Pathogenomes and virulence profiles of representative big six non-O157 serogroup Shiga toxin-producing Escherichia coli

Shiga toxin (Stx)-producing Escherichia coli (STEC) of non-O157:H7 serotypes are responsible for global and widespread human food-borne disease. Among these serogroups, O26, O45, O103, O111, O121, and O145 account for the majority of clinical infections and are colloquially referred to as the “Big Six.” The “Big Six” strain panel we sequenced and analyzed in this study are reference type cultures comprised of six strains representing each of the non-O157 STEC serogroups curated and distributed by the American Type Culture Collection (ATCC) as a resource to the research community under panel number ATCC MP-9. The application of long- and short-read hybrid sequencing yielded closed chromosomes and a total of 14 plasmids of diverse functions. Through high-resolution comparative phylogenomics, we cataloged the shared and strain-specific virulence and resistance gene content and established the close relationship of serogroup O26 and O103 strains featuring flagellar H-type 11. Virulence phenotyping revealed statistically significant differences in the Stx-production capabilities that we found to be correlated to the strain’s individual stx-status. Among the carried Stx1a, Stx2a, and Stx2d phages, the Stx2a phage is by far the most responsive upon RecA-mediated phage mobilization, and in consequence, stx2a + isolates produced the highest-level of toxin in this panel. The availability of high-quality closed genomes for this “Big Six” reference set, including carried plasmids, along with the recorded genomic virulence profiles and Stx-production phenotypes will provide a valuable foundation to further explore the plasticity in evolutionary trajectories in these emerging non-O157 STEC lineages, which are major culprits of human food-borne disease.


Introduction
Shiga toxin (Stx)-producing Escherichia coli (STEC) are distinguished from other E. coli pathovars (Kaper et al., 2004) by the production of a phage-borne cytotoxin (Smith et al., 2014;Krüger and Lucchesi, 2015;Zuppi et al., 2020) that is toxigenic toward renal endothelial (Obrig and Karpman, 2012) and intestinal epithelial cells (Schüller, 2011).Escherichia coli are historically classified by their variation in somatic O-and flagellar H-antigens (Orskov et al., 1977).Serotype O157:H7 is the dominant causative agent of STEC disease in the U.S. (Riley et al., 1983;Eppinger et al., 2011Eppinger et al., , 2013;;Sanjar et al., 2014;Rusconi et al., 2016).However, the incidence of non-O157 infections that feature different somatic antigens has been steadily increasing in recent years (Gould et al., 2013;Vishram et al., 2021;Glassman et al., 2022;Tarr et al., 2023).Among these, emerging serogroups O26, O45, O103, O111, O121, and O145 account for the majority of clinical non-O157 STEC infections in the US and are colloquially referred to as the "Big Six" (Eklund et al., 2001;Johnson et al., 2006;Bettelheim, 2007;Hadler et al., 2011;Hegde et al., 2012;Gould et al., 2013;Vishram et al., 2021).Disease in humans can progress to life-threatening complications, such as hemolytic uremic syndrome (HUS) and ultimately renal failure (Karmali et al., 1983;Majowicz et al., 2014).The disease has been linked to the amount and subtype of toxin produced (Donohue-Rolfe et al., 2000;Russo et al., 2016).STEC can harbor one or multiple Stx-bacteriophages featuring different combinations of stx-suballeles (Krüger and Lucchesi, 2015;Rusconi et al., 2016) that can also form hybrid toxins (Skinner et al., 2014).The most potent cytopathic toxins, Stx 2a and Stx 2d (Fuller et al., 2011;Hauser et al., 2020;McNichol et al., 2021), are prevalent in the Big Six serogroups (Jinnerot et al., 2020), and a strain's Stx-status is shaped by the dynamic Stx-phage acquisition, rather than by a common evolutionary history (Cowley et al., 2019;Nyong et al., 2020).Mobilization of Stx-prophages is triggered by diverse abiotic and biotic cues (Pacheco et al., 2012;Pacheco and Sperandio, 2012), and is required to produce toxin causing adverse toxigenic effects in murine STEC models (Nguyen and Sperandio, 2012;Tyler et al., 2013;Baumler and Sperandio, 2016;Balasubramanian et al., 2019;Rodríguez-Rubio et al., 2021).Triggering the RecA-dependent SOS-response with sublethal doses of mitomycin C (MMC) constitutes a major pathway of Stx − phage mobilization and is routinely used in public health laboratories to assess the pathogenic potential (Kimmitt et al., 2000).Besides Stx, another major virulence determinant is the locus of enterocyte effacement (LEE) packaged into a pathogenicity island, which encodes a type III secretion system (T3SS) along with its associated effectors, the outer membrane adhesin intimin (eae) and the translocated receptor (tir; Franzin and Sircili, 2015).The majority of the Big Six serogroups also carry serogroup-specific virulence plasmids along with an diverse array of additional plasmids (Caprioli et al., 2005;Ogura et al., 2009).In this study, we report the complete genomes and comprehensive analyses of the pathogenome composition along with Stx-production pathotypes of a Big Six reference strain panel representing each of the non-O157 STEC serogroups curated and distributed by the American Type Culture Collection (ATCC).The gathered pathogen information and recorded virulence traits provide a foundation to further elucidate the make-up and the evolutionary boundaries of these emerging non-O157 STEC.

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, United States).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 Qiagen Genomic-tip 100/G Kit (Qiagen, Inc., Valencia, CA, United States) according to the manufacturer's instructions.Genomic DNA was subjected to both long-read (Oxford Nanopore, Oxford, United Kingdom) and short-read (Illumina, Inc., San Diego, CA, United States) sequencing.For long-read Nanopore sequencing, gDNA was diluted to a concentration of 1.5 μg in 46 μL of nucleasefree water.The library was prepared using the Ligation Sequencing Kit (SQK-LSK109) with the Native Barcoding Expansion 1-12 (EXP-NBD104) according to the manufacturer's instructions and sequenced on a MinION with the R10.3 SpotON Flow Cell (FLO-MIN111).Paired-end short-read libraries were prepared with the Illumina Nextera XT DNA Library Preparation Kit and sequenced on the MiSeq platform using the MiSeq reagent Kit (v3) with 600-cycles.Sequence reads in the fastq format were imported into Galaxy v.22.05 (Community, 2022).Default parameters were used for all software unless specified otherwise.Quality control of fastq files was assessed using FastQC (v.0.74 + Galaxy0).2Nanopore and Illumina reads were used for hybrid assembly using Unicycler assembler (v.0.5.0 + Galaxy1; Wick et al., 2017).The chromosomal dnaA and plasmid repA genes, if applicable, were designated as the zero point of the closed molecules prior to annotation using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP; Tatusova et al., 2016).

Growth of cultures in LB and under phage mobilizing condition in LB + MMC
Strains were cultured overnight (o/n) at 37°C with shaking at 220 rpm in LB.Overnight LB cultures were diluted to an OD 600 of 0.03 in fresh LB media, grown to early-log phase (OD 600 ~0.3) at 37°C, and then subdivided into two subcultures, LB and LB + MMC.Triggering the RecA-dependent SOS-response with MMC constitutes a major pathway of Stx − phage mobilization (Kimmitt et al., 2000).Subculture LB + MMC was supplemented with MMC (Sigma-Aldrich, Saint Louis, MO, United States) at a final concentration of 0.5 μg/mL to mobilize the carried prophages, while subculture LB was used to evaluate spontaneous prophage mobilization.To confirm phage mobilization in MMC-treated cultures, growth curves were recorded in a 96-well plate (Corning 3,370, Corning Inc., Corning, NY, United States) on a BioTek Synergy H1 plate reader (BioTek Instruments, Inc., Winooski, VT, United States) recording OD 600 values for 6 h at 10 min intervals.All experiments were executed in two biological replicates.

PCR experiments
Primers and PCR-conditions are provided in Supplementary Table S3.LB and LB + MMC subcultures were grown for 6 h at 37°C with shaking at 220 rpm and then centrifuged at 5,000 g for 10 min: (1) Cell pellets were used to determine stxtranscripts levels, while (2) the supernatants were used to enumerate ΦStx-phage copies, targeting the phage-borne stx loci as follows: (1) Expression of stx genes RNA was purified using the PureLink RNA Mini kit (Invitrogen, Waltham, MA, United States), and RNA quantity and quality were measured with the NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States).Total RNA was treated with amplification grade DNase I (Invitrogen, Waltham, MA, United States), and reverse transcribed using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, United States).The stx-RT-qPCR was performed on the StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, United States) using the GoTaq qPCR Master Mix (Promega, Madison, WI, United States).( 2) Enumeration of ΦStx 1 -and ΦStx 2phage copies Supernatants were filtered through low-proteinbinding 0.22-μm-pore-size membrane filters (Millex-GP; Merck Millipore Ltd., Burlington, MA, United States), followed by DNase I (Invitrogen, Waltham, MA, United States) treatment for 15 min to remove bacterial gDNA.Lysate phage DNA was isolated using the QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA, United States), and eluted with 50 μL nuclease-free water.Phage numbers were determined by stx-qPCR on the StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, United States) using the GoTaq qPCR Master Mix (Promega, Madison, WI, United States).Standard curves for the stx transcripts and ΦStx-phage copy numbers were calculated using gBlocks (Integrated DNA Technologies (IDT), Coralville, Iowa, United States) in the RT-qPCR and qPCR experiments, respectively.

Stx-production pathotypes
The Stx-production phenotypes of the cultures were determined by recording the Stx titers through Enzyme-Linked ImmunoSorbent Assay (ELISA) under both spontaneous and MMC-induced conditions.Overnight (o/n) cultures were diluted to an OD 600 of 0.03 and grown to early-log phase (OD 600 ~0.3) in replenished LB media at 37°C.At this stage, cultures were split and incubated at 37°C for 6 h under non-induced and induced (0.5 μg/mL MMC) conditions.Toxin production was measured after harvesting 5 mL of each culture for parallel processing.To lyse bacterial cells and release produced Stx, cultures were treated with polymyxin B (Sigma-Aldrich, Saint Louis, MO, United States; 6 mg/mL 37°C, 10 min).Supernatants were collected after centrifugation (3,500 rpm, 10 min), filtered through 0.22 μm low protein-binding membrane filters (Millex-GP; Merck Millipore Ltd., Burlington, MA, United States) and diluted to measurable concentrations.Stx-production was measured using the Premier EHEC kit (Meridian Bioscience, Cincinnati, OH, United States) following the manufacturer's instructions.Titers were calculated using a standard curve generated from serial dilutions of purified Stx 2a (BEI Resources, NR-4478).Statistical significance was determined using Prism (v.9.5.0;GraphPad Software, San Diego, CA, United States).A two-way ANOVA with Sidak's multiple comparisons test was used to compare non-induced to MMC-induced conditions for each strain.Strain-tostrain comparisons were performed with a one-way ANOVA with Tukey's multiple comparisons test assessing each condition.

Pathogenome composition of MP-9 panel strains
In this study, we sequenced and comprehensively analyzed the pathogenomes and virulence traits of six non-O157 STEC strains.Strain panel MP-9 was obtained from ATCC, which is comprised of six strains representing each of the non-O157 STEC serogroups, colloquially referred to as the "Big Six" (Eklund et al., 2001;Johnson et al., 2006;Bettelheim, 2007;Hadler et al., 2011;Hegde et al., 2012;Gould et al., 2013;Vishram et al., 2021; Supplementary Table S1).STEC genomes house an extensive and partly repetitive phage complement that hampers assembly into closed genomes (Goldstein et al., 2019;Jaudou et al., 2022).In response, we applied a long-and short read sequencing hybrid strategy (Nyong et al., 2020;Allué-Guardia et al., 2022) that allowed us to provide the high-quality closed genomes, including carried plasmids (Figures 1, 2; Supplementary Figures S1, S2).The chromosomes have an average nucleotide identity of 98.8%, with a range from 97.6% to 99.8%, indicative of the substantial conserved chromosomal backbone of E. coli (Rasko et al., 2008;Jain et al., 2018).The chromosome size in this panel ranges from 5,288,508 to 5,840,137 bp with an average GC-content of 50.65%.When compared to non-pathogenic E. coli strain K-12 substrain MG1665, these STEC strains carry at least 648,833 bp of additional genetic information.Genome statistics along with strain-associated metadata are provided in Supplementary Table S1.
In Figure 1, we compared the chromosomes using strain BAA-2196 (O26:H11) as the designated reference.In comparison to E. coli strain K-12, the Big Six strains acquired multiple mobile genome elements (MGE), including the hallmark ΦStx-prophages, which are major contributors of STEC genome evolution and diversification (Lawrence and Ochman, 1998;Rasko et al., 2008;Robins-Browne et al., 2016).Individual comparisons referenced to each of the strains can be found in Supplementary Figure S1.The mobilome on the chromosomes consisting of prophages, genomic islands, and IS elements contributes 22.4% to 28.7% of sequence information, in line with the assessment in other STEC (Perna et al., 2001;Delannoy et al., 2017; Supplementary Table S4).Neither chromosomal nor plasmid-borne integrons were detected.The prophages account for 13.9 to 21.2% of the chromosome, followed by genomic islands (5.4 to 6.9%), and IS elements (0.8 to 2.5%).If plasmid-carried IS elements are considered, the percentage of IS elements increases by 1.2 to 2.7%.The IS elements in this panel showed variations in both prevalence and numbers (Supplementary Table S4).ISEScan detected 726 IS elements and categorized them into 16 known families and 40 clusters, indicative of the plasticity present in these non-O157 STEC (Supplementary Figure S3).Eight of the 40 clusters were present in the six isolates, though their respective numbers between the strains vary considerably.We further note that BAA-2196 (O26:H11) and BAA-2215 (O103:H11) strains feature similar copy numbers in shared IS clusters distinct from the remainder of strains indicative of their close relationship (Iguchi et al., 2012;Ju et al., 2012;Supplementary Figure S3; Supplementary Table S4).Thirteen elements of the IS3-168 cluster were found in each of BAA-2196 (O26:H11) and BAA-2215 (O103:H11), compared to an average of 55 copies in other strains.Inversely, the IS66-46 cluster was found to have 48 and 36 copies in BAA-2196 and BAA-2215, respectively, while other strains carry an average of eight copies.Further, eight clusters are strainspecific, and 24 clusters are present in a subset of strains.This may suggest different dynamics in the propagation of these elements.

Phylogenomic relatedness of ATCC MP-9 strains
The mobilome is comprised of prophages, genomic islands, IS elements, and plasmids, which evolve at different rates and can be acquired and secondarily lost and thus are often not indicative of evolutionary relationships.To investigate the phylogenomic boundaries of the individual strains, we established a phylogenomic framework inferred from targeted MLST and core genome MLST (cgMLST; Figure 3).As expected for this heterogenous set of serotypes, the strains belong to distinct STs with a total of 14,340 allelic changes and 926 InDels (Figure 3; Supplementary Table S5).Their shared inventory was computed at 4,304 genes comprised of 3,148 core and 908 accessory loci, indicative of the extended conserved E. coli backbone (Abu-Ali et al., 2009;Lim et al., 2010;Eppinger et al., 2011;Yin et al., 2015).High-resolution core genome MLST typing revealed a close phylogenetic relationship of serogroup O26:H11 and O103:H11 strains, as previously suggested by MLST-and genomewide single nucleotide polymorphisms (SNPs)-based analyses for these serogroups carrying flagellar antigens H2 and H11 (Iguchi et al., 2012;Ju et al., 2012).ST-21 (BAA-2196 O26:H11) and ST-723 (BAA2215 O103:H11) are only separated in their fumC allele and 357 allelic changes in the cgMLST analysis (Figure 3).This intimate relationship is reflected in the isolates' shared chromosomal and mobilome inventories, such as virulence genes, prophages, and LEE island organization, as discussed below.

FIGURE 5
Prevalence and distribution of chromosomal virulence determinants Percentage identities for each virulence gene identified in VFDB are visualized in a heatmap.The panel strains encode a total of 149 distinct virulence genes of which 113 are shared.Among these are toxin suballeles stx 1a , stx 2a , and stx 2d , the LEE genomic island, and siderophores, among others.The strain order reflects the inferred phylogenomic relationships.The hierarchical clustering of virulence genes based on their pair-wise distance is shown on the left.-Rubio et al., 2021;Fagerlund et al., 2022;Zhang et al., 2022;Yano et al., 2023).In total, seven chromosomal sites are occupied (Supplementary Table S4), some of which are known ΦStx-phage targets (Serra-Moreno et al., 2007;Eppinger et al., 2011;Bonanno et al., 2015;Rusconi et al., 2016;Allué-Guardia et al., 2022).The ΦStx-phage integrases have undergone evolution that allows them to target distinct insertion sites.Stx-phages tend to primarily integrate at a specific site; however, the integrase demonstrates the capacity to detect alternate insertion sites for integration if the preferred site is already occupied or absent (Groth and Calos, 2004;Serra-Moreno et al., 2007;Casjens and Hendrix, 2015;Henderson et al., 2021).ΦStx 2a phages are inserted into either arginine tRNA argW or NAD(P) H dehydrogenase wrbA, and the ΦStx 1a phage, in analogy to some ΦStx 2a in wrbA, or alternatively in peptide chain release factor prfC, outer membrane protein ompW, the tRNA-dihydrouridine synthase dusA, or tmRNA ssrA, while the ΦStx 2d phage is disrupting the spermidine uptake gene potC (Figure 6).As evident in the occupation status of wrbA by either ΦStx 1 or ΦStx 2 , there is no association between toxin suballele and insertion sites in line with previous observation (Groth and Calos, 2004;Serra-Moreno et al., 2007;Steyert et al., 2012;Henderson et al., 2021).

Comparison of Stx-virulence pathotypes
The actual disease outcome cannot be predicted from in silico virulence profiling, considering the complex interactions between infective agent, the host microbiota (Pruimboom-Brees et al., 2000;Gamage et al., 2006;Nguyen and Sperandio, 2012), and the infected patient (Wong et al., 2000;Dundas et al., 2001;Gould et al., 2009;Foster, 2013).Induction efficiency of the Stx-phages is positively correlated to Stx-production (Muniesa et al., 2004;Loś et al., 2009;Del Cogliano et al., 2018) and thus mobilization of Stx-phages is used as a means to assess the conferred pathogenic potential (Karch et al., 1999;Eppinger et al., 2022;Miyata et al., 2023).For the panel cultures, we recorded Stx-production traits under non-induced culture growth in LB and under phage mobilizing conditions by adding sublethal doses of MMC to the standard LB medium (Figure 8).In all cultures, toxin production was significantly elevated when grown in phageinducing LB + MMC media.LB titers were undistinguishable between the cultures.In contrast, we observed culture-level differences in Stx-production capabilities upon MMC treatment.More specifically, we noted a correlation of Stx-levels to the respective stx status patterns of the strains.The class of ΦStx 2a phages carry a highly potent cytotoxin (Fuller et al., 2011;Hauser et al., 2020;Pinto et al., 2021) and are known to mobilize upon activation of the SOS-response (Bonanno et al., 2016;Zhang et al., 2019;Eppinger et al., 2022).In consequence, the Stx titers of the three stx 2a + isolates were all found exacerbated (Figure 8).Strain BAA-2219 (O121:H19), carrying only stx 2a , is the highest-level producer followed by stx 1a /stx 2a + strains BAA-2440 (O111:H8) and BAA-2196 (O26:H11).Significantly lower and comparable titers were found in the remainder of strains: stx 1 strains BAA-2215 (O103:H11) and BAA-2193 (O45:H2), as well as stx 1a/ stx 2d strain BAA-2192 (O145:NM).One caveat using this methodology is that it cannot distinguish between the contribution of individual ΦStx-phages to the overall Stx titer (Skinner et al., 2014).For this reason, we further investigated the mobilization of individual ΦStxphages and resulting stx expression in the three strains that co-harbor ΦStx 1a , ΦStx 2a , and ΦStx 2d phages (Supplementary Figure S4).Both phages carried by stx 1a/ stx 2a + strains BAA-2196 (O26:H11) and BAA-2440 (O111:H8) respond to MMC treatment (Supplementary Figure S4).In the latter, ΦStx 2a copies and stx 2a transcripts exceed the respective ΦStx 1a numbers in both media, while in strain BAA-2196 the stx 1a and stx 2a transcript copies are comparable under non-induced growth in LB.In contrast, only the ΦStx 1a phage is significantly mobilized in stx 1a/ stx 2d + strain BAA-2192 (O145:NM), and in consequence stx 1a transcripts surpass stx 2d copies upon MMC induction, while stx 2d copies are more abundant under non-induced growth in LB.Our observations suggest a considerable heterogeneity in ΦStx-phage mobilization, even within the same ΦStx-phage subtype (Muniesa et al., 2004;Yano et al., 2023).Overall, we observed a positive correlation between phage mobilization, toxin transcript levels, and produced titers (Figure 8; Supplementary Figure S4); in analogy to other studies (de Sablet et al., 2008;Bielaszewska et al., 2012).

Discussion and conclusions
Non-O157 STEC are a heterogenous group of isolates.The clinically most relevant serogroups, O26, O103, O111, O45, O121, Comparison of LEE islands The complete LEE islands were extracted and compared in GeneSpy.Genes are colored according to their nucleotide homologies.The organization of the LEE1 to 5 operons is indicated above.The order of strains reflects their inferred phylogenomic position and is mirrored in the LEE island organization.and O145, are colloquially referred to as the "Big Six" due to the rising incidence of human infections.Integration of genome and virulence information for these emerging lineages is critical to improve risk assessment, biosurveillance, and prevention strategies (Franz et al., 2014;Eppinger and Cebula, 2015;Sadiq et al., 2015;Rusconi and Eppinger, 2016).Our study of these ATCC reference type cultures, comprised of six strains representing each of the non-O157 Big Six serogroups, can only provide a glimpse into the genome composition and virulence features.Our future efforts are directed to profile larger strain sets, anchored by the here presented genomes, in an attempt to capture the extent of plasticity found in the emerging human pathogenic Big Six serogroups.Comprehensive analyses of this panel highlight the distinct ΦStx-phage subtypes and their dissimilar phage mobilization patterns, likely associated with the plasticity of regulator regions relevant for replication (Ogura et al., 2015;Llarena et al., 2021;Allué-Guardia et al., 2022;Fagerlund et al., 2022), and intimately linked to Stx-production and Stx-conferred virulence.The different plasmid types and gene contents, including colicin types E3 and D and several antibiotics resistance determinants, provide only a glimpse into the genomic plasticity that can be found in this heterogenous panel of non-O157 STECs (Cortimiglia et al., 2020).Production of colicins and antibiotic resistance are major drivers of microbial evolution (Feldgarden and Riley, 1999;Leekitcharoenphon et al., 2021).Fitness effects mediated by colicins and antibiotic resistance determinants will impact a strain's individual evolutionary trajectory, and we note that antibiotic resistance and thus pathogenic potential among all STEC serogroups has increased over time and calls for enhanced biosurveillance (Mukherjee et al., 2021).The availability of closed high-quality genomes and carried plasmids of representative Big Six strains, along with insight into their pathogenome make-up and Stx-virulence pathotypes provides a foundation for the research community to broadly explore common and lineage-specific characteristics and evolutionary trajectories of these globally emerging human pathogenic non-O157 STEC lineages.

Data availability statement
The datasets presented in this study can be found in online repositories.The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.Variability in Stx-production The concentration of Stx 1 and Stx 2 produced under non-induced and MMC-induced phage mobilizing conditions was quantified by ELISA.Differences between the non-induced and MMC-induced phage mobilizing conditions for each strain were assessed using a twoway ANOVA, followed by Sidak's multiple comparisons.Statistical significance is denoted as *p < 0.05; **p < 0.005; ***p < 0.0005; and ****p < 0.00005.For strain-strain comparison under MMC-induced conditions, differences in toxin concentration are indicated by letters (a-d), with "a" denoting the highest concentration group, in a descending order determined by a one-way ANOVA with Tukey's multiple comparisons test.

FIGURE 1
FIGURE 1Comparison of ATCC MP-9 panel genomes BRIG comparison of six sequenced strains, along with Escherichia coli strains O157:H7 EC4115 and strain K-12 substrain MG1665, referenced to the 5,840,137 bp chromosome of BAA-2196 (O26:H11).CDS are presented on the +/−strands as blue arrows and functional annotations for virulence genes and other loci of interest are highlighted as shown in the legend.Query genomes are color-coded, and the order plotted in the circle reflects the inferred phylogenomic relationships.

FIGURE 2
FIGURE 2Comparison of a shared colicin plasmid BRIG comparison of a shared colicinogenic plasmid l present in serotypes O26:H11, O103:H11, and O111:H8 and referenced to the 6,673 bp plasmids of BAA-2196 pCol-O26-3.The plasmids are differentiated by a total of 30 SNPs and InDels.CDS are presented on the +/− strands as blue arrows.Query plasmids are plotted according to the strain's inferred phylogenomic relationships.

FIGURE 3
FIGURE 3Phylogenomic position of ATCC-MP9 strains The relatedness of panel strains, including O157:H7 strain EC4115, was determined using MLST in Ridom SeqSphere+: (A) targeted seven-gene MLST accessed in EnteroBase.Numbers on connecting branches indicate the number of genes with differing allele status, and (B) cgMLST-based phylogeny using the closed chromosome of E. coli strain K12 subst.MG1655 as seed.The shared gene inventory was determined at 4,304 genes, according to the inclusion/exclusion criteria of the SeqSphere+ Target Definer and is comprised of 3,148 core and 908 accessory loci.Colors denote ST-classifications established in (A).

TABLE 1
Molecules and accessions.