). Similarly, the LEE-H12-group contained both stx 2b + and stx- isolates. However, the draft status of the genomes hindered further investigation of the ΦStx status. Except for the H12 STEC, the LEE island is present in all other identified STEC phylogroups (H2, H6, H12, and H16) ( Fig. 7 ). The LEE island is also present enteropathogenic E. coli (EPEC) (H5) and a strain containing genes associated with both enterotoxigenic E. coli (ETEC) and EPEC (H8) ( Fig. 7 ). Over 20 intimins mediating the attachment to intestinal cells are currently described ( 58 , 202 – 206 ). Previous studies indicated a correlation between the H-antigen and the intimin subtype in LEE-positive E. coli ( 207 – 209 ). Indeed, we found a correlation between H-antigens and the five eae- suballeles present in serogroup O118. The intimin ε -subtype is associated with H2 STEC, exemplified by strain EC20017429, and LST strains of this group ( Fig. 7 ). This group also contains eae -negative strain PNUSAE161722, missing integral LEE components and effectors ( Fig. 7 ). Its position in the tree and intimate relationship to ST-17 LEE+ isolates may suggest secondary loss rather than an ancestral LEE-status. The H16-phylogroup features the β -intimin, represented by strains 2014C-3050, 2013C-4538, 12867, and 12089 ( Fig. 4 ). We further observed the following correlations H6- eae-ι , H5- eae - κ , and H8- eae - θ , in the other LEE+ phylogroups, though these were represented by only a few strains. Antimicrobial– and multidrug-resistance loci in serogroup O118 Antimicrobial-resistant and multidrug-resistant (MDR) E. coli O118 STEC strains have been previously reported ( 55 , 124 , 210 ). Consistent with these observations, AR/MDR strains were detected within STEC phylogroups H16 and H2 ( Fig. 8 ), and not in other STEC lineages. Among the resistant O118 STEC, we identified genes conferring resistance to ten antibiotic classes: β-Lactam, Chloramphenicol (CHL), Quinolone (QNL), Tetracycline (Tet), Polymyxin (PMX), Macrolide–Lincosamide–Streptogramin (MLS), Aminoglycoside, Sulfonamide, Trimethoprim, and Rifampin (RIF). Numerous MDR isolates, as defined by resistance to three or more antibiotic classes, were detected. A prominent three-class profile— tetracyclines, aminoglycosides, and sulfonamides—was observed frequently in H16 and H2 STEC, similar to the one observed in the MDRVC-Island of strain 12089 (H16, ST21) ( Fig. 5 ). We also noted that resistance gene presence and combinations followed phylogroup-specific patterns, as evident from the cgMLST-based phylogeny ( Fig. 8 ). However, due to the draft status of most genomes, we were limited in our ability to determine the genomic context, whether these AR loci are chromosomal ( Fig. 5 ), plasmid-borne ( 124 ) or phage-associated ( 13 ) ( Fig. 8 ). Download figure Open in new tab FIG 8 Phylogeny and distribution of antibiotic-resistance genes. Percentage identities for each antibiotic-resistance gene identified in ResFinder are visualized in a heatmap. The strains feature a total of 58 antibiotic-resistance genes that were classified into 10 antibiotic classes. Conclusion Whole-genome sequencing (WGS) typing strategies have significantly advanced our understanding of the genomic makeup and evolution of E. coli pathovars ( 12 , 211 , 212 ). The high-resolution framework established for the E. coli serogroup O118/O151 complex allowed us to define the phylogenomic boundaries and virulence traits of emerging O118 STEC. For partitioning O118 serogroup isolates, their H-antigen was a robust phylogenetic marker ( 213 ), although genetically unrelated E. coli strains may share H-types due to lateral acquisition ( 214 ). Among the delineated STEC O118 phylogroups, we observed substantial plasticity in virulence and resistance inventories, characterized by diverse stx and eae suballeles. The presence or absence of stx was shaped by dynamic ΦStx prophage acquisitions but did not necessarily reflect evolutionary relationships ( 201 ). Notably, we identified a secondary Stx prophage loss event in an stx -negative H2 subgroup branching from ST-17 STEC, suggesting that such losses, followed by potential reacquisition, may lead to transitional shifts in pathogenic potential ( 125 ). Our findings showed a strict correlation between the H-antigen and LEE-borne intimin subtype in STEC (H6/ eae-ι , H16/ eae-β , H2/ eae-ε ) and non-STEC (H5/ eae-κ , H8/ eae-θ ) isolates, reinforcing the hypothesis of evolutionary lineage sorting ( 207 , 215 – 217 ). We identified horizontally acquired pathogenicity-associated islands (PAI) that enhance virulence potential. Notable was the O-island 122 inserted into the LEE-island of clinical H2-strain EC20017429, a locus previously associated with severe disease outcomes ( 173 – 175 ). The discovered multifunctional island in clinical H16-strain 12089 enhances interbacterial competitiveness, virulence, stress adaptation, and may increase the strain’s fitness and pathogenic potential. However, disease outcome is multifactorial, resulting from the complex interplay between the infective agent, the host microbiota ( 218 – 221 ), and the infected individual ( 222 – 225 ), and thus cannot be reliably predicted in silico . The emergence of clinically relevant O118 STEC in both human and animal reservoirs, with the potential to cause severe foodborne disease, highlights the need for ongoing genomic surveillance. Insights into pathogenome plasticity and evolutionary trajectories, gained through comparative genomic approaches, are critical for informing public health strategies, risk assessment, and infection control efforts. Supplemental Figures FIG S1 Phylogenetic relationship inferred from Average Nucleotide Identities. UPGMA-clustered distance matrix of ANI values computed by an all-vs-all comparison of the 359 genomes. FIG S2 IS elements in the closed STEC O118 chromosomes. This heatmap shows the prevalence and distribution of the 726 cataloged IS elements closed H2 and H16 O118 STEC, which can be categorized into 16 known families and 40 clusters. As determined by MLST typing, individual strain relationships are also reflected in IS cluster types and copy numbers. FIG S3 Comparison of closed O118 genome architectures. Mauve-comparisons of chromosomes highlighting genomic rearrangements associated with hot spots for recombination. FIG S4 Pangenome analysis of serogroup O118 . The pangenome was computed with Roary, and the resulting gene presence/absence matrix was visualized in Phandango. The guide tree reflects the cgMLST tree topology, which partitions the isolates by H-type. FIG S5 Comparison of the ΦStx1a-prophages insertion sites in H2 strains. BLASTn-based comparison of the ΦStx 1a prophage insertion sites in H2 stx+/-isolates. The comparisons show that phage remnants were detected in related stx 1a negative phylogroups (H2, stx- ) with the same phage integrase type at torS . Supplemental Tables TABLE S1 Strain-associated metadata and genome statistics TABLE S2 Predicted prophage and mobilome gene content TABLE S3 Predicted virulence and antimicrobial resistance gene content TABLE S4 Sequence Types according to the Achtman MLST schema Supplemental Text Plasmid profiles of serogroup O118 E. coli The 359 analyzed closed and draft genomes identified 36 replicons, which provided a testament to the plasticity of plasmids in this group. Among these were incompatibility group families associated with virulence plasmids and colicins ( Table S2-6 ). The most prevalent replicon in STEC O118 was IncFIB(AP001918) of H2 strains (97.1%, 235/242) and of H16 strains (94.6%, 88/93). These usually 100 kb or larger low-copy plasmids have been associated with disseminating virulence genes in Enterobacteriaceae ( 226 ). Other Inc-types with shared distribution were colicin type Col156, present in H2 (42.6%, 103/242) and H16 (7.5%, 7/93) strains, as well as present in the pO111 plasmid in H16 (29.0%, 27/93) and H2 (14.5%, 35/242) strains. The presence of Col156 is often associated with plasmids that also carry antibiotic resistance genes ( 227 ). Carriage of virulence plasmid pO111 has been linked to severe gastrointestinal disease ( 228 ). Another noteworthy observation was that most STEC H16– and H2-phylogroups feature IncB/O/K/Z_3 groups (95.7% and 90.7%), and IncFII(pCoo) that is present in all H11-strains. Data Availability Statement The sequence data sets generated and analyzed in this study have been deposited in the Sequence Read Archive (SRA) and GenBank at NCBI under BioProject PRJNA1103183 ( https://www.ncbi.nlm.nih.gov/search/all/?term=PRJNA1103183 ). Accessions for genomic reads, assembled annotated chromosomes, and plasmids, along with strain-associated metadata, are provided in Table S1. Author Contributions Conceived and designed the experiments: JMB and ME. Analyzed the data: IR, SSK, JMB, and ME. Drafted the manuscript: ME. Contributed to and edited the manuscript: IR, SKK, JMB, and ME. All authors have read and agreed to the published version of this manuscript. Funding Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number SC1GM135110 to ME and the South Texas Center for Emerging Infectious Diseases (STCEID). Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgments This work received computational support from the High-Performance Computing Cluster (HPCC) operated by Tech Solutions at UTSA. The use of product and company names is necessary to accurately report the methods and results; however, the United States Department of Agriculture (USDA) neither guarantees nor warrants the standard of the products, and the use of names by the USDA implies no approval of the product to the exclusion of others that may also be suitable. The USDA is an equal opportunity provider and employer. We wish to thank Drs. Peter Iwen and Paul Fey of the University of Nebraska Medical Center College of Medicine and the Nebraska State Health Laboratory for providing E. coli strains 12089 and 12867. We would like to acknowledge Mariana Sainz Garcia, Felix Borrego, and Jacob Alford for assistance with data visualization. Bibliography 1. ↵ Stordeur P , China B , Charlier G , Roels S , Mainil J . 2000 . 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