Pathogenomes of Shiga Toxin Positive and Negative Escherichia coli O157:H7 Strains TT12A and TT12B: Comprehensive Phylogenomic Analysis Using Closed Genomes

Shiga toxin-producing Escherichia coli are zoonotic pathogens that cause food-borne human disease. Among these, the O157:H7 serotype has evolved from an enteropathogenic O55:H7 ancestor through the displacement of the somatic gene cluster and recurrent toxigenic conversion by Shiga toxin-converting bacteriophages. However, atypical strains that lack the Shiga toxin, the characteristic virulence hallmark, are circulating in this lineage. For this study, we analyzed the pathogenome and virulence inventories of the stx+ strain, TT12A, isolated from a patient with hemorrhagic colitis, and its respective co-isolated stx− strain, TT12B. Sequencing the genomes to closure proved critical to the cataloguing of subtle strain differentiating sequence and structural polymorphisms at a high-level of phylogenetic accuracy and resolution. Phylogenomic profiling revealed SNP and MLST profiles similar to the near clonal outbreak isolates. Their prophage inventories, however, were notably different. The attenuated atypical non-shigatoxigenic status of TT12B is explained by the absence of both the ΦStx1a- and ΦStx2a-prophages carried by TT12A, and we also recorded further alterations in the non-Stx prophage complement. Phenotypic characterization indicated that culture growth was directly impacted by the strains’ distinct lytic phage complement. Altogether, our phylogenomic and phenotypic analyses show that these intimately related isogenic strains are on divergent Stx(+/stx−) evolutionary paths.

This study analyzes the clinical O157:H7 isolate, TT12, which originated from a patient presenting with hemorrhagic colitis.When grown on selective media, the original study found that the isolates exhibited two distinct colony morphologies, designated as TT12A and TT12B [62].Subsequent stx PCR-interrogation indicated that TT12A and TT12B were Stx(+) and Stx(−), respectively.Further molecular analyses suggested that these strains were isogenic, but the results were not definitive, and not informed by genome sequences.For this study, we investigated the isolates' presumed isogenic status from a whole genome perspective making use of high-resolution comparative genomics techniques.The generation of high-quality closed genomes provided the basis for in-depth phylogenomic comparisons and allowed us to catalogue subtle strain-differentiating sequence and structural polymorphisms, which explain the atypical, non-shigatoxigenic status of strain TT12B.

Bacterial Strains Analyzed in This Study
Strain-associated metadata for TT12A and TT12B, along with other O157:H7 strains investigated in this study, can be found in Supplemental Table S1.The genome of TT12A was sequenced to closure in this study, while the co-isolated TT12B genome was previously sequenced by our group [68].

Genome Sequencing, Assembly, and Annotation
TT12A strain was cultured overnight at 37 • C with shaking at 220 rpm in lysogeny broth (LB) (Thermo Fisher Scientific, Asheville, NC, USA).The culture was then diluted to an 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 genomic DNA (gDNA) was extracted using the QIAamp DNA Mini Kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's instructions.Genomic DNA preparation was subjected to both long-read (Pacific Biosciences, Menlo Park, CA, USA) and short-read (Illumina, San Diego, CA, USA) sequencing.For longread sequencing on the PacBio RS II platform, gDNA was sheared into 20 kb fragments using g-TUBE (Covaris, Inc., Woburn, MA, USA).The library was prepared based on the 20 kb PacBio sample preparation protocol and sequenced using P6/C4 chemistry on four single-molecule real-time (SMRT) cells with a 240 min collection time.The continuous long-read data were de novo assembled using the PacBio hierarchical genome assembly process (HGAP v.3.0) with the default parameters in SMRT Analysis (v.2.3.0),including consensus polishing with Quiver [79].Long-reads were complemented with Illumina short-reads generated on the MiSeq platform.Paired-end libraries were prepared with the NxSeq AmpFREE Low DNA Library Kit (Lucigen, Middleton, WI, USA) with a 250 bp read length and sequenced using the MiSeq Reagent kit (v2) (500-cycle).Sequencing reads in the fastq format were imported into Galaxy [80], and the default software parameters were used for all analysis unless specified otherwise.FastQC (v.0.74 + Galaxy0) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc, accessed on 10 December 2023) and Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/, accessed on 10 December 2023) were used to determine read quality.Illumina reads were utilized for PacBio sequence error correction using Pilon (v.1.23)[81], and read-based SNP discovery as described below.The resulting contigs were evaluated with QUAST (v.5.2.0 + Galaxy1) [82].The chromosomal oriC (http://tubic.tju.edu.cn/Ori-Finder/,accessed on 10 December 2023) [83] 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) [84].

Core Genome SNP Phylogeny
To place strains TT12A and TT12B into their phylogenomic context, a custom-built cgSNP discovery pipeline [68,106,107], implemented on Galaxy [80], was applied.The chromosomal core genome is defined as a set of genic and intragenic regions that are not repeated and do not contain mobile elements, such as phages, genomic islands, IS elements, or plasmids, which evolve at different rates and are not indicative of evolutionary relationships.These regions were determined in the designated reference chromosome of E. coli strain EC4115 (GenBank accession: CP001164) [106] as described above.All mobile genetic elements were excluded from SNP discovery.Illumina reads were used for read-based SNP discovery.The modular pipeline contained the following workflow steps: (i) SNP discovery and typing Illumina reads were used for read-based SNP discovery and aligned to the designated reference with BWA-MEM (Galaxy v.0.7.17.2) [108].The resulting alignments were processed with FreeBayes (Galaxy v.0.4.1.0)[109] with the following threshold settings: mapping quality 30, base quality 30, coverage 10, and allelic frequency 0.75.For contigbased discovery, assemblies were aligned to the O157:H7 strain EC4115 reference molecules using NUCmer [98], followed by SNP prediction with delta-filter (v.4.0.0rc1 + Galaxy2) and show-snps distributed with the MUMmer package (v.4.0.0rc1 + Galaxy2) [98,110].The resulting SNP panel for each of the query genomes was used for further processing; (ii) SNP validation and filtering Catalogued SNPs from each genome were merged into a single SNP panel and SNPs located within the identified excluded regions were removed, as well as low-quality alignments or misalignments, non-uniformly distributed regions, and variant insertions and deletions (InDels), as previously described [106,107,111].SNPs were further curated by extracting the surrounding 40 nucleotides (nt) for each predicted SNP in the reference genome, followed by BLASTn of these fragments against the query genomes.SNPs with missing information ("no hits") or multiple hits were filtered out as well as ambiguous nucleotides; (iii) SNP annotation and chromosomal distribution The functional effects of SNPs were inferred from the reference genome annotation.Identified SNPs were classified into genic or intergenic by mapping the SNPs to the reference genome.The SNP-matrix tables were manipulated with Query Tabular Tool (Galaxy v.3.3.0][112]; (iv) SNP phylogeny The curated panel of high-quality SNPs served as a basis for phylogenetic reconstruction by maximum parsimony with PAUP (v.4.0) [113] with 100,000 bootstrap replicates.The majority-rule consensus SNP tree was visualized in Geneious (v.2022.2) [114] and decorated in iTol (v.6.5.8) [115].Calculation of the consistency index (CI) in Mesquite (v.3.6)[116] for each SNP allowed us to identify parsimony-informative SNPs and flag homoplastic SNPs as previously described [68,106,107,111,117,118].Locally collinear blocks between TT12A and TT12B chromosomes and plasmids were identified with progressive Mauve (v.2.4.1) [119] with the default settings in Geneious (v.2022.2) [114].Subtle sequence disambiguities comprising SNPs and InDels between the respective molecules were identified using "Find Variations/SNPs" in Geneious.A prediction of protein stability changes for single-site mutations was analyzed with MuPro (v.1.0)[120].

Core Genome MLST
The closed genomes of the representative O157:H7 strains (Supplemental Table S1) were imported into SeqSphere+ (v.8.3) (Ridom GmbH, Münster, Germany) for gene-by-gene alignment, allele calling, and comparison [121].A core genome MLST (cgMLST) schema was developed using the closed chromosome of E. coli K-12 substrain, MG1655, (GenBank accession U00096) [122] as a seed and queried against 11 closed genomes representing the 9 distinct O157:H7 phylogenetic clades [68,88,106,107].Core and accessory MLST targets were identified according to the inclusion/exclusion criteria of the SeqSphere+ Target Definer.The allele information from the defined core genome gene of the queried strains was used to establish phylogenetic hypotheses using the minimum-spanning method [123,124] with default settings in Ridom SeqSphere+ (v.8.3).

Bacterial Growth, Phage Induction, and Cell Viability
All experiments were executed with two biological replicates.TT12A and TT12B strains were cultured overnight (o/n) at 37 • C with shaking (220 rpm) in LB medium.Bacterial o/n cultures were diluted to an OD 600 of 0.03 in fresh LB medium and grown at 37 • C with shaking (220 rpm) to early-log phase (OD 600 ~0.3) and then divided into two subcultures, LB and LB + Mitomycin C (MMC).Triggering the RecA-dependent SOS response with MMC constitutes a major pathway of Stx phage induction and mobilization [125].Subculture LB + MMC was supplemented with MMC (Sigma-Aldrich, Saint Louis, MO, USA) at a final concentration of 0.5 µg/mL to mobilize carried prophages, while subculture LB was used to evaluate spontaneous prophage mobilization.Growth curves were recorded in a 96-well plate (Corning 3370, Corning Inc., Corning, NY, USA) at OD 600 on a BioTek Synergy H1 plate reader (BioTek Instruments, Inc., Winooski, VT, USA) for 16 hrs at 10 min intervals to assess prophage-induced bacterial lysis.

Prophage Profiling and Gene Expressions
PCR primer sequences, conditions, and amplicon lengths are provided in Supplemental Table S2.PCR was performed on gDNA preparations using the boiling extraction method [126] for strains TT12A and TT122B, processing three cultures each for the characteristic TT12A-and TT12B-specific morphology [62].To determine the orientation of an inversion in the shared Enterobacteria phage SfI-PP2, primers were designed with the NCBI primer design tool [127].PCR-amplicons were separated on a 1.5% agarose gel at 120V and examined in the GelDoc EZ Gel Imaging System and ImageLab (v.6.1)(BioRad, Hercules, CA, USA).

Mobilization of TT12A-Specific Prophages
To assess TT12A-specific phage mobilization upon MMC-induction, LB and LB + MMC subcultures were grown for 6 hrs at 37 • C with shaking (220 rpm) and then centrifuged at 5000× g for 10 min.Supernatants were filtered through low-protein-binding 0.22 µm pore size membrane filters (Millex-GP; Merck Millipore Ltd., Burlington, MA, USA) and treated with DNase I (Invitrogen, Waltham, MA, USA) for 15 min to remove bacterial gDNA.Phage DNA was extracted from the lysate using the QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA, USA), and eluted with 50 µL nuclease-free water.Phage mobilization was determined by qPCR targeting the phage-borne toxin genes stx1 and stx2 as well as ΦPP10-carried nleL gene on the StepOne Real-Time PCR System software (v 2.3) (Applied Biosystems, Foster City, CA, USA).Statistical significance was determined using Prism (v.9.5.0) (GraphPad Software, San Diego, CA, USA) with two-way ANOVA with Sidak's multiple comparisons test to compare non-induced to MMC-induced conditions.

Gene Expressions
Transcripts were quantified relative to the endogenous tufA gene by RT-qPCR [128].Cultures were grown in LB and LB + MMC for 6 hrs at 37 • C with shaking (220 rpm) then centrifuged (5000× g, 10 min).Cell pellets were used for total RNA purification using the PureLink RNA Mini kit (Invitrogen, Waltham, MA, USA).RNA quantity and quality were measured with the NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).Total RNA was treated with amplification grade DNase I (Invitrogen, Waltham, MA, USA), and reverse transcribed using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA).Targets for qPCR were the toxin genes stx1 and stx2 and the SOS-regulator recA.PCR reactions were performed on the StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using GoTaq qPCR Master Mix (Promega, Madison, WI, USA).Primer sequences, RT-qPCR conditions, and amplicon lengths are provided in Supplemental Table S2.Statistical significance was determined using Prism (v.9.5.0) (GraphPad Software, San Diego, CA, USA) with two-way ANOVA with Sidak's multiple comparisons test to compare results of non-induced to MMC-induced conditions for each strain.

Pathogenome Architectures and Mobilome Inventories of TT12A and TT12B
The proposed isogenic status for the O157:H7 isolates TT12A (stx+) and TT12B (stx−), was originally inferred from the PCR-based interrogation of the stx locus, along with a similar PFGE fragmentation pattern [62].To reassess the isolates' relationship, their closed genomes were subjected to high-resolution whole genome sequence typing (WGST) [107,129].Due to the homogeneity of prophage content and other repeats, E. coli O157:H7 genomes are known to assemble into fragmented drafts when only short-read sequencing technologies are applied [130,131].In response, we used PacBioRS long-read sequencing followed by error correction with Illumina short-reads.The resulting high-quality genomes allowed us to catalogue strain-differentiating sequence and structural polymorphisms at a high degree of phylogenetic resolution.Strain-associated metadata and genome statistics for the chromosomes and carried lineage-specific pO157 virulence plasmids are provided in Supplemental Table S1.Major drivers of the pathogenome diversification in the O157:H7 lineage are mobile genome elements and more prominently, the individual prophage contents [106,107,[132][133][134][135][136].As evident in the chromosome comparison in Figure 1, the TT12A and TT12B backbones are largely conserved and syntenic with an average nucleotide identity (ANI) of 99.99%.In comparison to non-pathogenic E. coli K12-type strains, O157:H7 acquired ΦStx and non-Stx prophages resulting in widespread genetic mosaicism [132,[137][138][139]. Bacteriophages target conserved chromosomal loci and undergo evolutionary acquisition, loss, and dissemination, which collectively shape a strain's individual gene inventory and pathogenicity traits [140][141][142].A substantial proportion of the TT12A and TT12B chromosomes is made up of prophages, accounting for 16% and 13.1%, respectively, of the total chromosomal sequence information.21 phages are shared; however, strain TT12A (5,501,009 bp) is distinguished from strain TT12B (5,335,866 bp) by the presence of three additional phages, ΦStx 1a , ΦStx 2a , and non-Stx Enterobacteria phage ΦBP-4795 prophage.The latter is partly homologous to the 57,930 bp ΦStx 1 -prophage carried by the E. coli O84:H4 strain with an average nucleotide identity of 45.3% [143].The direct evolutionary relationship of these Stx/non-Stx phages is unknown; however, alterations of the stx locus, resulting in confined loss or deletion of the entire phage, has been described in diverse STEC lineages [68].The absence of these three prophages, ΦStx 1a , ΦStx 2a , and non-Stx Enterobacter phage ΦBP-4795, in the strain TT12B results in a 165,143 bp larger genome of strain TT12A (Figure 1, Supplemental Table S3).The three strain-differentiating prophages along with an inversion in the shared ΦPP2 are visualized in their chromosomal context in Figure 2. In analogy to the chromosomes, the carried pO157 plasmids have an ANI of 99.98%, and differ in size by only 3 bp, again indicative of a close phylogenetic relationship of these strains (Figure 3).

Phylogenomic Position of TT12A and TT12B within the O157:H7 Lineage
In silico genotyping classified TT12A Stx(+) and its co-isolated TT12B Stx(−) strains as Sequence Type ST11, Lineage I, Clade 3.12 isolates (Supplemental Table S1) [88,91,121,144].To place the strains into the broader context of O157:H7/NM evolution [51][52][53][54][55][56], we constructed a phylogenetic hypothesis based on reference-based cgSNP discovery including representative O157:H7 strains for the nine distinct clades [68,88,106,107] (Supplemental Table S4).As evident in Figure 4, the overall tree topology reflects the general understanding of O157:H7/NM evolution from an enteropathogenic E. coli (EPEC) O55:H7 progenitor [51][52][53][54][55][56]145].Clade 3.12 strains TT12A, TT12B, and EDL933 form a cluster, and strains TT12A and TT12B were found to be indistinguishable on the chromosomal cgSNP level [62].This intimate relationship is further mirrored in the cgMLST analysis, with no allelic changes observed (Supplemental Figure S1, Supplemental Table S4), and along with the SNP information being consistent with an isogenic status.The strains' intimate relationship is also reflected in the IS element profiles.In total, ISEScan detected 87 IS elements and categorized them into 14 families and 27 clusters (Supplemental Table S3).The strains feature identical chromosomal or plasmid-borne profiles, not considering the two phage-borne IS elements that are part of the TT12A-specific Enterobacteria phage ΦBP-4795-PP10 (Figure 1, Supplemental Table S3), supporting an intimate relationship between these two strains.To build the clade-inclusive cgSNP phylogeny (Figure 4), we excluded SNPs within mobile elements and repeats, as the alignment of homologous regions of different evolutionary origins can introduce false positive signals and ultimately, phylogenetic inaccuracies [106,107].We thus used an alternative approach for these closely related isolates and processed all collinear blocks in their genomes to record the SNPs and InDels.This resulted in the detection of 16 chromosomal intragenic SNPs, all situated within two prophages, while no SNPs were recorded on the pO157 (Supplemental Table S4).In this context, we note that plasticity in the ΦStx-phage complement of O157:H7 and other STEC serotypes is well established [138].Particular variations within the ΦStx 2a -phages have been associated with altered toxin production capabilities [146][147][148][149]. Exploring the effects of such phage-to-phage variants on phage-host interactions or pathogenesis could deepen our understanding of the evolution of O157: H7 virulence.A single nonsynonymous (ns) SNP was detected in the shared ΦPP4-phage, while 15 SNPs were located within a single gene on the carried TT12A-ΦPP13 and TT12B-ΦPP11 prophages that code for the host specificity protein J [150].Though we cannot delineate the physiological effects of these variants, we note that 80% of SNPs are non-synonymous and are predicted to decrease protein stability in TT12B [120] with potential impacts on phage biology.
these two strains.To build the clade-inclusive cgSNP phylogeny (Figure 4), we excluded SNPs within mobile elements and repeats, as the alignment of homologous regions of different evolutionary origins can introduce false positive signals and ultimately, phylogenetic inaccuracies [106,107].We thus used an alternative approach for these closely related isolates and processed all collinear blocks in their genomes to record the SNPs and InDels.This resulted in the detection of 16 chromosomal intragenic SNPs, all situated within two prophages, while no SNPs were recorded on the pO157 (Supplemental Table S4).In this context, we note that plasticity in the ΦStx-phage complement of O157:H7 and other STEC serotypes is well established [138].Particular variations within the ΦStx2a-phages have been associated with altered toxin production capabilities [146][147][148][149]. Exploring the effects of such phage-to-phage variants on phage-host interactions or pathogenesis could deepen our understanding of the evolution of O157: H7 virulence.A single nonsynonymous (ns) SNP was detected in the shared ΦPP4-phage, while 15 SNPs were located within a single gene on the carried TT12A-ΦPP13 and TT12B-ΦPP11 prophages that code for the host specificity protein J [150].Though we cannot delineate the physiological effects of these variants, we note that 80% of SNPs are non-synonymous and are predicted to decrease protein stability in TT12B [120] with potential impacts on phage biology.
In addition, we detected 65 strain-differentiating InDels, 56 of which are chromosomal (Supplemental Table S4, Figures 1 and 3).The majority, 38 chromosomal and 8 plasmid-borne InDels, are located within homopolymer repeats, which are prone to dynamic expansion or shrinkage during short-term evolutionary terms [151].We also note here that 37 of the chromosomal and 1 of the plasmid InDels are associated with mobile genetic elements, including prophages, IS elements, and genomic islands (Supplemental Table S4).Phylogenomic position of TT12A and TT12B within the O157:H7 serotype Genome comparisons of TT12A and TT12B and the representative strains from clades 1 to 9 O157:H7 genomes yielded a total of 2,654 SNPs when referenced to strain EC4115.The tree shown is the majority-consensus tree of three equally parsimonious trees with a CI of 0.998.Trees were recovered using a heuristic search in PAUP with 100,000 bootstrap replicates.The tree is rooted to the clade 9 strain, PA48, visualized and decorated in iTol.Nodes are color-coded according to clade, and numbers of separating SNPs are shown.The prevalence of Stx-subtypes is indicated by black (stx+) and white (stx−) boxes.
In addition, we detected 65 strain-differentiating InDels, 56 of which are chromosomal (Supplemental Table S4, Figures 1 and 3).The majority, 38 chromosomal and 8 plasmidborne InDels, are located within homopolymer repeats, which are prone to dynamic expansion or shrinkage during short-term evolutionary terms [151].We also note here that 37 of the chromosomal and 1 of the plasmid InDels are associated with mobile genetic elements, including prophages, IS elements, and genomic islands (Supplemental Table S4).

Differences in Growth Phenotypes and Impact of the Prophage Inventories
We recorded growth in LB and under phage-mobilizing conditions in LB + MMC to determine the degree of prophage-induced lysis in TT12A and TT12B.Increased expression of recA post MMC treatment confirmed the successful SOS response activation in both cultures (Supplemental Figure S4A) [125,[190][191][192]. Culture growth of TT12A is considerably affected through phage lysis, unlike strain TT12B (Supplemental Figure S4B).The different growth phenotypes in the MMC-treated cultures thus seem to be mediated by the three TT12A-specific ΦStx 1a / 2a /non-Stx phages, all of which are known or predicted to be lytic in the case of Enterobacteria phage ΦBP-4795-PP10 (Figure 2, Supplemental Table S3).Even though another seven lytic phages are predicted as part of the shared phage complement (Supplemental Table S3), these phages are likely not mobilized through the SOS response pathway activation, as evident in the similar growth of TT12B in LB and LB + MMC media (Supplemental Figure S4B).

Conclusions
Whole genome sequence typing has proven to be invaluable for the identification and strain attribution of near clonal E. coli pathogen populations [40,107,193,194].Availability of high-resolution closed genomes allowed us to record subtle strain-level sequence and structural polymorphisms with high phylogenetic accuracy, demonstrating an isogenic relationship of these Stx(+/−) TT12A and TT12B isolates.The number of strain-differentiating SNPs is similar to the range reported for clonal O157:H7 outbreak strains [40,106,107,111,195].However, SNP data on its own are clearly insufficient to infer clonality in microbes without further assessing changes in genome structure and content.Dynamic phage acquisition and loss resulted in the strain-specific ΦStx and non-ΦStx prophage content, which may have occurred in a single or separate evolutionary events likely triggered by the mobilization and ultimately, loss of these phages.STECs have been intentionally cured from their Stx-phages by antibiotic or MMC addition to the growth medium [63,68,196].In this context, it is noteworthy that excised copies of the three TT12A-differentiating prophages ΦStx/non-Stx phages, absent in TT12B, were significantly increased when grown in the phage-inducing LB + MMC media (Supplemental Figure S5).Additionally, none of the insertion sites occupied in TT12A showed scarring that would indicate a former phage presence in TT12B.Alternative evolutionary scenarios can explain the Stx-phage absence in atypical O157:H7 strains, such as TT12B [51,68,197].We can only speculate about the events that gave rise to the TT12A and TT12B variants.Given the fact that these strains originate from a patient suffering from hemorrhagic colitis [62] along with the strains' established intimate phylogenetic relationship, the secondary loss of both ΦStx 1 / 2 prophages along with Enterobacteria phage ΦBP-4795-PP10 in TT12B during the course of infection, in a singular or in multiple events, seems likely.The ratio of Stx(+) to non-shigatoxigenic isolates is not known; however, dynamic loss of Stx-phages and potential re-acquisition may cause transitional stx(+/−) shifts in pathogenic potential [68,69,74].Bacteriophages control diverse bacterial biological functions.Stx has a dual role in human disease and spontaneous low-level Stx production is considered a form of bacterial altruism, promoting the toxin-dependent killing of eukaryotic predators and macrophages [29,[198][199][200][201][202][203][204][205].The ΦStx phage carriage has been associated with a number of virulence and fitness traits beyond Stx-production.The characterization of laboratory-engineered Stx-lysogens, often recovered in E. coli K12 backgrounds, indicated an impact on acid resistance, type III secretion, motility, and metabolism [206][207][208][209][210][211][212][213][214][215].The analyzed Stx(+/−) isogen cultures and genomes provide an excellent model to further investigate the impact of ΦStx-phage carriage in a native O157:H7 genome background that is not accounted for in the K12-engineered Stx-lysogens [206,208,210,215].Altogether, the genomic and phenotypic comparisons show that this isogen pair is intimately related, yet perhaps on a divergent evolutionary path.The role of the catalogued sequence and architectural polymorphisms, however, cannot be simply inferred from static genome comparison.Further investigations using transcriptomic and phenotypic profiling may provide greater insight into the role these variants may play in the physiology and pathogenicity of strains TT12A and TT12B.

Microorganisms 2024 , 23 Figure 1 .
Figure 1.Chromosome comparison of TT12A and TT12B BRIG comparison of the Stx(+) TT12A and Stx(−) TT12B genome architecture and gene content referenced to the larger Stx(+) genome.The comparison is further extended to include the EDL933 strain, clade 3.12, and the K-12 E. coli strain.CDSs are presented as arrows on the +/− strands, and functional annotations for the shared and three TT12A-specific prophages, virulence, and resistance genes, along with polymorphisms differentiating the two strains, are highlighted as shown in the legend.Chromosomal synteny in K-12 is disrupted by multiple prophages and other MGEs.

Figure 1 .
Figure 1.Chromosome comparison of TT12A and TT12B BRIG comparison of the Stx(+) TT12A and Stx(−) TT12B genome architecture and gene content referenced to the larger Stx(+) genome.The comparison is further extended to include the EDL933 strain, clade 3.12, and the K-12 E. coli strain.CDSs are presented as arrows on the +/− strands, and functional annotations for the shared and three TT12A-specific prophages, virulence, and resistance genes, along with polymorphisms differentiating the two strains, are highlighted as shown in the legend.Chromosomal synteny in K-12 is disrupted by multiple prophages and other MGEs.

Figure 2 .
Figure 2. Prophages and Stx-status of TT12A and TT12B BLASTn-based comparison of the prophage inventory and polymorphisms visualized in Easyfig.Strains are differentiated by the presence of three additional ΦStx-and Φnon-Stx prophages in the larger (stx+) TT12A strain at the following loci: (A) ΦStx2a-phage at wrbA: This locus is unoccupied in TT12B, while in strain A, a 63,250 kb ΦStx2a-prophage is inserted at wrbA, a preferred target locus for ΦStx2a-phage insertion in O157:H7.(B) ΦStx1a-phage at yehV: This locus is unoccupied in TT12B, while a 53,637 kb ΦStx1-prophage is inserted at yehV, a preferred target locus for ΦStx1a-phage insertion in O157:H7.(C) ΦPP10-Enterobacteria phage BP-4795 at potC: This locus is unoccupied in TT12B, while a 49,073 kb prophage is inserted at potC, a known target for phage insertion in O157:H7.(D) ΦPP2-Enterobacteria SfI phage: Chromosome assemblies feature an inversion within the shared ΦPP2-Enterobacteria SfI prophage.

Figure 2 .
Figure 2. Prophages and Stx-status of TT12A and TT12B BLASTn-based comparison of the prophage inventory and polymorphisms visualized in Easyfig.Strains are differentiated by the presence of three additional ΦStxand Φnon-Stx prophages in the larger (stx+) TT12A strain at the following loci: (A) ΦStx 2a -phage at wrbA: This locus is unoccupied in TT12B, while in strain A, a 63,250 kb ΦStx 2a -prophage is inserted at wrbA, a preferred target locus for ΦStx 2a -phage insertion in O157:H7.(B) ΦStx 1a -phage at yehV: This locus is unoccupied in TT12B, while a 53,637 kb ΦStx 1 -prophage is inserted at yehV, a preferred target locus for ΦStx 1a -phage insertion in O157:H7.(C) ΦPP10-Enterobacteria phage BP-4795 at potC: This locus is unoccupied in TT12B, while a 49,073 kb prophage is inserted at potC, a known target for phage insertion in O157:H7.(D) ΦPP2-Enterobacteria SfI phage: Chromosome assemblies feature an inversion within the shared ΦPP2-Enterobacteria SfI prophage.

Figure 3 .
Figure 3.Comparison of clade 3 pO157 plasmids BRIG comparison of the pO157 plasmid architecture and gene content.CDSs are presented as arrows on the +/− strands, and functional annotations for notable virulence determinants, such as EHEC hemolysin (hlyCABD), the serine protease (espP) along with plasmid differentiating polymorphisms, including SNPs and InDels, are highlighted as shown in the legend.

Figure 3 .
Figure 3.Comparison of clade 3 pO157 plasmids BRIG comparison of the pO157 plasmid architecture and gene content.CDSs are presented as arrows on the +/− strands, and functional annotations for notable virulence determinants, such as EHEC hemolysin (hlyCABD), the serine protease (espP) along with plasmid differentiating polymorphisms, including SNPs and InDels, are highlighted as shown in the legend.

Figure 4 .
Figure 4. Phylogenomic position of TT12A and TT12B within the O157:H7 serotype Genome comparisons of TT12A and TT12B and the representative strains from clades 1 to 9 O157:H7 genomes yielded a total of 2,654 SNPs when referenced to strain EC4115.The tree shown is the majorityconsensus tree of three equally parsimonious trees with a CI of 0.998.Trees were recovered using a heuristic search in PAUP with 100,000 bootstrap replicates.The tree is rooted to the clade 9 strain, PA48, visualized and decorated in iTol.Nodes are color-coded according to clade, and numbers of separating SNPs are shown.The prevalence of Stx-subtypes is indicated by black (stx+) and white (stx−) boxes.

Figure 4 .
Figure 4. Phylogenomic position of TT12A and TT12B within the O157:H7 serotype Genome comparisons of TT12A and TT12B and the representative strains from clades 1 to 9 O157:H7 genomes yielded a total of 2,654 SNPs when referenced to strain EC4115.The tree shown is the majority-consensus tree of three equally parsimonious trees with a CI of 0.998.Trees were recovered using a heuristic search in PAUP with 100,000 bootstrap replicates.The tree is rooted to the clade 9 strain, PA48, visualized and decorated in iTol.Nodes are color-coded according to clade, and numbers of separating SNPs are shown.The prevalence of Stx-subtypes is indicated by black (stx+) and white (stx−) boxes.

Figure 5 .
Figure 5. Virulence determinants in TT12A and TT12B A heatmap visualizing the percentage identities for each virulence determinant identified in TT12A and TT12B.Apart from Stx-phage contributed toxins and nleL, espN, and espK genes on ΦPP10 Enterobacteria phage BP-4795 only present in TT12A, the strains' virulence profiles are similar.

Figure 5 .
Figure 5. Virulence determinants in TT12A and TT12B A heatmap visualizing the percentage identities for each virulence determinant identified in TT12A and TT12B.Apart from Stx-phage contributed toxins and nleL, espN, and espK genes on ΦPP10 Enterobacteria phage BP-4795 only present in TT12A, the strains' virulence profiles are similar.