Characterization and comparative genome analysis of a new Tequatrovirus phage infecting Escherichia coli ST131

Characterization and comparative genome analysis of a new Tequatrovirus phage infecting Escherichia coli ST131 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Characterization and comparative genome analysis of a new Tequatrovirus phage infecting Escherichia coli ST131 Elsa Beurrier, Floriane Laumay, Juliette Hayer, Jacques Dainat, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7535326/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 20 You are reading this latest preprint version Abstract Objective The global rise of antimicrobial resistance with the spread of pathogenic multidrug-resistant clones such as Escherichia coli sequence type (ST) 131 represents a major challenge for public health, renewing interest in bacteriophages as therapeutical agents. Results We report the isolation and characterization of a new phage, Escherichia phage vB_EcoM-V1EC45, which was isolated from wastewater and was able to infect a clinical E. coli ST131 strain. Its genome is a 170,364 bp double-stranded DNA molecule annotated with 281 coding sequences and 9 tRNAs, including multiple genes involved in antidefense systems. V1EC45 is predicted to harbour a virulent lifestyle. Comparative genomics positioned V1EC45 within the Tequatrovirus genus, and the Tsx outer membrane bacterial protein is predicted to be used as the receptor-binding protein. This work highlights the value of using genomic, structural, and evolutionary analyses to support the directed development of targeted bacteriophage therapeutics. Antimicrobial resistance comparative genomics bacteriophage therapy receptor-binding protein outer membrane protein Tsx Figures Figure 1 Figure 2 Figure 3 Introduction Antimicrobial resistance (AMR) represents a major global health challenge, causing more than 1.2 million deaths annually and projected to exceed 10 million per year by 2050 [1, 2]. Among resistant pathogens, Escherichia coli sequence type (ST) 131 has emerged as a pandemic multidrug-resistant clone, distributed worldwide in both community and hospital settings and responsible for a wide range of infections [3]. Its combination of resistance and virulence determinants makes ST131 a major contributor to the global AMR threat [4]. Bacteriophages (phages) are being reconsidered as alternative or complementary therapeutics to antibiotics due to their lytic activity against specific bacterial hosts [5]. The genomic characterization of new phages infecting high-risk clones such as ST131 is thus of strategic interest for future phage therapy. Members of the Tequatrovirus genus (family Straboviridae ) are especially promising because of their large genomes, wide environmental distribution, and broad host range [6]. They have been tested in clinical trials [7] and are known to rely on receptor-binding proteins (RBPs) interacting with bacterial surface structures such as outer membrane proteins or lipopolysaccharides [8]. In this study, we characterized a new bacteriophage called Escherichia phage vB_EcoM-V1EC45 isolated from a clinical strain of E. coli ST131. We described its genomic architecture and anti-defense arsenal and, based on the exact similarity of its receptor binding protein ( gp38 ) compared to closely related Tequatrovirus , we predicted that Escherichia phage vB_EcoM-V1EC45 recognizes specifically its host through the Tsx outer membrane protein. Materials & Methods Bacteria and bacteriophages Wastewater samples were collected at the inflow of the Neuville-sur-Saône (France) treatment plant in January 2022 (GPS: 45.86871446486168, 4.839251994779392). After 0.22 µm filtration, samples were enriched with E. coli strains. Plaques with a small and clear morphology were obtained on clinical ST131 isolate 256G1 (provided by the French National Reference Center for Antibiotic Resistance), which was used as the isolation host. Five milliliters of filtered wastewater were mixed with 10 µL of an overnight culture of E. coli BLK16 [9] and 500 µL of 10X Tryptic Soy Broth (TSB). The mixture was incubated overnight at 37°C, 180 rpm. After centrifugation (4,000 × g, 15 min) and 0.22 µm PES filtration, the phage lysate was tested on clinical and laboratory E. coli strains. The highest titer (> 10⁹ PFU/mL) plaque assay [10] was obtained on strain 256G1 which was retained as the isolation host. The bacteriophage was purified using the double-layer agar method [10] that is that individual plaques were picked, suspended in 1 mL TSB, filtered, diluted, and re-plated. This step was repeated five times to ensure a pure isolate. After amplification in liquid culture, DNA was extracted with the DNA Extractor® WB kit (Fujifilm) [11]. DNA concentration was measured with Qubit™ and purity with NanoDrop™. Sequencing was performed on a NextSeq550 (Illumina) at the genEPII platform (Hospices Civils de Lyon), generating 2×150 bp reads. Libraries were prepared with the Illumina DNA Prep kit using a 1:10 scaled protocol. De novo bacteriophage assembly and annotation Raw reads were assessed with FastQC and cleaned with Fastp (adapter trimming, quality filtering; poly-G trimming disabled). Cleaned reads were assembled de novo with SPAdes. No mapping/subtraction against the host genome was performed. Depth estimation was refined with SAMtools. Contigs were screened by BLASTn against NCBI nt; those matching phages were curated. The major contig (170,364 bp) was polished with Pilon [12], yielding mean coverage of 1286X. Twenty-two additional contigs (332–937 bp) were low-quality fragments with little or no viral content. CheckV [13] classified the major contig as high-quality and complete (100% completeness) based on high-confidence Average Amino Acid Identity comparisons to known viral genomes. To evaluate the presence of gaps within the assemblies, two approaches were applied. First, the total number of ambiguous nucleotides (Ns) was counted in the sequences by scanning the FASTA files while excluding header lines. Additionally, the presence of longer stretches of ambiguous bases (≥ 3 consecutive Ns) was assessed to detect potential assembly gaps. As a complementary method, SeqKit [14]; none were detected, confirming a gapless assembly. The complete annotated genome was deposited in the European Nucleotide Archive ( https://www.ebi.ac.uk/ena/browser/home ) with the Sample Accession number SAMEA118794970 within the Study Accession PRJEB91440. The lifestyle of phage vB_EcoM-V1EC45 was predicted using the online tool PhageScope ( https://phagescope.deepomics.org/ ) [15]. The main assembled contig was submitted to BV-BRC ( https://www.bv-brc.org ) for bacteriophage-specific annotation and quality assessment. Open reading frames (ORFs) were classified into functional categories (structure, DNA metabolism, lysis, immunity, etc.) using custom R scripts. GC content visualization and calculation were performed with Proksee ( https://proksee.ca ) [16]. Putative horizontally transferred regions were detected with AlienHunter, run within Proksee, based on sequence composition deviations. Comparative genomics The phage genome was compared to other bacteriophages using BLASTn (NCBI) and to a sub-collection of related phages [8]. These genomes were annotated with Pharokka v1.7.5 [17], and resulting GFF files were analyzed with Roary [18] for pangenome reconstruction. Co-linearity, conserved blocks, and gene synteny were assessed with MAUVE [19] in Geneious Prime® 2025.1.3. Genes were categorized as total (0-100%), core (99–100%), soft-core (95–99%), shell (15–95%), or cloud (0–15%). Multiple sequence alignment of the core genes was performed with MAFFT [20] in the Roary workflow, and phylogeny was inferred under maximum likelihood with RAXML-NG. Bootstrapping stopped automatically after convergence (cutoff 3%, autoMRE) [21]. Detailed product references and software versions used in this study are provided in Supplementary Tables S1 and S2 and scripts or alignments are openly available at https://src.koda.cnrs.fr/remy.froissart/v1ec45 . Results and Discussion Phage isolation and genome analysis The isolated bacteriophage was named Escherichia phage vB_EcoM-V1EC45 (hereafter referred as V1EC45). Its genome length is 170,364 bp and the annotated genome map (Fig. 1 ) displays a modular organization with clusters of structural, replication, and lysis genes. AlienHunter detected atypical segments, but none exceeded the threshold (29.3), preventing firm conclusions on horizontal transfer. PhageScope further predicted a virulent lifestyle for V1EC45. Annotation identified 281 coding sequences (CDS), in which 136 CDS had assigned functions (Table S3 ) and nine tRNA genes and no rRNA genes. Detected genes were predicted to be involved in DNA metabolism (48; 17%), transcription (8; 2.8%), translation (10; 3.5%), structure including tail fibers (16; 5.7%) and other proteins (17; 6.0%), one receptor gene and one host-specificity gene, endonucleases (13; 4.6%), exonucleases (3; 1%), lysis (7; 2.5%), and host hijack/immunity functions (12; 4.2%). Additionally, 39 CDS (13.8%) encode hypothetical proteins, while 106 CDS (37.7%) were annotated as general “phage proteins.” The large fraction of uncharacterized ORFs highlights the poorly understood genomic diversity of bacteriophages. Anti-defense systems Like in many phages [22], V1EC45 carries genes counteracting bacterial antiviral defenses (detected with DefenseFinder [23]). First, anti-restriction systems with CDS 6 encoding an Arn-like protein [24] [25], structurally mimicking DNA; CDS 57 encodes Dam DNA adenine methyltransferase, protecting phage DNA at GATC motifs [25]. Second, anti-toxin-antitoxin (TA) systems with CDS 64 encoding TifA, neutralizing bacterial toxins [26]; CDS 65 encoding Dmd, a broad-spectrum antitoxin acting on RNases such as LsoA and RnlA [27, 28]. Third, anti-CBASS / cyclic messenger immunity with CDS 167 encoding Acb1, a nuclease degrading cyclic nucleotides (CBASS, Pycsar) [29, 30] and CDS 145 encoding Acb2, a nucleotide-binding protein acting as a “molecular sponge” [31]. Comparative genomics analysis Complete genome comparison against the NCBI collection NCBI retrieved 484 Tevenvirinae genomes with more than 38% coverage, including 73 Tequatrovirus sequences with more than 95% coverage. V1EC45 harboured a classical Tequatrovirus genome length, since the median of the genome length of 421 bacteriophages from this genus is 167,467 bp ± 6,364 bp, with a minimum size of 111,068 bp ( Escherichia phage UoN_LDJ77_1; OK570375) and a maximum size of 172,940 bp ( Escherichia phage vB_Eco_F26; OP870145). Mean GC content of V1EC45 genome was 35.3%, typical to a Tequatrovirus (median of 35.4% ± 0.1%). GC content was relatively homogeneous across V1EC45 genome except for regions harbouring more than 50% such as at loci coding for tRNAs or in the middle of the CDS 264 (coding for the receptor binding protein). BLASTn predicted Escherichia phage UTI-E4 to be the closest relative to V1EC45. VIRIDIC analysis confirmed four genomes (including UTI-E4) with 95% intergenomic similarity to V1EC45. A Tequatrovirus phylogenetic tree was reconstructed using 42 genomes (10 from the BASEL collection [8], 27 representatives from NCBI, and the four closest genomes; Table S4), leveraging the BASEL dataset that experimentally defined host receptors. Re-annotation of these 42 genomes with Pharokka and ANI analysis identified 888 genes: 130 core, 25 soft core, 236 shell, and 497 cloud. After concatenating the core genes, we aligned them at the nucleotide level (the alignment is available in our personal online supplementary data), and the Maximum-likelihood inference revealed five Tequatrovirus clades (bootstrap > 70%; Fig. 2 ). V1EC45 clustered in a clade rooted by Shigella phage CM8, grouping 14 phages with > 97% identity, including UTI-E4, corroborating BLASTn and VIRIDIC results. Whole-genome alignment between V1EC45 and UTI-E4 showed high CDS identity, one colinear block (Fig. S1 ), and some genomic differences: twenty CDS were unique to V1EC45 (10 phage and 7 hypothetical genes and three HNH endonucleases), and four CDS and one tRNA (tRNA-His) were unique to UTI-E4. Finally, four proteins were highly diverse between the two related phages (Table S5). Since V1EC45 was included within a clade grouping bacteriophages (namely WilhemHis, KarlGJung and Paracelsus) using the bacterial outer membrane protein Tsx as the primary receptor, we compared the sequence of the gene gp38 of all bacteriophages that were experimentally demonstrated to use Tsx as an RBP [8]. Indeed, gp38 has been described to code for the RPB [32] (a CDS upstream of the holin CDS and downstream of the long tail fiber distal subunit CDS within the bacteriophage genome). The alignment (Figure S2 ) revealed a strict similarity of the gp38 CDS of V1EC45 and those of KarlGJung and Paracelsus. Conversely, the alignment of the gp38 CDS of V1EC45 and the gp38 CDSs of five other phages using OmpC as an RBP showed very little similarity (Figure S3 ). Our results provide good confidence that V1EC45 uses Tsx as an RBP, but further experiments are still needed to definitively confirm this statement. The V1EC45 host range should be further investigated on a broader, phylogenetically structured collection of ST131 isolates (A, B, C1, C2). Indeed, multiple studies have shown that extraintestinal E. coli infections, particularly in the urinary tract, often involve coexisting clonal variants within the same patient [33, 34]. These intrahost coexisting genotypes challenge the efficacy of monovalent antimicrobial and bacteriophage therapies and highlight the need for bacteriophages capable of addressing polyclonal populations. In this context, identifying or creating bacteriophages able to infect diverse variants becomes urgent. Even if the natural host range is shaped by receptor compatibility, recent studies have demonstrated that experimental evolution can be used to broaden host specificity by selecting adaptive beneficial mutations [35]. When applied to V1EC45, such an approach could generate derivatives capable of targeting a wider spectrum of ST131 isolates while preserving lytic activity and genomic stability. Limitations This study has several limitations. First, the host range of bacteriophage V1EC45 was assessed on a limited set of E. coli ST131 isolates, preventing robust conclusions on its overall host range. Second, the prediction that V1EC45 uses the Tsx outer membrane protein as receptor-binding protein is based on sequence similarity, but awaits experimental confirmation. Finally, the therapeutic potential of V1EC45 has not been evaluated in relevant clinical or animal infection models. Further work is required to address these limitations and validate its suitability as a candidate for phage therapy. Abbreviations AMR antimicrobial resistance ANI Average nucleotide identity BV-BRC Bacterial and Viral Bioinformatics Resource Center CDS Coding DNA sequence CFU Colony forming unit LPS Lipopolysaccharides LTF Long tail fiber O-Ag O-antigen ORF Open reading frame mL milliliter µL microliter Phage Bacteriophage RBP Receptor-Binding Protein RCF relative centrifugal force PFU plaque forming unit ST Sequence type TSB Tryptic soy broth Declarations Conflict of interest The authors have no conflicts of interest to declare. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Funding Information This work was supported by the PHAG-ONE project (20-PAMR-009) and RF own funding. EB was supported by doctoral fellowships from Univ. Montpellier - ED GAIA. Author Contribution Conceptualization: Fl.L., Fr.L., M.M., C.K., R.F.; Methodology & Investigation: Fl.L., E.B., J.H., J.D., M.B., R.B., L.D., R.F.; Results analysis: R.F., E.B., M.B.; Writing: R.F. and E.B.; Revision: All. Acknowledgement This work also benefited from the genEPII sequencing platform (Hospices Civils de Lyon). We also acknowledge the ISO 9001-certified IRD itrop HPC platform (a member of the South Green Platform) for providing high-performance computing resources. We particularly thank Alexis Dereeper and the bioinformatics support team at Montpellier for their assistance throughout the analyses. This work benefited from computational resources made available via https://bioinfo.ird.fr and http://www.southgreen.fr. We also acknowledge UMR MIVEGEC for providing an efficient and supportive working environment throughout this project. Data Availability The complete genome sequence generated and analyzed during the current study is available in the European Nucleotide Archive repository under the Study Accession number PRJEB91440 ( https://www.ebi.ac.uk/ena/browser/view/PRJEB91440 ), with the raw reads available under the accession number ERR15340316 and the assembled and annotated genome available under the Sample Accession number SAMEA118794970. The supplemental data (such as scripts, alignments, sequences) that support the findings of this study are also openly available on our git-server https://src.koda.cnrs.fr/remy.froissart/v1ec45 . References 1. Murray CJL, Ikuta KS, Sharara F, et al (2022) Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. The Lancet 399:629–655 DOI: 10.1016/S0140-6736(21)02724-0 2. Price R (2016) O’Neill report on antimicrobial resistance: funding for antimicrobial specialists should be improved. 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11:55:04","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":298958,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7535326/v1/1b9623e1d71aabb538418268.html"},{"id":93134335,"identity":"061ea023-219c-4fb0-9503-0e1bf08ed9ce","added_by":"auto","created_at":"2025-10-09 12:03:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnnotated genomic map of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEscherichia \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ephage vB_EcoM-V1EC45\u003c/strong\u003e. Starting from the top lane, colored arrows represent open reading frames (ORFs) annotated according to their predicted functions (see detailed annotation in Table S3) and distributed across both DNA strands (Lane 1: positive strand; Lane 2: negative strand). Each arrow color corresponds to a distinct functional category indicated on the right of the figure. The black profile below the genomic map (Lane 3) indicates the local GC content as a percentage centered around 35%. The light purple bands (Lane 4) represent the Alien Hunter predictions. The base scale (kbp) is shown beneath the genomic map.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7535326/v1/c30fc9fa785c323a91493c7b.png"},{"id":93133319,"identity":"2c57f285-a0cb-4330-8ce9-3efc8164db02","added_by":"auto","created_at":"2025-10-09 11:55:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":118626,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMidpoint-rooted maximum-likelihood phylogeny of the core genomes of 42 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTequatrovirus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ebacteriophages\u003c/strong\u003e. Phylogenetic relationships were inferred at the nucleotide level via core-genome alignment (130 genes, 81,356 alignment sites, 7,871 patterns and 87.17% invariant) with the GTR+FO+Imodel. Nonparametric bootstrap support is indicated on top of each branch (after more than 600 replicated trees). The phylogenetic tree was designed via Figtree v1.4.4. The names of bacteriophages are labeled according to their specificity to a bacterial outer membrane protein used as a receptor (Tsx in red, FadL in green, OmpA in yellow, OmpC in blue, OmpF in purple and undetermined in black). The scale bar represents 5 accepted substitutions per 1000 nucleotides.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7535326/v1/6ef317f8b945b374f1f63569.png"},{"id":93134334,"identity":"7d085a79-bc72-4878-9262-40aa9307bab7","added_by":"auto","created_at":"2025-10-09 12:03:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":22969,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative genomics analysis between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEscherichia \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ephage UTI-E4 (top) and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEscherichia \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ephage vB_EcoM-V1EC45 (bottom) via EasyFig\u003c/strong\u003e. Each orange arrow represents a coding sequence. Lines connecting the genome maps indicate gene-level identity, ranging from 69% to 100%, represented by varying shades of gray. See Table S3 for details in the present/absent CDS.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7535326/v1/1da62b31e5c3f06060659591.jpg"},{"id":93137630,"identity":"60e72358-e006-43e0-9138-20e7a136f524","added_by":"auto","created_at":"2025-10-09 12:27:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":846848,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7535326/v1/757bf828-b737-4c1a-b063-ce280fcc66c9.pdf"},{"id":93133326,"identity":"274f83a4-0240-43a7-bf2c-404a927d95a8","added_by":"auto","created_at":"2025-10-09 11:55:03","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1743952,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalOnlinematerials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7535326/v1/caf412efc91d153389b9895e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization and comparative genome analysis of a new Tequatrovirus phage infecting Escherichia coli ST131","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntimicrobial resistance (AMR) represents a major global health challenge, causing more than 1.2\u0026nbsp;million deaths annually and projected to exceed 10\u0026nbsp;million per year by 2050 [1, 2]. Among resistant pathogens, \u003cem\u003eEscherichia coli\u003c/em\u003e sequence type (ST) 131 has emerged as a pandemic multidrug-resistant clone, distributed worldwide in both community and hospital settings and responsible for a wide range of infections [3]. Its combination of resistance and virulence determinants makes ST131 a major contributor to the global AMR threat [4].\u003c/p\u003e\u003cp\u003eBacteriophages (phages) are being reconsidered as alternative or complementary therapeutics to antibiotics due to their lytic activity against specific bacterial hosts [5]. The genomic characterization of new phages infecting high-risk clones such as ST131 is thus of strategic interest for future phage therapy. Members of the \u003cem\u003eTequatrovirus\u003c/em\u003e genus (family \u003cem\u003eStraboviridae\u003c/em\u003e) are especially promising because of their large genomes, wide environmental distribution, and broad host range [6]. They have been tested in clinical trials [7] and are known to rely on receptor-binding proteins (RBPs) interacting with bacterial surface structures such as outer membrane proteins or lipopolysaccharides [8].\u003c/p\u003e\u003cp\u003eIn this study, we characterized a new bacteriophage called \u003cem\u003eEscherichia\u003c/em\u003e phage vB_EcoM-V1EC45 isolated from a clinical strain of \u003cem\u003eE. coli\u003c/em\u003e ST131. We described its genomic architecture and anti-defense arsenal and, based on the exact similarity of its receptor binding protein (\u003cem\u003egp38\u003c/em\u003e) compared to closely related \u003cem\u003eTequatrovirus\u003c/em\u003e, we predicted that \u003cem\u003eEscherichia\u003c/em\u003e phage vB_EcoM-V1EC45 recognizes specifically its host through the Tsx outer membrane protein.\u003c/p\u003e"},{"header":"Materials \u0026 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eBacteria and bacteriophages\u003c/h2\u003e\n\u003cp\u003eWastewater samples were collected at the inflow of the Neuville-sur-Sa\u0026ocirc;ne (France) treatment plant in January 2022 (GPS: 45.86871446486168, 4.839251994779392). After 0.22 \u0026micro;m filtration, samples were enriched with \u003cem\u003eE. coli\u003c/em\u003e strains. Plaques with a small and clear morphology were obtained on clinical ST131 isolate 256G1 (provided by the French National Reference Center for Antibiotic Resistance), which was used as the isolation host.\u003c/p\u003e\n\u003cp\u003eFive milliliters of filtered wastewater were mixed with 10 \u0026micro;L of an overnight culture of \u003cem\u003eE. coli\u003c/em\u003e BLK16 [9] and 500 \u0026micro;L of 10X Tryptic Soy Broth (TSB). The mixture was incubated overnight at 37\u0026deg;C, 180 rpm. After centrifugation (4,000 \u0026times; g, 15 min) and 0.22 \u0026micro;m PES filtration, the phage lysate was tested on clinical and laboratory \u003cem\u003eE. coli\u003c/em\u003e strains. The highest titer (\u0026gt;\u0026thinsp;10⁹ PFU/mL) plaque assay [10] was obtained on strain 256G1 which was retained as the isolation host.\u003c/p\u003e\n\u003cp\u003eThe bacteriophage was purified using the double-layer agar method [10] that is that individual plaques were picked, suspended in 1 mL TSB, filtered, diluted, and re-plated. This step was repeated five times to ensure a pure isolate.\u003c/p\u003e\n\u003cp\u003eAfter amplification in liquid culture, DNA was extracted with the DNA Extractor\u0026reg; WB kit (Fujifilm) [11]. DNA concentration was measured with Qubit\u0026trade; and purity with NanoDrop\u0026trade;. Sequencing was performed on a NextSeq550 (Illumina) at the genEPII platform (Hospices Civils de Lyon), generating 2\u0026times;150 bp reads. Libraries were prepared with the Illumina DNA Prep kit using a 1:10 scaled protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDe novo bacteriophage assembly and annotation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw reads were assessed with FastQC and cleaned with Fastp (adapter trimming, quality filtering; poly-G trimming disabled). Cleaned reads were assembled de novo with SPAdes. No mapping/subtraction against the host genome was performed. Depth estimation was refined with SAMtools.\u003c/p\u003e\n\u003cp\u003eContigs were screened by BLASTn against NCBI nt; those matching phages were curated. The major contig (170,364 bp) was polished with Pilon [12], yielding mean coverage of 1286X. Twenty-two additional contigs (332\u0026ndash;937 bp) were low-quality fragments with little or no viral content. CheckV [13] classified the major contig as high-quality and complete (100% completeness) based on high-confidence Average Amino Acid Identity comparisons to known viral genomes. To evaluate the presence of gaps within the assemblies, two approaches were applied. First, the total number of ambiguous nucleotides (Ns) was counted in the sequences by scanning the FASTA files while excluding header lines. Additionally, the presence of longer stretches of ambiguous bases (\u0026ge;\u0026thinsp;3 consecutive Ns) was assessed to detect potential assembly gaps. As a complementary method, SeqKit [14]; none were detected, confirming a gapless assembly. The complete annotated genome was deposited in the European Nucleotide Archive (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/ena/browser/home\u003c/span\u003e\u003c/span\u003e) with the Sample Accession number SAMEA118794970 within the Study Accession PRJEB91440. The lifestyle of phage vB_EcoM-V1EC45 was predicted using the online tool PhageScope (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phagescope.deepomics.org/\u003c/span\u003e\u003c/span\u003e) [15].\u003c/p\u003e\n\u003cp\u003eThe main assembled contig was submitted to BV-BRC (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bv-brc.org\u003c/span\u003e\u003c/span\u003e) for bacteriophage-specific annotation and quality assessment. Open reading frames (ORFs) were classified into functional categories (structure, DNA metabolism, lysis, immunity, etc.) using custom R scripts. GC content visualization and calculation were performed with Proksee (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://proksee.ca\u003c/span\u003e\u003c/span\u003e) [16]. Putative horizontally transferred regions were detected with AlienHunter, run within Proksee, based on sequence composition deviations.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eComparative genomics\u003c/h3\u003e\n\u003cp\u003eThe phage genome was compared to other bacteriophages using BLASTn (NCBI) and to a sub-collection of related phages [8]. These genomes were annotated with Pharokka v1.7.5 [17], and resulting GFF files were analyzed with Roary [18] for pangenome reconstruction. Co-linearity, conserved blocks, and gene synteny were assessed with MAUVE [19] in Geneious Prime\u0026reg; 2025.1.3. Genes were categorized as total (0-100%), core (99\u0026ndash;100%), soft-core (95\u0026ndash;99%), shell (15\u0026ndash;95%), or cloud (0\u0026ndash;15%). Multiple sequence alignment of the core genes was performed with MAFFT [20] in the Roary workflow, and phylogeny was inferred under maximum likelihood with RAXML-NG. Bootstrapping stopped automatically after convergence (cutoff 3%, autoMRE) [21].\u003c/p\u003e\n\u003cp\u003eDetailed product references and software versions used in this study are provided in Supplementary Tables S1 and S2 and scripts or alignments are openly available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://src.koda.cnrs.fr/remy.froissart/v1ec45\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003ePhage isolation and genome analysis\u003c/h2\u003e\u003cp\u003eThe isolated bacteriophage was named \u003cem\u003eEscherichia\u003c/em\u003e phage vB_EcoM-V1EC45 (hereafter referred as V1EC45). Its genome length is 170,364 bp and the annotated genome map (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) displays a modular organization with clusters of structural, replication, and lysis genes. AlienHunter detected atypical segments, but none exceeded the threshold (29.3), preventing firm conclusions on horizontal transfer. PhageScope further predicted a virulent lifestyle for V1EC45.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnnotation identified 281 coding sequences (CDS), in which 136 CDS had assigned functions (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) and nine tRNA genes and no rRNA genes. Detected genes were predicted to be involved in DNA metabolism (48; 17%), transcription (8; 2.8%), translation (10; 3.5%), structure including tail fibers (16; 5.7%) and other proteins (17; 6.0%), one receptor gene and one host-specificity gene, endonucleases (13; 4.6%), exonucleases (3; 1%), lysis (7; 2.5%), and host hijack/immunity functions (12; 4.2%). Additionally, 39 CDS (13.8%) encode hypothetical proteins, while 106 CDS (37.7%) were annotated as general \u0026ldquo;phage proteins.\u0026rdquo; The large fraction of uncharacterized ORFs highlights the poorly understood genomic diversity of bacteriophages.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnti-defense systems\u003c/h3\u003e\n\u003cp\u003eLike in many phages [22], V1EC45 carries genes counteracting bacterial antiviral defenses (detected with DefenseFinder [23]). First, anti-restriction systems with CDS 6 encoding an Arn-like protein [24] [25], structurally mimicking DNA; CDS 57 encodes Dam DNA adenine methyltransferase, protecting phage DNA at GATC motifs [25]. Second, anti-toxin-antitoxin (TA) systems with CDS 64 encoding TifA, neutralizing bacterial toxins [26]; CDS 65 encoding Dmd, a broad-spectrum antitoxin acting on RNases such as LsoA and RnlA [27, 28]. Third, anti-CBASS / cyclic messenger immunity with CDS 167 encoding Acb1, a nuclease degrading cyclic nucleotides (CBASS, Pycsar) [29, 30] and CDS 145 encoding Acb2, a nucleotide-binding protein acting as a \u0026ldquo;molecular sponge\u0026rdquo; [31].\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eComparative genomics analysis\u003c/h2\u003e\u003cp\u003eComplete genome comparison against the NCBI collection NCBI retrieved 484 \u003cem\u003eTevenvirinae\u003c/em\u003e genomes with more than 38% coverage, including 73 \u003cem\u003eTequatrovirus\u003c/em\u003e sequences with more than 95% coverage. V1EC45 harboured a classical \u003cem\u003eTequatrovirus\u003c/em\u003e genome length, since the median of the genome length of 421 bacteriophages from this genus is 167,467 bp\u0026thinsp;\u0026plusmn;\u0026thinsp;6,364 bp, with a minimum size of 111,068 bp (\u003cem\u003eEscherichia\u003c/em\u003e phage UoN_LDJ77_1; OK570375) and a maximum size of 172,940 bp (\u003cem\u003eEscherichia\u003c/em\u003e phage vB_Eco_F26; OP870145). Mean GC content of V1EC45 genome was 35.3%, typical to a \u003cem\u003eTequatrovirus\u003c/em\u003e (median of 35.4% \u0026plusmn; 0.1%). GC content was relatively homogeneous across V1EC45 genome except for regions harbouring more than 50% such as at loci coding for tRNAs or in the middle of the CDS 264 (coding for the receptor binding protein).\u003c/p\u003e\u003cp\u003eBLASTn predicted \u003cem\u003eEscherichia\u003c/em\u003e phage UTI-E4 to be the closest relative to V1EC45. VIRIDIC analysis confirmed four genomes (including UTI-E4) with 95% intergenomic similarity to V1EC45. A \u003cem\u003eTequatrovirus\u003c/em\u003e phylogenetic tree was reconstructed using 42 genomes (10 from the BASEL collection [8], 27 representatives from NCBI, and the four closest genomes; Table S4), leveraging the BASEL dataset that experimentally defined host receptors.\u003c/p\u003e\u003cp\u003eRe-annotation of these 42 genomes with Pharokka and ANI analysis identified 888 genes: 130 core, 25 soft core, 236 shell, and 497 cloud. After concatenating the core genes, we aligned them at the nucleotide level (the alignment is available in our personal online supplementary data), and the Maximum-likelihood inference revealed five \u003cem\u003eTequatrovirus\u003c/em\u003e clades (bootstrap\u0026thinsp;\u0026gt;\u0026thinsp;70%; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eV1EC45 clustered in a clade rooted by Shigella phage CM8, grouping 14 phages with \u0026gt;\u0026thinsp;97% identity, including UTI-E4, corroborating BLASTn and VIRIDIC results. Whole-genome alignment between V1EC45 and UTI-E4 showed high CDS identity, one colinear block (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and some genomic differences: twenty CDS were unique to V1EC45 (10 phage and 7 hypothetical genes and three HNH endonucleases), and four CDS and one tRNA (tRNA-His) were unique to UTI-E4. Finally, four proteins were highly diverse between the two related phages (Table S5).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSince V1EC45 was included within a clade grouping bacteriophages (namely WilhemHis, KarlGJung and Paracelsus) using the bacterial outer membrane protein Tsx as the primary receptor, we compared the sequence of the gene gp38 of all bacteriophages that were experimentally demonstrated to use Tsx as an RBP [8]. Indeed, gp38 has been described to code for the RPB [32] (a CDS upstream of the holin CDS and downstream of the long tail fiber distal subunit CDS within the bacteriophage genome). The alignment (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) revealed a strict similarity of the gp38 CDS of V1EC45 and those of KarlGJung and Paracelsus. Conversely, the alignment of the gp38 CDS of V1EC45 and the gp38 CDSs of five other phages using OmpC as an RBP showed very little similarity (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Our results provide good confidence that V1EC45 uses Tsx as an RBP, but further experiments are still needed to definitively confirm this statement.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe V1EC45 host range should be further investigated on a broader, phylogenetically structured collection of ST131 isolates (A, B, C1, C2). Indeed, multiple studies have shown that extraintestinal E. coli infections, particularly in the urinary tract, often involve coexisting clonal variants within the same patient [33, 34]. These intrahost coexisting genotypes challenge the efficacy of monovalent antimicrobial and bacteriophage therapies and highlight the need for bacteriophages capable of addressing polyclonal populations. In this context, identifying or creating bacteriophages able to infect diverse variants becomes urgent. Even if the natural host range is shaped by receptor compatibility, recent studies have demonstrated that experimental evolution can be used to broaden host specificity by selecting adaptive beneficial mutations [35]. When applied to V1EC45, such an approach could generate derivatives capable of targeting a wider spectrum of ST131 isolates while preserving lytic activity and genomic stability.\u003c/p\u003e\u003c/div\u003e"},{"header":"Limitations","content":"\u003cp\u003eThis study has several limitations. First, the host range of bacteriophage V1EC45 was assessed on a limited set of \u003cem\u003eE. coli\u003c/em\u003e ST131 isolates, preventing robust conclusions on its overall host range. Second, the prediction that V1EC45 uses the Tsx outer membrane protein as receptor-binding protein is based on sequence similarity, but awaits experimental confirmation. Finally, the therapeutic potential of V1EC45 has not been evaluated in relevant clinical or animal infection models. Further work is required to address these limitations and validate its suitability as a candidate for phage therapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAMR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eantimicrobial resistance\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eANI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAverage nucleotide identity\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBV-BRC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBacterial and Viral Bioinformatics Resource Center\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCDS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCoding DNA sequence\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCFU\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eColony forming unit\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLPS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLipopolysaccharides\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLTF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLong tail fiber\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eO-Ag\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eO-antigen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eORF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOpen reading frame\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emilliliter\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u0026micro;L\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emicroliter\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePhage\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBacteriophage\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRBP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eReceptor-Binding Protein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRCF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003erelative centrifugal force\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePFU\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eplaque forming unit\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eST\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSequence type\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTSB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTryptic soy broth\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding Information\u003c/h2\u003e\u003cp\u003eThis work was supported by the PHAG-ONE project (20-PAMR-009) and RF own funding. EB was supported by doctoral fellowships from Univ. Montpellier - ED GAIA.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: Fl.L., Fr.L., M.M., C.K., R.F.; Methodology \u0026amp; Investigation: Fl.L., E.B., J.H., J.D., M.B., R.B., L.D., R.F.; Results analysis: R.F., E.B., M.B.; Writing: R.F. and E.B.; Revision: All.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work also benefited from the genEPII sequencing platform (Hospices Civils de Lyon). We also acknowledge the ISO 9001-certified IRD itrop HPC platform (a member of the South Green Platform) for providing high-performance computing resources. We particularly thank Alexis Dereeper and the bioinformatics support team at Montpellier for their assistance throughout the analyses. This work benefited from computational resources made available via https://bioinfo.ird.fr and http://www.southgreen.fr. We also acknowledge UMR MIVEGEC for providing an efficient and supportive working environment throughout this project.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe complete genome sequence generated and analyzed during the current study is available in the European Nucleotide Archive repository under the Study Accession number PRJEB91440 ( https://www.ebi.ac.uk/ena/browser/view/PRJEB91440 ), with the raw reads available under the accession number ERR15340316 and the assembled and annotated genome available under the Sample Accession number SAMEA118794970. The supplemental data (such as scripts, alignments, sequences) that support the findings of this study are also openly available on our git-server https://src.koda.cnrs.fr/remy.froissart/v1ec45 .\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e1. 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Kolenda C, Medina M, Bonhomme M, Rousseau C, Laumay F, Martins-Simoes P, Ferry T, Laurent F (2022) \u0026laquo; Phage training \u0026raquo; ou \u0026eacute;volution dirig\u0026eacute;e de bact\u0026eacute;riophages anti-Staphylococcus aureus : extension du spectre d\u0026rsquo;activit\u0026eacute; \u0026agrave; Staphylococcus epidermidis multi-r\u0026eacute;sistant aux antibiotiques. M\u0026eacute;decine Mal Infect Form 1:S70 DOI: 10.1016/j.mmifmc.2022.03.152\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e12. Walker BJ, Abeel T, Shea T, et al (2014) Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement. PLOS ONE 9:e112963 DOI: 10.1371/journal.pone.0112963\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e13. Nayfach S, Camargo AP, Schulz F, Eloe-Fadrosh E, Roux S, Kyrpides NC (2021) CheckV assesses the quality and completeness of metagenome-assembled viral genomes. 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Cao Yao JC, Garcia Cehic D, Quer J, M\u0026eacute;ndez JN, Gorr\u0026iacute;n AD, Hevia LG, Fern\u0026aacute;ndez MTT (2024) Complete Genome Sequences of Four Mycobacteriophages Involved in Directed Evolution against Undisputed Mycobacterium abscessus Clinical Strains. Microorganisms 12:374 DOI: 10.3390/microorganisms12020374\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e32. Trojet SN, Caumont-Sarcos A, Perrody E, Comeau AM, Krisch HM (2011) The gp38 Adhesins of the T4 Superfamily: A Complex Modular Determinant of the Phage\u0026rsquo;s Host Specificity. Genome Biol Evol 3:674\u0026ndash;686 DOI: 10.1093/gbe/evr059\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e33. Didelot X, Walker AS, Peto TE, Crook DW, Wilson DJ (2016) Within-host evolution of bacterial pathogens. Nat Rev Microbiol 14:150\u0026ndash;162 DOI: 10.1038/nrmicro.2015.13\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e34. Levert M, Zamfir O, Clermont O, et al (2010) Molecular and Evolutionary Bases of Within-Patient Genotypic and Phenotypic Diversity in Escherichia coli Extraintestinal Infections. PLOS Pathog 6:e1001125 DOI: 10.1371/journal.ppat.1001125\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e35. Chen M, Zhang L, Abdelgader SA, Yu L, Xu J, Yao H, Lu C, Zhang W (2017) Alterations in gp37 Expand the Host Range of a T4-Like Phage. Appl Environ Microbiol 83:e01576-17 DOI: 10.1128/AEM.01576-17\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSupplemental Online materials\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Antimicrobial resistance, comparative genomics, bacteriophage therapy, receptor-binding protein, outer membrane protein Tsx","lastPublishedDoi":"10.21203/rs.3.rs-7535326/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7535326/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eThe global rise of antimicrobial resistance with the spread of pathogenic multidrug-resistant clones such as \u003cem\u003eEscherichia coli\u003c/em\u003e sequence type (ST) 131 represents a major challenge for public health, renewing interest in bacteriophages as therapeutical agents.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe report the isolation and characterization of a new phage, \u003cem\u003eEscherichia\u003c/em\u003e phage vB_EcoM-V1EC45, which was isolated from wastewater and was able to infect a clinical \u003cem\u003eE. coli\u003c/em\u003e ST131 strain. Its genome is a 170,364 bp double-stranded DNA molecule annotated with 281 coding sequences and 9 tRNAs, including multiple genes involved in antidefense systems. V1EC45 is predicted to harbour a virulent lifestyle. Comparative genomics positioned V1EC45 within the \u003cem\u003eTequatrovirus\u003c/em\u003e genus, and the Tsx outer membrane bacterial protein is predicted to be used as the receptor-binding protein. This work highlights the value of using genomic, structural, and evolutionary analyses to support the directed development of targeted bacteriophage therapeutics.\u003c/p\u003e","manuscriptTitle":"Characterization and comparative genome analysis of a new Tequatrovirus phage infecting Escherichia coli ST131","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-09 11:54:58","doi":"10.21203/rs.3.rs-7535326/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-15T08:33:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-02T08:44:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T06:43:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-20T06:07:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-16T09:55:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-15T21:32:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"10454419120278999976231105844952985855","date":"2025-10-15T20:20:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-15T19:00:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336118571095120547572247395372752248303","date":"2025-10-15T06:01:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18354492145883477055824088737405996507","date":"2025-10-15T04:20:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3464365045675327266551560358990164199","date":"2025-10-13T15:43:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332041061677928377619123036336029733771","date":"2025-10-13T06:23:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296596868048198379258615480551360384547","date":"2025-10-13T05:43:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-01T12:02:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"306031562062487890598854335447619236258","date":"2025-09-27T11:45:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-27T11:22:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-26T10:22:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-25T09:06:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-24T16:24:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2025-09-24T16:19:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d1417691-9086-431b-919f-689cd02689ec","owner":[],"postedDate":"October 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-24T10:26:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-09 11:54:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7535326","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7535326","identity":"rs-7535326","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}