Diverse lytic bacteriophages from India reveal genomic signatures and therapeutic potential against MDR Escherichia coli

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With conventional antibiotics increasingly ineffective against multidrug-resistant (MDR) strains, alternative solutions are urgently needed. Lytic bacteriophages, known for their host specificity and potent antibacterial activity, offer a promising therapeutic option. However, limited genomic data on phages from diverse ecological contexts hinders comprehensive understanding of their diversity and functional potential. Methods This study aimed to isolate, characterize, and assess the therapeutic potential of lytic bacteriophages targeting MDR E. coli isolated from livestock. Phages were enriched from sewage samples using an MDR E. coli host. Plaque morphology was assessed for lytic characteristics. Thermal and pH stability were assessed under controlled incubation conditions. Host range was determined against 20 MDR E. coli strains from cattle, buffalo, and goat. Whole genome sequencing and annotation were performed to determine genetic features, taxonomic classification, safety, and phylogenetic relationships. Results Three lytic phages BASU E2, BASU E7, and BASU E10 were successfully isolated, producing clear plaques indicative of strong lytic activity. All phages remained viable at 37°C and 42°C and within pH 6–9, but were inactivated at 80°C and at highly acidic or alkaline conditions. BASU E7 exhibited the broadest host range, lysing 75% of the tested strains. Genome analysis revealed double-stranded DNA genomes of approximately 157 kb (BASU E2), 50 kb (BASU E7), and 48 kb (BASU E10), with G + C content ranging from 44.62–45.47%. Taxonomically, BASU E2 was classified under Ackermannviridae , while BASU E7 and BASU E10 belonged to Siphoviridae , all within Caudovirales . All phages were predicted to be strictly lytic, lacking genes associated with lysogeny, antibiotic resistance, or virulence. Phylogenetic analysis showed distinct cluster of BASU E2, suggesting divergent evolutionary origin from BASU E7 and BASU E10. Conclusion This study presents the isolation of genetically safe, environmentally stable, and broadly active lytic phages effective against MDR E. coli of animal origin. These findings highlight the potential of these phages for use in veterinary phage therapy. However, further in vivo validation and the development of phage cocktails are necessary to advance their clinical application. Multidrug-resistant Escherichia coli phage therapy veterinary pathogens phage genome analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background AMR is a major threat to public and animal health in the 21st century. It was estimated that worldwide 4·71 million deaths were linked with bacterial AMR in 2021 including 1·14 million deaths attributable to bacterial AMR [ 1 ]. E. coli ranks among the topmost five bacterial pathogens accountable for all infection-related deaths and is among the topmost three pathogens associated with AMR-attributable mortality [ 2 ]. The World Health Organization has recognized E. coli as one of the 12 bacterial species posing a critical threat to human wellbeing due to its rapidly increasing antimicrobial resistance [ 3 ]. Infections caused by resistant E. coli strains are challenging to treat in both human and veterinary medicine across the globe [ 4 ]. As many antimicrobial drugs used in human medicine are also utilized in veterinary medicine and indiscriminate misuse can result in therapeutic failure and increased health consequences in both animals and humans [ 5 ]. A pan India surveillance of AMR in livestock revealed that E. coli isolates of food-animal origin were found more frequently resistant to commonly used antibiotics [ 6 ]. The development and approval of new antibacterial agents, including antibiotics, have not kept pace with the rising prevalence of antibiotic-resistant bacteria [ 7 ]. Alternative therapeutic approaches are urgently needed to expand treatment options against antibiotic-resistant bacteria. Bacteriophages are emerging as promising therapeutic agents and viable alternatives to antibiotics, with substantial progress being made in their effective clinical application [ 8 ]. Phages are viruses that selectively infect and lyse bacteria, and they are found ubiquitously in various environments [ 9 ]. They offer several advantages over antibiotics, including potent antibacterial activity, high efficacy in targeting infections without disrupting the normal microbiota, and minimal side effects [ 10 ]. Phages can be engineered to enhance their efficacy by delivering antimicrobial agents directly to the site of infection [ 11 ] or can also be used synergistically with antibiotics [ 12 ]. The use of phage combinations (phage cocktails) has been shown to efficiently treat bacterial infections, together with those caused by MDR strains, and reduce resistance and may be used to reduce the potential for bacteria to evolve phage resistance [ 13 ]. However, the lack of region-specific phage data, particularly from animal and environmental reservoirs in India, limits our understanding of phage diversity and their therapeutic potential. This study addresses this gap by isolating and characterizing lytic phages targeting MDR E. coli from livestock sources, contributing to the development of safe and effective alternatives to antibiotics. Methods Bacterial strains MDR E. coli strains maintained at the Department of Veterinary Public Health and Epidemiology, Bihar Veterinary College, Patna, India served as the bacterial hosts for this study (Fig. 1 ). Among these, the MDR E. coli strain BUE4, previously isolated from bovine urine, was employed for the enrichment of sewage samples to facilitate bacteriophage isolation. All MDR E. coli strains were subjected to rigorous characterization, including assessments of purity, colony morphology, and standard biochemical and molecular identification protocols. These verified strains were subsequently used to perform bacteriophage susceptibility assays. Isolation and purification of lytic Escherichia phages Approximately 50 mL of sewage samples (n = 10) were collected from livestock farms affiliated with Bihar Animal Sciences University, Patna, India, for phage isolation. The sewage samples were processed using an enrichment-based protocol, following the methodology outlined previously [ 14 ]. Lytic bacteriophages were identified by the formation of clear plaques on a soft agar overlay, indicating bacterial lysis. These phages were subsequently purified through three successive passage of plaque isolation to ensure clonal purity. The naming of the isolated phages followed the guidelines proposed previously [ 15 ], incorporating the full host genus name, the term "phage," and a distinct identifier. Phages sensitivity test Phage sensitivity assays were conducted using the spot test method on top agar overlays seeded with 20 MDR E. coli strains. For each assay, 10 µl of phage lysate was applied onto the surface of the bacterial lawn and allowed to adsorb completely. The plates were incubated at 37°C for 18–24 hr and subsequently examined for the presence of clear lytic zones, which signified phage infectivity and host susceptibility. The resulting sensitivity profiles were analyzed and presented using Gene Cluster 3.0 and Java TreeView software, as discussed previously [ 14 ]. Phages thermal and pH stability The thermal stability of the isolated bacteriophages was assessed by incubating 1 mL aliquots of phage lysate (10⁷ PFU/mL) at four different temperatures: 37°C, 42°C, 65°C, and 80°C. The samples were maintained in a water bath at the designated temperatures for 60 minutes, then rapidly cooled on ice for 10 minutes to halt further thermal activity. Subsequently, serial 10-fold dilutions were prepared, and the remaining viable phage particles were quantified using the double-layer agar plaque assay. For pH stability evaluation, 100 µL of phage lysate (10⁸ PFU/mL) was mixed with 900 µL of SM (Salt Magnesium) buffer adjusted to various pH values (3.0, 6.0, 7.5, 9.0, and 12.0). The mixtures were incubated at 37°C for 60 minutes. After incubation, phage titers were determined using the agar overlay method to assess the impact of pH on phage viability. Results are expressed as mean log₁₀ PFU/mL ± standard error, calculated from three independent experiments. Each complete experiment was repeated three times (n = 3). One-way analysis of variance (ANOVA) was performed to identify significant differences among treatment means, and statistical significance (p < 0.05) was determined using Tukey’s Honest Significant Difference (HSD) test. Whole genome sequencing, assembly and annotation Genomes of phages were isolated using the Qigen DNA isolation kit. The concentration of extracted genomic DNA was measured using a NanoDrop. Genomic DNA was sent to Wipro Life Science Lab, Kolkata for whole genomic sequencing using IlluminaNextSeq 2000 (Illumina, San Diego, CA). The sequencing library was prepared and sequencing was performed using the TruSeq Nano DNA prep kit using paired-end 2 × 300-bp reads. Quality assessment was conducted using FastQC v0.11.9 [ 16 ]. The adapter was trimmed using Trimmomatic v0.39 and low-quality reads with Qscore < 20 was removed [ 17 ]. Further contaminated reads were also filtered out using BWA v0.7.17 [ 18 ]. Trimmed and filtered sequence reads were used to perform denovo assembly with MetaviralSPAdes with the default parameters [ 19 ]. Assembly quality and completeness check was performed with CheckV tool which checks for host genome contamination [ 20 ]. Assembled contigues of phages were further checked in the Phager web server for identification or phage contig ( https://phagecompass.ku.dk/ ) and contamination was removed manually. Genomes were submitted to PHASTEST for rapid preliminary gene calling and annotation [ 21 ]. Assembled draft genomes were annotated using the Edge Bioinformatics Server in Prokka and Pharokka [ 22 , 23 ]. Additionally, the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST, http://blast.ncbi.nlm.nlm.nih.gov/ ) was utilized for sequence similarity alignment. For assigning recent taxonomy to a bacteriophage at the genus and species level taxMyPhage server was used [ 24 ]. Blastn was used to compare the whole genome sequences of phages with those in the NCBI database, and the phages with the closest sequences in GenBank were identified. The circular maps of the phage genomes were generated using PHASTEST [ 21 ]. The putative transfer RNA (tRNA) encoding genes were predicted using tRNAscan-SE [ 25 ]. The presence of resistance genes and virulence genes was examined against the NCBI, CARD, and VFDB databases through ABRicate v.0.8.13. The ResFinder platforms were employed to identify the presence of resistance genes ( https://cge.cbs.dtu.dk/services/ResFinder/ ). Phage Lifestyle Phages were further classified using PhageCampass ( https://ptax.ku.dk/ ). In addition, the phage proteomes were analyzed using PhageGE [ 26 ] and phageLeads [ 27 ] a computational classification algorithm trained to predict phage lifestyles. Putative host and phage promoters in intergenic regions were identified using PhagePromoter [ 28 ] ( https://galaxy.bio.di.uminho.pt/ ). Putative rho-independent terminators occurring in intergenic regions were determined using ARNold which computes the free energy of the predicted terminator stem-loops [ 29 ]. Phylogenetic analysis Available genome sequences of phages of E. coli , Salmonella , and Shigella spp. were retrieved from the NCBI database (Supplementary Table XLS1). The whole-genome phylogenetic trees were constructed using the VICTOR (viral comparison and tree building online resource) [ 30 ]. The analysis was conducted under settings recommended for prokaryotic viruses. Phylogenetic tree construction is based on the GBDP method that is specifically configured for prokaryotic viruses. The program calculates intergenomic distances from BLAST + hits using GBDP (including 100 pseudo bootstrap replicates) and uses them to infer a balanced minimum evolution tree with branch support via FASTME including subtree pruning and regrafting post-processing [ 30 ]. Taxon boundaries at the species, genus, and family level were estimated automatically by VICTOR. A genomic network tree was constructed using the PhageClouds (( https://phagecompass.ku.dk/ ) [ 31 ]. PhageClouds supported the search of related phages among all complete phage genomes from GenBank. Result Phage Isolation and Morphological Characterization Three lytic bacteriophages- Escherichia phage BASU E2, BASU E7, and BASU E10 were successfully isolated using multidrug-resistant E. coli strains as host bacteria. Upon infection, all three phages produced distinct, clear, and circular plaques ranging in size from approximately 0.5 to 1 mm in diameter on the bacterial lawn, indicative of their lytic activity (Fig. 2 ). Phages thermal and pH stability The thermal stability of the isolated phages was assessed by incubating phage lysates at various temperatures (37°C, 42°C, 65°C, and 80°C) for up to 60 min. Phage titers remained relatively stable at 37°C and 42°C, with statistically significant reduction observed at 42°C in compare with stability at 37°C (p < 0.05). However, a marked decline in phage viability was recorded following exposure to 65°C, and complete inactivation occurred at 80°C within 60 min of incubation (Fig. 3 a). Likewise, phage stability under varying pH conditions (ranging from pH 3 to 12) was evaluated by incubating phage lysates for 60 min at 37°C in SM buffer adjusted to the respective pH values. Phage viability was well maintained between pH 6 and 9, showing significant loss in infectivity in compare with pH 7.5 (p 12) led to a complete loss of phage activity (Fig. 3 b). Phages sensitivity test The lytic activity of Escherichia phage BASU E2, BASU E7, and BASU E10 was evaluated against 20 multidrug-resistant E. coli isolates obtained from cattle milk, buffalo milk, and goat rectal swabs, as illustrated in Fig. 4 . Among the three phages, BASU E7 exhibited the broadest host range, lysing 75% (15/20) of the tested strains. BASU E2 demonstrated lytic activity against 65% (13/20) of isolates, while BASU E10 was effective against 55% (11/20) of the E. coli strains. Assembly and annotation All three phage genomes were assembled and the quality of each phage genome was good. Isolated phages (BASU E2, E7, and E10) have a linear double-stranded DNA genome with lengths of 157,938bp, 50,255, and 48,147 bp respectively, and GC contents of 44.62%, 45.47%, and 45.08% respectively (Table 1 ). The genomes contained 204, 77, and 70 putative ORFs. Genomes were predicted for fiber protein, tail protein, head protein, portal protein, holing, neck protein, and putative Endolysin) other than hypothetical proteins (Supplementary Table XLS2). Phage BASU E2 belongs to the family Ackermannviridae and the other two phages BASU E7 and E10 belong to Siphoviridae in NCBI BLAST-based classification (Table 2 ). In ICTV's latest classification, all phages were classified into the family Drexlerviridae and the genus Tunavirus (Table 3 ). Different tRNA genes were found in the genomes using the tRNA scan-SE program (Table 4 ). The genome of phages was visualized using PHASTEST (Fig. 5 a, 5 b, and 5 c). Compared with the ABRicate databases, no virulence or antibiotic resistance genes and virulence genes were identified in the genomes. Table 1 Assembly and annotation summary of Escherichia phages BAUE2 BASUE7 BASUE10 GC% 44.62% 45.47% 45.08% Contigs 1 1 1 Total bp 157,938 bp 50,255 bp 48,147 tRNA 4 0 0 Total CDS 204 77 70 Hypothetical protein 193 74 69 Function assigned 11 3 1 Table 2 Classification of Escherichia phages by EDGE Server. Phage contigs were mapped to reference genomes in the NCBI database using minimap2, and taxonomic inference was performed based on sequence similarity. BASUE2 BASUE7 BASUE10 Super kingdom Viruses Viruses Viruses Order Caudovirales Caudovirales Caudovirales Family Ackermannviridae Siphoviridae Siphoviridae Genus Cba120virus T1virus T1virus Species Salmonella phage GG32 Escherichia coli virus ADB2 Shigella virus Psf2 Table 3 Classification of Escherichia phages based on the latest International Committee on the Taxonomy of Viruses (ICTV) taxonomy using the TaxmyPHAGE server. BASUE2 BASUE7 BASUE10 Kingdom Heunggongvirae Heunggongvirae Heunggongvirae Phylum Uroviricota Uroviricota Uroviricota Class Caudoviricetes Caudoviricetes Caudoviricetes Order Pantevenvirales Not Defined Not Defined Family Ackermannviridae Drexlerviridae Drexlerviridae Subfamily Cvivirinae Tunavirinae Tunavirinae Genus Kuttervirus Tunavirus Tunavirus Species Kuttervirus PM10 Tunavirus new_name Tunavirus new_name Table 4 Prediction of tRNA genes in the genomes of Escherichia phages Predicted tRNA genes BASUE2 BASUE7 BASUE10 tRNAs decoding Standard 20 AA 4 Met, tyr, Asn and Ser 0 0 Selenocysteine tRNAs (TCA) 0 0 0 Possible suppressor tRNAs (CTA, TTA, TCA) 0 0 0 tRNAs with undetermined/unknown isotypes 1 0 0 Predicted pseudogenes 0 0 0 Total tRNAs 5 0 0 Phage life Style Phage lifestyle was predicted to be virulent and have lytic activity. Integrase genes were not identified in any genomes (Table 5 ). Prediction in PhageLeads could not find any predicted temperate lifestyle genes. Putative host and phage promoters were identified. BASU E2, BASU E7 and BASU E10 phages were predicated of 25, 37, and 30 host promoter sequences (Supplementary Table XLS3). BASU E7 was predicted of 3 phage promoter sequences also. All phages were predicted for 43 putative rho-independent terminators (Supplementary Table XLS4). Table 5 Phage lifestyle assessment of Escherichia phages Phage PhageGE Lytic Score PhageGE Temperate Score PHASTEST Integrase BASUE2 0.986 0.014 No BASUE7 0.986 0.014 No BASUE10 0.9 0.1 No Phylogenetic analysis Phylogenetic analysis revealed the isolated BASU E2 phage cluster separately from the other two phages BASU E7 and E10 (Fig. 6 ). BASU E7 and E10 phages formed one cluster with Escherichia phage Tls, Enterobacter phage F20, Escherichia phage EB49, Escherichia phage Jk06, Escherichia phage Rtp and Enterobacter phage F20 while BASU E2 clustered with Escherichia phage PhaxI, Escherichia phage Cba120 and Shigella phage Ag3. A genomic interaction graph of phage with closet 26 phage based on BLAST search in NCBI with threshold = 0.15 was prepared (Fig. 7 a, 7 b, 7 c). BASU phages showed close interaction with Escherichia phage, Enterobacter phage, and Shigella phage. Discussion In recent times, the overuse of antibiotics has resulted in the emergence of multi-drug resistance against E. coli , which is negatively impacting food safety, animals and human health. Here we report the isolation, host range characterization, and genome sequencing of three phages against multidrug resistance E. coli isolated from animal clinical samples. The isolation of Escherichia phages BASU E2, E7, and E10 using MDR E. coli strains demonstrates their potential as lytic phages for therapeutic applications. The clear, circular plaques (0.5–1 mm) indicate strong lytic activity, a hallmark of phages capable of efficient bacterial lysis [ 32 , 33 ]. Such morphology suggests rapid infection cycles and efficient host recognition [ 34 , 35 ]. Using MDR strains as hosts enhances clinical relevance, aligning with current efforts to combat antibiotic resistance through phage therapy [ 36 , 37 ]. The variation in plaque size may reflect differences in phage replication dynamics, warranting further investigation [ 38 ]. The isolated phages demonstrated good thermal stability at physiological and mildly elevated temperatures (37°C and 42°C), retaining infectivity with significant titer reduction, consistent with the behaviour of other robust lytic phages [ 39 ]. However, exposure to higher temperatures (65°C and especially 80°C) significantly reduced or ended phage activity, likely due to capsid protein denaturation or DNA degradation [ 40 ]. Similarly, phage viability was stable within a neutral to mildly alkaline pH range (pH 6–9), which is favourable for therapeutic applications and environmental use [ 41 ]. Extreme pH values (≤ 3 or ≥ 12) caused complete inactivation, as also observed in other studies, likely due to damage to structural proteins or genome integrity [ 42 ]. These results highlight the importance of environmental conditions in phage application and formulation strategies. The differential lytic activity exhibited by Escherichia phages BASU E2, E7, and E10 against MDR E. coli strains underscores their therapeutic potential and host specificity. BASU E7, with a lytic profile covering 75% of the tested strains, demonstrated the broadest host range, making it a promising candidate for phage therapy or biocontrol applications [ 43 ]. Host range variation among phages is influenced by tail fiber or receptor-binding protein specificity, which determines the phage’s ability to recognize and infect diverse E. coli strains [ 38 , 33 ]. The activity against E. coli strains isolated from different animal sources (cattle, buffalo, goat) highlights the cross-strain efficacy of these phages, which is critical for field applications in veterinary settings. However, the moderate range observed for BASU E2 and E10 (65% and 55%, respectively) also reflects the narrow-host-range nature of many phages, suggesting that a cocktail approach may enhance therapeutic coverage [ 44 ]. The genome of BASU E2 and BASU E7 was predicated to be intact. Analysis indicated no tRNA in phages BASU E7 and BASU E10. tRNA-encoding genes were not identified in the genomes, suggesting that they rely on the host's tRNA for protein synthesis [ 45 ]. The presence of tRNA genes in the BASU E2 phage genome indicates that it may optimize protein synthesis during infection, potentially possesses a broader host range, and could be evolutionarily adapted for enhanced survival across various hosts or environmental conditions [ 46 ]. Genome analysis found no antibiotic resistance genes, or virulence factors, suggesting that the phage may be safe for phage therapy applications. No integrase gene predicated in all three phages is suggestive of lytic nature and suitability for therapy applications. Genomic analysis revealed that all three phages belong to the class of Caudoviricetes , a class belonging to the tailed phage that represents the highest percentage of the phage sequences submitted in the NCBI database [ 47 ]. Through all phages found effective in killing E. coli strains, BLAST analysis of the genome of phages BASU E2, BASU E7, and BASU E9 revealed the highest similarity to Salmonella phage, E. coli phage, and Shigella phage, respectively. BASU E2 phage shares a high similarity (99%) with Salmonella phage Chennai (MN953776.1), BASU E7 phage shares a high similarity (95%) with Escherichia phage phi2013 (MT427400.3), BASUE10 phage shares a high similarity (93%) with Shigella phage vB_SsoS-ISF002 (MF093736.1) on BLAST analysis. Previously wide host range of coli phage was reported against the Enterobacteria, Proteus mirabilis, Shigella dysenteriae , and Salmonella strains [ 48 ]. Multi-host-specific phages provide better agents for treating complicated and multi-etiological infections and food safety. Each predicted ORF was BLAST in NCBI for functional assignment. The functional ORFs were grouped into 3 categories: host lysis, DNA replication and metabolism, and phage packaging and structure. The host lysis category contained ORFs, of lysis proteins holin, putative class II holin, and lysozyme. DNA replication and metabolism category contains ORF of DNA helicase, endonuclease, DNA polymerase I, single-strand DNA-binding protein, and several others involved in DNA repair and replication. Within the genome of all phages tail-associated genes for tail formation and interactions with hosts were present. Tail spike and phage tail fiber genes significantly determined the host range [ 49 ]. These factors likely contribute to the phage's ability to lyse E. coli strains. The phage packaging and structure category had predicted ORFs, of the terminase subunits, structural proteins for the head and tail, and proteins involved in tail assembly and packaging. Besides other non-assigned hypothetical proteins, ORF was also predicated. Whole genome-based Phylogenetic tree analysis clustered the BASU E2 phage in one cluster and BASU E7 and BASU E10 in another separate cluster. This indicates that the genomes of BASU E7 and BASU E10 are similar and have a close evolutionary relationship, possibly originating from the same ancestor. Furthermore, BASU E2 showed high homology with other Salmonella phages indicating a new type of phage. The genomic interaction graph of phage showed close interaction with Escherichia phage, Enterobacteria phage, and Shigella phage. It allows identifying clusters of closely related phages that target a specific bacterial host, giving a visual overview of phages that are associated with it. The high lytic effects of three different phages against MDR E. coli indicate its possibility to formulate a phage cocktail. Cocktail phages are more effective in the treatment of complicated and multi-etiological infections and in controlling food hygiene [ 34 ]. Conclusion The current investigation highlights the therapeutic promise of bacteriophages as a targeted intervention against MDR E. coli , particularly in veterinary contexts. By integrating phenotypic, environmental, and genomic assessments, the study provides a comprehensive framework for evaluating phage candidates for translational application. The absence of undesirable genetic elements such as virulence or resistance determinants, along with bioinformatic predictions supporting a strictly lytic lifestyle, underscores the biosafety and clinical relevance of the phage isolates. Moreover, the phylogenomic divergence among the phages reflects underlying evolutionary trajectories and suggests complementary potential when used in combination. These findings contribute to the expanding phage repertoire in the Indian context and support the rational design of phage-based biotherapeutics for use in One Health frameworks. Future in vivo validation, stability testing under field conditions, and formulation into tailored phage cocktails will be pivotal in advancing these candidates toward therapeutic implementation. Declarations Ethics approval and consent to participate Not applicable Clinical trial number Not applicable Consent for publication Not applicable Data availability The raw sequencing datasets of the phages have been deposited in the NCBI SRA database under BioProject ID PRJNA1147012, and the corresponding nucleotide sequences have been submitted to GenBank under Submission ID 2953970. Competing interests The authors declare that they have no competing interests. Funding This study was financially supported by Bihar Animal Sciences University, Patna, India, through a university-funded project entitled ‘Isolation and Characterization of Bacteriophages Targeting Foodborne Pathogens from Sewage Samples of Livestock Farms’. Authors' contributions Anjay and P.K. designed the experiments. Anjay, P.K., A.P., R.K.J., and A.J. analyzed the data and drafted the manuscript. B., S.S.R., S.K., A., and B.D. critically revised and arranged the manuscript. 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Pseudomonas aeruginosa PA5oct jumbo phage: a novel perspective for phage therapy in cystic fibrosis. Sci Rep. 2017;7(1):1–15. https://doi.org/10.1038/s41598-017-03368-6 . Merabishvili M, Vervaet C, Pirnay JP, De Vos D, Verbeken G, Mast J, et al. Stability of bacteriophages in different pH and temperature conditions. PLoS ONE. 2009;4(7):e5936. https://doi.org/10.1371/journal.pone.0005936 . Kutter E, Sulakvelidze A, editors. Bacteriophages: Biology and Applications. CRC; 2005. Chan BK, Abedon ST, Loc-Carrillo C. Phage cocktails and the future of phage therapy. Future Microbiol. 2013;8(6):769–83. https://doi.org/10.2217/fmb.13.47 . Drulis-Kawa Z, Majkowska-Skrobek G, Maciejewska B. Bacteriophages and phage-derived proteins—application approaches. Curr Med Chem. 2012;22(14):1757–73. https://doi.org/10.2174/092986712800099767 . Cao Y, Zhang Y, Lan W, Sun X. Characterization of vB_VpaP_MGD2, a newly isolated bacteriophage with biocontrol potential against multidrug-resistant Vibrio parahaemolyticus . Arch Virol. 2021;166:413–26. https://doi.org/10.1007/s00705-020-04846-9 . Lomeli-Ortega CO, Balcázar JL. Why tRNA acquisition could be relevant to bacteriophages? Microb Biotechnol. 2024;17(4):e14464. https://doi.org/10.1111/1751-7915.14464 . Zhu Y, Shang J, Peng C, Sun Y. Phage family classification under Caudoviricetes : A review of current tools using the latest ICTV classification framework. Front Microbiol. 2022;13:1032186. https://doi.org/10.3389/fmicb.2022.1032186 . Goodridge L, Gallaccio A, Griffiths MW. Morphological, host range, and genetic characterization of two coliphages. Appl Environ Microbiol. 2003;69(9):5364–71. https://doi.org/10.1128/AEM.69.9.5364-5371.2003 . Taslem Mourosi J, Awe A, Guo W, Batra H, Ganesh H, Wu X, et al. Understanding bacteriophage tail fiber interaction with host surface receptor: the key blueprint for reprogramming phage host range. Int J Mol Sci. 2022;23(20):12146. https://doi.org/10.3390/ijms232012146 . Additional Declarations No competing interests reported. Supplementary Files SupplementryTableXLS1.xlsx SupplementryTableXLS2.xlsx SupplementrytableXLS3.xlsx SupplementryTableXLS4.xlsx Cite Share Download PDF Status: Published Journal Publication published 07 Feb, 2026 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 14 Aug, 2025 Reviews received at journal 07 Aug, 2025 Reviewers agreed at journal 04 Aug, 2025 Reviews received at journal 02 Aug, 2025 Reviewers agreed at journal 30 Jul, 2025 Reviewers invited by journal 13 May, 2025 Editor assigned by journal 09 May, 2025 Submission checks completed at journal 09 May, 2025 First submitted to journal 30 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6564877","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":455861748,"identity":"8e8927e9-bbbe-439f-b022-9aff5ebbebb5","order_by":0,"name":"Anjay Anjay","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIie2RMQuCUBDHXwjP5ar1pLCv8ECwoKGvkosttjZFS+AUNvctmpyDozdJs9ASBA1NgeAo+WxX24LeD477D/eDO44xjeYXOZWFVTKqOPxC4XyuIrRTKjgI1ZqV3pUet8mavP1olz3T9QSYSedjnWJd/LFASd4h7MbTQJaLge+ndYpImIvIyRGyGzsBLxUEt0Exc8SCnJmEhxMUrRRw0QrJFhyM+zJsoVgJrNCKFjZK3zWWEQJvuqWXmPEA8yn0t3TPgnxj902StYrCQLZVnX9e2jSu6LzYplJfbaY1Go3m/3gDublDf1r32/wAAAAASUVORK5CYII=","orcid":"","institution":"Bihar Animal Sciences University","correspondingAuthor":true,"prefix":"","firstName":"Anjay","middleName":"","lastName":"Anjay","suffix":""},{"id":455861749,"identity":"949cd691-2c58-419a-a7b0-c54d13b97ac5","order_by":1,"name":"Purushottam Kaushik","email":"","orcid":"","institution":"Bihar Animal Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Purushottam","middleName":"","lastName":"Kaushik","suffix":""},{"id":455861750,"identity":"16e0c07a-4df2-458d-a702-85d462fdca2d","order_by":2,"name":"Awadhesh Prajapati","email":"","orcid":"","institution":"Bihar Animal Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Awadhesh","middleName":"","lastName":"Prajapati","suffix":""},{"id":455861751,"identity":"413fe016-a556-4a28-9e65-b834f85a3fe8","order_by":3,"name":"Rohit Kumar Jaiswal","email":"","orcid":"","institution":"Bihar Animal Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Rohit","middleName":"Kumar","lastName":"Jaiswal","suffix":""},{"id":455861752,"identity":"d108d34c-4f16-41f8-a2f4-83fd81655dcf","order_by":4,"name":"Bhoomika Bhoomika","email":"","orcid":"","institution":"Bihar Animal Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Bhoomika","middleName":"","lastName":"Bhoomika","suffix":""},{"id":455861753,"identity":"305406fc-fc6e-45c6-9792-2db4bcf65f01","order_by":5,"name":"Seuli Saha Roy","email":"","orcid":"","institution":"Bihar Animal Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Seuli","middleName":"Saha","lastName":"Roy","suffix":""},{"id":455861754,"identity":"23d8737a-66e5-4c31-b639-c24cd2b87802","order_by":6,"name":"Ajeet Kumar","email":"","orcid":"","institution":"Bihar Animal Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Ajeet","middleName":"","lastName":"Kumar","suffix":""},{"id":455861755,"identity":"3adbb9f1-916f-4f7c-80fd-2c7ff4dd9e31","order_by":7,"name":"Sudha Kumari","email":"","orcid":"","institution":"Bihar Animal Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Sudha","middleName":"","lastName":"Kumari","suffix":""},{"id":455861756,"identity":"da9d5795-edca-4f6e-9131-c217b01da220","order_by":8,"name":"Archana Archana","email":"","orcid":"","institution":"Bihar Animal Sciences University","correspondingAuthor":false,"prefix":"","firstName":"Archana","middleName":"","lastName":"Archana","suffix":""},{"id":455861757,"identity":"e1be8fad-8c0c-43b1-9c6b-e80d8f926cf5","order_by":9,"name":"Baleshwari Dixit","email":"","orcid":"","institution":"Nanaji Deshmukh Veterinary Science University","correspondingAuthor":false,"prefix":"","firstName":"Baleshwari","middleName":"","lastName":"Dixit","suffix":""}],"badges":[],"createdAt":"2025-04-30 12:31:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6564877/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6564877/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-026-04780-8","type":"published","date":"2026-02-07T15:57:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82744851,"identity":"bef1d40d-fcd5-4aea-a80b-b879e22d5c97","added_by":"auto","created_at":"2025-05-14 18:13:00","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":131202,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEscherichia coli \u003c/em\u003estrains with their source of isolation and antibiotic-resistant pattern. The right vertical axis lists the MDR \u003cem\u003eE. coli\u003c/em\u003e isolates ID with their source of isolation and top of the figure is labelled with antibiotics. Resistance is represented as dark black colour.\u003c/p\u003e","description":"","filename":"Fig1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6564877/v1/5de7a8fd9cc9ab095e8d470d.jpeg"},{"id":82744854,"identity":"f3005ef1-eb6f-4d9e-9087-05bc08b50a0a","added_by":"auto","created_at":"2025-05-14 18:13:00","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1397896,"visible":true,"origin":"","legend":"\u003cp\u003ePlaque morphology of\u003cem\u003e Escherichia \u003c/em\u003ephage BASU E2 (2A), \u003cem\u003eEscherichia\u003c/em\u003e phage BASU E7 (2B)\u003cem\u003e \u003c/em\u003eand \u003cem\u003eEscherichia\u003c/em\u003ephage BASU E10 (2C).\u003c/p\u003e","description":"","filename":"Fig.2a2b2c.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6564877/v1/751cdc1e54fb07be4823a18f.jpeg"},{"id":82745006,"identity":"d5c22f69-3279-4d5c-8ecf-d4b0035b2aa3","added_by":"auto","created_at":"2025-05-14 18:21:04","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":661974,"visible":true,"origin":"","legend":"\u003cp\u003eThermal stability (3a) and pH stability (3b) of \u003cem\u003eEscherichia \u003c/em\u003ephages.\u003c/p\u003e","description":"","filename":"Fig3ab.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6564877/v1/5629cdda3351265ef6c3808f.jpeg"},{"id":82744856,"identity":"247bd3fd-d203-4657-9d5a-e6357314755e","added_by":"auto","created_at":"2025-05-14 18:13:00","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":46938,"visible":true,"origin":"","legend":"\u003cp\u003ePhages sensitivity pattern against multi drug resistant \u003cem\u003eE. coli\u003c/em\u003e strains. The right vertical axis lists the phages name and bottom of the figure is labelled with the \u003cem\u003eE. coli\u003c/em\u003e isolates ID. Lysis is represented as dark black colour and no lysis is as blank.\u003c/p\u003e","description":"","filename":"Fig.4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6564877/v1/6a0d08cf943afc6079f4c282.jpeg"},{"id":82744983,"identity":"4a1db269-6cbf-4b9c-812d-5ee7136cfbac","added_by":"auto","created_at":"2025-05-14 18:21:00","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":364674,"visible":true,"origin":"","legend":"\u003cp\u003eCircular genome maps of \u003cem\u003eEscherichia\u003c/em\u003e phages BASUE2 (5a), BASUE7 (5b), and BASUE10 (5c) generated using the Proksee server. The concentric rings (from innermost to outermost) represent GC skew, GC content, and predicted coding sequences (CDSs). Transfer RNA genes (tRNAs, shown in red) were identified only in the BASU E2 genome. Functional annotations of the CDSs are detailed in Supplementary Tables.\u003c/p\u003e","description":"","filename":"Fig5a.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6564877/v1/7c3cfba56f5d4b9cf141ee76.jpeg"},{"id":82744869,"identity":"24ab2478-d81b-4156-be8a-e6dcdb1cef08","added_by":"auto","created_at":"2025-05-14 18:13:00","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":92655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eGenome-wide phylogenetic analysis of Escherichia phages.\u003c/em\u003e The phylogenetic tree was constructed using the VICTOR tool based on the d0 formula (GBDP distance), comprising 40 phage genomes including the BASU phages. Color coding represents taxonomic classification according to the ICTV taxonomy, highlighting the phylogenetic placement and relatedness of the BASU phages within the broader phage community.\u003c/p\u003e","description":"","filename":"Fig6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6564877/v1/bce4b149283f0de70443a8ff.jpeg"},{"id":82744863,"identity":"12c94374-4263-40fd-8de9-4ed87f0dd3b5","added_by":"auto","created_at":"2025-05-14 18:13:00","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":364760,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eGenomic network graphs generated through PhageClauds illustrating the interactions of Escherichia phages BASUE2 (7a), BASUE7 (7b), and BASUE10 (7c) with 26 closely related phages from the GenBank database.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig7a.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6564877/v1/6af91a2ff59b85c01b4b36b6.jpeg"},{"id":102234007,"identity":"3bd0ea8a-9fca-47ab-a8cf-27738b8c4276","added_by":"auto","created_at":"2026-02-09 16:03:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4056805,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6564877/v1/96c4b4fb-0aae-4965-9a68-e0f76ec8e75a.pdf"},{"id":82744850,"identity":"98932221-9acd-4144-a1ca-92577f314283","added_by":"auto","created_at":"2025-05-14 18:13:00","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14033,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementryTableXLS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6564877/v1/45d1aace899faaaf0d5dd4e6.xlsx"},{"id":82745624,"identity":"a54cd071-6966-4ef1-9107-8dbb537d04ce","added_by":"auto","created_at":"2025-05-14 18:29:00","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":51470,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementryTableXLS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6564877/v1/aa1c8fb845a11437c884d96b.xlsx"},{"id":82745622,"identity":"e3464de0-6fb1-4a09-b51c-2abfea417fb3","added_by":"auto","created_at":"2025-05-14 18:29:00","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14197,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementrytableXLS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6564877/v1/db539d41e2190daf74f62b70.xlsx"},{"id":82744987,"identity":"d8da1667-b882-4047-a5aa-b3fc0eaeb138","added_by":"auto","created_at":"2025-05-14 18:21:00","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16317,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementryTableXLS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6564877/v1/fb6b0b59eae6fb67b0898acb.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Diverse lytic bacteriophages from India reveal genomic signatures and therapeutic potential against MDR Escherichia coli","fulltext":[{"header":"Background","content":"\u003cp\u003eAMR is a major threat to public and animal health in the 21st century. It was estimated that worldwide 4\u0026middot;71\u0026nbsp;million deaths were linked with bacterial AMR in 2021 including 1\u0026middot;14\u0026nbsp;million deaths attributable to bacterial AMR [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. \u003cem\u003eE. coli\u003c/em\u003e ranks among the topmost five bacterial pathogens accountable for all infection-related deaths and is among the topmost three pathogens associated with AMR-attributable mortality [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The World Health Organization has recognized \u003cem\u003eE. coli\u003c/em\u003e as one of the 12 bacterial species posing a critical threat to human wellbeing due to its rapidly increasing antimicrobial resistance [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Infections caused by resistant \u003cem\u003eE. coli\u003c/em\u003e strains are challenging to treat in both human and veterinary medicine across the globe [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. As many antimicrobial drugs used in human medicine are also utilized in veterinary medicine and indiscriminate misuse can result in therapeutic failure and increased health consequences in both animals and humans [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. A pan India surveillance of AMR in livestock revealed that \u003cem\u003eE. coli\u003c/em\u003e isolates of food-animal origin were found more frequently resistant to commonly used antibiotics [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The development and approval of new antibacterial agents, including antibiotics, have not kept pace with the rising prevalence of antibiotic-resistant bacteria [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlternative therapeutic approaches are urgently needed to expand treatment options against antibiotic-resistant bacteria. Bacteriophages are emerging as promising therapeutic agents and viable alternatives to antibiotics, with substantial progress being made in their effective clinical application [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Phages are viruses that selectively infect and lyse bacteria, and they are found ubiquitously in various environments [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. They offer several advantages over antibiotics, including potent antibacterial activity, high efficacy in targeting infections without disrupting the normal microbiota, and minimal side effects [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Phages can be engineered to enhance their efficacy by delivering antimicrobial agents directly to the site of infection [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] or can also be used synergistically with antibiotics [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The use of phage combinations (phage cocktails) has been shown to efficiently treat bacterial infections, together with those caused by MDR strains, and reduce resistance and may be used to reduce the potential for bacteria to evolve phage resistance [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, the lack of region-specific phage data, particularly from animal and environmental reservoirs in India, limits our understanding of phage diversity and their therapeutic potential. This study addresses this gap by isolating and characterizing lytic phages targeting MDR \u003cem\u003eE. coli\u003c/em\u003e from livestock sources, contributing to the development of safe and effective alternatives to antibiotics.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains\u003c/h2\u003e \u003cp\u003eMDR \u003cem\u003eE. coli\u003c/em\u003e strains maintained at the Department of Veterinary Public Health and Epidemiology, Bihar Veterinary College, Patna, India served as the bacterial hosts for this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among these, the MDR \u003cem\u003eE. coli\u003c/em\u003e strain BUE4, previously isolated from bovine urine, was employed for the enrichment of sewage samples to facilitate bacteriophage isolation. All MDR \u003cem\u003eE. coli\u003c/em\u003e strains were subjected to rigorous characterization, including assessments of purity, colony morphology, and standard biochemical and molecular identification protocols. These verified strains were subsequently used to perform bacteriophage susceptibility assays.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIsolation and purification of lytic\u003c/b\u003e \u003cb\u003eEscherichia\u003c/b\u003e \u003cb\u003ephages\u003c/b\u003e\u003c/p\u003e \u003cp\u003eApproximately 50 mL of sewage samples (n\u0026thinsp;=\u0026thinsp;10) were collected from livestock farms affiliated with Bihar Animal Sciences University, Patna, India, for phage isolation. The sewage samples were processed using an enrichment-based protocol, following the methodology outlined previously [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Lytic bacteriophages were identified by the formation of clear plaques on a soft agar overlay, indicating bacterial lysis. These phages were subsequently purified through three successive passage of plaque isolation to ensure clonal purity. The naming of the isolated phages followed the guidelines proposed previously [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], incorporating the full host genus name, the term \"phage,\" and a distinct identifier.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhages sensitivity test\u003c/h3\u003e\n\u003cp\u003ePhage sensitivity assays were conducted using the spot test method on top agar overlays seeded with 20 MDR \u003cem\u003eE. coli\u003c/em\u003e strains. For each assay, 10 \u0026micro;l of phage lysate was applied onto the surface of the bacterial lawn and allowed to adsorb completely. The plates were incubated at 37\u0026deg;C for 18\u0026ndash;24 hr and subsequently examined for the presence of clear lytic zones, which signified phage infectivity and host susceptibility. The resulting sensitivity profiles were analyzed and presented using Gene Cluster 3.0 and Java TreeView software, as discussed previously [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003ePhages thermal and pH stability\u003c/h3\u003e\n\u003cp\u003eThe thermal stability of the isolated bacteriophages was assessed by incubating 1 mL aliquots of phage lysate (10⁷ PFU/mL) at four different temperatures: 37\u0026deg;C, 42\u0026deg;C, 65\u0026deg;C, and 80\u0026deg;C. The samples were maintained in a water bath at the designated temperatures for 60 minutes, then rapidly cooled on ice for 10 minutes to halt further thermal activity. Subsequently, serial 10-fold dilutions were prepared, and the remaining viable phage particles were quantified using the double-layer agar plaque assay.\u003c/p\u003e \u003cp\u003eFor pH stability evaluation, 100 \u0026micro;L of phage lysate (10⁸ PFU/mL) was mixed with 900 \u0026micro;L of SM (Salt Magnesium) buffer adjusted to various pH values (3.0, 6.0, 7.5, 9.0, and 12.0). The mixtures were incubated at 37\u0026deg;C for 60 minutes. After incubation, phage titers were determined using the agar overlay method to assess the impact of pH on phage viability.\u003c/p\u003e \u003cp\u003eResults are expressed as mean log₁₀ PFU/mL\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error, calculated from three independent experiments. Each complete experiment was repeated three times (n\u0026thinsp;=\u0026thinsp;3). One-way analysis of variance (ANOVA) was performed to identify significant differences among treatment means, and statistical significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was determined using Tukey\u0026rsquo;s Honest Significant Difference (HSD) test.\u003c/p\u003e\n\u003ch3\u003eWhole genome sequencing, assembly and annotation\u003c/h3\u003e\n\u003cp\u003eGenomes of phages were isolated using the Qigen DNA isolation kit. The concentration of extracted genomic DNA was measured using a NanoDrop. Genomic DNA was sent to Wipro Life Science Lab, Kolkata for whole genomic sequencing using IlluminaNextSeq 2000 (Illumina, San Diego, CA). The sequencing library was prepared and sequencing was performed using the TruSeq Nano DNA prep kit using paired-end 2 \u0026times; 300-bp reads. Quality assessment was conducted using FastQC v0.11.9 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The adapter was trimmed using Trimmomatic v0.39 and low-quality reads with Qscore\u0026thinsp;\u0026lt;\u0026thinsp;20 was removed [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Further contaminated reads were also filtered out using BWA v0.7.17 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Trimmed and filtered sequence reads were used to perform denovo assembly with MetaviralSPAdes with the default parameters [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Assembly quality and completeness check was performed with CheckV tool which checks for host genome contamination [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Assembled contigues of phages were further checked in the Phager web server for identification or phage contig (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phagecompass.ku.dk/\u003c/span\u003e\u003cspan address=\"https://phagecompass.ku.dk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and contamination was removed manually. Genomes were submitted to PHASTEST for rapid preliminary gene calling and annotation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Assembled draft genomes were annotated using the Edge Bioinformatics Server in Prokka and Pharokka [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Additionally, the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://blast.ncbi.nlm.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"http://blast.ncbi.nlm.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized for sequence similarity alignment. For assigning recent taxonomy to a bacteriophage at the genus and species level taxMyPhage server was used [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Blastn was used to compare the whole genome sequences of phages with those in the NCBI database, and the phages with the closest sequences in GenBank were identified. The circular maps of the phage genomes were generated using PHASTEST [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The putative transfer RNA (tRNA) encoding genes were predicted using tRNAscan-SE [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The presence of resistance genes and virulence genes was examined against the NCBI, CARD, and VFDB databases through ABRicate v.0.8.13. The ResFinder platforms were employed to identify the presence of resistance genes (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.cbs.dtu.dk/services/ResFinder/\u003c/span\u003e\u003cspan address=\"https://cge.cbs.dtu.dk/services/ResFinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003ePhage Lifestyle\u003c/h3\u003e\n\u003cp\u003ePhages were further classified using PhageCampass (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ptax.ku.dk/\u003c/span\u003e\u003cspan address=\"https://ptax.ku.dk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). In addition, the phage proteomes were analyzed using PhageGE [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and phageLeads [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] a computational classification algorithm trained to predict phage lifestyles. Putative host and phage promoters in intergenic regions were identified using PhagePromoter [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://galaxy.bio.di.uminho.pt/\u003c/span\u003e\u003cspan address=\"https://galaxy.bio.di.uminho.pt/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Putative rho-independent terminators occurring in intergenic regions were determined using ARNold which computes the free energy of the predicted terminator stem-loops [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eAvailable genome sequences of phages of \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eSalmonella\u003c/em\u003e, and \u003cem\u003eShigella\u003c/em\u003e spp. were retrieved from the NCBI database (Supplementary Table XLS1). The whole-genome phylogenetic trees were constructed using the VICTOR (viral comparison and tree building online resource) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The analysis was conducted under settings recommended for prokaryotic viruses. Phylogenetic tree construction is based on the GBDP method that is specifically configured for prokaryotic viruses. The program calculates intergenomic distances from BLAST\u0026thinsp;+\u0026thinsp;hits using GBDP (including 100 pseudo bootstrap replicates) and uses them to infer a balanced minimum evolution tree with branch support via FASTME including subtree pruning and regrafting post-processing [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Taxon boundaries at the species, genus, and family level were estimated automatically by VICTOR. A genomic network tree was constructed using the PhageClouds ((\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phagecompass.ku.dk/\u003c/span\u003e\u003cspan address=\"https://phagecompass.ku.dk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. PhageClouds supported the search of related phages among all complete phage genomes from GenBank.\u003c/p\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePhage Isolation and Morphological Characterization\u003c/h2\u003e \u003cp\u003eThree lytic bacteriophages- \u003cem\u003eEscherichia\u003c/em\u003e phage BASU E2, BASU E7, and BASU E10 were successfully isolated using multidrug-resistant \u003cem\u003eE. coli\u003c/em\u003e strains as host bacteria. Upon infection, all three phages produced distinct, clear, and circular plaques ranging in size from approximately 0.5 to 1 mm in diameter on the bacterial lawn, indicative of their lytic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhages thermal and pH stability\u003c/h2\u003e \u003cp\u003eThe thermal stability of the isolated phages was assessed by incubating phage lysates at various temperatures (37\u0026deg;C, 42\u0026deg;C, 65\u0026deg;C, and 80\u0026deg;C) for up to 60 min. Phage titers remained relatively stable at 37\u0026deg;C and 42\u0026deg;C, with statistically significant reduction observed at 42\u0026deg;C in compare with stability at 37\u0026deg;C (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, a marked decline in phage viability was recorded following exposure to 65\u0026deg;C, and complete inactivation occurred at 80\u0026deg;C within 60 min of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLikewise, phage stability under varying pH conditions (ranging from pH 3 to 12) was evaluated by incubating phage lysates for 60 min at 37\u0026deg;C in SM buffer adjusted to the respective pH values. Phage viability was well maintained between pH 6 and 9, showing significant loss in infectivity in compare with pH 7.5 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, exposure to highly acidic (pH 3) or highly alkaline conditions (pH\u0026thinsp;\u0026gt;\u0026thinsp;12) led to a complete loss of phage activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhages sensitivity test\u003c/h2\u003e \u003cp\u003eThe lytic activity of \u003cem\u003eEscherichia\u003c/em\u003e phage BASU E2, BASU E7, and BASU E10 was evaluated against 20 multidrug-resistant \u003cem\u003eE. coli\u003c/em\u003e isolates obtained from cattle milk, buffalo milk, and goat rectal swabs, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Among the three phages, BASU E7 exhibited the broadest host range, lysing 75% (15/20) of the tested strains. BASU E2 demonstrated lytic activity against 65% (13/20) of isolates, while BASU E10 was effective against 55% (11/20) of the \u003cem\u003eE. coli\u003c/em\u003e strains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAssembly and annotation\u003c/h2\u003e \u003cp\u003eAll three phage genomes were assembled and the quality of each phage genome was good. Isolated phages (BASU E2, E7, and E10) have a linear double-stranded DNA genome with lengths of 157,938bp, 50,255, and 48,147 bp respectively, and GC contents of 44.62%, 45.47%, and 45.08% respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The genomes contained 204, 77, and 70 putative ORFs. Genomes were predicted for fiber protein, tail protein, head protein, portal protein, holing, neck protein, and putative Endolysin) other than hypothetical proteins (Supplementary Table XLS2). Phage BASU E2 belongs to the family \u003cem\u003eAckermannviridae\u003c/em\u003e and the other two phages BASU E7 and E10 belong to \u003cem\u003eSiphoviridae\u003c/em\u003e in NCBI BLAST-based classification (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In ICTV's latest classification, all phages were classified into the family \u003cem\u003eDrexlerviridae\u003c/em\u003e and the genus \u003cem\u003eTunavirus\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Different tRNA genes were found in the genomes using the tRNA scan-SE program (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The genome of phages was visualized using PHASTEST (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Compared with the ABRicate databases, no virulence or antibiotic resistance genes and virulence genes were identified in the genomes.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAssembly and annotation summary of \u003cem\u003eEscherichia\u003c/em\u003e phages\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBAUE2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBASUE7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBASUE10\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGC%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.62%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.47%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.08%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eContigs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e157,938 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50,255 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48,147\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal CDS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypothetical protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e193\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFunction assigned\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClassification of \u003cem\u003eEscherichia\u003c/em\u003e phages by EDGE Server. Phage contigs were mapped to reference genomes in the NCBI database using minimap2, and taxonomic inference was performed based on sequence similarity.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBASUE2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBASUE7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBASUE10\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSuper kingdom\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eViruses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eViruses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eViruses\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOrder\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCaudovirales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eCaudovirales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaudovirales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFamily\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAckermannviridae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSiphoviridae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eSiphoviridae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGenus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCba120virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT1virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT1virus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSpecies\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e phage GG32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e virus ADB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eShigella\u003c/em\u003e virus Psf2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClassification of \u003cem\u003eEscherichia\u003c/em\u003e phages based on the latest International Committee on the Taxonomy of Viruses (ICTV) taxonomy using the TaxmyPHAGE server.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBASUE2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBASUE7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBASUE10\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKingdom\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHeunggongvirae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHeunggongvirae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eHeunggongvirae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhylum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eUroviricota\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eUroviricota\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eUroviricota\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClass\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCaudoviricetes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eCaudoviricetes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eCaudoviricetes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrder\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePantevenvirales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNot Defined\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNot Defined\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFamily\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAckermannviridae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDrexlerviridae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eDrexlerviridae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubfamily\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCvivirinae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eTunavirinae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eTunavirinae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eKuttervirus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eTunavirus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eTunavirus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKuttervirus PM10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTunavirus new_name\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTunavirus new_name\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrediction of tRNA genes in the genomes of \u003cem\u003eEscherichia\u003c/em\u003e phages\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePredicted tRNA genes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBASUE2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBASUE7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBASUE10\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etRNAs decoding Standard 20 AA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 Met, tyr, Asn and Ser\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSelenocysteine tRNAs (TCA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePossible suppressor tRNAs (CTA, TTA, TCA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etRNAs with undetermined/unknown isotypes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePredicted pseudogenes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal tRNAs\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePhage life Style\u003c/h2\u003e \u003cp\u003ePhage lifestyle was predicted to be virulent and have lytic activity. Integrase genes were not identified in any genomes (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Prediction in PhageLeads could not find any predicted temperate lifestyle genes. Putative host and phage promoters were identified. BASU E2, BASU E7 and BASU E10 phages were predicated of 25, 37, and 30 host promoter sequences (Supplementary Table XLS3). BASU E7 was predicted of 3 phage promoter sequences also. All phages were predicted for 43 putative rho-independent terminators (Supplementary Table XLS4).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhage lifestyle assessment of \u003cem\u003eEscherichia\u003c/em\u003e phages\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhageGE\u003c/p\u003e \u003cp\u003eLytic Score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhageGE\u003c/p\u003e \u003cp\u003eTemperate Score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePHASTEST Integrase\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBASUE2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.986\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBASUE7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.986\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBASUE10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003ePhylogenetic analysis revealed the isolated BASU E2 phage cluster separately from the other two phages BASU E7 and E10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). BASU E7 and E10 phages formed one cluster with \u003cem\u003eEscherichia\u003c/em\u003e phage Tls, \u003cem\u003eEnterobacter\u003c/em\u003e phage F20, \u003cem\u003eEscherichia\u003c/em\u003e phage EB49, \u003cem\u003eEscherichia\u003c/em\u003e phage Jk06, \u003cem\u003eEscherichia\u003c/em\u003e phage Rtp and \u003cem\u003eEnterobacter\u003c/em\u003e phage F20 while BASU E2 clustered with \u003cem\u003eEscherichia\u003c/em\u003e phage PhaxI, \u003cem\u003eEscherichia\u003c/em\u003e phage Cba120 and \u003cem\u003eShigella\u003c/em\u003e phage Ag3. A genomic interaction graph of phage with closet 26 phage based on BLAST search in NCBI with threshold\u0026thinsp;=\u0026thinsp;0.15 was prepared (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). BASU phages showed close interaction with \u003cem\u003eEscherichia\u003c/em\u003e phage, \u003cem\u003eEnterobacter\u003c/em\u003e phage, and \u003cem\u003eShigella\u003c/em\u003e phage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent times, the overuse of antibiotics has resulted in the emergence of multi-drug resistance against \u003cem\u003eE. coli\u003c/em\u003e, which is negatively impacting food safety, animals and human health. Here we report the isolation, host range characterization, and genome sequencing of three phages against multidrug resistance \u003cem\u003eE. coli\u003c/em\u003e isolated from animal clinical samples. The isolation of \u003cem\u003eEscherichia\u003c/em\u003e phages BASU E2, E7, and E10 using MDR \u003cem\u003eE. coli\u003c/em\u003e strains demonstrates their potential as lytic phages for therapeutic applications. The clear, circular plaques (0.5\u0026ndash;1 mm) indicate strong lytic activity, a hallmark of phages capable of efficient bacterial lysis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Such morphology suggests rapid infection cycles and efficient host recognition [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Using MDR strains as hosts enhances clinical relevance, aligning with current efforts to combat antibiotic resistance through phage therapy [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The variation in plaque size may reflect differences in phage replication dynamics, warranting further investigation [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe isolated phages demonstrated good thermal stability at physiological and mildly elevated temperatures (37\u0026deg;C and 42\u0026deg;C), retaining infectivity with significant titer reduction, consistent with the behaviour of other robust lytic phages [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, exposure to higher temperatures (65\u0026deg;C and especially 80\u0026deg;C) significantly reduced or ended phage activity, likely due to capsid protein denaturation or DNA degradation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Similarly, phage viability was stable within a neutral to mildly alkaline pH range (pH 6\u0026ndash;9), which is favourable for therapeutic applications and environmental use [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Extreme pH values (\u0026le;\u0026thinsp;3 or \u0026ge;\u0026thinsp;12) caused complete inactivation, as also observed in other studies, likely due to damage to structural proteins or genome integrity [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. These results highlight the importance of environmental conditions in phage application and formulation strategies.\u003c/p\u003e \u003cp\u003eThe differential lytic activity exhibited by \u003cem\u003eEscherichia\u003c/em\u003e phages BASU E2, E7, and E10 against MDR \u003cem\u003eE. coli\u003c/em\u003e strains underscores their therapeutic potential and host specificity. BASU E7, with a lytic profile covering 75% of the tested strains, demonstrated the broadest host range, making it a promising candidate for phage therapy or biocontrol applications [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Host range variation among phages is influenced by tail fiber or receptor-binding protein specificity, which determines the phage\u0026rsquo;s ability to recognize and infect diverse \u003cem\u003eE. coli\u003c/em\u003e strains [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe activity against \u003cem\u003eE. coli\u003c/em\u003e strains isolated from different animal sources (cattle, buffalo, goat) highlights the cross-strain efficacy of these phages, which is critical for field applications in veterinary settings. However, the moderate range observed for BASU E2 and E10 (65% and 55%, respectively) also reflects the narrow-host-range nature of many phages, suggesting that a cocktail approach may enhance therapeutic coverage [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genome of BASU E2 and BASU E7 was predicated to be intact. Analysis indicated no tRNA in phages BASU E7 and BASU E10. tRNA-encoding genes were not identified in the genomes, suggesting that they rely on the host's tRNA for protein synthesis [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The presence of tRNA genes in the BASU E2 phage genome indicates that it may optimize protein synthesis during infection, potentially possesses a broader host range, and could be evolutionarily adapted for enhanced survival across various hosts or environmental conditions [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Genome analysis found no antibiotic resistance genes, or virulence factors, suggesting that the phage may be safe for phage therapy applications. No integrase gene predicated in all three phages is suggestive of lytic nature and suitability for therapy applications. Genomic analysis revealed that all three phages belong to the class of \u003cem\u003eCaudoviricetes\u003c/em\u003e, a class belonging to the tailed phage that represents the highest percentage of the phage sequences submitted in the NCBI database [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThrough all phages found effective in killing \u003cem\u003eE. coli\u003c/em\u003e strains, BLAST analysis of the genome of phages BASU E2, BASU E7, and BASU E9 revealed the highest similarity to \u003cem\u003eSalmonella\u003c/em\u003e phage, \u003cem\u003eE. coli\u003c/em\u003e phage, and \u003cem\u003eShigella\u003c/em\u003e phage, respectively. BASU E2 phage shares a high similarity (99%) with \u003cem\u003eSalmonella\u003c/em\u003e phage Chennai (MN953776.1), BASU E7 phage shares a high similarity (95%) with \u003cem\u003eEscherichia\u003c/em\u003e phage phi2013 (MT427400.3), BASUE10 phage shares a high similarity (93%) with \u003cem\u003eShigella\u003c/em\u003e phage vB_SsoS-ISF002 (MF093736.1) on BLAST analysis. Previously wide host range of coli phage was reported against the \u003cem\u003eEnterobacteria, Proteus mirabilis, Shigella dysenteriae\u003c/em\u003e, and \u003cem\u003eSalmonella\u003c/em\u003e strains [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Multi-host-specific phages provide better agents for treating complicated and multi-etiological infections and food safety.\u003c/p\u003e \u003cp\u003eEach predicted ORF was BLAST in NCBI for functional assignment. The functional ORFs were grouped into 3 categories: host lysis, DNA replication and metabolism, and phage packaging and structure. The host lysis category contained ORFs, of lysis proteins holin, putative class II holin, and lysozyme. DNA replication and metabolism category contains ORF of DNA helicase, endonuclease, DNA polymerase I, single-strand DNA-binding protein, and several others involved in DNA repair and replication. Within the genome of all phages tail-associated genes for tail formation and interactions with hosts were present. Tail spike and phage tail fiber genes significantly determined the host range [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. These factors likely contribute to the phage's ability to lyse \u003cem\u003eE. coli\u003c/em\u003e strains. The phage packaging and structure category had predicted ORFs, of the terminase subunits, structural proteins for the head and tail, and proteins involved in tail assembly and packaging. Besides other non-assigned hypothetical proteins, ORF was also predicated.\u003c/p\u003e \u003cp\u003eWhole genome-based Phylogenetic tree analysis clustered the BASU E2 phage in one cluster and BASU E7 and BASU E10 in another separate cluster. This indicates that the genomes of BASU E7 and BASU E10 are similar and have a close evolutionary relationship, possibly originating from the same ancestor. Furthermore, BASU E2 showed high homology with other Salmonella phages indicating a new type of phage. The genomic interaction graph of phage showed close interaction with Escherichia phage, Enterobacteria phage, and Shigella phage. It allows identifying clusters of closely related phages that target a specific bacterial host, giving a visual overview of phages that are associated with it. The high lytic effects of three different phages against MDR \u003cem\u003eE. coli\u003c/em\u003e indicate its possibility to formulate a phage cocktail. Cocktail phages are more effective in the treatment of complicated and multi-etiological infections and in controlling food hygiene [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe current investigation highlights the therapeutic promise of bacteriophages as a targeted intervention against MDR \u003cem\u003eE. coli\u003c/em\u003e, particularly in veterinary contexts. By integrating phenotypic, environmental, and genomic assessments, the study provides a comprehensive framework for evaluating phage candidates for translational application. The absence of undesirable genetic elements such as virulence or resistance determinants, along with bioinformatic predictions supporting a strictly lytic lifestyle, underscores the biosafety and clinical relevance of the phage isolates. Moreover, the phylogenomic divergence among the phages reflects underlying evolutionary trajectories and suggests complementary potential when used in combination. These findings contribute to the expanding phage repertoire in the Indian context and support the rational design of phage-based biotherapeutics for use in One Health frameworks. Future in vivo validation, stability testing under field conditions, and formulation into tailored phage cocktails will be pivotal in advancing these candidates toward therapeutic implementation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequencing datasets of the phages have been deposited in the NCBI SRA database under BioProject ID PRJNA1147012, and the corresponding nucleotide sequences have been submitted to GenBank under Submission ID 2953970.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by Bihar Animal Sciences University, Patna, India, through a university-funded project entitled \u0026lsquo;Isolation and Characterization of Bacteriophages Targeting Foodborne Pathogens from Sewage Samples of Livestock Farms\u0026rsquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnjay and P.K. designed the experiments. Anjay, P.K., A.P., R.K.J., and A.J. analyzed the data and drafted the manuscript. B., S.S.R., S.K., A., and B.D. critically revised and arranged the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNaghavi M, Vollset SE, Ikuta KS, Swetschinski LR, Gray AP, Wool EE, et al. Global burden of bacterial antimicrobial resistance 1990\u0026ndash;2021: a systematic analysis with forecasts to 2050. Lancet. 2024;404(10459):1199\u0026ndash;226. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0140-6736(24)00267-3\u003c/span\u003e\u003cspan address=\"10.1016/S0140-6736(24)00267-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang C, Fu X, Liu Y, Zhao H, Wang G. 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Int J Mol Sci. 2022;23(20):12146. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms232012146\u003c/span\u003e\u003cspan address=\"10.3390/ijms232012146\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\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-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Multidrug-resistant Escherichia coli, phage therapy, veterinary pathogens, phage genome analysis","lastPublishedDoi":"10.21203/rs.3.rs-6564877/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6564877/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe rise of antimicrobial resistance (AMR) in \u003cem\u003eEscherichia coli\u003c/em\u003e, particularly among animal-derived isolates, poses a major threat to public and veterinary health. With conventional antibiotics increasingly ineffective against multidrug-resistant (MDR) strains, alternative solutions are urgently needed. Lytic bacteriophages, known for their host specificity and potent antibacterial activity, offer a promising therapeutic option. However, limited genomic data on phages from diverse ecological contexts hinders comprehensive understanding of their diversity and functional potential.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study aimed to isolate, characterize, and assess the therapeutic potential of lytic bacteriophages targeting MDR \u003cem\u003eE. coli\u003c/em\u003e isolated from livestock. Phages were enriched from sewage samples using an MDR \u003cem\u003eE. coli\u003c/em\u003e host. Plaque morphology was assessed for lytic characteristics. Thermal and pH stability were assessed under controlled incubation conditions. Host range was determined against 20 MDR \u003cem\u003eE. coli\u003c/em\u003e strains from cattle, buffalo, and goat. Whole genome sequencing and annotation were performed to determine genetic features, taxonomic classification, safety, and phylogenetic relationships.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThree lytic phages BASU E2, BASU E7, and BASU E10 were successfully isolated, producing clear plaques indicative of strong lytic activity. All phages remained viable at 37\u0026deg;C and 42\u0026deg;C and within pH 6\u0026ndash;9, but were inactivated at 80\u0026deg;C and at highly acidic or alkaline conditions. BASU E7 exhibited the broadest host range, lysing 75% of the tested strains. Genome analysis revealed double-stranded DNA genomes of approximately 157 kb (BASU E2), 50 kb (BASU E7), and 48 kb (BASU E10), with G\u0026thinsp;+\u0026thinsp;C content ranging from 44.62\u0026ndash;45.47%. Taxonomically, BASU E2 was classified under \u003cem\u003eAckermannviridae\u003c/em\u003e, while BASU E7 and BASU E10 belonged to \u003cem\u003eSiphoviridae\u003c/em\u003e, all within \u003cem\u003eCaudovirales\u003c/em\u003e. All phages were predicted to be strictly lytic, lacking genes associated with lysogeny, antibiotic resistance, or virulence. Phylogenetic analysis showed distinct cluster of BASU E2, suggesting divergent evolutionary origin from BASU E7 and BASU E10.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study presents the isolation of genetically safe, environmentally stable, and broadly active lytic phages effective against MDR \u003cem\u003eE. coli\u003c/em\u003e of animal origin. These findings highlight the potential of these phages for use in veterinary phage therapy. However, further in vivo validation and the development of phage cocktails are necessary to advance their clinical application.\u003c/p\u003e","manuscriptTitle":"Diverse lytic bacteriophages from India reveal genomic signatures and therapeutic potential against MDR Escherichia coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-14 18:12:55","doi":"10.21203/rs.3.rs-6564877/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-14T07:36:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-08T00:09:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"220449950863252276382001296355005463461","date":"2025-08-05T01:43:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-02T06:25:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"215600451795882463739600107561481030904","date":"2025-07-30T14:16:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-13T05:17:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-09T05:55:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-09T05:53:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-04-30T12:08:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f25cbb67-e9b1-46e3-b2cc-1b6faf050177","owner":[],"postedDate":"May 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:00:50+00:00","versionOfRecord":{"articleIdentity":"rs-6564877","link":"https://doi.org/10.1186/s12866-026-04780-8","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2026-02-07 15:57:17","publishedOnDateReadable":"February 7th, 2026"},"versionCreatedAt":"2025-05-14 18:12:55","video":"","vorDoi":"10.1186/s12866-026-04780-8","vorDoiUrl":"https://doi.org/10.1186/s12866-026-04780-8","workflowStages":[]},"version":"v1","identity":"rs-6564877","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6564877","identity":"rs-6564877","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}