Isolation and Molecular characterization of a Novel Bacteriophage SVV09-A: Targeted to Staphylococcus ureilyticus from Diabetic Wounds

Isolation and Molecular characterization of a Novel Bacteriophage SVV09-A: Targeted to Staphylococcus ureilyticus from Diabetic Wounds | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Isolation and Molecular characterization of a Novel Bacteriophage SVV09-A: Targeted to Staphylococcus ureilyticus from Diabetic Wounds Lakshmi Sharvani K.S, Pritam kanti Guha, Swetha Vallabhaneni, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8409739/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The pathogenic strains of Staphylococcus ureilyticus are one of the causes of frequently associated nosocomial infections in the hospital environment. The increasing antibiotic resistance in CoNS frequently results in treatment failures, highlighting the pressing requirement for new eradication methods. The present study mainly focused on isolation, and physiological characterization of phages from sewage water that target Staphylococcus ureilyticus , along with molecular characterization (whole genome sequencing) of phages and insilico analysis of its endolysin, including phylogenetic studies, open reading frames, and 3D model. Using the double-layer agar method, several phages were isolated, and the phage that has exhibited the broadest host range was selected. The current study reveals the genome of the lytic phage Staphylococcus ureilyticus , designated SVV09-A, which has been examined and annotated. Further analysis indicated that the phage has exhibited optimal activity at pH levels between 6 and 8 and within a temperature range of 30–37°C; manganese metal ions have shown great impact on phage adsorption rate. The whole genome sequence (WGS) of the phage SVV09-A was determined, revealing a linear DNA of 58,797 bp, with a G + C content of 46.9, and the phage was classified within the order Caudovirales. ORF analysis revealed 336 ORFs and uncovered functions for 67 genes. The estimated endolysin gene of phage SVV09-A had a length of 705 bp, which corresponds to 234 amino acids (~ 25.33 kDa). These findings offer a structural and functional basis for endolysin, establishing a framework for upcoming in vitro and in vivo efficacy research targeting multidrug-resistant Gram-positive infections. Phages Endolysin In silico Staphylococcus ureilyticus Transmission electron microscopy Whole genome sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Staphylococcus ureilyticus , a significant cause of wound and blood stream infections is a gram-positive bacterium, previously known as Staphylococcus cohni subsp ureilyticus. In 1975, scientists found Staphylococcus cohnii (SC) on human skin for the first time and demonstrated that Staphylococcus cohni subsp ureilyticus has a broader host range from primates to humans. It was previously considered as a commensal organism but due to its potential to transmit infections through aerosols and its resistance to different antibiotics, it is recognized as a potential Multi drug resistant (MDR) organism ( 1 ). It is one of the causes of frequently associated nosocomial infections in the hospital environment. These strains can lead to various invasive infections including bacteraemia, septicaemia, endocarditis especially in immunocompromised patients( 2 ). Antibiotic resistance poses a significant threat to global health, compromising the treatment of infections caused by pathogens such as Pseudomonas aeruginosa , Acinetobacter baumannii , Klebsiella pneumoniae , and methicillin-resistant Staphylococcus aureus (MRSA). Bacteriophage therapy, once overshadowed by the advent of antibiotics, has regained attention as an alternative and adjunct treatment option. Phages offer a targeted approach to combating bacterial infections and hold potential in mitigating the spread and impact of MDR pathogens. ( 3 ). Bacteriophages, or phages, are viruses that specifically infect and lyse bacteria and have gained renewed attention as potential alternatives or adjuncts to antibiotics in the era of multidrug-resistant (MDR) pathogens. Their therapeutic value arises from several key features: high specificity for target bacterial strains, the ability to replicate at the site of infection, and the capacity to degrade biofilms through phage-encoded depolymerases. Unlike antibiotics, phages bypass conventional resistance mechanisms such as efflux pumps and β-lactamase activity, making MDR organisms susceptible to phage attack ( 4 – 5 ). However, despite their promise, phage therapy faces significant challenges, including a narrow host range requiring strain-specific selection or phage cocktails, the potential development of phage-resistant bacterial mutants, and regulatory hurdles arising from the need for individualized formulations. Additional limitations include possible immune neutralization of phages, difficulties in ensuring stability during storage and delivery, and concerns about horizontal gene transfer if lysogenic or poorly characterized phages are used ( 6 – 8 ). Overall, bacteriophage therapy offers a compelling, biologically precise approach to combating MDR infections, but broader clinical integration will depend on standardized manufacturing, rigorous genomic screening of phages, optimized delivery strategies, and large-scale clinical trials to validate safety and efficacy. Bacteria employ a diverse set of defense mechanisms to resist bacteriophage infection, acting at multiple stages of the phage replication cycle. the most fundamental strategies include adsorption inhibition, blocking of genome entry using superinfection exclusion systems, abortive infection (Abi) systems. Within this defense mechanisms, endolysins—phage-encoded peptidoglycan hydrolases—play a critical role in the terminal phase of the lytic cycle. Endolysins degrade the bacterial cell wall from within following phage replication, enabling cell lysis and release of progeny virions( 9 ). Because they target highly conserved cell wall structures, endolysins are remarkably potent and are often effective even against metabolically inactive cells or biofilm-embedded bacteria. Their modular architecture, incorporating catalytic and cell-wall binding domains, contributes to substrate specificity and high lytic efficiency. Bacterial resistance to endolysins is generally less common than resistance to antibiotics or adsorption-blocking mechanisms, but bacteria can still modulate susceptibility. Structural alterations such as peptidoglycan O-acetylation, changes in cross-linking density, and modifications in teichoic acid composition can reduce endolysin accessibility or enzymatic efficiency. Biofilm matrices also physically restrict diffusion of lytic enzymes. Yet, the importance of endolysins extends beyond phage biology: their potent, targeted lytic activity has spurred interest in their development as antibacterial agents (“enzybiotics”), particularly against multidrug-resistant Gram-positive pathogens( 10 – 13 ). Their low propensity to induce resistance, ability to function independently of phage infection, and effectiveness against stationary-phase cells have positioned endolysins as promising adjuncts or alternatives in antimicrobial therapy( 14 ). Despite these mechanisms, phages continuously evolve counterdefenses, including anti-CRISPR proteins, modified receptor-binding proteins, epigenetic adaptations, and genome modifications that evade R–M digestion. This ongoing coevolutionary arms race shapes both microbial ecosystems and the outcomes of therapeutic phage applications. To mitigate resistance during phage therapy, strategies such as phage cocktails, sequential phage application, and combined phage–antibiotic therapy are commonly employed( 15 – 19 ). The aim of present study was the isolation and characterization of Staphylococcus ureilyticus phages active against a wide range of infections. The article discusses the isolation and characterization of lytic phage specific to Staphylococcus ureilyticus employing next generation sequencing. The therapeutic potential of these phages by proving their capacity to eliminate MDR bacteria have also been investigated. We further tried to understand the structure of putative endolysin protein using in silico analysis. Materials and methods Isolation of Staphylococcus bacterial strains Among the wound samples isolated from the patients at RIMS, Kadapa, many bacterial spp have been isolated and identified. From the list, we selected only the potential pathogenic i.e., Staphylococcus sps based on morphological, different culture methods (mannitol salt agar, blood agar) physiological and molecular characterization. Before collecting the samples, the wounds were cleaned with phosphate-buffered saline (PBS), and pus samples were obtained of any gender and age with sterile swabs and brought to the laboratory within 1 hour to avoid the wound swabs drying up. The isolates were identified as Staphylococcus sps by the application of culture and standard biochemical tests for identification of Staphylococcus sps as follows: Gram-staining, catalase, Indole, oxidase, mannitol, sucrose and lactose fermentation. The MDR clinical isolates were selected for further analysis. The S taphylococcus spp were preserved at -80 C in Luria Broth medium containing 30% glycerol until further processing. Isolation of SVV09 -A phages Sewage samples for phage isolation were collected from the sources of public ponds in and around hospitals in Kadapa. The bacterial strain was grown overnight in Luria Bertani broth (LB) at 37°C with albumin. LB soft agar overlays were utilized for phage experiments (including isolation and plaque counting). A double-layer agar method was employed for phage isolation and propagation. Sewage water was centrifuged to remove debris, followed by inoculation with Staphylococcus ureilyticus , incubation, and filtration(20-24). A plaque assay was used for detection. Phage purification Phage propagation involves incubating isolated phages with particular host bacteria, followed by the centrifugation and filtration of the upper agar layer. To isolate phages, we choose one plaque and placed it into salt magnesium buffer (SM) combined with Manganese chloride (MnCl2) followed by centrifugation to settle debris, then filtered and the double layer agar technique was employed. Following 18-24 hours, the earlier procedures were repeated to isolate phages. The clear phages were kept in suitable SM buffer with minor adjustments (including the addition of 0.002% w/v gelatin and albumin) at 4◦C and with 30% glycerol at −80◦C Host Range Analysis The isolated phage host range was tested on several pathogenic bacterial strains. The bacterial strains that were tested ( Staphylococcus ureilyticus strains and Staphylococcus aureus strains) were clinical pathogens and wound samples collected at the Rajiv Gandhi Institute of Medical Sciences in Kadapa. The susceptibility of the phage was evaluated using the spot assay method. Plates were inverted and incubated overnight, then examined for plaque presence with negative control. In summary, 100 µl of overnight bacterial host cultures (10 8 –10 9 CFU/ml) were combined with 2.5 ml of 0.7 % soft agar at 45 °C. The mixture was subsequently introduced to a 1.5% solid agar plate with the addition of MnCl 2 metal ion to increase the adsorption on double layer agar plates. Following solidification, 10 µl aliquots of phage suspension (1.0 × 10 8 PFU) were applied to the lawn of host bacteria. The plates were dried and incubated at 37°C for a duration of 18-24 hours(25-26). The area of clearance observed at the site of phage inoculation indicated that the host was susceptible to the corresponding phage. Temperature, pH, and the impact of metal ions on the phage adsorption rate were among the other external variables that were examined. Effect of temperature on the stability and viability of the phages Thermal stability assessments were conducted to evaluate the effect of environmental factors on phage development, as stability is vital for the preservation of lytic phages(27). In this experiment, phage filtrates at a concentration of 1 × 10^9 PFU/ml were prepared in microcentrifuge tubes and exposed to varying temperatures: 30°C, 37°C (control), and 45°C. The samples were incubated for durations of 10, 30, 60, and 90 minutes. After incubation, the double layer agar technique was employed to assess the lytic efficacy of the phages under different temperature conditions, with the results being compared to those of the control at 37°C (24,28-29). Stability of phages at different pH The effects of varying acidic and alkaline pH levels on phages were systematically examined. Purified phages, maintained at known concentrations, were prepared in SM buffer across a range of pH values, specifically pH 2, pH 4, pH 6, pH 7, and pH 8. This incubation occurred for one hour at a controlled temperature of 37 °C, as referenced in (24,30). Subsequently, the phage lysate was serially diluted (up to a 10-fold dilution) with SM buffer, combining 1 mL of the diluted solution with 0.5 mL of host culture ( S. ureilyticus ) at an optical density (O.D.) of 0.6. This mixture was then incubated for 30 minutes under the initial conditions previously mentioned. Phages cultivated in the pH 7 solution were designated as the control group for this experiment. Effect of metal ions on phage adsorption Research was conducted to investigate the influence of calcium, magnesium, zinc, and manganese ions on the adsorption of phages. The primary objective was to assess how divalent metal ions affect the rate of phage adsorption, utilizing solutions of CaCl2, MgSO4, ZnCl2, and MnCl2 (31,32). An overnight culture of S. ureilyticus was prepared, achieving an O.D. of 0.6. This culture was then distributed into four autoclaved flasks, with 25 ml designated for each flask. One set of flasks was inoculated with 500 μL of phage containing 1x10^9 PFU, serving as the control group. The remaining set received 500 μL of phage in conjunction with 250 μL of 10 mM solutions of CaCl2, MgSO4, ZnCl2, and MnCl2. The flasks were incubated under continuous shaking at 120 rpm and 37°C. Samples were collected from both groups at specified intervals: 10, 20, 30, 40, 50, and 60 minutes. Transmission Electron Microscopy We conducted transmission electron microscopy (TEM) analysis to enhance our understanding of the structural determinants of the phage regarding its infectivity and host range, following the protocol (33). Transmission electron microscopy (TEM) was utilized for the identification and classification of bacteriophages at Jamia Hamdard University, New Delhi. Bacteriophage isolate was applied to the grids (carbon film copper grids) and negatively stained using 2% uranyl acetate. The stained samples were dried with filter paper and examined with a transmission electron microscope. Phage classification and identification were performed in accordance with the International Committee on Taxonomy of Viruses recommendations Phage DNA extraction Genomic DNA was isolated utilizing the Phenol-Chloroform extraction technique in conjunction with CTAB (35). To effectively degrade the bacterial genome, the sample treated with DNase and RNase were subjected to heating at 37 °C for a duration of one hour. Following this incubation, the enzymes were inactivated by further heating at 75 °C for ten minutes. A total of 0.5 mL of a 1% SDS solution was added to the remaining 500 µL of phage lysate. To denature the phage protein capsids, a 5 µL aliquot of proteinase K (at a concentration of 20 mg/mL) was added, and the mixture was incubated overnight at 56 °C. After the digestion, 1 mL of a phenol–chloroform–isoamyl alcohol solution was placed, and the mixture was centrifuged for five minutes at 6000 rpm. A quarter volume of sodium acetate was then incorporated, along with an equal volume of isopropanol and followed by centrifugation for fifteen minutes at 13,000 rpm, the supernatant was collected. These steps were followed by two sequential washing by the addition of 1 mL of 70% ethanol, and further centrifugate the content for two minutes at 13,000 rpm. Finally, the DNA pellet was air dried (36,37). Phage DNA purification and Quantification The phage genomic DNA was extracted and quantified using both the phenol-chloroform extraction method and the CTAB method for column purification. The evaluation was conducted with a Nanodrop™ Lite Spectrophotometer (Thermo Fisher Scientific Limited) to determine the DNA concentration, which was measured at 260 nm and yielded a total reading of 46.7 ng/µL. Furthermore, the purity, quality, and size of the DNA were analyzed through agarose gel electrophoresis. Whole genome Illumina sequencing of SVV09-A phage: Phage DNA was sequenced utilizing Illumina technology on the HiSeq 2500 platform (Illumina, USA) at Eurofins Pvt. Ltd. in Bangalore. This sequencing achieved a genome coverage of 30X, generating a total of 3.5 GB of data for the sample. Library preparation and Data analysis Paired end sequencing was prepared from the DNA sample using NEB Next® Ultra TM II FS DNA Library Preparation kit for Illumina. 100ng of DNA sample was processed for enzymatic fragmentation using FS enzyme mix supplied in the kit to generate a mean fragment distribution of 200-300bp. The fragmentated DNA samples were then subjected to end-repair and adapter ligation as per the kit recommendation. The adapter ligated products were purified using AMPureXP beads and processed for PCR amplification with the index primers to facilitate the hybridization onto a flow cell. The purified PCR-enriched libraries were evaluated on the 4200 Tape Station System (Agilent Technologies) utilizing high sensitivity D1000 screen tape according to the manufacturer's guidelines. The PE (Pair End) Illumina libraries were introduced to the NovaSeq X Plus for cluster creation and were utilized to sequence the entire genome of the phages. The high-quality PE reads of the sample were assembled using metaviral SPAdes assembler (v3.15.5). Insilco analysis of SVV09-A phage Genome validation, Gene Prediction and Phylogeny In order to evaluate the completeness of phage genome, the genome was validated using PhageScope. The assessment of completeness was conducted utilizing Average Amino acid Identity method (AAI). Gene prediction was performed using GeneMarkS (phage). The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) used to manually annotate the predicted proteins’ specific functions. Circular plot was generated using Proksee which helps in the identification of gene position, GC content, GC Skew. The Mega 11 tool was additionally utilized to generate a diagram illustrating the comparative analysis of phage genomes alongside their related homologs. Open reading frames (ORFs) Identification The ORF Finder tool from NCBI was utilized to identify possible open reading frames (ORFs) in the phage DNA sequence. The software offers the range of each ORF along with the translation of its associated protein(38, 39). Endolysin gene structural identification and Phylogenetic analysis To analyse the structural features and evolutionary relationships of the endolysin gene, we performed a BLASTP search for the endolysin protein sequence to identify homologous sequences across different species. NCBI tools, were used for pairwise alignments of similar sequences from various organisms (40). We retrieved FASTA format sequences closely related to the target endolysin protein and filtered them based on alignment and similarity scores to ensure their relevance to the endolysin protein family(38, 41). Additionally, the identification of transmembrane helices within the protein was conducted using DeepTMHMM version 1.0, ensuring a comprehensive understanding of the protein’s structural and functional properties(38). The endolysin protein sequence was meticulously analyzed to determine its secondary structure using the SOPMA algorithm, a method described (42) and further utilized (43). To assess the protein's physical and chemical properties, and other parameters were calculated through the Expasy-Protparam tool (44,45). These parameters included the theoretical isoelectric point, which indicates the pH at which the protein has no net charge, and the molecular weight, reflecting the size of the protein in Daltons. Additionally, we determined the total counts of positive and negative amino acid residues, the extinction coefficient, which is important for spectroscopic analyses, the protein's half-life in vivo, the instability index suggesting the stability of the protein in a cellular environment, the aliphatic index reflecting the proportion of hydrophobic residues, and the grand average hydropathy (GRAVY) score, which provides insights into the hydrophilic or hydrophobic nature of the protein. To predict the protein's folding state, we employed the FoldIndex program (46), which utilizes the amino acid composition to estimate the likelihood of a given conformation. A three-dimensional representation of the endolysin protein was constructed in the Swiss-Model environment, a platform known for its ability to generate homology models based on template sequences. Following the model generation, we conducted a thorough analysis to identify the model with the highest sequence identity to the chosen template. This selection was based on comparative metrics, including the GMQE (Global Model Quality Estimate) score and the QMEAN score, both of which provide quantitative measures of model reliability. Elevated GMQE and QMEAN (Qualitative Model Energy Analysis) scores are indicative of a high-quality model, suggesting that it closely resembles the true structure of the protein (47). Finally, to assess the conformational viability of the predicted model, we generated a Ramachandran plot using the RAMPAGE server (48). This plot provides visual insights into the distribution of dihedral angles in the protein structure, allowing us to validate the sterically allowed conformations of amino acid residues. Through these comprehensive analyses, we gained valuable understanding of the structural and functional aspects of the endolysin protein. Results Isolation of Bacteria A total of 120 samples were collected from wound patients with Staphylococcus ureilyticus identification and characterization performed using a combination of biochemical and molecular characterization as well as antibiotic sensitivity testing. The prevalence of Staphylococcus ureilyticus was found to be 20.33%. Antibiotic susceptibility tests indicated that 96% of Staphylococcus ureilyticus was found to be resistant to Chloramphenicol while oxacillin exhibited lowest resistance with only 50% effectiveness. The most predominant bacteria Staphylococcus ureilyticus was selected for further analysis. The nucleotide sequences of S. ureilyticus SVV09 were deposited at the GenBank database under the accession number OR625452. Isolation, purification and amplification of SVV09-A phages Phages isolated against S. ureilyticus SVV09 strain, large to medium-sized plaques are observed. Further, Phage titre was determined for examining the specific phage of interest and the selected phages were purified using plaque method and the purified phages were stored in an appropriate SM buffer with the addition of 0.002% w/v gelatine and 0.2g albumin at 4 ◦ C and with 30% glycerol at −80 ◦ C which remained stable for several months. No significant changes in PFU/ml, phage viability, and lytic efficiency were observed after storage. We assessed the host range of phage isolates through spot tests involving S. ureilyticus SVV09 and with other Staphylococcus aureus strains. Findings indicated that some phages displayed lytic properties against Staphylococcus ureilyticus and other Staphylococcus aureus strains evaluated as demonstrated by the inhibition zones (Fig. 1) and. Each of the phages underwent testing for lytic activity. The phage SVV09-A exhibited extensive lytic activity, lysing half of the evaluated Staphylococcus strains, resulting in its choice for additional characterization The primary external factor influencing phage stability is pH, while temperature impacts the lytic activity of SVV09-A . They were only fluctuating using various pH ranges, and more stable phages were achieved within a pH range of 6 to 8, although low activity was detected at pH 2.0 and pH 4 (Fig:2). Subsequently, thermal stability findings indicated that temperature is a key factor influencing phage effectiveness in phage therapy. The impact of temperature on the phages exhibited different outcomes (Fig:3 ). Findings indicated that the phage exhibited activity at the temperatures examined. Phage SVV09-A exhibited nearly the same activity at 30 ºC and 37 ºC, showing no considerable decrease in plaque formation. The assessment of the phages' stability and viability at a high temperature of 45 ºC over various time intervals was conducted. Phage incubation for 10 to 90 minutes led to decreased phage proliferation at 45 ºC. Consequently, these findings indicate that temperatures of 37 ºC and a pH of 7 positively influence the stability of phage SVV09-A. The impact of divalent metal ions (MgSO4, CaCl2, ZnCl2, and MnCl2) on the adsorption rate of phages has been extensively investigated and documented. The data indicates that ZnCl2 and MnCl2 ions increased the adsorption rate of SVV09-A phage (Fig: 4), significantly boosting the adsorption rate of phages as shown by the PFU measured at regular intervals of 10, 20, 30, 40, 50, and 60 minutes ( Fig : 5). These findings indicate that the influence of metal ions enhances both the infectivity rate and the burst size. Genomic analysis of SVV09-A phage The DNA of the phage was subjected to meticulous analysis through agarose gel electrophoresis, vividly depicted (Fig 6A). This process yielded a clear visual representation of the DNA fragments, allowing for an insightful examination of its structure. Subsequently, the purified DNA underwent sophisticated whole genome sequencing, executed on the state-of-the-art Illumina platform, utilizing a 2x150 bp chemistry configuration that promises unparalleled accuracy and depth of coverage. The complete genome of the SVV09-A phage was scrutinized using the cutting-edge PhageScope technology. This examination revealed a striking confirmation of the phage's genomic integrity, validated by the average amino acid identity technique, which showcases the high fidelity of its genetic material. This particular phage houses a linear DNA, a design that provides resilience and stability. To explore its exquisite structural characteristics, scientists employed a transmission electron microscope, which unveiled the details of the phage's architecture, as illustrated in (Fig 6B). The SVV09-A phage boasts a captivating morphology: its icosahedral head, with a diameter of approximately 44 nanometers, embodies a geometric form that enhances its ability to attach to and penetrate host cells. Extending from this head is a long, flexible, non-contractile tail, measuring around 231 nanometers, which plays a critical role in the phage's intricate infection process. Based on these morphological characteristics, the phage is classified within the Siphoviridae family, Caudovirales order known for its tailed phages, which encompasses many well-known phages recognized for their complex life cycles, renowned for its array of phages that engage in complex interplay with their bacterial hosts. For future reference and potential applications, the nucleotide sequences of SVV09-A have been carefully documented and submitted to the BANKIT database, accessible under the specific accession number PV593182. The GeneMarkS (Phage) software was employed to explore and identify a range of functional genes within the phage genome, particularly those associated with essential functions like endolysins, which are enzymes that degrade bacterial cell walls, and tail proteins, which are crucial for virus attachment to host cells. Through this comprehensive analysis, a total of 67 distinct genes were identified. The average length of these genes was approximately 807 base pairs (bp), demonstrating significant variability in gene size; the longest gene measured an impressive 4,338 bp, while the shortest was only 123 bp. A large portion of the identified genes exhibited homology with the Proteus phage vB_PmiS_Jing313, suggesting a shared evolutionary history and functional similarities. To visualize these findings, a circular plot was created using the Proksee software, which is represented (Fig: 7). The results from BLAST hit analysis revealed that the SVV09-A phage shares notable similarities with the Proteus phage vB_PmiS_NotEvenPhaged, as depicted ( Fig: S1). Lastly, to provide a detailed comparison of the sequences, a multiple sequence alignment of the SVV09-A phage was produced, which is displayed (Fig: S2). This alignment helps to highlight variations and conserved regions among the sequences, offering insights into the functional significance of these genes within the phage biology The ORF Finder tool has successfully provided a remarkable total of 336 potential open reading frames (ORFs) (Fig: S3) throughout the complete genomic sequence of the phage of which 67 genes were functionally assigned. Furthermore, detailed positional data is available, indicating both the starting and ending nucleotide positions of each ORF. This information is crucial for understanding the length of each ORF, with measurements reported in terms of the number of amino acids and nucleotides (Fig S4). Each identified ORF has been meticulously assigned a distinct numerical identifier, which facilitates easy reference and tracking. The tool provides comprehensive information for each ORF, including its precise location within the genome, the direction of the DNA strand on which it is located, and the corresponding reading frame. These extensive findings provide significant insight into the potential protein-coding regions present within the investigated DNA sequence, offering a valuable foundation for future research and deeper exploration of the phage genetic capabilities. These findings shed light on potential protein-coding regions in the DNA sequence being investigated. The BLAST hit reveals a fascinating similarity between test protein and the endolysin found in the Proteus phage PM87 (YP_009997985.1) (Fig: S5). The multiple sequence alignment of endolysin protein was shown with maximum sequence identity orthologous sequences with a E-value 2e-170 (Fig: S6) Interestingly, the prediction of transmembrane helices identified a single TMhelix site within the endolysin, situated between amino acids 207 and 222, suggesting a strategic role in membrane interactions. The structure and function of a protein are significantly influenced by various physical and chemical characteristics. In this study, the coding sequence for endolysin was determined to be 705 base pairs in length, resulting in a peptide composed of 234 amino acids. The anticipated secondary structure in SOPMA (Self-Optimized Prediction method With Alignment) analysis reveals a complex arrangement, featuring 90 alpha helices, 135 random coils, and 9 extended strands, as illustrated (Fig: 8 and Table 1). Upon conducting in silico folding analysis, we discovered that the protein contains three distinct unfolded segments. Notably, the longest of these segments comprises 33 consecutive residues. Collectively, these unfolded regions consist of 63 amino acids, highlighting a significant portion of the protein's structure that deviates from a stable conformation (Fig: 9). Table 1 SOPMA analysis of Endolysin protein No of Amino acids Alpha Helix Extended Strand Random Coil 234 Amino acids 90 Aminoacids (38.46%) 9 (3.85%) 135 (57.69%) The ProtParam tool provided essential insights into the protein’s composition, revealing that it comprises 42% non-polar hydrophobic amino acids, indicating a propensity for hydrophobic interactions, and 58% polar hydrophilic amino acids, which are likely to engage in hydrogen bonding and interactions with the aqueous environment. Additionally, the molar extinction coefficient at 280 nm was measured to be 36,900 M⁻¹ cm⁻¹ under both conditions: when all cysteine residues are involved in disulfide bonds and when they are in their reduced form, suggesting a consistent behavior in absorbance due to structural integrity. A detailed analysis yielded an instability index of 21.40, indicating that the protein may be relatively stable, coupled with an aliphatic index of 88.72, which suggests a high degree of hydrophobicity that could influence the protein’s thermal stability. Additionally, the grand average of hydropathicity was measured at -0.251, providing further insight into the protein's overall hydropathy (Table 2). Three-dimensional (3D) configurations of proteins are of paramount importance for elucidating their functional roles, dynamics, and potential interactions with ligands. To ensure the reliability of our 3D protein model, we evaluated the positioning of residues in relation to the permissible regions of the Ramachandran plot, which serves as a hallmark for assessing stereochemical integrity. This analysis is critical in confirming the reliability of our constructed model and its applicability in further research. The sequence demonstrated alignment with fourteen distinct templates, revealing a broad range of similarity percentages between 8.00% and 55.77%. To identify the most suitable model, we carefully evaluated several criteria, including the maximum sequence similarity, Global Model Quality Estimation (GMQE), and QMEAN scores. GMQE serves as a comprehensive quality assessment tool that synthesizes various characteristics derived from the alignment with the target template, showcasing the model's reliability. On the other hand, QMEAN is a sophisticated scoring function that assesses both global and local quality metrics, allowing us to gauge the structural integrity of the model effectively. Among the analyzed templates, the one exhibiting the highest sequence similarity of 53.16%, unveiled that the folding topology of the proposed endolysin closely mirrors that of the endolysin from Enterobacteriophage enc34, showcasing an impressive 99.96% confidence, with the query sequence also recorded the best GMQE score of 0.54 and the most favorable QMEAN score of 0.73. This template was subsequently chosen for further analysis and the corresponding PDB file was obtained. To assess the stereochemical quality of the model, we utilized the RAMPAGE server to generate a Ramachandran plot. The results revealed that an impressive 152 out of 157 residues, constituting 96.82% of the total, are located within the most favored regions, indicative of a well-structured model. Additionally, 4 residues (2.55%) fell within the allowed region, suggesting acceptable stereochemistry, while only one residue, specifically A20 Glycine, was identified in the outlier region (Fig. 10). This analysis underscores the model's overall high quality and reliability for further applications. Table 2: Prot param analysis of endolysin protein Theoretical isoelectric point Molecular weight(kDa) Non-polar Hydrophobic residues Polar hydrophilic residues Negatively charged residues Positively charged residues Extinction coefficients at 280 nm (Cys oxidized form) Extinction coefficients at 280 nm (Cys reduces form) Aliphatic index Grand average of hydropathicity (GRAVY): Instability index (II) The estimated half-life is: 9.18 25.5 42% 58% 23 28 36900 M -1 cm -1 36900 M -1 cm -1 88.72 -0.251 21.40 30 hours (mammalian reticulocytes, in vitro). >20 hours (yeast, in vivo). >10 hours (Escherichia coli, in vivo). Discussion Infections of diabetic wounds pose formidable challenges for healthcare providers, often deepened by the insidious presence of coagulase-negative Staphylococci (CoNS), intricate mixed microbial communities, and opportunistic fungi. The alarming rise in antibiotic resistance, now recognized as a pressing global health crisis, significantly complicates the path to effective treatment ( 49 ). CoNS are particularly troublesome, harboring the mecA gene and the cfr gene, which alter adenine residues at various sites. This genetic alteration interferes with the efficacy of at least five distinct classes of antibiotics, including oxazolidinones, phenicols, lincosamides, pleuromutilins, and streptogramin A ( 50 ). The battle against these resilient pathogens is a vivid reminder of the intricate and often daunting landscape of modern medicine. The identification and prevention of coagulase-negative staphylococci (CoNS) are critical in medical settings. This study concentrated on the isolation and identification of Staphylococcus ureilyticus strains derived from samples of diabetic wounds, as well as the characterization of phages that exhibit efficacy against these bacteria. Among the isolated strains, the Staphylococcus spp constituted the most prevalent group, with CoNS accounting for 24%. The notable frequency of Staphylococcus ureilyticus identified in our study aligns with findings (Soldera et al. 2013), which emphasized the role of Staphylococcus ureilyticus as a frequent colonizer within healthcare environments. Together, these findings highlight the critical need for prompt monitoring and intervention strategies in wound care units, as infections caused by CoNS can lead to serious and potentially life-threatening complications for affected patients. Culture and biochemical tests were utilized in the identification of S. ureilyticus. They are Gram-positive cocci, negative coagulase, and positive catalase, which aligns with findings from another research ( 51 ). The positive catalase test indicated the presence of hydrogen peroxide-decomposing enzymes typical of staphylococci, while a negative coagulase test differentiated it from other species, particularly Staphylococcus aureus . Furthermore, the ability of the isolate to ferment mannitol supported its identification as Staphylococcus ureilyticus , consistent with findings reported in other studies ( 1 ), Despite being time-consuming, these techniques continue to be very effective for precise identification. We identified a Staphylococcus phage obtained from sewage water in hospitals, specifically the phage SVV09-A . The phage showed a broad range of host specificity. Following isolation, Whole-genome sequencing uncovered key genes essential for phage activity, and open reading frame (ORF) analysis identified 336 potential coding sequences. The proteins found in the SVV09-A phage genomes include DNA primase, DNA helicase, exonuclease, transcriptional regulator, tail fiber, and endolysin. These proteins are crucial for the effective replication and spread of phages within host bacteria genome sequencing uncovered few of the critical genes necessary for phage activity, while ORF analysis identified 336 possible coding sequences with a putative endolysin ORF was found to be ORF 315. The phage SVV09-A exhibited an unexpectedly high genomic similarity (97.13%) to a previously described Proteus phage vB_PmiS_NotEvenPhaged and we also observed that the endolysin gene of a Staphylococcus -infecting bacteriophage is 100% identical to the endolysin of a phage reported to infect Proteus spp. Such cross-genera genomic conservation is uncommon, particularly because Staphylococcus and Proteus occupy distinct phylogenetic and ecological niches and represent Gram-positive and Gram-negative bacteria, respectively. However, several well-characterized evolutionary features of bacteriophage biology provide plausible explanations for this observation. First, endolysins are among the most highly conserved proteins in the phage genome, primarily due to their essential role in hydrolyzing the bacterial peptidoglycan to allow progeny release. The catalytic domains responsible for cleaving glycosidic or amide bonds in peptidoglycan are structurally constrained, and even minor amino acid substitutions can significantly impair enzymatic activity ( 61 – 62 ). As a result, endolysins often evolve under strong purifying selection, preserving identical catalytic sequences across diverse phage taxa. Reports of nearly identical endolysins in phages infecting different bacterial genera support the idea that these enzymes retain conserved functionality despite host divergence ( 63 ). Second, endolysins belong to the lysis module, one of the most frequently exchanged genetic regions in bacteriophage genomes. Phages readily undergo horizontal gene transfer and modular recombination, particularly in environmental reservoirs rich in microbial diversity, such as wastewater, soil, and the animal microbiome ( 64 – 65 ). The holin–endolysin cassette often moves as a functional unit, enabling phages infecting unrelated hosts to acquire identical lysis genes. Thus, the complete identity between the Staphylococcus and Proteus phage endolysins may reflect recent or recurrent module exchange events rather than shared host specificity. This modular evolution allows phages infecting different genera to retain near-identical genomic backbones while varying only in genes required for host recognition. Thus, the high overall sequence similarity observed here may reflect a shared evolutionary lineage, with divergence occurring primarily in host-range determinant genes such as tail fibers or receptor-binding proteins. Third, although Staphylococcus and Proteus differ in cell envelope structure, the core architecture of peptidoglycan—including the MurNAc–GlcNAc backbone and conserved peptide crosslinks—remains sufficiently similar to allow a shared enzymatic mechanism of degradation. Many endolysins target bonds that are broadly conserved across Gram-positive and Gram-negative bacteria ( 59 , 66 ). Consequently, identical catalytic domains may function effectively in phages infecting different bacterial phyla, enabling such enzymes to be maintained across wide evolutionary distances. Collectively, these findings underscore the modular, mosaic, and functionally constrained nature of phage genomes, particularly within the lysis system. The perfect identity between the two endolysin genes suggests strong structural and biochemical conservation of the enzyme, combined with the capacity of phages to exchange key functional modules across taxonomic boundaries. These results highlight that phage genomic similarity—and especially endolysin conservation—does not necessarily correlate with host specificity and should be interpreted with caution when inferring phage evolutionary relationships or potential host range. Endolysins have shown significant promise as potent antibacterial agents, particularly against predominant Gram-positive pathogens such as Staphylococcus aureus . These phage-derived enzymes could potentially replace traditional antibiotics in the treatment of various bacterial infections ( 52 ). Recent studies mentioned that endolysin treatment is often more clinically effective than conventional antibiotic therapies ( 53 ). The unique specificity of endolysins towards their target bacteria underscores an evolutionary adaptation that enables them to efficiently degrade cell walls exhibiting distinct structural features. Among the templates we reviewed, the one with the highest sequence similarity at 53.16% showed that the suggested endolysin's folding structure is very similar to that of the endolysin from Enterobacteriophage enc34, with a confidence level of 99.96%. The query sequence also achieved the highest GMQE score of 0.54 and the best QMEAN score of 0.73 and these findings are similar to other studies ( 54 ). Typically, endolysins are composed of multiple domains, including specialized cell wall binding domains (CBDs) that specifically recognize and bind to unique molecular components. For instance, they target the glycine-rich peptidoglycan layer characteristic of Staphylococcus , facilitating the precise lysis of these bacteria as well as other related streptococci ( 55 ). These insights not only illuminate the mechanism of action for endolysins but also raise important considerations for their broader therapeutic applicability ( 56 ). To enhance their effectiveness against a wider array of bacterial species, it is essential to explore engineering strategies that can improve endolysin permeability. This would help to overcome the inherent host specificity of these enzymes and broaden their potential use in combating resistant bacterial infections. The proteins and genes identified in the current study likely serve a crucial role in evading the formidable defences of Staphylococcus ureilyticus . Hence the remarkable ability positions SVV09-A phage as an exceptional candidate for phage therapy, especially in the battle against stubborn infections which resist conventional antibiotics. Conclusion The incidence of bacterial infections is escalating globally on a daily basis. Staphylococcus ureilyticus has emerged as a predominant pathogen associated with wound infections. This bacterium has been identified in various wound specimens obtained from patients with hospital-acquired infections in RIMS, Kadapa. Experimental findings indicate that the isolated phage exhibit complete lytic activity, as evidenced by the presence of clear lysis zones on the isolated host bacteria. Phages specific to Staphylococcus ureilyticus were successfully isolated and purified, and their host range against S taphylococcus strains were identified. Among these, a phage exhibiting a broad host range and robust lytic activity was selected and characterized. The SVV09-A phage is classified within the Siphoviridae family, belongs to Caudovirales order with a documented genome size of 58.80 kb based on structural characteristics. The stability of the phage was assessed in relation to various factors, including temperature, pH levels, and the presence of metal ions. This research emphasizes the critical need for evaluating safety protocols associated with phage therapies, ensuring their viability as potential therapeutic options. Furthermore, bioinformatics and molecular research methodologies were employed to enhance our understanding of gene sequences and their potential applications in therapy. The proposed endolysins demonstrate stable structures, distinct charge characteristics, and unique elemental compositions, suggesting a role in complex cellular mechanisms. A comprehensive understanding of phages and the biology of lytic enzymes may facilitate the development of innovative treatments for bacterial resistance to various pharmacological agents. The phage SVV09-A shows in vitro lytic activity against its host strain, significant stability, extensive efficacy and its endolysin appears to be stable enzyme in silico which requires further investigation for addressing multi-drug-resistant Staphylococcus ureilyticus. Our analysis revealed that the endolysin of a Staphylococcus -infecting bacteriophage is identical at the nucleotide and amino acid levels to the endolysin encoded by a phage reported to infect Proteus spp. Bacteriophage genomes evolve through extensive modular exchange, yet core lysis functions are thought to be shaped primarily by host-specific constraints. Here we show that a Staphylococcus phage encodes an endolysin that is identical to that of a phage reported to infect Proteus , two distantly related bacterial genera. This unexpected conservation highlights the strong functional constraints acting on endolysins and reveals that essential lysis modules can move across broad taxonomic boundaries. Our findings refine current understanding of phage genome modularity, challenge assumptions that link gene content to host identity, and provide insight into the evolutionary and functional stability of enzymes with therapeutic potential. Abbreviations MDR: Multi drug resistant CoNS: Coagulase-negative Staphylococci pH: Potential of Hydrogen LB: Luria Bertani Broth PFU: Plaque forming Unit MnCl2: Manganese Chloride ZnCl2: Zinc Chloride CaCl2: Calcium Chloride MgSO4: Magnesium Sulphate SM: Salt magnesium AMR: Antimicrobial resistance DNA: Deoxy ribonucleic acid dsDNA: Double stranded Deoxy ribonucleic acid rRNA: Ribosomal ribonucleic acid PCR: Polymerase Chain reaction TM helix: Transmembrane helix DEEPTMHMM: A Deep Learning Model for Transmembrane Topology Prediction and Classification NEB: New England Biolabs FS: Fragment size BP: Base Pair GRAVY: Grand average hydropathy GMQE: Global Model Quality Estimate QMEAN: Qualitative Model Energy Analysis SOPMA: Self-Optimized Prediction method With Alignment NGS: Next Generation Sequencing WGS: Whole Genome Sequencing ORF: Open reading Frame CBD: Cell wall binding domain MSA: Multiple sequence alignment Declarations Supplementary material: The supplementary figures are available in attached pdf. Funding: This study was a part of the project supported the Rashtriya Uchchattar Shiksha Abhiyan (RUSA) (File No: RUSA-ANU/YVU/Research project-24/Sanction order/2024) Acknowledgements: The authors would like to acknowledge Rashtriya Uchchattar Shiksha Abhiyan (RUSA) for funding, additionally, the Department of Microbiology at Yogi Vemana University in Kadapa for their assistance during the research Author Contributions: Author Contribution Statement; Lakshmi Sharvani K.S has carried out the experiments and prepared the original draft. Vijaya Lakshmi D and Vijaya Raghava Prasad D conceived, planned and supervised the study. Pritam Kanti Guha reviewed and edited the manuscript. Swetha Vallabhaneni, Guru Prasad C., Renuka Pallem, Vaishnavi R., Krishna Vamsi M reviewed the manuscript. All authors have read and approved the final manuscript. Ethical approval: The work has been conducted at the Department of Microbiology, Yogi Vemana University, Kadapa, after receiving ethical approval from the Institutional Ethical Committee (IEC) (Ref. No. 01/IEC/DVR/YVU/2022-23, dated 24.04.2023). Consent for Publication: All authors agreed to the final version of manuscript and give their consent to publish. Competing Interests: The authors declare no competing interests. References Kwon, H., Park, S. Y., Kim, et al. (2022). Characterization of a Lytic bacteriophage vB_SurP-PSU3 Infecting Staphylococcus ureilyticus and Its Efficacy Against Biofilm. Frontiers in microbiology , 13 , 925866. https://doi.org/10.3389/fmicb.2022.925866 Lavecchia, A., Chiara, M., De Virgilio, C., et al. (2021). Comparative Genomics Suggests a Taxonomic Revision of the Staphylococcus cohnii Species Complex. Genome biology and evolution , 13 (4), evab020. https://doi.org/10.1093/gbe/evab020 English, B. K, and A. H. Gaur. (2010). 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16:38:53","extension":"xml","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":176491,"visible":true,"origin":"","legend":"","description":"","filename":"c6fe58fba52a4a27814c10e21e7969b51structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8409739/v1/c71e8b3da3fcc30228fbb0f8.xml"},{"id":99286950,"identity":"ecca5b94-9716-47fe-a52b-4ba0b5767d37","added_by":"auto","created_at":"2025-12-31 09:32:44","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":199301,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8409739/v1/c8336ee89ff7a8bd5ff52ee7.html"},{"id":99286916,"identity":"2b9ddb5b-560a-4921-b852-658badca9355","added_by":"auto","created_at":"2025-12-31 09:32:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":742072,"visible":true,"origin":"","legend":"\u003cp\u003eDouble layer agar plates showing plaque formed by phages, isolated from sewage water against \u003cem\u003eStaphylococcus ureilyticus SVV09\u003c/em\u003e. Phage has shown large clear zone.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8409739/v1/ec7b87548157eadc515749ea.png"},{"id":99286917,"identity":"c5e9379a-42ec-4e24-9e45-127c1f9add99","added_by":"auto","created_at":"2025-12-31 09:32:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22896,"visible":true,"origin":"","legend":"\u003cp\u003epH effect on \u003cem\u003eSVV09-A phage\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8409739/v1/6b53b7f397e699106e0e0d2c.png"},{"id":99286918,"identity":"81230f53-fcad-4320-a8b3-3858087f2585","added_by":"auto","created_at":"2025-12-31 09:32:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27702,"visible":true,"origin":"","legend":"\u003cp\u003eTemperature effect on \u003cem\u003eSVV09-A \u003c/em\u003ephage\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8409739/v1/bea9fda84725ee76f374f1ae.png"},{"id":99320682,"identity":"1f871bc0-40dc-4261-a22c-abbf88f472cf","added_by":"auto","created_at":"2025-12-31 16:38:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":248432,"visible":true,"origin":"","legend":"\u003cp\u003ePictorial representation of the impact of metal ions (MnCl2: Manganese chloride, ZnCl2: Zinc Chloride, Ca: calcium chloride, MgSO4: Magnesium sulphate) on adsorption rate of phage of which “Mn and Zn” metals has significant impact on phage adsorption \u0026nbsp;while “Ca and Mg” has less impact on phage adsorption.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8409739/v1/46a3b80149777dfa5f9c318d.png"},{"id":99320663,"identity":"5fe14303-6c35-406b-8824-dafea9c254d2","added_by":"auto","created_at":"2025-12-31 16:38:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":90730,"visible":true,"origin":"","legend":"\u003cp\u003eA, B, C, D shows impact of metal ions (Ca, Mg, Mn, Zn) respectively on \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8409739/v1/32489604142afef14a4909d4.png"},{"id":99286925,"identity":"c78adbd0-181e-4a39-bd4b-1a151d1ef8b6","added_by":"auto","created_at":"2025-12-31 09:32:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":329464,"visible":true,"origin":"","legend":"\u003cp\u003eA. Phage DNA sample on 0.8% Agarose-gel. \u003cstrong\u003eB. \u003c/strong\u003eElectron Micrographs of the\u003cem\u003eS. ureilyticus\u003c/em\u003e \u003cem\u003eSVV09-A\u003c/em\u003e phage.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8409739/v1/d34ded841669c41afe205dc2.png"},{"id":99320840,"identity":"47de1ca6-8de2-469c-9ebe-1d2958cd1b19","added_by":"auto","created_at":"2025-12-31 16:38:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1032133,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of circular plot representing the position of genes and GC content of \u003cem\u003eSVV09-A phage\u003c/em\u003e (PV593182.1) using Proksee\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8409739/v1/c5d11d93d14bda03e6849e77.png"},{"id":99286921,"identity":"33a05bf7-43c7-457a-868d-e99a1919b320","added_by":"auto","created_at":"2025-12-31 09:32:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":425379,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted secondary structure of endolysin. Blue vertical lines indicate alpha helix; yellow vertical lines represent random coil. Amino acids involved in specific secondary structures: ‘e’ indicates extended strand, ‘c’ indicates random coil, ‘h’ indicates alpha helix.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8409739/v1/88550d8e104812a21a417410.png"},{"id":99320725,"identity":"e1b92d5f-7e6f-4e4c-bc49-a61736dacfdb","added_by":"auto","created_at":"2025-12-31 16:38:52","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":170315,"visible":true,"origin":"","legend":"\u003cp\u003eA 3D model of the Endolysin protein was created with SWISS MODEL. A. representing its predicted folding structure. Positive and negative values on the Y-axis indicate regions of folding and unfolding. The figures on the X-axis denote amino acid residue numbers. B The optimal model was chosen according to the greatest sequence identity with the template, along with the highest GMQE and QMEAN scores. Greater GMQE and QMEAN scores signify enhanced reliability of the constructed model.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8409739/v1/6fd2c73eed272d07025a3d39.png"},{"id":99321313,"identity":"0fabc4c9-e35b-440a-ab23-666ca3e273b2","added_by":"auto","created_at":"2025-12-31 16:39:20","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":210948,"visible":true,"origin":"","legend":"\u003cp\u003eRamachandran plot for putative endolysin protein model. Cyan, blue and red (dots/triangles) represent torsion angles of favoured, allowed and disallowed regions respectively; dot represents residues other than glycine and triangles represents glycine\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-8409739/v1/54f5423c2deb698b7c881f82.png"},{"id":106093542,"identity":"09835b23-aebd-4c8d-90a7-c80ba9c37b97","added_by":"auto","created_at":"2026-04-03 11:37:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4373427,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8409739/v1/f032caf1-da2c-44c9-a4f9-338b2e7291c2.pdf"},{"id":99320003,"identity":"38b47da1-6488-4419-b1b9-4bd080ad85e4","added_by":"auto","created_at":"2025-12-31 16:38:05","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3759114,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigues.docx","url":"https://assets-eu.researchsquare.com/files/rs-8409739/v1/58653e25cf535ad9453a4043.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation and Molecular characterization of a Novel Bacteriophage SVV09-A: Targeted to Staphylococcus ureilyticus from Diabetic Wounds","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e, a significant cause of wound and blood stream infections is a gram-positive bacterium, previously known as \u003cem\u003eStaphylococcus cohni subsp ureilyticus.\u003c/em\u003e In 1975, scientists found \u003cem\u003eStaphylococcus cohnii\u003c/em\u003e (SC) on human skin for the first time and demonstrated that \u003cem\u003eStaphylococcus cohni subsp ureilyticus\u003c/em\u003e has a broader host range from primates to humans. It was previously considered as a commensal organism but due to its potential to transmit infections through aerosols and its resistance to different antibiotics, it is recognized as a potential Multi drug resistant (MDR) organism (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It is one of the causes of frequently associated nosocomial infections in the hospital environment. These strains can lead to various invasive infections including bacteraemia, septicaemia, endocarditis especially in immunocompromised patients(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAntibiotic resistance poses a significant threat to global health, compromising the treatment of infections caused by pathogens such as \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, and methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA). Bacteriophage therapy, once overshadowed by the advent of antibiotics, has regained attention as an alternative and adjunct treatment option. Phages offer a targeted approach to combating bacterial infections and hold potential in mitigating the spread and impact of MDR pathogens. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Bacteriophages, or phages, are viruses that specifically infect and lyse bacteria and have gained renewed attention as potential alternatives or adjuncts to antibiotics in the era of multidrug-resistant (MDR) pathogens. Their therapeutic value arises from several key features: high specificity for target bacterial strains, the ability to replicate at the site of infection, and the capacity to degrade biofilms through phage-encoded depolymerases. Unlike antibiotics, phages bypass conventional resistance mechanisms such as efflux pumps and β-lactamase activity, making MDR organisms susceptible to phage attack (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). However, despite their promise, phage therapy faces significant challenges, including a narrow host range requiring strain-specific selection or phage cocktails, the potential development of phage-resistant bacterial mutants, and regulatory hurdles arising from the need for individualized formulations. Additional limitations include possible immune neutralization of phages, difficulties in ensuring stability during storage and delivery, and concerns about horizontal gene transfer if lysogenic or poorly characterized phages are used (\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Overall, bacteriophage therapy offers a compelling, biologically precise approach to combating MDR infections, but broader clinical integration will depend on standardized manufacturing, rigorous genomic screening of phages, optimized delivery strategies, and large-scale clinical trials to validate safety and efficacy.\u003c/p\u003e \u003cp\u003eBacteria employ a diverse set of defense mechanisms to resist bacteriophage infection, acting at multiple stages of the phage replication cycle. the most fundamental strategies include adsorption inhibition, blocking of genome entry using superinfection exclusion systems, abortive infection (Abi) systems. Within this defense mechanisms, endolysins\u0026mdash;phage-encoded peptidoglycan hydrolases\u0026mdash;play a critical role in the terminal phase of the lytic cycle. Endolysins degrade the bacterial cell wall from within following phage replication, enabling cell lysis and release of progeny virions(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Because they target highly conserved cell wall structures, endolysins are remarkably potent and are often effective even against metabolically inactive cells or biofilm-embedded bacteria. Their modular architecture, incorporating catalytic and cell-wall binding domains, contributes to substrate specificity and high lytic efficiency.\u003c/p\u003e \u003cp\u003eBacterial resistance to endolysins is generally less common than resistance to antibiotics or adsorption-blocking mechanisms, but bacteria can still modulate susceptibility. Structural alterations such as peptidoglycan O-acetylation, changes in cross-linking density, and modifications in teichoic acid composition can reduce endolysin accessibility or enzymatic efficiency. Biofilm matrices also physically restrict diffusion of lytic enzymes. Yet, the importance of endolysins extends beyond phage biology: their potent, targeted lytic activity has spurred interest in their development as antibacterial agents (\u0026ldquo;enzybiotics\u0026rdquo;), particularly against multidrug-resistant Gram-positive pathogens(\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Their low propensity to induce resistance, ability to function independently of phage infection, and effectiveness against stationary-phase cells have positioned endolysins as promising adjuncts or alternatives in antimicrobial therapy(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite these mechanisms, phages continuously evolve counterdefenses, including anti-CRISPR proteins, modified receptor-binding proteins, epigenetic adaptations, and genome modifications that evade R\u0026ndash;M digestion. This ongoing coevolutionary arms race shapes both microbial ecosystems and the outcomes of therapeutic phage applications. To mitigate resistance during phage therapy, strategies such as phage cocktails, sequential phage application, and combined phage\u0026ndash;antibiotic therapy are commonly employed(\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe aim of present study was the isolation and characterization of \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e phages active against a wide range of infections. The article discusses the isolation and characterization of lytic phage specific to \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e employing next generation sequencing. The therapeutic potential of these phages by proving their capacity to eliminate MDR bacteria have also been investigated. We further tried to understand the structure of putative endolysin protein using in silico analysis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eIsolation of \u003cem\u003eStaphylococcus\u003c/em\u003e bacterial strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the wound samples isolated from the patients at RIMS, Kadapa, many bacterial spp have been isolated and identified. From the list, we selected only the potential pathogenic i.e., \u003cem\u003eStaphylococcus sps\u0026nbsp;\u003c/em\u003ebased on morphological, different culture methods (mannitol salt agar, blood agar) physiological and molecular characterization. Before collecting the samples, the wounds were cleaned with phosphate-buffered saline (PBS), and pus samples were obtained of any gender and age with sterile swabs and brought to the laboratory within 1 hour to avoid the wound swabs drying up. The isolates were identified as \u003cem\u003eStaphylococcus\u003c/em\u003e \u003cem\u003esps\u003c/em\u003e by the application of culture and standard biochemical tests for identification of \u003cem\u003eStaphylococcus\u003c/em\u003e \u003cem\u003esps\u003c/em\u003e as follows: Gram-staining, catalase, Indole, oxidase, mannitol, sucrose and lactose fermentation. The MDR clinical isolates were selected for further analysis. The S\u003cem\u003etaphylococcus spp\u0026nbsp;\u003c/em\u003ewere preserved at -80 C in Luria Broth medium\u0026nbsp;containing 30% glycerol until further processing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of \u003cem\u003eSVV09\u003c/em\u003e-A phages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSewage samples for phage isolation were collected from the sources of public ponds in and around hospitals in Kadapa. The bacterial strain was grown overnight in Luria Bertani broth (LB) at 37\u0026deg;C with albumin. LB soft agar overlays were utilized for phage experiments (including isolation and plaque counting). A double-layer agar method was employed for phage isolation and propagation. Sewage water was centrifuged to remove debris, followed by inoculation with \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e, incubation, and filtration(20-24). A plaque assay was used for detection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhage purification\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhage propagation involves incubating isolated phages with particular host bacteria, followed by the centrifugation and filtration of the upper agar layer. To isolate phages, we choose one plaque and placed it into salt magnesium buffer (SM) combined with Manganese chloride (MnCl2) followed by centrifugation to settle debris, then filtered and the double layer agar technique was employed. Following 18-24 hours, the earlier procedures were repeated to isolate phages. The clear phages were kept in suitable SM buffer with minor adjustments (including the addition of 0.002% w/v gelatin and albumin) at 4◦C and with 30% glycerol at \u0026minus;80◦C\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHost Range Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe isolated phage host range was tested on several pathogenic bacterial strains. The bacterial strains that were tested \u003cem\u003e( Staphylococcus ureilyticus\u003c/em\u003e strains and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e strains) were clinical pathogens and wound samples collected at the Rajiv Gandhi Institute of Medical Sciences in Kadapa. The susceptibility of the phage was evaluated using the spot assay method. Plates were inverted and incubated overnight, then examined for plaque presence with negative control. In summary, 100 \u0026micro;l of overnight bacterial host cultures (10\u003csup\u003e8\u003c/sup\u003e \u0026ndash;10\u003csup\u003e9\u003c/sup\u003e CFU/ml) were combined with 2.5 ml of 0.7 % soft agar at 45 \u0026deg;C. The mixture was subsequently introduced to a 1.5% solid agar plate with the addition of MnCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003emetal ion to increase the adsorption on double layer agar plates. Following solidification, 10 \u0026micro;l aliquots of phage suspension (1.0 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e PFU) were applied to the lawn of host bacteria. The plates were dried and incubated at 37\u0026deg;C for a duration of 18-24 hours(25-26). The area of clearance observed at the site of phage inoculation indicated that the host was susceptible to the corresponding phage.\u003c/p\u003e\n\u003cp\u003eTemperature, pH, and the impact of metal ions on the phage adsorption rate were among the other external variables that were examined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of temperature on the stability and viability of the phages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThermal stability assessments were conducted to evaluate the effect of environmental factors on phage development, as stability is vital for the preservation of lytic phages(27). In this experiment, phage filtrates at a concentration of 1 \u0026times; 10^9 PFU/ml were prepared in microcentrifuge tubes and exposed to varying temperatures: 30\u0026deg;C, 37\u0026deg;C (control), and 45\u0026deg;C. The samples were incubated for durations of 10, 30, 60, and 90 minutes. After incubation, the double layer agar technique was employed to assess the lytic efficacy of the phages under different temperature conditions, with the results being compared to those of the control at 37\u0026deg;C (24,28-29).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStability of phages at different pH\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of varying acidic and alkaline pH levels on phages were systematically examined. Purified phages, maintained at known concentrations, were prepared in SM buffer across a range of pH values, specifically pH 2, pH 4, pH 6, pH 7, and pH 8. This incubation occurred for one hour at a controlled temperature of 37 \u0026deg;C, as referenced in (24,30). Subsequently, the phage lysate was serially diluted (up to a 10-fold dilution) with SM buffer, combining 1 mL of the diluted solution with 0.5 mL of host culture (\u003cem\u003eS. ureilyticus\u003c/em\u003e) at an optical density (O.D.) of 0.6. This mixture was then incubated for 30 minutes under the initial conditions previously mentioned. Phages cultivated in the pH 7 solution were designated as the control group for this experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of metal ions on phage adsorption\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearch was conducted to investigate the influence of calcium, magnesium, zinc, and manganese ions on the adsorption of phages. The primary objective was to assess how divalent metal ions affect the rate of phage adsorption, utilizing solutions of CaCl2, MgSO4, ZnCl2, and MnCl2 (31,32). An overnight culture of \u003cem\u003eS. ureilyticus\u003c/em\u003e was prepared, achieving an O.D. of 0.6. This culture was then distributed into four autoclaved flasks, with 25 ml designated for each flask. One set of flasks was inoculated with 500 \u0026mu;L of phage containing 1x10^9 PFU, serving as the control group. The remaining set received 500 \u0026mu;L of phage in conjunction with 250 \u0026mu;L of 10 mM solutions of CaCl2, MgSO4, ZnCl2, and MnCl2. The flasks were incubated under continuous shaking at 120 rpm and 37\u0026deg;C. Samples were collected from both groups at specified intervals: 10, 20, 30, 40, 50, and 60 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission Electron Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted transmission electron microscopy (TEM) analysis to enhance our understanding of the structural determinants of the phage regarding its infectivity and host range, following the protocol (33). Transmission electron microscopy (TEM) was utilized for the identification and classification of bacteriophages at Jamia Hamdard University, New Delhi. Bacteriophage isolate was applied to the grids (carbon film copper grids) and negatively stained using 2% uranyl acetate. The stained samples were dried with filter paper and examined with a transmission electron microscope. Phage classification and identification were performed in accordance with the International Committee on Taxonomy of Viruses recommendations\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhage DNA extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA was isolated utilizing the Phenol-Chloroform extraction technique in conjunction with CTAB (35). To effectively degrade the bacterial genome, the sample treated with DNase and RNase were subjected to heating at 37 \u0026deg;C for a duration of one hour. Following this incubation, the enzymes were inactivated by further heating at 75 \u0026deg;C for ten minutes. A total of 0.5 mL of a 1% SDS solution was added to the remaining 500 \u0026micro;L of phage lysate. To denature the phage protein capsids, a 5 \u0026micro;L aliquot of proteinase K (at a concentration of 20 mg/mL) was added, and the mixture was incubated overnight at 56 \u0026deg;C. After the digestion, 1 mL of a phenol\u0026ndash;chloroform\u0026ndash;isoamyl alcohol solution was placed, and the mixture was centrifuged for five minutes at 6000 rpm. \u0026nbsp;A quarter volume of sodium acetate was then incorporated, along with an equal volume of isopropanol and followed by centrifugation for fifteen minutes at 13,000 rpm, the supernatant was collected. These steps were followed by two sequential washing by the addition of 1 mL of 70% ethanol, and further centrifugate the content for two minutes at 13,000 rpm. Finally, the DNA pellet was air dried (36,37).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhage DNA purification and Quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phage genomic DNA was extracted and quantified using both the phenol-chloroform extraction method and the CTAB method for column purification. The evaluation was conducted with a Nanodrop\u0026trade; Lite Spectrophotometer (Thermo Fisher Scientific Limited) to determine the DNA concentration, which was measured at 260 nm and yielded a total reading of 46.7 ng/\u0026micro;L. Furthermore, the purity, quality, and size of the DNA were analyzed through agarose gel electrophoresis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole genome Illumina sequencing of \u003cem\u003eSVV09-A\u0026nbsp;\u003c/em\u003ephage:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhage DNA was sequenced utilizing Illumina technology on the HiSeq 2500 platform (Illumina, USA) at Eurofins Pvt. Ltd. in Bangalore. This sequencing achieved a genome coverage of 30X, generating a total of 3.5 GB of data for the sample.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLibrary preparation and Data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePaired end sequencing was prepared from the DNA sample using NEB Next\u0026reg; Ultra\u003csup\u003eTM\u003c/sup\u003e II FS DNA Library Preparation kit for Illumina. 100ng of DNA sample was processed for enzymatic fragmentation using FS enzyme mix supplied in the kit to generate a mean fragment distribution of 200-300bp. The fragmentated DNA samples were then subjected to end-repair and adapter ligation as per the kit recommendation. The adapter ligated products were purified using AMPureXP beads and processed for PCR amplification with the index primers to facilitate the hybridization onto a flow cell. The purified PCR-enriched libraries were evaluated on the 4200 Tape Station System (Agilent Technologies) utilizing high sensitivity D1000 screen tape according to the manufacturer\u0026apos;s guidelines. The PE (Pair End) Illumina libraries were introduced to the NovaSeq X Plus for cluster creation and were utilized to sequence the entire genome of the phages. The high-quality PE reads of the sample were assembled using metaviral SPAdes assembler (v3.15.5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInsilco analysis of \u003cem\u003eSVV09-A phage\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome validation, Gene Prediction and Phylogeny\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to evaluate the completeness of phage genome, the genome was validated using PhageScope. The assessment of completeness was conducted utilizing Average Amino acid Identity method (AAI). Gene prediction was performed using GeneMarkS (phage).\u0026nbsp;The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) used to manually annotate the predicted proteins\u0026rsquo; specific functions. Circular plot was generated using Proksee which helps in the identification of gene position, GC content, GC Skew. The Mega 11 tool was additionally utilized to generate a diagram illustrating the comparative analysis of phage genomes alongside their related homologs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen reading frames (ORFs) Identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ORF Finder tool from NCBI was utilized to identify possible open reading frames (ORFs) in the phage DNA sequence. The software offers the range of each ORF along with the translation of its associated protein(38, 39).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEndolysin gene structural identification and Phylogenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyse the structural features and evolutionary relationships of the endolysin gene, we performed a BLASTP search for the endolysin protein sequence to identify homologous sequences across different species. NCBI tools, were used for pairwise alignments of similar sequences from various organisms (40). We retrieved FASTA format sequences closely related to the target endolysin protein and filtered them based on alignment and similarity scores to ensure their relevance to the endolysin protein family(38, 41). Additionally, the identification of transmembrane helices within the protein was conducted using DeepTMHMM version 1.0, ensuring a comprehensive understanding of the protein\u0026rsquo;s structural and functional properties(38).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; The endolysin protein sequence was meticulously analyzed to determine its secondary structure using the SOPMA algorithm, a method described \u0026nbsp; (42) and further utilized (43). To assess the protein\u0026apos;s physical and chemical properties, and other parameters were calculated through the Expasy-Protparam tool (44,45). These parameters included the theoretical isoelectric point, which indicates the pH at which the protein has no net charge, and the molecular weight, reflecting the size of the protein in Daltons. Additionally, \u0026nbsp;we determined the total counts of positive and negative amino acid residues, the extinction coefficient, which is important for spectroscopic analyses, the protein\u0026apos;s half-life in vivo, the instability index suggesting the stability of the protein in a cellular environment, the aliphatic index reflecting the proportion of hydrophobic residues, and the grand average hydropathy (GRAVY) score, which provides insights into the hydrophilic or hydrophobic nature of the protein. To predict the protein\u0026apos;s folding state, we employed the FoldIndex program (46), which utilizes the amino acid composition to estimate the likelihood of a given conformation. A three-dimensional representation of the endolysin protein was constructed in the Swiss-Model environment, a platform known for its ability to generate homology models based on template sequences. Following the model generation, we conducted a thorough analysis to identify the model with the highest sequence identity to the chosen template. This selection was based on comparative metrics, including the GMQE (Global Model Quality Estimate) score and the QMEAN score, both of which provide quantitative measures of model reliability. Elevated GMQE and QMEAN (Qualitative Model Energy Analysis) scores are indicative of a high-quality model, suggesting that it closely resembles the true structure of the protein (47). Finally, to assess the conformational viability of the predicted model, we generated a Ramachandran plot using the RAMPAGE server (48). This plot provides visual insights into the distribution of dihedral angles in the protein structure, allowing us to validate the sterically allowed conformations of amino acid residues. Through these comprehensive analyses, we gained valuable understanding of the structural and functional aspects of the endolysin protein.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIsolation of Bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 120 samples were collected from wound patients with \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e identification and characterization performed using a combination of biochemical and molecular characterization as well as antibiotic sensitivity testing. The prevalence of \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e was found to be 20.33%. Antibiotic susceptibility tests indicated that 96% of \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e was found to be resistant to Chloramphenicol while oxacillin exhibited lowest resistance with only 50% effectiveness. The most predominant bacteria \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e was selected for further analysis. The nucleotide sequences of \u003cem\u003eS. ureilyticus\u003c/em\u003e \u003cem\u003eSVV09\u003c/em\u003e were deposited at the GenBank database under the accession number OR625452.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation, purification and amplification of \u003cem\u003eSVV09-A\u003c/em\u003e phages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhages isolated against \u003cem\u003eS. ureilyticus SVV09\u003c/em\u003e strain, large to medium-sized plaques are observed. Further, Phage titre was determined for examining the specific phage of interest and the selected phages were purified using plaque method and the purified phages were stored in an appropriate SM buffer with the addition of 0.002% w/v gelatine and 0.2g albumin at 4\u003csup\u003e◦\u003c/sup\u003eC and with 30% glycerol at \u0026minus;80\u003csup\u003e◦\u003c/sup\u003eC which remained stable for several months. \u0026nbsp; No significant changes in PFU/ml, phage viability, and lytic efficiency were observed after storage. We assessed the host range of phage isolates through spot tests involving S. ureilyticus SVV09 and with other \u003cem\u003eStaphylococcus aureus\u003c/em\u003e strains. Findings indicated that some phages displayed lytic properties against \u003cem\u003eStaphylococcus ureilyticus\u0026nbsp;\u003c/em\u003eand other \u003cem\u003eStaphylococcus aureus\u003c/em\u003e strains evaluated as demonstrated by the inhibition zones (Fig. 1) and. Each of the phages underwent testing for lytic activity. The phage \u003cem\u003eSVV09-A\u003c/em\u003e exhibited extensive lytic activity, lysing half of the evaluated \u003cem\u003eStaphylococcus\u003c/em\u003e strains, resulting in its choice for additional characterization\u003c/p\u003e\n\u003cp\u003eThe primary external factor influencing phage stability is pH, while temperature impacts the lytic activity of \u003cem\u003eSVV09-A\u003c/em\u003e. They were only fluctuating using various pH ranges, and more stable phages were achieved within a pH range of 6 to 8, although low activity was detected at pH 2.0 and pH 4 (Fig:2). Subsequently, thermal stability findings indicated that temperature is a key factor influencing phage effectiveness in phage therapy. The impact of temperature on the phages exhibited different outcomes (Fig:3 ). Findings indicated that the phage exhibited activity at the temperatures examined. Phage \u003cem\u003eSVV09-A\u003c/em\u003e exhibited nearly the same activity at 30 \u0026ordm;C and 37 \u0026ordm;C, showing no considerable decrease in plaque formation. The assessment of the phages\u0026apos; stability and viability at a high temperature of 45 \u0026ordm;C over various time intervals was conducted. Phage incubation for 10 to 90 minutes led to decreased phage proliferation at 45 \u0026ordm;C. Consequently, these findings indicate that temperatures of 37 \u0026ordm;C and a pH of 7 positively influence the stability of phage \u003cem\u003eSVV09-A.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe impact of divalent metal ions (MgSO4, CaCl2, ZnCl2, and MnCl2) on the adsorption rate of phages has been extensively investigated and documented. The data indicates that ZnCl2 and MnCl2 ions increased the adsorption rate of\u003cem\u003e\u0026nbsp;SVV09-A\u0026nbsp;\u003c/em\u003ephage (Fig: 4), significantly boosting the adsorption rate of phages as shown by the PFU measured at regular intervals of 10, 20, 30, 40, 50, and 60 minutes ( Fig : 5). These findings indicate that the influence of metal ions enhances both the infectivity rate and the burst size.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenomic analysis of \u003cem\u003eSVV09-A\u003c/em\u003e phage \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DNA of the phage was subjected to meticulous analysis through agarose gel electrophoresis, vividly depicted (Fig 6A). This process yielded a clear visual representation of the DNA fragments, allowing for an insightful examination of its structure. Subsequently, the purified DNA underwent sophisticated whole genome sequencing, executed on the state-of-the-art Illumina platform, utilizing a 2x150 bp chemistry configuration that promises unparalleled accuracy and depth of coverage. The complete genome of the \u003cem\u003eSVV09-A\u003c/em\u003e phage was scrutinized using the cutting-edge PhageScope technology. This examination revealed a striking confirmation of the phage\u0026apos;s genomic integrity, validated by the average amino acid identity technique, which showcases the high fidelity of its genetic material. This particular phage houses a linear DNA, a design that provides resilience and stability. To explore its exquisite structural characteristics, scientists employed a transmission electron microscope, which unveiled the details of the phage\u0026apos;s architecture, as illustrated in (Fig 6B). The \u003cem\u003eSVV09-A\u003c/em\u003e phage boasts a captivating morphology: its icosahedral head, with a diameter of approximately 44 nanometers, embodies a geometric form that enhances its ability to attach to and penetrate host cells. Extending from this head is a long, flexible, non-contractile tail, measuring around 231 nanometers, which plays a critical role in the phage\u0026apos;s intricate infection process. Based on these morphological characteristics, the phage is classified within the Siphoviridae family, Caudovirales order known for its tailed phages, which encompasses many well-known phages recognized for their complex life cycles, renowned for its array of phages that engage in complex interplay with their bacterial hosts. For future reference and potential applications, the nucleotide sequences of \u003cem\u003eSVV09-A\u003c/em\u003e have been carefully documented and submitted to the BANKIT database, accessible under the specific accession number PV593182.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe GeneMarkS (Phage) software was employed to explore and identify a range of functional genes within the phage genome, particularly those associated with essential functions like endolysins, which are enzymes that degrade bacterial cell walls, and tail proteins, which are crucial for virus attachment to host cells. Through this comprehensive analysis, a total of 67 distinct genes were identified. The average length of these genes was approximately 807 base pairs (bp), demonstrating significant variability in gene size; the longest gene measured an impressive 4,338 bp, while the shortest was only 123 bp. A large portion of the identified genes exhibited homology with the Proteus phage vB_PmiS_Jing313, suggesting a shared evolutionary history and functional similarities. To visualize these findings, a circular plot was created using the Proksee software, which is represented (Fig: 7). The results from BLAST hit analysis revealed that the \u003cem\u003eSVV09-A\u003c/em\u003e phage shares notable similarities with the Proteus phage vB_PmiS_NotEvenPhaged, as depicted ( Fig: S1). Lastly, to provide a detailed comparison of the sequences, a multiple sequence alignment of the \u003cem\u003eSVV09-A\u003c/em\u003e phage was produced, which is displayed \u0026nbsp;(Fig: S2). This alignment helps to highlight variations and conserved regions among the sequences, offering insights into the functional significance of these genes within the phage biology\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe ORF Finder tool has successfully provided a remarkable total of 336 potential open reading frames (ORFs) (Fig: S3) throughout the complete genomic sequence of the phage of which 67 genes were functionally assigned. Furthermore, detailed positional data is available, indicating both the starting and ending nucleotide positions of each ORF. This information is crucial for understanding the length of each ORF, with measurements reported in terms of the number of amino acids and nucleotides (Fig S4). Each identified ORF has been meticulously assigned a distinct numerical identifier, which facilitates easy reference and tracking. The tool provides comprehensive information for each ORF, including its precise location within the genome, the direction of the DNA strand on which it is located, and the corresponding reading frame. These extensive findings provide significant insight into the potential protein-coding regions present within the investigated DNA sequence, offering a valuable foundation for future research and deeper exploration of the phage genetic capabilities. These findings shed light on potential protein-coding regions in the DNA sequence being investigated.\u003c/p\u003e\n\u003cp\u003eThe BLAST hit reveals a fascinating similarity between test protein and the endolysin found in the Proteus phage PM87 (YP_009997985.1) (Fig: S5). The multiple sequence alignment of endolysin protein was shown with maximum sequence identity orthologous sequences with a E-value 2e-170 (Fig: S6) Interestingly, the prediction of transmembrane helices identified a single TMhelix site within the endolysin, situated between amino acids 207 and 222, suggesting a strategic role in membrane interactions.\u003c/p\u003e\n\u003cp\u003eThe structure and function of a protein are significantly influenced by various physical and chemical characteristics. In this study, the coding sequence for endolysin was determined to be 705 base pairs in length, resulting in a peptide composed of 234 amino acids. The anticipated secondary structure in SOPMA (Self-Optimized Prediction method With Alignment) analysis reveals a complex arrangement, featuring 90 alpha helices, 135 random coils, and 9 extended strands, as illustrated (Fig: 8 and Table 1). Upon conducting in silico folding analysis, we discovered that the protein contains three distinct unfolded segments. Notably, the longest of these segments comprises 33 consecutive residues. Collectively, these unfolded regions consist of 63 amino acids, highlighting a significant portion of the protein\u0026apos;s structure that deviates from a stable conformation (Fig: 9).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1 \u0026thinsp;SOPMA analysis of Endolysin protein\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 43.3121%;\"\u003e\n \u003cp\u003eNo of Amino acids\u003c/p\u003e\n \u003cp\u003eAlpha Helix\u003c/p\u003e\n \u003cp\u003eExtended Strand\u003c/p\u003e\n \u003cp\u003eRandom Coil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56.6879%;\"\u003e\n \u003cp\u003e234 Amino acids\u003c/p\u003e\n \u003cp\u003e90 Aminoacids (38.46%)\u003c/p\u003e\n \u003cp\u003e9 (3.85%)\u003c/p\u003e\n \u003cp\u003e135 (57.69%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe ProtParam tool provided essential insights into the protein\u0026rsquo;s composition, revealing that it comprises 42% non-polar hydrophobic amino acids, indicating a propensity for hydrophobic interactions, and 58% polar hydrophilic amino acids, which are likely to engage in hydrogen bonding and interactions with the aqueous environment. Additionally, the molar extinction coefficient at 280 nm was measured to be 36,900 M⁻\u0026sup1; cm⁻\u0026sup1; under both conditions: when all cysteine residues are involved in disulfide bonds and when they are in their reduced form, suggesting a consistent behavior in absorbance due to structural integrity. A detailed analysis yielded an instability index of 21.40, indicating that the protein may be relatively stable, coupled with an aliphatic index of 88.72, which suggests a high degree of hydrophobicity that could influence the protein\u0026rsquo;s thermal stability. Additionally, the grand average of hydropathicity was measured at -0.251, providing further insight into the protein\u0026apos;s overall hydropathy (Table 2). Three-dimensional (3D) configurations of proteins are of paramount importance for elucidating their functional roles, dynamics, and potential interactions with ligands. To ensure the reliability of our 3D protein model, we evaluated the positioning of residues in relation to the permissible regions of the Ramachandran plot, which serves as a hallmark for assessing stereochemical integrity. This analysis is critical in confirming the reliability of our constructed model and its applicability in further research. The sequence demonstrated alignment with fourteen distinct templates, revealing a broad range of similarity percentages between 8.00% and 55.77%. To identify the most suitable model, we carefully evaluated several criteria, including the maximum sequence similarity, Global Model Quality Estimation (GMQE), and QMEAN scores. GMQE serves as a comprehensive quality assessment tool that synthesizes various characteristics derived from the alignment with the target template, showcasing the model\u0026apos;s reliability. On the other hand, QMEAN is a sophisticated scoring function that assesses both global and local quality metrics, allowing us to gauge the structural integrity of the model effectively. Among the analyzed templates, the one exhibiting the highest sequence similarity of 53.16%, unveiled that the folding topology of the proposed endolysin closely mirrors that of the endolysin from Enterobacteriophage enc34, showcasing an impressive 99.96% confidence, with the query sequence also recorded the best GMQE score of 0.54 and the most favorable QMEAN score of 0.73. This template was subsequently chosen for further analysis and the corresponding PDB file was obtained. To assess the stereochemical quality of the model, we utilized the RAMPAGE server to generate a Ramachandran plot. The results revealed that an impressive 152 out of 157 residues, constituting 96.82% of the total, are located within the most favored regions, indicative of a well-structured model. Additionally, 4 residues (2.55%) fell within the allowed region, suggesting acceptable stereochemistry, while only one residue, specifically A20 Glycine, was identified in the outlier region (Fig. 10). This analysis underscores the model\u0026apos;s overall high quality and reliability for further applications.\u003c/p\u003e\n\u003cp\u003eTable 2: Prot param analysis of endolysin protein\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"648\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003eTheoretical isoelectric point\u003c/p\u003e\n \u003cp\u003eMolecular weight(kDa)\u003c/p\u003e\n \u003cp\u003eNon-polar Hydrophobic residues\u003c/p\u003e\n \u003cp\u003ePolar hydrophilic residues\u003c/p\u003e\n \u003cp\u003eNegatively charged residues\u003c/p\u003e\n \u003cp\u003ePositively charged residues\u003c/p\u003e\n \u003cp\u003eExtinction coefficients at 280 nm (Cys oxidized form)\u003c/p\u003e\n \u003cp\u003eExtinction coefficients at 280 nm (Cys reduces form)\u003c/p\u003e\n \u003cp\u003eAliphatic index\u003c/p\u003e\n \u003cp\u003eGrand average of hydropathicity (GRAVY):\u003c/p\u003e\n \u003cp\u003eInstability index (II)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eThe estimated half-life is:\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50%;\"\u003e\n \u003cp\u003e9.18\u003c/p\u003e\n \u003cp\u003e25.5\u003c/p\u003e\n \u003cp\u003e42%\u003c/p\u003e\n \u003cp\u003e58%\u003c/p\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003cp\u003e36900 M\u003csup\u003e-1\u003c/sup\u003e cm\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e36900 M\u003csup\u003e-1\u003c/sup\u003e cm\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e88.72\u003c/p\u003e\n \u003cp\u003e-0.251\u003c/p\u003e\n \u003cp\u003e21.40\u003c/p\u003e\n \u003cp\u003e30 hours (mammalian reticulocytes, in vitro).\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026gt;20 hours (yeast, in vivo).\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026gt;10 hours (Escherichia coli, in vivo).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eInfections of diabetic wounds pose formidable challenges for healthcare providers, often deepened by the insidious presence of coagulase-negative Staphylococci (CoNS), intricate mixed microbial communities, and opportunistic fungi. The alarming rise in antibiotic resistance, now recognized as a pressing global health crisis, significantly complicates the path to effective treatment (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). CoNS are particularly troublesome, harboring the mecA gene and the cfr gene, which alter adenine residues at various sites. This genetic alteration interferes with the efficacy of at least five distinct classes of antibiotics, including oxazolidinones, phenicols, lincosamides, pleuromutilins, and streptogramin A (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). The battle against these resilient pathogens is a vivid reminder of the intricate and often daunting landscape of modern medicine. The identification and prevention of coagulase-negative staphylococci (CoNS) are critical in medical settings. This study concentrated on the isolation and identification of \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e strains derived from samples of diabetic wounds, as well as the characterization of phages that exhibit efficacy against these bacteria. Among the isolated strains, the \u003cem\u003eStaphylococcus spp\u003c/em\u003e constituted the most prevalent group, with CoNS accounting for 24%. The notable frequency of \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e identified in our study aligns with findings (Soldera et al. 2013), which emphasized the role of \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e as a frequent colonizer within healthcare environments. Together, these findings highlight the critical need for prompt monitoring and intervention strategies in wound care units, as infections caused by CoNS can lead to serious and potentially life-threatening complications for affected patients. Culture and biochemical tests were utilized in the identification of S. ureilyticus. They are Gram-positive cocci, negative coagulase, and positive catalase, which aligns with findings from another research (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). The positive catalase test indicated the presence of hydrogen peroxide-decomposing enzymes typical of staphylococci, while a negative coagulase test differentiated it from other species, particularly \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Furthermore, the ability of the isolate to ferment mannitol supported its identification as \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e, consistent with findings reported in other studies (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), Despite being time-consuming, these techniques continue to be very effective for precise identification.\u003c/p\u003e \u003cp\u003eWe identified a Staphylococcus phage obtained from sewage water in hospitals, specifically the phage \u003cem\u003eSVV09-A\u003c/em\u003e. The phage showed a broad range of host specificity. Following isolation, Whole-genome sequencing uncovered key genes essential for phage activity, and open reading frame (ORF) analysis identified 336 potential coding sequences. The proteins found in the \u003cem\u003eSVV09-A\u003c/em\u003e phage genomes include DNA primase, DNA helicase, exonuclease, transcriptional regulator, tail fiber, and endolysin. These proteins are crucial for the effective replication and spread of phages within host bacteria genome sequencing uncovered few of the critical genes necessary for phage activity, while ORF analysis identified 336 possible coding sequences with a putative endolysin ORF was found to be ORF 315. The phage SVV09-A exhibited an unexpectedly high genomic similarity (97.13%) to a previously described Proteus phage vB_PmiS_NotEvenPhaged and we also observed that the endolysin gene of a \u003cem\u003eStaphylococcus\u003c/em\u003e-infecting bacteriophage is 100% identical to the endolysin of a phage reported to infect \u003cem\u003eProteus spp.\u003c/em\u003e Such cross-genera genomic conservation is uncommon, particularly because \u003cem\u003eStaphylococcus\u003c/em\u003e and \u003cem\u003eProteus\u003c/em\u003e occupy distinct phylogenetic and ecological niches and represent Gram-positive and Gram-negative bacteria, respectively. However, several well-characterized evolutionary features of bacteriophage biology provide plausible explanations for this observation.\u003c/p\u003e \u003cp\u003eFirst, endolysins are among the most highly conserved proteins in the phage genome, primarily due to their essential role in hydrolyzing the bacterial peptidoglycan to allow progeny release. The catalytic domains responsible for cleaving glycosidic or amide bonds in peptidoglycan are structurally constrained, and even minor amino acid substitutions can significantly impair enzymatic activity (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). As a result, endolysins often evolve under strong purifying selection, preserving identical catalytic sequences across diverse phage taxa. Reports of nearly identical endolysins in phages infecting different bacterial genera support the idea that these enzymes retain conserved functionality despite host divergence (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSecond, endolysins belong to the lysis module, one of the most frequently exchanged genetic regions in bacteriophage genomes. Phages readily undergo horizontal gene transfer and modular recombination, particularly in environmental reservoirs rich in microbial diversity, such as wastewater, soil, and the animal microbiome (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). The holin\u0026ndash;endolysin cassette often moves as a functional unit, enabling phages infecting unrelated hosts to acquire identical lysis genes. Thus, the complete identity between the \u003cem\u003eStaphylococcus\u003c/em\u003e and \u003cem\u003eProteus\u003c/em\u003e phage endolysins may reflect recent or recurrent module exchange events rather than shared host specificity. This modular evolution allows phages infecting different genera to retain near-identical genomic backbones while varying only in genes required for host recognition. Thus, the high overall sequence similarity observed here may reflect a shared evolutionary lineage, with divergence occurring primarily in host-range determinant genes such as tail fibers or receptor-binding proteins.\u003c/p\u003e \u003cp\u003eThird, although \u003cem\u003eStaphylococcus\u003c/em\u003e and \u003cem\u003eProteus\u003c/em\u003e differ in cell envelope structure, the core architecture of peptidoglycan\u0026mdash;including the MurNAc\u0026ndash;GlcNAc backbone and conserved peptide crosslinks\u0026mdash;remains sufficiently similar to allow a shared enzymatic mechanism of degradation. Many endolysins target bonds that are broadly conserved across Gram-positive and Gram-negative bacteria (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). Consequently, identical catalytic domains may function effectively in phages infecting different bacterial phyla, enabling such enzymes to be maintained across wide evolutionary distances.\u003c/p\u003e \u003cp\u003eCollectively, these findings underscore the modular, mosaic, and functionally constrained nature of phage genomes, particularly within the lysis system. The perfect identity between the two endolysin genes suggests strong structural and biochemical conservation of the enzyme, combined with the capacity of phages to exchange key functional modules across taxonomic boundaries. These results highlight that phage genomic similarity\u0026mdash;and especially endolysin conservation\u0026mdash;does not necessarily correlate with host specificity and should be interpreted with caution when inferring phage evolutionary relationships or potential host range.\u003c/p\u003e \u003cp\u003eEndolysins have shown significant promise as potent antibacterial agents, particularly against predominant Gram-positive pathogens such as \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. These phage-derived enzymes could potentially replace traditional antibiotics in the treatment of various bacterial infections (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Recent studies mentioned that endolysin treatment is often more clinically effective than conventional antibiotic therapies (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). The unique specificity of endolysins towards their target bacteria underscores an evolutionary adaptation that enables them to efficiently degrade cell walls exhibiting distinct structural features. Among the templates we reviewed, the one with the highest sequence similarity at 53.16% showed that the suggested endolysin's folding structure is very similar to that of the endolysin from Enterobacteriophage enc34, with a confidence level of 99.96%. The query sequence also achieved the highest GMQE score of 0.54 and the best QMEAN score of 0.73 and these findings are similar to other studies (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTypically, endolysins are composed of multiple domains, including specialized cell wall binding domains (CBDs) that specifically recognize and bind to unique molecular components. For instance, they target the glycine-rich peptidoglycan layer characteristic of \u003cem\u003eStaphylococcus\u003c/em\u003e, facilitating the precise lysis of these bacteria as well as other related streptococci (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). These insights not only illuminate the mechanism of action for endolysins but also raise important considerations for their broader therapeutic applicability (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). To enhance their effectiveness against a wider array of bacterial species, it is essential to explore engineering strategies that can improve endolysin permeability. This would help to overcome the inherent host specificity of these enzymes and broaden their potential use in combating resistant bacterial infections. The proteins and genes identified in the current study likely serve a crucial role in evading the formidable defences of \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e. Hence the remarkable ability positions \u003cem\u003eSVV09-A\u003c/em\u003e phage as an exceptional candidate for phage therapy, especially in the battle against stubborn infections which resist conventional antibiotics.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe incidence of bacterial infections is escalating globally on a daily basis. \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e has emerged as a predominant pathogen associated with wound infections. This bacterium has been identified in various wound specimens obtained from patients with hospital-acquired infections in RIMS, Kadapa. Experimental findings indicate that the isolated phage exhibit complete lytic activity, as evidenced by the presence of clear lysis zones on the isolated host bacteria. Phages specific to \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e were successfully isolated and purified, and their host range against S\u003cem\u003etaphylococcus\u003c/em\u003e strains were identified. Among these, a phage exhibiting a broad host range and robust lytic activity was selected and characterized. The \u003cem\u003eSVV09-A\u003c/em\u003e phage is classified within the \u003cem\u003eSiphoviridae\u003c/em\u003e family, belongs to \u003cem\u003eCaudovirales\u003c/em\u003e order with a documented genome size of 58.80 kb based on structural characteristics. The stability of the phage was assessed in relation to various factors, including temperature, pH levels, and the presence of metal ions. This research emphasizes the critical need for evaluating safety protocols associated with phage therapies, ensuring their viability as potential therapeutic options. Furthermore, bioinformatics and molecular research methodologies were employed to enhance our understanding of gene sequences and their potential applications in therapy. The proposed endolysins demonstrate stable structures, distinct charge characteristics, and unique elemental compositions, suggesting a role in complex cellular mechanisms. A comprehensive understanding of phages and the biology of lytic enzymes may facilitate the development of innovative treatments for bacterial resistance to various pharmacological agents. The phage \u003cem\u003eSVV09-A\u003c/em\u003e shows in vitro lytic activity against its host strain, significant stability, extensive efficacy and its endolysin appears to be stable enzyme in silico which requires further investigation for addressing multi-drug-resistant \u003cem\u003eStaphylococcus ureilyticus.\u003c/em\u003e Our analysis revealed that the endolysin of a \u003cem\u003eStaphylococcus\u003c/em\u003e-infecting bacteriophage is identical at the nucleotide and amino acid levels to the endolysin encoded by a phage reported to infect \u003cem\u003eProteus\u003c/em\u003e spp. Bacteriophage genomes evolve through extensive modular exchange, yet core lysis functions are thought to be shaped primarily by host-specific constraints. Here we show that a \u003cem\u003eStaphylococcus\u003c/em\u003e phage encodes an endolysin that is identical to that of a phage reported to infect \u003cem\u003eProteus\u003c/em\u003e, two distantly related bacterial genera. This unexpected conservation highlights the strong functional constraints acting on endolysins and reveals that essential lysis modules can move across broad taxonomic boundaries. Our findings refine current understanding of phage genome modularity, challenge assumptions that link gene content to host identity, and provide insight into the evolutionary and functional stability of enzymes with therapeutic potential.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMDR: Multi drug resistant\u003c/p\u003e\n\u003cp\u003eCoNS: Coagulase-negative Staphylococci\u003c/p\u003e\n\u003cp\u003epH: Potential of Hydrogen\u003c/p\u003e\n\u003cp\u003eLB: Luria Bertani Broth\u003c/p\u003e\n\u003cp\u003ePFU: Plaque forming Unit\u003c/p\u003e\n\u003cp\u003eMnCl2: Manganese Chloride\u003c/p\u003e\n\u003cp\u003eZnCl2: Zinc Chloride\u003c/p\u003e\n\u003cp\u003eCaCl2: Calcium Chloride\u003c/p\u003e\n\u003cp\u003eMgSO4: Magnesium Sulphate\u003c/p\u003e\n\u003cp\u003eSM: Salt magnesium\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAMR: Antimicrobial resistance\u003c/p\u003e\n\u003cp\u003eDNA: Deoxy ribonucleic acid\u003c/p\u003e\n\u003cp\u003edsDNA: Double stranded Deoxy ribonucleic acid\u003c/p\u003e\n\u003cp\u003erRNA: Ribosomal ribonucleic acid\u003c/p\u003e\n\u003cp\u003ePCR: Polymerase Chain reaction\u003c/p\u003e\n\u003cp\u003eTM helix: Transmembrane helix\u003c/p\u003e\n\u003cp\u003eDEEPTMHMM: A Deep Learning Model for Transmembrane Topology Prediction and Classification\u003c/p\u003e\n\u003cp\u003eNEB: New England Biolabs\u003c/p\u003e\n\u003cp\u003eFS: Fragment size\u003c/p\u003e\n\u003cp\u003eBP: Base Pair\u003c/p\u003e\n\u003cp\u003eGRAVY: Grand average hydropathy\u003c/p\u003e\n\u003cp\u003eGMQE: Global Model Quality Estimate\u003c/p\u003e\n\u003cp\u003eQMEAN: Qualitative Model Energy Analysis\u003c/p\u003e\n\u003cp\u003eSOPMA: Self-Optimized Prediction method With Alignment\u003c/p\u003e\n\u003cp\u003eNGS: Next Generation Sequencing\u003c/p\u003e\n\u003cp\u003eWGS: Whole Genome Sequencing\u003c/p\u003e\n\u003cp\u003eORF: Open reading Frame\u003c/p\u003e\n\u003cp\u003eCBD: Cell wall binding domain\u003c/p\u003e\n\u003cp\u003eMSA: Multiple sequence alignment\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary material:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supplementary figures are available in attached pdf.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was a part of the project supported the Rashtriya Uchchattar Shiksha Abhiyan (RUSA) (File No: RUSA-ANU/YVU/Research project-24/Sanction order/2024)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge Rashtriya Uchchattar Shiksha Abhiyan (RUSA) for funding, \u0026nbsp;additionally, the Department of Microbiology at Yogi Vemana University in Kadapa for their assistance during \u0026nbsp;the research\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthor Contribution Statement; Lakshmi Sharvani K.S has carried out the experiments and prepared the original draft. Vijaya Lakshmi D and Vijaya Raghava Prasad D conceived, planned and supervised the study. Pritam Kanti Guha reviewed and edited the manuscript. Swetha Vallabhaneni, Guru Prasad C., Renuka Pallem, Vaishnavi R., Krishna Vamsi M reviewed the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work has been conducted at the Department of Microbiology, Yogi Vemana University, Kadapa, after receiving ethical approval from the Institutional Ethical Committee (IEC) (Ref. No. 01/IEC/DVR/YVU/2022-23, dated 24.04.2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agreed to the final version of manuscript and give their consent to publish.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKwon, H., Park, S. Y., Kim, et al. (2022). Characterization of a Lytic bacteriophage vB_SurP-PSU3 Infecting \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e and Its Efficacy Against Biofilm. \u003cem\u003eFrontiers in microbiology\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e, 925866. https://doi.org/10.3389/fmicb.2022.925866\u003c/li\u003e\n\u003cli\u003eLavecchia, A., Chiara, M., De Virgilio, C., et al. (2021). 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Peptidoglycan structure and architecture. \u003cem\u003eFEMS microbiology reviews\u003c/em\u003e, \u003cem\u003e32\u003c/em\u003e(2), 149\u0026ndash;167. https://doi.org/10.1111/j.1574-6976.2007.00094.x\u003c/li\u003e\n\u003c/ol\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Phages, Endolysin, In silico, Staphylococcus ureilyticus, Transmission electron microscopy, Whole genome sequencing","lastPublishedDoi":"10.21203/rs.3.rs-8409739/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8409739/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe pathogenic strains of \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e are one of the causes of frequently associated nosocomial infections in the hospital environment. The increasing antibiotic resistance in CoNS frequently results in treatment failures, highlighting the pressing requirement for new eradication methods. The present study mainly focused on isolation, and physiological characterization of phages from sewage water that target \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e, along with molecular characterization (whole genome sequencing) of phages and insilico analysis of its endolysin, including phylogenetic studies, open reading frames, and 3D model. Using the double-layer agar method, several phages were isolated, and the phage that has exhibited the broadest host range was selected. The current study reveals the genome of the lytic phage \u003cem\u003eStaphylococcus ureilyticus\u003c/em\u003e, designated SVV09-A, which has been examined and annotated. Further analysis indicated that the phage has exhibited optimal activity at pH levels between 6 and 8 and within a temperature range of 30\u0026ndash;37\u0026deg;C; manganese metal ions have shown great impact on phage adsorption rate. The whole genome sequence (WGS) of the phage \u003cem\u003eSVV09-A\u003c/em\u003e was determined, revealing a linear DNA of 58,797 bp, with a G\u0026thinsp;+\u0026thinsp;C content of 46.9, and the phage was classified within the order Caudovirales. ORF analysis revealed 336 ORFs and uncovered functions for 67 genes. The estimated endolysin gene of phage \u003cem\u003eSVV09-A\u003c/em\u003e had a length of 705 bp, which corresponds to 234 amino acids (~\u0026thinsp;25.33 kDa). These findings offer a structural and functional basis for endolysin, establishing a framework for upcoming in vitro and in vivo efficacy research targeting multidrug-resistant Gram-positive infections.\u003c/p\u003e","manuscriptTitle":"Isolation and Molecular characterization of a Novel Bacteriophage SVV09-A: Targeted to Staphylococcus ureilyticus from Diabetic Wounds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-31 09:32:39","doi":"10.21203/rs.3.rs-8409739/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5e333f6d-9b66-4fb7-bc97-342990debf36","owner":[],"postedDate":"December 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-02T11:41:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-31 09:32:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8409739","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8409739","identity":"rs-8409739","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}