Genomic Insights into Halo KS-7: An AI-Driven Characterization of a Novel Halophilic Bacteriophage Targeting Carbapenem-Resistant Klebsiella pneumoniae

Genomic Insights into Halo KS-7: An AI-Driven Characterization of a Novel Halophilic Bacteriophage Targeting Carbapenem-Resistant Klebsiella pneumoniae | 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 Article Genomic Insights into Halo KS-7: An AI-Driven Characterization of a Novel Halophilic Bacteriophage Targeting Carbapenem-Resistant Klebsiella pneumoniae Sahar Abed, Masoumeh Beig, Sepideh Soltani, Morvarid Shafiei, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6534345/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 Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a multidrug-resistant (MDR) pathogen causing severe infections in immunocompromised patients, prompting the exploration of alternative therapies like bacteriophage therapy. In this study, we isolated and characterized a novel halophilic lytic bacteriophage, Halo KS-7, targeting K. pneumoniae , and used an AI-driven annotation pipeline in Python to analyze its genome and therapeutic potential. Bacteriophages were isolated from Hospital wastewater, purified through plaque isolation, and confirmed using the double-layer agar method. Morphological analysis via transmission electron microscopy (TEM) and plaque assays assessed lytic activity. DNA sequencing was done using Illumina HiSeq 2000, followed by genome assembly, AI-guided annotation, gene prediction, protein function classification, and comparative genomics using CLC Genomics Workbench. We also evaluated host range, temperature stability, pH sensitivity, and salt stress tolerance to assess therapeutic potential. Halo KS-7 exhibited strong lytic activity against CRKP and was classified as a Myoviridae bacteriophage by TEM. Phenotypic assays demonstrated optimal activity at 37 °C and neutral pH, effective activity from pH 4–10, and enhanced performance in high-salinity conditions. Its 58.716 kb linear dsDNA genome (44.4% G+C) contains 49 predicted ORFs, lacks integrase, lysogeny, or antibiotic-resistance genes, and includes three tRNA genes (tRNATyr, tRNAPro, and tRNAAsn). It also includes a toxin gene and auxiliary factors like MazG, pyrophosphatase, and HNH endonucleases that enhance bacterial killing without promoting horizontal gene transfer or resistance. Functional annotation assigned ~65% of ORFs to structural, replication, and packaging roles. Comparative genomics showed moderate similarity to other Myoviridae but with distinct accessory features, emphasizing its novelty and therapeutic value. Halo KS-7 is a novel, strictly lytic bacteriophage with strong antibacterial activity and stress resilience, supporting its use as a promising biocontrol agent against CRKP and its potential for clinical development in managing MDR infections. Biological sciences/Microbiology/Bacteria Biological sciences/Microbiology/Bacteriology Biological sciences/Microbiology/Phage biology Carbapenem-resistant Klebsiella pneumoniae (CRKP) bacteriophage therapy Halo KS-7 artificial intelligence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Multidrug-resistant (MDR) Klebsiella pneumoniae is a major global health threat, causing various infections such as pneumonia and bloodstream infections ( 1 , 2 ). Carbapenem-resistant K. pneumoniae (CRKP), a particularly concerning pathogen, has been classified as a critical priority by the World Health Organization due to its high antibiotic resistance and limited treatment options ( 3 ) ( 4 ). This bacterium's ability to form biofilms, its high antibiotic resistance, and its potential for horizontal gene transfer (HGT) complicate treatment ( 5 ). As traditional antibiotics become less effective, alternative therapeutic approaches are urgently needed ( 6 ). Bacteriophages are emerging as a promising solution for antibiotic-resistant bacteria. They offer advantages like high specificity, targeting antibiotic-resistant strains, and minimal disruption to the host microbiota ( 7 – 9 ). Some bacteriophages, particularly those targeting K. pneumoniae , also produce depolymerase that degrades the bacterial capsule, improving immune clearance and bacteriophage effectiveness ( 10 , 11 ). Bacteriophages isolated from sources such as sewage and clinical samples may exhibit considerable genomic diversity, with variations ranging from small 40 kb podoviruses to large 170 kb myoviruses ( 12 – 14 ). Continued isolation and genomic analysis of new bacteriophages are critical for advancing bacteriophage therapy and understanding bacteriophage-host–host interactions. In particular, bacteriophages from extreme environments such as high-salt (halophilic) conditions may offer unique features, including improved environmental or physiological stability and expanded host range ( 15 ). Halophilic bacteriophages adapted to high-salt environments are highly stable under thermal, pH, and desiccation stress. Their unique genomes offer opportunities for genetic engineering and help improve our understanding of bacteriophage-host interactions. Notably, these bacteriophages also have potential practical uses, such as biocontrol agents in salt-rich food production and stable topical treatments for MDR infections under challenging environments like chronic wounds ( 16 ). However, they remain underexplored in non-halophilic hosts like K. pneumoniae . Advances in artificial intelligence (AI) and bioinformatics have revolutionized bacteriophage research, enabling efficient genome annotation and identification of potentially useful therapeutic genetic elements in bacteriophages, ( 17 ) such as holins, important for host lysis and MazG-like pyrophosphatases which can neutralize host defenses to ensure virus ( 18 ). While some studies have explored bacteriophages targeting MDR K. pneumoniae , few have systematically utilized computational pipelines to evaluate their genomic features and therapeutic relevance ( 19 – 21 ). Limited bacteriophage databases and a lack of understanding of host specificity and infection mechanisms continue to hinder the advancement of bacteriophage therapy ( 21 – 23 ). These gaps show the need for continued discovery and genomic investigation of novel bacteriophages, particularly those with unique ecological adaptations. While CLC Genomics Workbench (Qiagen, Hilden, Germany) is a popular commercial tool known for its intuitive interface and integrated bioinformatics functionalities, it is limited in flexibility, transparency, and scalability, making it less suitable for researchers who need greater control over their workflows ( 24 ). Python-based pipelines offer a powerful open-source alternative that allows for customization and transparency, enabling researchers to fine-tune their analysis, from preprocessing and quality filtering to genome assembly and annotation ( 25 ). Python's seamless integration with libraries like Biopython, pandas, matplotlib, and NumPy supports advanced data analysis, machine learning, and high-quality visualizations, offering greater precision and adaptability than commercial software ( 26 ). Additionally, Python promotes reproducibility through script-based workflows and can be easily automated to handle large datasets without costly licenses ( 27 ). Python's scalability allows workflows to be deployed on various platforms, including local machines, high-performance clusters, and cloud infrastructure. Moreover, unlike proprietary tools with significant financial barriers, Python is cost-effective and freely available. Here, we used a Python-driven pipeline to characterize the novel halophilic bacteriophage Halo KS-7, demonstrating Python's robustness, adaptability, and versatility of open-source solutions. This allows us to identify genes associated with therapeutic relevance. By comparing this genome to other known K. pneumoniae bacteriophages, we aim to identify distinctive traits that could support its potential as a biocontrol agent. This study seeks to demonstrate the value of integrating wet-lab techniques with computational tools in discovering and evaluating promising bacteriophage candidates. 2. Results 2.1. Isolation and Characterization of a Halophilic K. pneumoniae Bacteriophage The Halo KS-7 bacteriophage was specifically isolated to target CRKP. The spot assay results, illustrated in Figure 1A, and the DLA assay outcomes presented in Figure 1B unequivocally affirmed its lytic activity. TEM analysis revealed that Halo KS-7 exhibits a morphology consistent with the Caudovirales order (Figure 2). 3.5.1. Host Range Determination Halo KS-7 exhibited a broad host range across the tested CRKP isolates. In assays involving 30 clinical CRKP strains, clear plaques were observed in 57% of the strains, confirming the bacteriophage's selective and targeted lytic activity. 3.5.2. Temperature Stability Figure 3A illustrates the thermal stability of Halo KS-7. The bacteriophage exhibited optimal lytic activity at 37°C and maintained good activity in the range of 4°C to 60°C, with no significant drop in titer (within 0.5 log of the initial). At 70°C, a notable decrease in lytic activity was observed. However, it did not reach complete inactivation, indicating that Halo KS-7 has moderate thermal stability, similar to many colibacteriophages but slightly lower than some extremophilic bacteriophages. 3.5.3. pH Sensitivity The influence of pH on Halo KS-7 bacteriophage’s lytic activity is seen in Figure 3B. The bacteriophage has good lytic activity at pH between 4 and 10; its highest activity was observed at pH = 7. At pH below 4, bacteriophage activity was completely stopped (pH = 2, no activity). As pH increased and the environment became more alkaline, the lytic activity of the bacteriophage decreased so that at pH = 14, its activity reached almost zero. At pH = 10, bacteriophage activity was still relatively high but decreased compared to its optimal value at pH = 7. These data indicate that the bacteriophage is most stable and effective in neutral and slightly alkaline conditions (pH = 7 to 10), while it is extremely vulnerable in highly acidic (pH = 2) and highly alkaline (pH = 14) environments. 3.5.4. Salt Tolerance Figure 4A depicts the response of Halo KS-7 to varying salt concentrations. The bacteriophage exhibited increased lytic activity as NaCl concentration in the medium rose. At 5% NaCl, the bacteriophage titer was 4.36 log PFU/mL, which increased to 6.8 log PFU/mL at 10% NaCl. The highest activity, 8.1 log PFU/mL, was at 15% NaCl. These findings suggest that elevated NaCl concentrations led to the formation of larger, sharper plaques (Figure 4B), showing the beneficial effect of salt on bacteriophage lytic activity. Accordingly, Halo KS-7 is classified as a halophilic bacteriophage, demonstrating optimal lytic activity in saline environments. 2.2. Whole Genome Sequencing and Bioinformatics Analysis: Genome Sequencing and Assembly A commercial column-based DNA extraction protocol ensured material purity without bacterial genomic contamination. The genome sequence data was assembled de novo. The complete genome sequence of Halo KS-7 has been deposited in GenBank under accession number PV026551 (https://www.ncbi.nlm.nih.gov/search/all/?term=PV026551). The Halo KS-7 bacteriophage genome has a genome size of 58,716 bp. It comprises 49 predicted ORFs, with 64.47% related to genes encoding putative functional proteins. The GC content of Halo KS-7 is 44.4%, and its closest related organism is Klebsiella bacteriophage vB_KpP_FBKp27 (accession number: NC_068555), with a 90.81% identity and 94% query coverage (Figure 5). 2.3. Functional Annotation and Genomic Analysis Figure 6A presents a comparative genomic alignment of multiple K. pneumoniae strains, showing the organization of genomic features across different isolates. The diagram shows colored bars representing various genomic regions, with each strain labeled on the left (e.g., OX335406, OP030736, BK050257, PV026551, and NC_068555). The connecting lines between the bars indicate similarity and homology between these genomic regions across strains, with each segment representing specific gene sequences or other functional genomic features. The scale on the top indicates the genomic coordinates, ranging from 0 to 60,000 base pairs. This comparison illustrates conserved and variable genomic regions across the strains, providing insights into their genomic diversity and evolutionary relationships. The visualization is crucial for identifying homologous gene clusters and potential regions of genetic variation and understanding the genomic structure of K. pneumoniae isolates. Figure 6B provides a heatmap showing pairwise genome similarity percentages. The ANI values confirm that Halo KS-7 (PV026551) shares only moderate sequence identity (75.42%–78.34%) with its closest relatives. The comparison with NC_068555, which has the highest alignment score (90.81% identity but only 94% query coverage), suggests that Halo KS-7 is genetically distinct. Syntactic analysis and ANI calculations demonstrate that Halo KS-7 exhibits significant genetic divergence from known bacteriophages. The unique ORFs and moderate sequence similarity with its closest relatives strongly support its classification as a novel bacteriophage. Holin and spanin genes associated with host cell lysis were detected among the functional genes identified. Genes encoding HNH endonucleases and exonucleases were present, along with a RuvC-like Holliday junction resolvase and a Sak4-like single-stranded DNA annealing protein. A MazG-like pyrophosphatase, single-stranded DNA-binding proteins, ribonucleotide reductase, ATPase, and RNA polymerase genes were also identified. These functional genes collectively suggest a genetic repertoire potentially supporting bacteriophage replication, survival, and host interaction under various environmental conditions. Halo KS-7’s genome encodes three tRNA genes (tRNA Tyr , tRNA Pro , and tRNA Asn ). These likely enhance translational efficiency by supplementing the host’s tRNA pool, particularly when specific tRNAs are in short supply during infection. 2.4. Therapeutic Potential and Genetic Safety Considerations Genomic analysis indicates that Halo KS-7 is exclusively lytic and lacks lysogenic properties, as well as genes associated with antibiotic resistance or virulence factors that make it an excellent candidate for therapeutic applications. Identifying key genes involved in cell wall degradation, adaptation to high-salinity environments, and enhanced bacteriophage efficacy underscores its potential to combat antibiotic-resistant infections. Some of the Halo KS-7 bacteriophage genes and their functional roles in therapeutic applications are shown in Table 1. Genome analysis of Halo KS-7 revealed a putative toxin gene located between nucleotide positions 39,942 and 40,232, encoding a 96 amino acid protein (MAYQNNSAASRNNNTSSANAGEAPERNIGGYLNIGVRGRDGQVRRLGQGGRGIALREDHAVEGKVLEFLRAQGIEALDEHLVITFGDAKALENFEL). The amino acid sequence derived from the gene exhibited similarity to a toxin found in Klebsiella , as indicated by BLASTp analysis. However, the toxin could not be categorized into a specific family. Structural analysis using InterProScan identified a colicin-like motif within the protein sequence, which is typically associated with pore-forming activity and bactericidal effects. 3. Discussion In this study, we identified Halo KS-7 as a novel halophilic bacteriophage infecting K. pneumoniae , with several characteristics distinguishing it from previously described bacteriophages. Notably, it’s strictly lytic nature and halotolerance provide advantages in therapeutic contexts, especially in environments where high salt concentrations may otherwise inhibit other bacteriophage activities. The bacteriophage’s genome encodes various auxiliary enzymes, including holins and spanins, which may enhance its ability to lyse bacterial cells efficiently. Another notable feature is the presence of an auxiliary metabolic gene, MazG, encoded by gene gp_39. Auxiliary metabolic genes are typically of host origin and are co-opted by bacteriophages to manipulate host metabolic pathways in ways that support viral replication. MazG genes have been predominantly identified in marine bacteriophages and are associated with lytic and temperate lifestyles ( 28 ). They are thought to facilitate bacteriophage propagation in nutrient-limited environments by modulating host nucleotide metabolism, thus aiding replication in starved cells ( 29 ). Given this context, the presence of MazG in Halo KS-7 suggests it may possess the capacity to alter host metabolism and potentially interfere with host bacteriophage defense systems. However, further investigation is required to elucidate the specific mechanisms involved in Halo KS-7 's interaction with its host. A toxin-like gene in the lytic Halo KS-7 bacteriophage has significant implications for both its biological function and potential therapeutic applications. Unlike temperate bacteriophages, which can integrate their genomes into host bacteria and enter a lysogenic state, Halo KS-7's strictly lytic nature ensures that it does not undergo lysogenic conversion or genome integration. This characteristic significantly reduces the risks typically associated with temperate bacteriophages, such as the potential HGT of toxin genes, which could raise biosafety concerns. Recent studies challenge the long-held assumption that strictly lytic bacteriophages operate in predictable and straightforward ways, especially in vivo. Evidence suggests that even these bacteriophages may exhibit unexpected behaviors, such as host-switching or direct interaction with mammalian immune cells, which could have critical implications for bacteriophage therapy ( 30 ). Moreover, some lytic bacteriophages appear capable of mediating HGT, including the mobilization of virulence factors, raising potential safety concerns. Bacteriophage capsids can also elicit immune responses, and the limited understanding of bacteriophage pharmacodynamics in mammalian systems adds further complexity to their therapeutic use ( 30 ). A compelling example is the crAss001 bacteriophage, classified as lytic but capable of long-term coexistence with its Bacteroides host in liquid culture without eliminating the bacterial population ( 31 ). This observation aligns with the concept of pseudolysogeny or carrier state, where a stable equilibrium is maintained despite the absence of lysogeny genes. The persistence of crAss-like bacteriophages in the gut, even after fecal microbiota transplantation, supports this view. It has been proposed that such stability might involve cryptic mechanisms, possibly through host-encoded transposons or quorum sensing pathways, allowing bacteriophages to persist without classical integration machinery ( 31 , 32 ). Further supporting this idea is φPDS1, a lytic bacteriophage infecting Parabacteroides distasonis , which demonstrates efficient replication and plaque formation but fails to clear its host in liquid cultures. Over time, a significant rise in resistant bacterial subpopulations (~ 22% to ~ 95%) was observed, likely due to phase-variable expression of surface receptors. This results in a heterogeneous bacterial population, with some cells being permissive and others resistant, enabling prolonged bacteriophage-host coexistence ( 29 ) Such findings emphasize that bacteriophage persistence may not always require lysogeny but can instead result from selective pressures and population-level adaptations. Interestingly, despite extensive research, no known bacteriophage has successfully eradicated its host population in natural settings. This suggests that stable coexistence, rather than eradication, might be a more evolutionarily favored strategy ( 32 ). Some bacteriophages, like VP882, can even sense host population density via quorum sensing systems and modulate their lytic behavior accordingly, indicating a higher level of environmental responsiveness than previously assumed ( 32 ). Finally, interactions between bacteriophages, bacteria, and the mammalian host extend far beyond simple lysis. In murine models, immune-mediated inflammation has been shown to induce probacteriophage activation, exacerbating disease through enhanced lysogenic conversion. In contrast, host vaccination reduced this bacteriophage-induced pathogenicity without impacting bacterial load ( 33 ). These findings highlight that bacteriophages can shape host immunity and disease outcomes, whether lytic or temperat,e reinforcing the need to account for bacteriophage–host–immune system dynamics in therapeutic and ecological contexts. Together, these observations underscore the complexity of bacteriophage biology in vivo. Even bacteriophages traditionally considered lytic may engage in cryptic persistence, population-level balancing, or immune modulation, complicating their application in clinical settings and challenging our conventional dichotomy of lytic versus temperate lifestyles. The toxin-like gene in Halo KS-7 likely serves a beneficial role in enhancing the bacteriophage's ability to infect and lyse bacterial hosts. This toxin is proposed to function similarly to bacteriocins, antimicrobial proteins that disrupt bacterial membranes or interfere with vital intracellular processes. The identified colicin-like motif suggests that the toxin operates similarly to a bacteriocin, a class of proteins known for pore-forming and bactericidal activities. Specifically, the toxin may facilitate the breakdown of the host cell membrane or inhibit cellular functions, increasing infection efficiency. This feature likely complements the bacteriophage’s primary lytic enzymes, such as holins, which are responsible for breaking down the bacterial cell wall. Together, these components may enhance the bacteriophage’s capacity to rapidly kill target bacteria by disrupting the membrane and attacking the bacterial cell from multiple angles. Moreover, this toxin gene reflects an evolutionary refinement in bacteriophage design, favoring more precise and efficient bacterial killing mechanisms. Incorporating this auxiliary lysis gene could enhance the bacteriophage's therapeutic efficacy, particularly in targeting K. pneumoniae , without the associated risks of gene transfer to non-pathogenic microorganisms. In this way, Halo KS-7 may be a bacteriophage therapy tool optimized for selective bacterial eradication while safeguarding the microbiome. The toxin-like gene could provide Halo KS-7 a competitive advantage by enhancing its ability to outcompete other microorganisms in the same ecological niche. Since Halo KS-7 cannot undergo lysogenic conversion, this toxin gene may be a critical survival mechanism, particularly in environments where rapid bacterial turnover or competition with other microbial species is common. Overall, the toxin-like gene in Halo KS-7 appears to offer notable advantages, enhancing its ability to infect and lyse bacterial hosts. However, potential limitations or undesirable effects may arise, particularly if the toxin’s action is not fully controlled in therapeutic applications. Given Halo KS-7's strong genetic and functional capabilities, any limitations associated with the toxin gene could be effectively addressed using precise gene-editing techniques. Approaches such as CRISPR-Cas or recombineering could remove the toxin gene while preserving the bacteriophage’s lytic activity and antibacterial efficacy. These modifications would enable the safe and effective clinical use of Halo KS-7 as a therapeutic agent for treating MDR bacterial infections. Our findings accord with and extend recent research on Klebsiella bacteriophages. For instance, Peng et al. (2025) ( 13 ) isolated a lytic bacteriophage vB_Kp_XP4 that targets hypervirulent K1 K. pneumoniae , noting its potent activity and absence of undesirable genes – similar to Halo KS-7 in its strictly lytic nature. Mirza et al. (2025) ( 34 ) characterized bacteriophage vbKpUKJ_2 from hospital sewage, which showed a broad host range (infecting ~ 43% of tested K. pneumoniae isolates) and high thermal/pH stability, aligning with Halo KS-7’s robustness. Unlike vbKpUKJ_2 (a Drexlerviridae siphovirus with ~ 45 kb genome) ( 34 ), Halo KS-7 is a myovirus with a slightly larger genome (58.716 kb) and likely a different evolutionary origin. The Halo KS-7 phage demonstrated attributes relevant to clinical therapy and food safety applications. Similar findings were reported by Chen et al. (2023) ( 12 ), who characterized bacteriophage vB_KpP_HS106 against K. pneumoniae K2, showing stability across pH 4–12 and temperatures from 4–50°C, comparable to Halo KS-7’s stability profile. While vB_KpP_HS106 was primarily studied for food safety purposes, Halo KS-7’s characteristics suggest the potential for broader applications, including clinical therapy and food safety. A distinctive aspect of Halo KS-7 is its halophilic adaptation. Bacteriophages requiring or tolerating high salt are more commonly associated with halophilic bacterial hosts (e.g., Halomonas , Vibrio in marine settings) than Klebsiella . Halo KS-7’s survival in up to 10–15% NaCl without losing much activity is remarkable; by comparison, most enteric bacteriophages are significantly inactivated beyond ~ 5% NaCl due to osmotic damage to the capsid. This property might translate into advantages for therapeutic formulation. For example, bacteriophage preparations often contain stabilizers like salts or sugars. Halo KS-7 might remain potent in the broader range of formulations or specific infection sites (such as hypertonic cystic fibrosis sputum). While halotolerance is not a typical requirement for bacteriophage therapy, it illustrates the diverse stress tolerances bacteriophages can evolve, which might correlate with other traits like desiccation resistance or shelf stability. The bacteriophage genome encodes several auxiliary enzymes that enhance infectivity and offer promising therapeutic applications. Among them, the holin gene is critical in orchestrating bacterial lysis. Holins form-controlled pores in the bacterial inner membrane, allowing them to reach the peptidoglycan layer and initiate cell wall degradation. This precise timing and mechanism significantly improve lysis efficiency and accelerate the bacteriophage replication cycle, which is advantageous in therapeutic settings ( 35 ). The Rz-like process further supports the lytic spanin, facilitating the final step of bacterial cell lysis by disrupting the outer membrane. Complete membrane disruption ensures full bacterial collapse, minimizing the survival of residual or partially lysed cells, which is a key factor in effective infection clearance ( 36 ). Additionally, the bacteriophage encodes a MazG-like pyrophosphatase, an enzyme known to interfere with bacterial stress responses. By hydrolyzing nucleotide alarmones such as (p)ppGpp, this enzyme disrupts the stringent response which is a defense mechanism activated under nutrient limitation or antibiotic exposure. This interference may sensitize bacteria to bacteriophage attack and reduce the formation of persister cells, thereby enhancing the overall efficacy of bacteriophage therapy ( 18 ). The presence of a bacteriophage-encoded RNA polymerase further underscores its adaptability. This enzyme allows the transcription of bacteriophage genes independently of the host’s transcriptional machinery, which is advantageous when the host is under metabolic stress or suppresses foreign gene expression ( 37 ). Lastly, the ribonucleotide reductase gene converts ribonucleotides into deoxyribonucleotides, ensuring a sufficient supply of DNA precursors for viral replication. This is especially beneficial in nutrient-limited environments such as biofilms or infected tissues, where nucleotide availability may be restricted ( 38 ). Together, these enzymes constitute a strategic genetic arsenal that enhances bacteriophage replication, promotes efficient host cell lysis, and improves resilience under stress. Their presence suggests strong potential for therapeutic use, especially in targeting persistent or MDR bacterial infections. Intriguingly, the Halo KS-7 genome harbors three tRNA (tRNA Tyr , tRNA Pro , and tRNA Asn ) genes, suggesting an adaptive strategy to optimize translational efficiency when host tRNA pools are limiting. The bacteriophage can sustain high-level protein synthesis during infection by supplying its tRNAs, promoting rapid replication and potent lytic activity. Although minimal-genome approaches often eschew accessory elements, recent work shows that standalone tRNA genes pose negligible biosafety risks when unlinked to lysogeny or mobile elements. Because Halo KS-7 lacks integrase and follows a strictly lytic lifecycle, these tRNAs likely represent evolutionary fine-tuning for efficient host takeover rather than a hazard. Such features underscore the therapeutic potential of Halo KS-7 against CRKP, especially in challenging clinical contexts. Using an AI-driven pipeline was instrumental in parsing Halo KS-7’s genome. Bacteriophage genomes typically have a high percentage of ORFs (genes with no known homologs), and Halo KS-7 was no exception (~ 35% hypothetical proteins). By leveraging a protein language model and large training datasets, we could predict likely functions for some previously uncharacterized ORFs, adding to the functional annotation. For example, the AI pipeline suggested that one small hypothetical protein had features of a DNA-binding transcriptional activator, which was confirmed by finding a helix-turn-helix motif. This demonstrates how modern AI tools can complement traditional BLAST-based annotation, a point echoed in recent reviews ( 17 ). While AI annotations must be validated (to avoid misassignments), the approach accelerated our understanding of Halo KS-7’s genetic makeup. As AI models are trained on more bacteriophage data, their accuracy for function prediction will improve, potentially unveiling new anti-bacterial proteins encoded by bacteriophages. In Halo KS-7, beyond known categories, the function of ~ 27 ORFs remains unknown – these could include novel anti-host factors or structural proteins that warrant further study, possibly through proteomics or structural biology. Halo KS-7 meets several key criteria for a therapeutic bacteriophage candidate. First, it is strictly lytic and lacks genes for lysogeny, minimizing safety concerns ( 13 , 34 ). Second, it has a relatively broad action against diverse K. pneumoniae strains, including MDR and hypervirulent types, which is essential given the genetic diversity of K. pneumoniae clinical isolates ( 39 ). Third, the bacteriophage genome encodes several enzymes such as holins, spanins, MazG-like pyrophosphatase, RNA polymerase, and ribonucleotide reductase that collectively enhance its ability to infect and lyse bacterial cells. These enzymes enable efficient membrane disruption, suppress bacterial stress responses, and replication of bacteriophage under nutrient-limited or stressful conditions ( 18 , 38 , 40 – 42 ). Their presence suggests the bacteriophage is well-suited for therapeutic applications, potentially maintaining stability and activity across various infection environments and delivery routes such as inhalation or topical use. Fourth, its stability in a range of conditions implies it could be formulated for various routes of administration (inhalation for pneumonia, topical for wound infections, etc.) without rapid inactivation. Recent in vivo studies give optimism that bacteriophages like Halo KS-7 could be effective. For example, Gan et al. (2022) ( 43 ) demonstrated that two lytic bacteriophages could rescue mice from fatal K. pneumoniae pneumonia, significantly reducing bacterial load and inflammation. Similarly, Duarte et al. (2022) ( 44 ) reported successful compassionate use of bacteriophages in a human patient with recurrent Klebsiella urinary infection. These cases, alongside rigorous laboratory evaluations, suggest that bacteriophages are moving from bench to bedside for K. pneumoniae . Halo KS-7’s broad host range would be advantageous for treating infections where the specific capsular type is unknown or mixed Klebsiella populations are present. In a practical scenario, Halo KS-7 could be used as part of a bacteriophage cocktail to preempt resistance – combining it with other bacteriophages targeting complementary receptors. Indeed, bacteriophage cocktails have broadened the overall killing spectrum and delayed the emergence of bacteriophage-resistant mutants ( 14 ). While this study provides a comprehensive analysis of Halo KS-7, there are some limitations. We did not perform in vivo experiments, so the bacteriophage’s efficacy and immunogenicity in animal infection models remain to be tested. Given its promising in vitro profile, testing Halo KS-7 in a mouse pneumonia model, similar to Gan et al. and Li et al., ( 43 , 45 ) would be a logical step to confirm therapeutic potential and safety (e.g., absence of immunopathology). From a genomic standpoint, Halo KS-7 underscores how much bacteriophage genetic diversity remains undiscovered. The AI pipeline flagged a number of unique ORFs; characterizing these (via knockout mutagenesis or structural studies) could reveal novel functions. One speculative but intriguing ORF encodes a small protein of 90 amino acids with a repeated motif; such proteins sometimes act as inhibitors of host processes (e.g., protease inhibitors or nucleotide sequestration proteins). Uncovering any anti-host mechanisms could augment our understanding of how bacteriophages subvert Klebsiella during infection. Furthermore, exploring the relationship of Halo KS-7 with the rare bacteriophages it is related to (like the “vB_KpnM_Saline”) could help define a new bacteriophage genus. Given the mosaic nature of bacteriophage genomes, the presence or absence of specific functional modules such as lysis-related or host takeover genes, shows the importance of analyzing bacteriophage genes individually and in the context of the complete genome. Comprehensive characterization of Halo KS-7 reveals a potent lytic bacteriophage with distinctive halophilic adaptation and a broad host range. Isolated from a clinical setting and thoroughly examined through laboratory assays and an AI-assisted genomic annotation pipeline, Halo KS-7 represents a novel addition to the growing repertoire of bacteriophages with translational promise. Genomic analysis confirmed the absence of antibioticresistance, integrase, or recombinase gene, underscoring its strictly lytic lifecycle and lack of lysogenic potential. Intriguingly, the genome also harbors three tRNA genes (tRNA Tyr , tRNA Pro , and tRNA Asn ), which likely optimize translational efficiency when host tRNA pools are limited, sustaining rapid protein synthesis and enhancing lytic activity. The presence of a toxin gene and auxiliary factors such as MazG, like pyrophosphatase and HNH endonucleases, further suggest built-in enhancements to bacterial killing without promoting HGT or antimicrobial resistance (AMR), reinforcing its therapeutic safety. Finally, the application of AI in genomic annotation was instrumental in rapidly distinguishing both beneficial and undesirable elements, showcasing the power of computational tools in modern bacteriophage research. As AMR continues to escalate, bacteriophages like Halo KS-7 offer a timely and targeted alternative for infection control. Future directions will focus on assessing its efficacy in vivo infection models, exploring formulation strategies, and evaluating its role within bacteriophage cocktails or as a source of recombinant enzymes. Incorporating such bacteriophages into therapeutic pipelines and clinical trials will be crucial to realizing their clinical impact. Our findings contribute to the broader understanding of bacteriophage diversity and function, laying the groundwork for further development of Halo KS-7 in combating critical K. pneumoniae infections. 4. Materials and Methods 4.1. Bacteriophage Isolation and Host Bacteria Halo KS-7 was isolated from wastewater at Shahriar Hospital in Tehran, Iran. The sewage sample was centrifugated (10,000 × g, 10 min) and filtered through a 0.22 µm membrane. The host bacterium, K. pneumoniae , was cultured in Luria-Bertani (LB) agar at 37˚C until the logarithmic growth phase was reached. The wastewater sample (50 mL) was enriched with an equal volume of exponential-phase K. pneumoniae culture and incubated at 37°C for 24 hours with gentle agitation. The host K. pneumoniae isolates were carbapenem-resistant clinical strains previously isolated from diabetic foot ulcers of hospital patients. The resulting bacteriophage lysate underwent purification via three successive cycles of single-plaque selection and co-culturing. Bacteriophage isolation was verified using the double-layer agar (DLA) method ( 46 ). The plate was incubated at 37˚C for 24 h, and clear plaques indicated a successful bacteriophage infection. Bacteriophage titration involved tenfold serial dilutions in SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris (pH = 7.5), and 0.002% gelatin (w/v)). Bacteriophage titers were determined by the double agar overlay plaque assay and reported as plaque-forming units per milliliter (PFU/mL) ( 5 ). 4.2. Morphological Characterization by Transmission Electron Microscopy (TEM) Following standard protocols, isolated bacteriophage particles were analyzed using transmission electron microscopy (TEM) ( 47 ). Briefly, 10 µL of purified bacteriophage suspension was applied to a carbon-coated copper grid and allowed to adsorb for 3–5 minutes. The sample was then negatively stained with 1% (w/v) uranyl acetate (pH = 7). Imaging was performed using a Zeiss LEO 906 TEM (Carl Zeiss LEO EM 906 E, Germany) operating at an accelerating voltage of 100 kV ( 48 , 49 ). 4.3. Host Range Determination The host range of Halo KS-7 was evaluated on a panel of bacterial strains to assess its lytic spectrum. This panel included 30 CRKP strains ( 50 ). Host range spot tests were conducted by spotting 10 µL of high-titer bacteriophage (~ 10^9 PFU/mL) onto lawns of each bacterial strain on appropriate agar. Plates were incubated overnight at 37°C, and lysis was assessed qualitatively based on plaque morphology. Transparent or opaque plaques indicated susceptibility to Halo KS-7, reflecting lytic activity. In contrast, the absence of plaques or only faint clearing suggested resistance to the bacteriophage. 4.4. Physical and Stability Tests The stability of Halo KS-7 was evaluated under a range of conditions representing physiological and extreme environments. To assess thermal stability, bacteriophage lysates (~ 10⁸ PFU/mL in SM buffer) were incubated for 1 hour at (-20°C, 4°C, 37°C, 50°C, 60°C, and 70°C), followed by titration using plaque assays on LB agar (Wang et al., 2016). For pH stability, aliquots were adjusted to pH values ( 2 , 4 , 7 , 10 , and 14 ) using SM buffer modified with HCl or NaOH and incubated at room temperature for 1 hour before titration. Halotolerance was assessed by incubating bacteriophage lysates in SM buffer supplemented with varying NaCl concentrations (5%, 10%, and 15%) at 25°C for 24 hours, followed by titer determination using the double agar layer (DLA) method ( 51 ). All experiments were performed in triplicate. 4.5. Bacteriophage DNA Extraction and Genome Sequencing According to the manufacturer's instructions, the bacteriophage genome was extracted using a commercial DNA extraction kit (DNA Pure, FAVOR-GEN, Iran). Quality and purity of the extracted DNA were verified through optical density measurements, agarose gel electrophoresis, and a PCR assay using bacterial 16S rRNA primers to confirm the absence of bacterial genomic contamination ( 52 ). An Illumina library was constructed from 100 ng of purified Halo KS-7 DNA for whole-genome sequencing (WGS) using a Nextera XT Library Preparation Kit. Paired-end sequencing (2 × 150 bp) was carried out on the Illumina HiSeq platform. Raw sequencing reads were subjected to quality assessment using FastQC software, followed by adapter removal and quality filtering with Trimmomatic. High-quality reads were de novo assembled using SPAdes v3.15.2 with the "careful" mode enabled to reduce mismatches and short indels. Assembly quality was evaluated, resulting in a single contiguous bacteriophage genome. Any remaining gaps or ambiguous regions were resolved via Sanger sequencing of PCR-amplified fragments. The finalized genome was confirmed to be a linear double-stranded DNA molecule with defined termini, as determined by read coverage analysis and identification of putative terminal repeats ( 53 ). 4.6. Bioinformatics Analysis This study used two complementary approaches to analyze bacteriophage sequencing data. The first approach utilized an AI-enabled pipeline for viral detection and validation from next-generation sequencing data ( 54 ). This pipeline integrates advanced bioinformatics tools with AI to efficiently identify viral sequences and de novo genome assembly, offering a comprehensive view of bacteriophage genetic diversity. The second approach involved using CLC Genomics Workbench 22, a widely adopted platform that supports read processing, mapping, variant calling, and genome assembly through an intuitive graphical interface and robust analytical capabilities. 4.6.1. AI-Driven Genome Annotation Pipeline In this study, the WGS data of bacteriophage Halo KS-7 was analyzed using a bioinformatics pipeline based on methods described by Ghorbani et al. (2024) ( 54 ). Raw sequencing reads were initially assessed for quality using FastQC, then trimming with Trimmomatic to remove low-quality bases and adapter sequences. High-quality reads were then assembled de novo using SPAdes, generating a complete genome sequence. The assembly was refined and validated through multiple alignment checks and coverage assessments. Genome annotation was performed using Prokka, which identified open reading frames (ORFs), tRNAs, and other genomic elements. Functional annotation of predicted ORFs was conducted through comparative analysis against NCBI nr, Pfam, and InterProScan databases. Comparative genomic tools, including Blastn and VICTOR, were employed to evaluate taxonomic classification and genomic similarity to other bacteriophages. Conserved domains, virulence factors, and potential antibiotic resistance genes were screened using PHASTER and the Comprehensive Antibiotic Resistance Database. All computational analyses were executed using Python scripts provided in the referenced study, ensuring reproducibility and alignment with established methodologies. This automated workflow streamlined the read processing, genome assembly, annotation, and functional characterization processes. Overall, the results offered in-depth insights into the genetic architecture, structural features, and evolutionary lineage of Halo KS-7, confirming its potential applications ( 54 ). 4.6.2. Analysis Using CLC Genomics Workbench 22 Sequencing reads were trimmed using CLC Genomics Workbench 22 (QIAGEN) with standard parameters to remove adapter sequences, ambiguous nucleotides, and low-quality bases. Specifically, bases with quality scores ≤ 5, reads shorter than 15 nucleotides and reads containing more than two ambiguous nucleotides were removed ( 55 ). The cleaned reads were assembled de novo using default parameters (word size: 15 nt). Assembled contigs were subjected to nucleotide BLAST analysis using Geneious version 22 (Biomatters, New Zealand). The contig with the highest similarity to viral sequences in the GenBank database was selected for further study to identify putative bacteriophages. Confirmation was performed using the VirusDetect tool ( http://virusdetect.feilab.net/cgi-bin/virusdetect/vdo_home.cgi ) , and results were independently validated by mapping cleaned reads back to the viral genome identified in the BLAST results. Genome annotation was carried out using the CLC Microbial Genomics Module, leveraging a reference genome and integrated BLAST tools. Annotation parameters included a default similarity threshold of 95% and an E-value cutoff of 0.0001 ( 55 ). The finalized genome sequence was then aligned with selected bacteriophage genomes from the NCBI database using the Whole Genome Alignment plugin in CLC Genomics Workbench ( 56 ), providing insights into phylogenetic relationships and genomic conservation. Declarations Author contributions SA: conceptualization, data curation, formal analysis, investigation, methodology, software, visualization, writing – original draft, writing – review & editing, validation, project administration. MB: conceptualization, formal analysis, validation, software, data curation, writing – original draft, writing–review & editing, visualization. SS: conceptualization, visualization, investigation, data curation, writing – original draft. MSH: conceptualization, methodology, project administration, supervision, validation, writing – review and editing. PS: validation, writing – review & editing. MP: visualization, investigation. AHS: validation, writing – review & editing. Data availability statement The datasets generated during the current study are available in the GenBank repository (accession number PV02655, https://www.ncbi.nlm.nih.gov/search/all/?term=PV026551). Competing Interests Statement The authors have declared that no competing interests exist. Funding The authors declare that the research was conducted without any commercial or financial relationships that could potentially create a conflict of interest. Ethical Considerations The collection of wastewater samples for this study was conducted in accordance with the ethical guidelines set by the Pasteur Institute of Iran under the ethical code IR.PII.AEC.1403.008. Permission was obtained for the collection of wastewater samples by Shahriar Hospital, with no identifiable patient data collected during the research. References Yuan Y, Wang J, Yao Z, Ma B, Li Y, Yan W, et al. Risk Factors for Carbapenem-Resistant Klebsiella pneumoniae Bloodstream Infections and Outcomes. Infect Drug Resist. 2020;13:207-15. Beig M, Aghamohammad S, Majidzadeh N, Asforooshani MK, Rezaie N, Abed S, et al. Antibiotic resistance rates in hypervirulent Klebsiella pneumoniae strains: A systematic review and meta-analysis. J Glob Antimicrob Resist. 2024;38:376-88. Beig M, Arabestani MR. Investigation of MexAB-OprM efflux pump gene expression in clinical isolates of pseudomonas aeruginosa isolated from Intensive Care Unit. Iranian Journal of Medical Microbiology. 2019;13(2):142-50. Mohammadpour D, Memar MY, Leylabadlo HE, Ghotaslou A, Ghotaslou R. Carbapenem-Resistant Klebsiella pneumoniae: A comprehensive review of phenotypic and genotypic methods for detection. The Microbe. 2025;6:100246. Sadeqi S, Michniewski S, Nikkhahi F, Jameson E, Marashi SMA. Isolation of new Klebsiella pneumoniae phage PSKP16. Iran J Microbiol. 2023;15(1):62-8. Chinemerem Nwobodo D, Ugwu MC, Oliseloke Anie C, Al-Ouqaili MTS, Chinedu Ikem J, Victor Chigozie U, et al. Antibiotic resistance: The challenges and some emerging strategies for tackling a global menace. J Clin Lab Anal. 2022;36(9):e24655. Sadeqi S, Hashemi Shahraki A, Nikkhahi F, Javadi A, Amin Marashi SM. Application of bacteriophage cocktails for reducing the bacterial load of nosocomial pathogens in hospital wastewater. Iran J Microbiol. 2022;14(3):395-401. Goodarzi F, Hallajzadeh M, Sholeh M, Talebi M, Mahabadi VP, Amirmozafari N. Biological characteristics and anti-biofilm activity of a lytic phage against vancomycin-resistant Enterococcus faecium. Iran J Microbiol. 2021;13(5):691-702. Goodarzi F, Hallajzadeh M, Sholeh M, Talebi M, Pirhajati Mahabadi V, Amirmozafari N. Anti-biofilm Activity of a Lytic Phage Against Vancomycin-Resistant Enterococcus faecalis. Iran J Pathol. 2022;17(3):285-93. Zurabov F, Glazunov E, Kochetova T, Uskevich V, Popova V. Bacteriophages with depolymerase activity in the control of antibiotic resistant Klebsiella pneumoniae biofilms. Sci Rep. 2023;13(1):15188. Mohammadi M, Mehrisadat M, Masoumeh B, Somaieh N, Mina Y, and Monfared FN. The Role of Nanotechnology in Overcoming Barriers to Phage Therapy: an Overview. Future Virology. 2023;18(6):387-98. Chen C, Tao Z, Li T, Chen H, Zhao Y, Sun X. Isolation and characterization of novel bacteriophage vB_KpP_HS106 for Klebsiella pneumonia K2 and applications in foods. Front Microbiol. 2023;14:1227147. Peng X, Chang J, Zhang H, Li X, Zhang C, Jiao S, et al. Isolation, characterization, and genomic analysis of a novel bacteriophage vB_Kp_XP4 targeting hypervirulent and multidrug-resistant Klebsiella pneumoniae. Front Microbiol. 2025;16:1491961. Chen H, Liu H, Gong Y, Dunstan RA, Ma Z, Zhou C, et al. A Klebsiella-phage cocktail to broaden the host range and delay bacteriophage resistance both in vitro and in vivo. NPJ Biofilms Microbiomes. 2024;10(1):127. Manuel RD, Snyder JC. The Expanding Diversity of Viruses from Extreme Environments. Int J Mol Sci. 2024;25(6). Motlagh AM, Bhattacharjee AS, Coutinho FH, Dutilh BE, Casjens SR, Goel RK. Insights of Phage-Host Interaction in Hypersaline Ecosystem through Metagenomics Analyses. Front Microbiol. 2017;8:352. Flamholz ZN, Li C, Kelly L. Improving viral annotation with artificial intelligence. mBio. 2024;15(10):e0320623. Ho P, Chen Y, Biswas S, Canfield E, Abdolvahabi A, Feldman DE. Bacteriophage antidefense genes that neutralize TIR and STING immune responses. Cell Rep. 2023;42(4):112305. Gholizadeh O, Ghaleh HEG, Tat M, Ranjbar R, Dorostkar R. The potential use of bacteriophages as antibacterial agents against Klebsiella pneumoniae. Virol J. 2024;21(1):191. Chen Q, Zhang F, Bai J, Che Q, Xiang L, Zhang Z, et al. Bacteriophage-resistant carbapenem-resistant Klebsiella pneumoniae shows reduced antibiotic resistance and virulence. Int J Antimicrob Agents. 2024;64(2):107221. Wang Q, Chen R, Liu H, Liu Y, Li J, Wang Y, et al. Isolation and characterization of lytic bacteriophage vB_KpnP_23: A promising antimicrobial candidate against carbapenem-resistant Klebsiella pneumoniae. Virus Res. 2024;350:199473. Parmar KM, Gaikwad SL, Dhakephalkar PK, Kothari R, Singh RP. Intriguing Interaction of Bacteriophage-Host Association: An Understanding in the Era of Omics. Front Microbiol. 2017;8:559. Narayanan MP, Kumar A, Kumar Verma G, Bairwa A, Mirza AA, Goyal B. Efficacy of Bacteriophages in Wound Healing: An Updated Review. Cureus. 2024;16(10):e71542. Abdi G, Jain M, Barwant M, Tendulkar R, Tendulkar M, Tariq M, et al. Unveiling the dynamic role of bioinformatics in automation for efficient and accurate data processing and interpretation. Advances in Bioinformatics: Springer; 2024. p. 279-319. Rost HL, Sachsenberg T, Aiche S, Bielow C, Weisser H, Aicheler F, et al. OpenMS: a flexible open-source software platform for mass spectrometry data analysis. Nat Methods. 2016;13(9):741-8. Sundaram J, Gowri K, Devaraju S, Gokuldev S, Jayaprakash S, Anandaram H, et al. An Exploration of Python Libraries in Machine Learning Models for Data Science. Advanced Interdisciplinary Applications of Machine Learning Python Libraries for Data Science: IGI Global; 2023. p. 1-31. Djaffardjy M, Marchment G, Sebe C, Blanchet R, Bellajhame K, Gaignard A, et al. Developing and reusing bioinformatics data analysis pipelines using scientific workflow systems. Comput Struct Biotechnol J. 2023;21:2075-85. Zhai Z, Zhang Z, Zhao G, Liu X, Qin F, Zhao Y. Genomic Characterization of Two Novel RCA Phages Reveals New Insights into the Diversity and Evolution of Marine Viruses. Microbiol Spectr. 2021;9(2):e0123921. Cortes-Martin A, Denise R, Guerin E, Stockdale SR, Draper LA, Ross RP, et al. Isolation and characterization of a novel lytic Parabacteroides distasonis bacteriophage phiPDS1 from the human gut. Gut Microbes. 2024;16(1):2298254. Sutton TDS, Hill C. Gut Bacteriophage: Current Understanding and Challenges. Front Endocrinol (Lausanne). 2019;10:784. Shkoporov AN, Khokhlova EV, Fitzgerald CB, Stockdale SR, Draper LA, Ross RP, et al. PhiCrAss001 represents the most abundant bacteriophage family in the human gut and infects Bacteroides intestinalis. Nat Commun. 2018;9(1):4781. Guerin E, Hill C. Shining Light on Human Gut Bacteriophages. Front Cell Infect Microbiol. 2020;10:481. Wahida A, Tang F, Barr JJ. Rethinking phage-bacteria-eukaryotic relationships and their influence on human health. Cell Host Microbe. 2021;29(5):681-8. Mirza KA, Tchatchiashvili T, Marquet M, Nietzsche S, Pletz MW, Makarewicz O. Characterization and genome analysis of novel Klebsiella pneumoniae phage vbKpUKJ_2 isolated from hospital sewage water. BMC Microbiol. 2025;25(1):96. Young R. Phage lysis: do we have the hole story yet? Curr Opin Microbiol. 2013;16(6):790-7. Summer EJ, Berry J, Tran TA, Niu L, Struck DK, Young R. Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts. J Mol Biol. 2007;373(5):1098-112. Miller ES, Kutter E, Mosig G, Arisaka F, Kunisawa T, Ruger W. Bacteriophage T4 genome. Microbiol Mol Biol Rev. 2003;67(1):86-156, table of contents. Cheng W, Chen Q, Ren Y, Zhang Y, Lu L, Gui L, et al. The identification of viral ribonucleotide reductase encoded by ORF23 and ORF141 genes and effect on CyHV-2 replication. Front Microbiol. 2023;14:1154840. Lobocka M, Dabrowska K, Gorski A. Engineered Bacteriophage Therapeutics: Rationale, Challenges and Future. BioDrugs. 2021;35(3):255-80. Wang I-N, Smith DL, Young R. Holins: The Protein Clocks of Bacteriophage Infections. Annual Review of Microbiology. 2000;54(Volume 54, 2000):799-825. Berry J, Summer EJ, Struck DK, Young R. The final step in the phage infection cycle: the Rz and Rz1 lysis proteins link the inner and outer membranes. Mol Microbiol. 2008;70(2):341-51. Yang H, Ma Y, Wang Y, Yang H, Shen W, Chen X. Transcription regulation mechanisms of bacteriophages: recent advances and future prospects. Bioengineered. 2014;5(5):300-4. Gan L, Fu H, Tian Z, Cui J, Yan C, Xue G, et al. Bacteriophage Effectively Rescues Pneumonia Caused by Prevalent Multidrug-Resistant Klebsiella pneumoniae in the Early Stage. Microbiol Spectr. 2022;10(5):e0235822. Duarte J, Maximo C, Costa P, Oliveira V, Gomes NCM, Romalde JL, et al. Potential of an Isolated Bacteriophage to Inactivate Klebsiella pneumoniae: Preliminary Studies to Control Urinary Tract Infections. Antibiotics (Basel). 2024;13(2):195. Li P, Guo G, Zheng X, Xu S, Zhou Y, Qin X, et al. Therapeutic efficacy of a K5-specific phage and depolymerase against Klebsiella pneumoniae in a mouse model of infection. Vet Res. 2024;55(1):59. Zadeh RG, Zadeh MH, Sholeh M, Asgharzadeh S, Darbandi A, Fashami P. Bacteriophage and probiotic therapy in the treatment of Pseudomonas aeruginosa burn infection and their synergistic effect. Egypt J Exp Biol. 2023;19:21-9. Alduhaidhawi AHM, AlHuchaimi SN, Al-Mayah TA, Al-Ouqaili MTS, Alkafaas SS, Muthupandian S, et al. Prevalence of CRISPR-Cas Systems and Their Possible Association with Antibiotic Resistance in Enterococcus faecalis and Enterococcus faecium Collected from Hospital Wastewater. Infect Drug Resist. 2022;15:1143-54. Abed S, Sholeh M, Khazani Asforooshani M, Shafiei M, Hashemi Shahraki A, Nasr S. Insights into the novel Enterococcus faecalis phage: A comprehensive genome analysis. PLoS One. 2024;19(5):e0301292. Khazani Asforooshani M, Elikaei A, Abed S, Shafiei M, Barzi SM, Solgi H, et al. A novel Enterococcus faecium phage EF-M80: unveiling the effects of hydrogel-encapsulated phage on wound infection healing. Front Microbiol. 2024;15:1416971. Yu P, Mathieu J, Li M, Dai Z, Alvarez PJ. Isolation of Polyvalent Bacteriophages by Sequential Multiple-Host Approaches. Appl Environ Microbiol. 2016;82(3):808-15. Komijani M, Bouzari M, Rahimi F. Detection and characterization of a novel lytic bacteriophage (vB-KpneM-Isf48) against Klebsiella pneumoniae isolates from infected wounds carrying antibiotic-resistance genes (TEM, SHV, and CTX-M). 2017. Gygli SM, Keller PM, Ballif M, Blochliger N, Homke R, Reinhard M, et al. Whole-Genome Sequencing for Drug Resistance Profile Prediction in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2019;63(4):10.1128/aac. 02175-18. Bosworth A, Robson J, Lawrence B, Casey AL, Fair A, Khanam S, et al. Deployment of whole genome next-generation sequencing of SARS-CoV-2 in a military maritime setting. BMJ Mil Health. 2024;170(e2):e144-e9. Ghorbani A, Rostami M, Guzzi PH. AI-enabled pipeline for virus detection, validation, and SNP discovery from next-generation sequencing data. Front Genet. 2024;15:1492752. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 2012;6(8):1621-4. Misu M, Yoshikawa T, Sugimoto S, Takamatsu Y, Kurosu T, Ouji Y, et al. Rapid whole genome sequencing methods for RNA viruses. Front Microbiol. 2023;14:1137086. Table Table 1. Key Functional Genes in Halo KS-7 Bacteriophage and Their Potential Roles Therapy. Gene Name Function Benefit to Bacteriophage Therapy Reference Holin CDS Creates pores in the bacterial membrane, allowing endolysins to access the peptidoglycan. Enhances bacterial lysis efficiency, accelerating infection. (40) Rz-like spanin CDS Facilitates the final step of cell lysis by disrupting the outer membrane. Ensures complete bacterial lysis, preventing persistence. (41) MazG-like pyrophosphatase CDS Inhibits bacterial stress response by degrading nucleotide alarmones. Reduces bacterial resistance mechanisms. Ensures survival of bacteriophages within the bacterial cell. (18) RNA polymerase CDS Transcribes bacteriophage genes independently of host machinery. Enables efficient viral gene expression even in stressed hosts. (42) ribonucleotide reductase CDS Converts ribonucleotides to deoxyribonucleotides for DNA synthesis. Supports rapid bacteriophage replication in nutrient-limited conditions. (38) Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6534345","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":453848167,"identity":"fd59f9d4-4e71-4569-8f08-6a7c852af9f7","order_by":0,"name":"Sahar Abed","email":"","orcid":"","institution":"Department of Microbial Biotechnology, Faculty of Basic Sciences and Advanced Technologies in Biology, University of Science and Culture, Tehran, Iran","correspondingAuthor":false,"prefix":"","firstName":"Sahar","middleName":"","lastName":"Abed","suffix":""},{"id":453848168,"identity":"cd6f57bb-b817-4025-a9ae-95f4e899dfc0","order_by":1,"name":"Masoumeh Beig","email":"","orcid":"","institution":"Pasteur Institute of Iran","correspondingAuthor":false,"prefix":"","firstName":"Masoumeh","middleName":"","lastName":"Beig","suffix":""},{"id":453848169,"identity":"1e907e8f-e436-4a76-bea6-c85616ded3ce","order_by":2,"name":"Sepideh Soltani","email":"","orcid":"","institution":"Pasteur Institute of Iran","correspondingAuthor":false,"prefix":"","firstName":"Sepideh","middleName":"","lastName":"Soltani","suffix":""},{"id":453848170,"identity":"8c7f7eb7-c935-4172-88f1-98a32e6d6915","order_by":3,"name":"Morvarid Shafiei","email":"data:image/png;base64,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","orcid":"","institution":"Pasteur Institute of Iran","correspondingAuthor":true,"prefix":"","firstName":"Morvarid","middleName":"","lastName":"Shafiei","suffix":""},{"id":453848171,"identity":"dfb0de40-4fcf-48e4-97e7-9b9e3c3aa85b","order_by":4,"name":"Peter Speck","email":"","orcid":"","institution":"College of Science and Engineering, Flinders University, Bedford Park, South Australia","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Speck","suffix":""},{"id":453848172,"identity":"8cf9917f-c78f-442b-9178-30fdb3f741ce","order_by":5,"name":"Mohaddeseh Pahlevani","email":"","orcid":"","institution":"Pasteur Institute of Iran","correspondingAuthor":false,"prefix":"","firstName":"Mohaddeseh","middleName":"","lastName":"Pahlevani","suffix":""},{"id":453848173,"identity":"db920b0a-e815-4453-a83e-e7ab6be316ee","order_by":6,"name":"Abdolrazagh Hashemi Shahraki","email":"","orcid":"","institution":"Division of Pulmonary, Critical Care and Sleep, College of Medicine-Jacksonville, University of Florida, Florida, USA","correspondingAuthor":false,"prefix":"","firstName":"Abdolrazagh","middleName":"Hashemi","lastName":"Shahraki","suffix":""}],"badges":[],"createdAt":"2025-04-26 10:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6534345/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6534345/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82605008,"identity":"9be0d6ea-9a55-4d7a-b7cc-c911c5f0cf72","added_by":"auto","created_at":"2025-05-13 09:59:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":15853340,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLytic activity of bacteriophage Halo KS-7 against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKlebsiella pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cbr\u003e\n\u003cstrong\u003e(A)\u003c/strong\u003e Spot test showing a clear lysis zone on a bacterial lawn. \u003cstrong\u003e(B)\u003c/strong\u003e Plaque assay revealing discrete plaques formed by Halo KS-7.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6534345/v1/a795cd1d12ecfb065ffa24e6.png"},{"id":82602761,"identity":"cb0b55f6-2d2b-46f0-8e84-5534b9134665","added_by":"auto","created_at":"2025-05-13 09:51:44","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":53231,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElectron microscopy analysis of Halo KS-7 bacteriophage morphology. \u003c/strong\u003eTransmission electron micrograph of the Halo KS-7 bacteriophage, negatively stained with 2% (w/v) uranyl acetate. The image highlights the bacteriophage’s characteristic structure, including the head and tail, with a scale bar representing 60 nm.\u003c/p\u003e","description":"","filename":"Figure2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6534345/v1/2c99dbb62878d52ebc82f682.jpeg"},{"id":82602762,"identity":"0323aab6-b931-4264-b911-84d6cf284654","added_by":"auto","created_at":"2025-05-13 09:51:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2032897,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStability of Halo KS-7 bacteriophage under different temperature and pH conditions.\u003c/strong\u003e\u003cbr\u003e\n\u003cstrong\u003e(A)\u003c/strong\u003e Thermal stability of the bacteriophage assessed at various temperatures ranging from −20 °C to 70 °C. The highest bacteriophage titre was observed at 37 °C, significantly decreasing at extreme temperatures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e pH stability profile of the bacteriophage evaluated across a pH range of 2 to 14. Optimal bacteriophage stability was recorded at pH = 7, with reduced titres under highly acidic and alkaline conditions. Bacteriophage titres are expressed as log PFU/ml. Error bars represent the standard deviation from triplicate experiments.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6534345/v1/4af3f9cd505c368538f5108d.jpg"},{"id":82602765,"identity":"c76b8f02-9cc9-485d-b159-105bb439452c","added_by":"auto","created_at":"2025-05-13 09:51:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2150202,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of NaCl concentration on the stability and infectivity of Halo KS-7 bacteriophage.\u003c/strong\u003e\u003cbr\u003e\n\u003cstrong\u003e(A)\u003c/strong\u003e Bacteriophage titre (log PFU/ml) measured after incubation at different NaCl concentrations (5%, 10%, and 15% w/v). The results indicate enhanced bacteriophage stability with increasing salinity, with the highest titre observed at 15% NaCl. Error bars represent standard deviations from triplicate experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Plaque formation assay showing the lytic activity of Halo KS-7 on a host lawn, confirming infectivity under high-salt conditions.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6534345/v1/9f7167fa089a43fccfaf8355.jpg"},{"id":82602773,"identity":"73cff3c0-e902-4bb7-8a2e-ba0523ac8afd","added_by":"auto","created_at":"2025-05-13 09:51:45","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5014354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWhole-genome sequencing and bioinformatic analysis of Halo KS-7 bacteriophage.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnnotated genome map of the Halo KS-7 bacteriophage. The genome consists of 49 predicted ORFs, depicted as arrows indicating direction of transcription. Functional annotation was performed using bioinformatic tools, categorizing the ORFs into proposed functional modules including replication, structural assembly, packaging, lysis, and hypothetical proteins. Several ORFs encode proteins with conserved domains, such as DNA primase, RNA polymerase, terminase subunits, portal protein, and holins, while others remain annotated as hypothetical due to lack of significant homology.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6534345/v1/394e03d1a0294a1a20a39d15.jpg"},{"id":82602777,"identity":"11535aec-ea9e-4b97-8fc1-cd0a5647b31e","added_by":"auto","created_at":"2025-05-13 09:51:45","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3958551,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative genomic and nucleotide identity analysis of Halo KS-7 and related bacteriophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Synteny analysis of bacteriophage genomes illustrating gene organization and conserved regions across multiple bacteriophages. Color-coded blocks and connecting lines represent homologous sequences, while gene rearrangements highlight genomic divergence in Halo KS-7.\u003c/p\u003e\n\u003cp\u003e(B) Heatmap showing pairwise average nucleotide identity (ANI) percentages among the analyzed bacteriophages. The moderate ANI values between Halo KS-7 and its closest relatives support its designation as a novel bacteriophage.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6534345/v1/b2c20dccb0b3768e3888fc17.jpg"},{"id":84689822,"identity":"3678d556-a41d-4c59-b71e-514f0a9451cc","added_by":"auto","created_at":"2025-06-16 09:32:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":30128455,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6534345/v1/e41020fe-6362-43d7-b79b-41b8688933b3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genomic Insights into Halo KS-7: An AI-Driven Characterization of a Novel Halophilic Bacteriophage Targeting Carbapenem-Resistant Klebsiella pneumoniae","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMultidrug-resistant (MDR) \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e is a major global health threat, causing various infections such as pneumonia and bloodstream infections (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Carbapenem-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e (CRKP), a particularly concerning pathogen, has been classified as a critical priority by the World Health Organization due to its high antibiotic resistance and limited treatment options (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). This bacterium's ability to form biofilms, its high antibiotic resistance, and its potential for horizontal gene transfer (HGT) complicate treatment (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). As traditional antibiotics become less effective, alternative therapeutic approaches are urgently needed (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBacteriophages are emerging as a promising solution for antibiotic-resistant bacteria. They offer advantages like high specificity, targeting antibiotic-resistant strains, and minimal disruption to the host microbiota (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Some bacteriophages, particularly those targeting \u003cem\u003eK. pneumoniae\u003c/em\u003e, also produce depolymerase that degrades the bacterial capsule, improving immune clearance and bacteriophage effectiveness (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBacteriophages isolated from sources such as sewage and clinical samples may exhibit considerable genomic diversity, with variations ranging from small 40 kb podoviruses to large 170 kb myoviruses (\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Continued isolation and genomic analysis of new bacteriophages are critical for advancing bacteriophage therapy and understanding bacteriophage-host\u0026ndash;host interactions. In particular, bacteriophages from extreme environments such as high-salt (halophilic) conditions may offer unique features, including improved environmental or physiological stability and expanded host range (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHalophilic bacteriophages adapted to high-salt environments are highly stable under thermal, pH, and desiccation stress. Their unique genomes offer opportunities for genetic engineering and help improve our understanding of bacteriophage-host interactions. Notably, these bacteriophages also have potential practical uses, such as biocontrol agents in salt-rich food production and stable topical treatments for MDR infections under challenging environments like chronic wounds (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, they remain underexplored in non-halophilic hosts like \u003cem\u003eK. pneumoniae\u003c/em\u003e. Advances in artificial intelligence (AI) and bioinformatics have revolutionized bacteriophage research, enabling efficient genome annotation and identification of potentially useful therapeutic genetic elements in bacteriophages, (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) such as holins, important for host lysis and MazG-like pyrophosphatases which can neutralize host defenses to ensure virus (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile some studies have explored bacteriophages targeting MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e, few have systematically utilized computational pipelines to evaluate their genomic features and therapeutic relevance (\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Limited bacteriophage databases and a lack of understanding of host specificity and infection mechanisms continue to hinder the advancement of bacteriophage therapy (\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). These gaps show the need for continued discovery and genomic investigation of novel bacteriophages, particularly those with unique ecological adaptations.\u003c/p\u003e \u003cp\u003eWhile CLC Genomics Workbench (Qiagen, Hilden, Germany) is a popular commercial tool known for its intuitive interface and integrated bioinformatics functionalities, it is limited in flexibility, transparency, and scalability, making it less suitable for researchers who need greater control over their workflows (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Python-based pipelines offer a powerful open-source alternative that allows for customization and transparency, enabling researchers to fine-tune their analysis, from preprocessing and quality filtering to genome assembly and annotation (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Python's seamless integration with libraries like Biopython, pandas, matplotlib, and NumPy supports advanced data analysis, machine learning, and high-quality visualizations, offering greater precision and adaptability than commercial software (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Additionally, Python promotes reproducibility through script-based workflows and can be easily automated to handle large datasets without costly licenses (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Python's scalability allows workflows to be deployed on various platforms, including local machines, high-performance clusters, and cloud infrastructure. Moreover, unlike proprietary tools with significant financial barriers, Python is cost-effective and freely available. Here, we used a Python-driven pipeline to characterize the novel halophilic bacteriophage Halo KS-7, demonstrating Python's robustness, adaptability, and versatility of open-source solutions. This allows us to identify genes associated with therapeutic relevance. By comparing this genome to other known \u003cem\u003eK. pneumoniae\u003c/em\u003e bacteriophages, we aim to identify distinctive traits that could support its potential as a biocontrol agent. This study seeks to demonstrate the value of integrating wet-lab techniques with computational tools in discovering and evaluating promising bacteriophage candidates.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e2.1. Isolation and Characterization of a Halophilic \u003cem\u003eK. pneumoniae\u003c/em\u003e Bacteriophage\u003c/p\u003e\n\u003cp\u003eThe Halo KS-7 bacteriophage was specifically isolated to target CRKP. The spot assay results, illustrated in Figure 1A, and the DLA assay outcomes presented in Figure 1B unequivocally affirmed its lytic activity. TEM analysis revealed that Halo KS-7 exhibits a morphology consistent with the \u003cem\u003eCaudovirales\u003c/em\u003e order (Figure 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.1. Host Range Determination\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Halo KS-7 exhibited a broad host range across the tested CRKP isolates. In assays involving 30 clinical CRKP strains, clear plaques were observed in 57% of the strains, confirming the bacteriophage\u0026apos;s selective and targeted lytic activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.2. Temperature Stability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 3A illustrates the thermal stability of Halo KS-7. The bacteriophage exhibited optimal lytic activity at 37\u0026deg;C and maintained good activity in the range of 4\u0026deg;C to 60\u0026deg;C, with no significant drop in titer (within 0.5 log of the initial). At 70\u0026deg;C, a notable decrease in lytic activity was observed. However, it did not reach complete inactivation, indicating that Halo KS-7 has moderate thermal stability, similar to many colibacteriophages but slightly lower than some extremophilic bacteriophages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.3. pH Sensitivity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe influence of pH on Halo KS-7 bacteriophage\u0026rsquo;s lytic activity is seen in Figure 3B.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e The bacteriophage has good lytic activity at pH between 4 and 10; its highest activity was observed at pH = 7. At pH below 4, bacteriophage activity was completely stopped (pH = 2, no activity). As pH increased and the environment became more alkaline, the lytic activity of the bacteriophage decreased so that at pH = 14, its activity reached almost zero. At pH = 10, bacteriophage activity was still relatively high but decreased compared to its optimal value at pH = 7. These data indicate that the bacteriophage is most stable and effective in neutral and slightly alkaline conditions (pH = 7 to 10), while it is extremely vulnerable in highly acidic (pH = 2) and highly alkaline (pH = 14) environments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.4. Salt Tolerance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 4A depicts the response of Halo KS-7 to varying salt concentrations. The bacteriophage exhibited increased lytic activity as NaCl concentration in the medium rose. At 5% NaCl, the bacteriophage titer was 4.36 log PFU/mL, which increased to 6.8 log PFU/mL at 10% NaCl. The highest activity, 8.1 log PFU/mL, was at 15% NaCl.\u003c/p\u003e\n\u003cp\u003eThese findings suggest that elevated NaCl concentrations led to the formation of larger, sharper plaques (Figure 4B), showing the beneficial effect of salt on bacteriophage lytic activity. Accordingly, Halo KS-7 is classified as a halophilic bacteriophage, demonstrating optimal lytic activity in saline environments.\u003c/p\u003e\n\u003cp\u003e2.2. Whole Genome Sequencing and Bioinformatics Analysis: Genome Sequencing and Assembly\u003c/p\u003e\n\u003cp\u003eA commercial column-based DNA extraction protocol ensured material purity without bacterial genomic contamination. The genome sequence data was assembled de novo. The complete genome sequence of Halo KS-7 has been deposited in GenBank under accession number PV026551 (https://www.ncbi.nlm.nih.gov/search/all/?term=PV026551). The Halo KS-7 bacteriophage genome has a genome size of 58,716 bp. It comprises 49 predicted ORFs, with 64.47% related to genes encoding putative functional proteins. The GC content of Halo KS-7 is 44.4%, and its closest related organism is \u003cem\u003eKlebsiella\u003c/em\u003e bacteriophage vB_KpP_FBKp27 (accession number: NC_068555), with a 90.81% identity and 94% query coverage (Figure 5).\u003c/p\u003e\n\u003cp\u003e2.3. Functional Annotation and Genomic Analysis\u003c/p\u003e\n\u003cp\u003eFigure 6A presents a comparative genomic alignment of multiple \u003cem\u003eK. pneumoniae\u003c/em\u003e strains, showing the organization of genomic features across different isolates. The diagram shows colored bars representing various genomic regions, with each strain labeled on the left (e.g., OX335406, OP030736, BK050257, PV026551, and NC_068555). The connecting lines between the bars indicate similarity and homology between these genomic regions across strains, with each segment representing specific gene sequences or other functional genomic features. The scale on the top indicates the genomic coordinates, ranging from 0 to 60,000 base pairs. This comparison illustrates conserved and variable genomic regions across the strains, providing insights into their genomic diversity and evolutionary relationships. The visualization is crucial for identifying homologous gene clusters and potential regions of genetic variation and understanding the genomic structure of \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates.\u003c/p\u003e\n\u003cp\u003eFigure 6B provides a heatmap showing pairwise genome similarity percentages. The ANI values confirm that Halo KS-7 (PV026551) shares only moderate sequence identity (75.42%\u0026ndash;78.34%) with its closest relatives. The comparison with NC_068555, which has the highest alignment score (90.81% identity but only 94% query coverage), suggests that Halo KS-7 is genetically distinct. Syntactic analysis and ANI calculations demonstrate that Halo KS-7 exhibits significant genetic divergence from known bacteriophages. The unique ORFs and moderate sequence similarity with its closest relatives strongly support its classification as a novel bacteriophage. Holin and spanin genes associated with host cell lysis were detected among the functional genes identified. Genes encoding HNH endonucleases and exonucleases were present, along with a RuvC-like Holliday junction resolvase and a Sak4-like single-stranded DNA annealing protein. A MazG-like pyrophosphatase, single-stranded DNA-binding proteins, ribonucleotide reductase, ATPase, and RNA polymerase genes were also identified. These functional genes collectively suggest a genetic repertoire potentially supporting bacteriophage replication, survival, and host interaction under various environmental conditions.\u003c/p\u003e\n\u003cp\u003eHalo KS-7\u0026rsquo;s genome encodes three tRNA genes (tRNA\u003csup\u003eTyr\u003c/sup\u003e, tRNA\u003csup\u003ePro\u003c/sup\u003e, and tRNA\u003csup\u003eAsn\u003c/sup\u003e). These likely enhance translational efficiency by supplementing the host\u0026rsquo;s tRNA pool, particularly when specific tRNAs are in short supply during infection.\u003c/p\u003e\n\u003cp\u003e2.4. Therapeutic Potential and Genetic Safety Considerations\u003c/p\u003e\n\u003cp\u003eGenomic analysis indicates that Halo KS-7 is exclusively lytic and lacks lysogenic properties, as well as genes associated with antibiotic resistance or virulence factors that make it an excellent candidate for therapeutic applications. Identifying key genes involved in cell wall degradation, adaptation to high-salinity environments, and enhanced bacteriophage efficacy underscores its potential to combat antibiotic-resistant infections. Some of the Halo KS-7 bacteriophage genes and their functional roles in therapeutic applications are shown in Table 1.\u003c/p\u003e\n\u003cp\u003eGenome analysis of Halo KS-7 revealed a putative toxin gene located between nucleotide positions 39,942 and 40,232, encoding a 96 amino acid protein (MAYQNNSAASRNNNTSSANAGEAPERNIGGYLNIGVRGRDGQVRRLGQGGRGIALREDHAVEGKVLEFLRAQGIEALDEHLVITFGDAKALENFEL). The amino acid sequence derived from the gene exhibited similarity to a toxin found in \u003cem\u003eKlebsiella\u003c/em\u003e, as indicated by BLASTp analysis. However, the toxin could not be categorized into a specific family. Structural analysis using InterProScan identified a colicin-like motif within the protein sequence, which is typically associated with pore-forming activity and bactericidal effects.\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eIn this study, we identified Halo KS-7 as a novel halophilic bacteriophage infecting \u003cem\u003eK. pneumoniae\u003c/em\u003e, with several characteristics distinguishing it from previously described bacteriophages. Notably, it\u0026rsquo;s strictly lytic nature and halotolerance provide advantages in therapeutic contexts, especially in environments where high salt concentrations may otherwise inhibit other bacteriophage activities. The bacteriophage\u0026rsquo;s genome encodes various auxiliary enzymes, including holins and spanins, which may enhance its ability to lyse bacterial cells efficiently.\u003c/p\u003e \u003cp\u003eAnother notable feature is the presence of an auxiliary metabolic gene, MazG, encoded by gene gp_39. Auxiliary metabolic genes are typically of host origin and are co-opted by bacteriophages to manipulate host metabolic pathways in ways that support viral replication. MazG genes have been predominantly identified in marine bacteriophages and are associated with lytic and temperate lifestyles (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). They are thought to facilitate bacteriophage propagation in nutrient-limited environments by modulating host nucleotide metabolism, thus aiding replication in starved cells (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Given this context, the presence of MazG in Halo KS-7 suggests it may possess the capacity to alter host metabolism and potentially interfere with host bacteriophage defense systems. However, further investigation is required to elucidate the specific mechanisms involved in Halo KS-7 's interaction with its host.\u003c/p\u003e \u003cp\u003eA toxin-like gene in the lytic Halo KS-7 bacteriophage has significant implications for both its biological function and potential therapeutic applications. Unlike temperate bacteriophages, which can integrate their genomes into host bacteria and enter a lysogenic state, Halo KS-7's strictly lytic nature ensures that it does not undergo lysogenic conversion or genome integration. This characteristic significantly reduces the risks typically associated with temperate bacteriophages, such as the potential HGT of toxin genes, which could raise biosafety concerns.\u003c/p\u003e \u003cp\u003eRecent studies challenge the long-held assumption that strictly lytic bacteriophages operate in predictable and straightforward ways, especially in vivo. Evidence suggests that even these bacteriophages may exhibit unexpected behaviors, such as host-switching or direct interaction with mammalian immune cells, which could have critical implications for bacteriophage therapy (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Moreover, some lytic bacteriophages appear capable of mediating HGT, including the mobilization of virulence factors, raising potential safety concerns. Bacteriophage capsids can also elicit immune responses, and the limited understanding of bacteriophage pharmacodynamics in mammalian systems adds further complexity to their therapeutic use (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA compelling example is the crAss001 bacteriophage, classified as lytic but capable of long-term coexistence with its Bacteroides host in liquid culture without eliminating the bacterial population (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). This observation aligns with the concept of pseudolysogeny or carrier state, where a stable equilibrium is maintained despite the absence of lysogeny genes. The persistence of crAss-like bacteriophages in the gut, even after fecal microbiota transplantation, supports this view. It has been proposed that such stability might involve cryptic mechanisms, possibly through host-encoded transposons or quorum sensing pathways, allowing bacteriophages to persist without classical integration machinery (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurther supporting this idea is φPDS1, a lytic bacteriophage infecting \u003cem\u003eParabacteroides distasonis\u003c/em\u003e, which demonstrates efficient replication and plaque formation but fails to clear its host in liquid cultures. Over time, a significant rise in resistant bacterial subpopulations (~\u0026thinsp;22% to ~\u0026thinsp;95%) was observed, likely due to phase-variable expression of surface receptors. This results in a heterogeneous bacterial population, with some cells being permissive and others resistant, enabling prolonged bacteriophage-host coexistence (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) Such findings emphasize that bacteriophage persistence may not always require lysogeny but can instead result from selective pressures and population-level adaptations.\u003c/p\u003e \u003cp\u003eInterestingly, despite extensive research, no known bacteriophage has successfully eradicated its host population in natural settings. This suggests that stable coexistence, rather than eradication, might be a more evolutionarily favored strategy (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Some bacteriophages, like VP882, can even sense host population density via quorum sensing systems and modulate their lytic behavior accordingly, indicating a higher level of environmental responsiveness than previously assumed (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFinally, interactions between bacteriophages, bacteria, and the mammalian host extend far beyond simple lysis. In murine models, immune-mediated inflammation has been shown to induce probacteriophage activation, exacerbating disease through enhanced lysogenic conversion. In contrast, host vaccination reduced this bacteriophage-induced pathogenicity without impacting bacterial load (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). These findings highlight that bacteriophages can shape host immunity and disease outcomes, whether lytic or temperat,e reinforcing the need to account for bacteriophage\u0026ndash;host\u0026ndash;immune system dynamics in therapeutic and ecological contexts.\u003c/p\u003e \u003cp\u003eTogether, these observations underscore the complexity of bacteriophage biology in vivo. Even bacteriophages traditionally considered lytic may engage in cryptic persistence, population-level balancing, or immune modulation, complicating their application in clinical settings and challenging our conventional dichotomy of lytic versus temperate lifestyles.\u003c/p\u003e \u003cp\u003eThe toxin-like gene in Halo KS-7 likely serves a beneficial role in enhancing the bacteriophage's ability to infect and lyse bacterial hosts. This toxin is proposed to function similarly to bacteriocins, antimicrobial proteins that disrupt bacterial membranes or interfere with vital intracellular processes. The identified colicin-like motif suggests that the toxin operates similarly to a bacteriocin, a class of proteins known for pore-forming and bactericidal activities. Specifically, the toxin may facilitate the breakdown of the host cell membrane or inhibit cellular functions, increasing infection efficiency. This feature likely complements the bacteriophage\u0026rsquo;s primary lytic enzymes, such as holins, which are responsible for breaking down the bacterial cell wall. Together, these components may enhance the bacteriophage\u0026rsquo;s capacity to rapidly kill target bacteria by disrupting the membrane and attacking the bacterial cell from multiple angles.\u003c/p\u003e \u003cp\u003eMoreover, this toxin gene reflects an evolutionary refinement in bacteriophage design, favoring more precise and efficient bacterial killing mechanisms. Incorporating this auxiliary lysis gene could enhance the bacteriophage's therapeutic efficacy, particularly in targeting \u003cem\u003eK. pneumoniae\u003c/em\u003e, without the associated risks of gene transfer to non-pathogenic microorganisms. In this way, Halo KS-7 may be a bacteriophage therapy tool optimized for selective bacterial eradication while safeguarding the microbiome.\u003c/p\u003e \u003cp\u003eThe toxin-like gene could provide Halo KS-7 a competitive advantage by enhancing its ability to outcompete other microorganisms in the same ecological niche. Since Halo KS-7 cannot undergo lysogenic conversion, this toxin gene may be a critical survival mechanism, particularly in environments where rapid bacterial turnover or competition with other microbial species is common.\u003c/p\u003e \u003cp\u003eOverall, the toxin-like gene in Halo KS-7 appears to offer notable advantages, enhancing its ability to infect and lyse bacterial hosts. However, potential limitations or undesirable effects may arise, particularly if the toxin\u0026rsquo;s action is not fully controlled in therapeutic applications. Given Halo KS-7's strong genetic and functional capabilities, any limitations associated with the toxin gene could be effectively addressed using precise gene-editing techniques. Approaches such as CRISPR-Cas or recombineering could remove the toxin gene while preserving the bacteriophage\u0026rsquo;s lytic activity and antibacterial efficacy. These modifications would enable the safe and effective clinical use of Halo KS-7 as a therapeutic agent for treating MDR bacterial infections.\u003c/p\u003e \u003cp\u003eOur findings accord with and extend recent research on \u003cem\u003eKlebsiella\u003c/em\u003e bacteriophages. For instance, Peng et al. (2025) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) isolated a lytic bacteriophage vB_Kp_XP4 that targets hypervirulent K1 \u003cem\u003eK. pneumoniae\u003c/em\u003e, noting its potent activity and absence of undesirable genes \u0026ndash; similar to Halo KS-7 in its strictly lytic nature. Mirza et al. (2025) (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) characterized bacteriophage vbKpUKJ_2 from hospital sewage, which showed a broad host range (infecting\u0026thinsp;~\u0026thinsp;43% of tested \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates) and high thermal/pH stability, aligning with Halo KS-7\u0026rsquo;s robustness. Unlike vbKpUKJ_2 (a Drexlerviridae siphovirus with ~\u0026thinsp;45 kb genome) (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), Halo KS-7 is a myovirus with a slightly larger genome (58.716 kb) and likely a different evolutionary origin. The Halo KS-7 phage demonstrated attributes relevant to clinical therapy and food safety applications. Similar findings were reported by Chen et al. (2023) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), who characterized bacteriophage vB_KpP_HS106 against \u003cem\u003eK. pneumoniae\u003c/em\u003e K2, showing stability across pH 4\u0026ndash;12 and temperatures from 4\u0026ndash;50\u0026deg;C, comparable to Halo KS-7\u0026rsquo;s stability profile. While vB_KpP_HS106 was primarily studied for food safety purposes, Halo KS-7\u0026rsquo;s characteristics suggest the potential for broader applications, including clinical therapy and food safety. A distinctive aspect of Halo KS-7 is its halophilic adaptation. Bacteriophages requiring or tolerating high salt are more commonly associated with halophilic bacterial hosts (e.g., \u003cem\u003eHalomonas\u003c/em\u003e, \u003cem\u003eVibrio\u003c/em\u003e in marine settings) than \u003cem\u003eKlebsiella\u003c/em\u003e. Halo KS-7\u0026rsquo;s survival in up to 10\u0026ndash;15% NaCl without losing much activity is remarkable; by comparison, most enteric bacteriophages are significantly inactivated beyond ~\u0026thinsp;5% NaCl due to osmotic damage to the capsid. This property might translate into advantages for therapeutic formulation. For example, bacteriophage preparations often contain stabilizers like salts or sugars. Halo KS-7 might remain potent in the broader range of formulations or specific infection sites (such as hypertonic cystic fibrosis sputum). While halotolerance is not a typical requirement for bacteriophage therapy, it illustrates the diverse stress tolerances bacteriophages can evolve, which might correlate with other traits like desiccation resistance or shelf stability.\u003c/p\u003e \u003cp\u003eThe bacteriophage genome encodes several auxiliary enzymes that enhance infectivity and offer promising therapeutic applications. Among them, the holin gene is critical in orchestrating bacterial lysis. Holins form-controlled pores in the bacterial inner membrane, allowing them to reach the peptidoglycan layer and initiate cell wall degradation. This precise timing and mechanism significantly improve lysis efficiency and accelerate the bacteriophage replication cycle, which is advantageous in therapeutic settings (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Rz-like process further supports the lytic spanin, facilitating the final step of bacterial cell lysis by disrupting the outer membrane. Complete membrane disruption ensures full bacterial collapse, minimizing the survival of residual or partially lysed cells, which is a key factor in effective infection clearance (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdditionally, the bacteriophage encodes a MazG-like pyrophosphatase, an enzyme known to interfere with bacterial stress responses. By hydrolyzing nucleotide alarmones such as (p)ppGpp, this enzyme disrupts the stringent response which is a defense mechanism activated under nutrient limitation or antibiotic exposure. This interference may sensitize bacteria to bacteriophage attack and reduce the formation of persister cells, thereby enhancing the overall efficacy of bacteriophage therapy (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe presence of a bacteriophage-encoded RNA polymerase further underscores its adaptability. This enzyme allows the transcription of bacteriophage genes independently of the host\u0026rsquo;s transcriptional machinery, which is advantageous when the host is under metabolic stress or suppresses foreign gene expression (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLastly, the ribonucleotide reductase gene converts ribonucleotides into deoxyribonucleotides, ensuring a sufficient supply of DNA precursors for viral replication. This is especially beneficial in nutrient-limited environments such as biofilms or infected tissues, where nucleotide availability may be restricted (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTogether, these enzymes constitute a strategic genetic arsenal that enhances bacteriophage replication, promotes efficient host cell lysis, and improves resilience under stress. Their presence suggests strong potential for therapeutic use, especially in targeting persistent or MDR bacterial infections.\u003c/p\u003e \u003cp\u003eIntriguingly, the Halo KS-7 genome harbors three tRNA (tRNA\u003csup\u003eTyr\u003c/sup\u003e, tRNA\u003csup\u003ePro\u003c/sup\u003e, and tRNA\u003csup\u003eAsn\u003c/sup\u003e) genes, suggesting an adaptive strategy to optimize translational efficiency when host tRNA pools are limiting. The bacteriophage can sustain high-level protein synthesis during infection by supplying its tRNAs, promoting rapid replication and potent lytic activity. Although minimal-genome approaches often eschew accessory elements, recent work shows that standalone tRNA genes pose negligible biosafety risks when unlinked to lysogeny or mobile elements. Because Halo KS-7 lacks integrase and follows a strictly lytic lifecycle, these tRNAs likely represent evolutionary fine-tuning for efficient host takeover rather than a hazard. Such features underscore the therapeutic potential of Halo KS-7 against CRKP, especially in challenging clinical contexts.\u003c/p\u003e \u003cp\u003eUsing an AI-driven pipeline was instrumental in parsing Halo KS-7\u0026rsquo;s genome. Bacteriophage genomes typically have a high percentage of ORFs (genes with no known homologs), and Halo KS-7 was no exception (~\u0026thinsp;35% hypothetical proteins). By leveraging a protein language model and large training datasets, we could predict likely functions for some previously uncharacterized ORFs, adding to the functional annotation. For example, the AI pipeline suggested that one small hypothetical protein had features of a DNA-binding transcriptional activator, which was confirmed by finding a helix-turn-helix motif. This demonstrates how modern AI tools can complement traditional BLAST-based annotation, a point echoed in recent reviews (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). While AI annotations must be validated (to avoid misassignments), the approach accelerated our understanding of Halo KS-7\u0026rsquo;s genetic makeup. As AI models are trained on more bacteriophage data, their accuracy for function prediction will improve, potentially unveiling new anti-bacterial proteins encoded by bacteriophages. In Halo KS-7, beyond known categories, the function of ~\u0026thinsp;27 ORFs remains unknown \u0026ndash; these could include novel anti-host factors or structural proteins that warrant further study, possibly through proteomics or structural biology.\u003c/p\u003e \u003cp\u003eHalo KS-7 meets several key criteria for a therapeutic bacteriophage candidate. First, it is strictly lytic and lacks genes for lysogeny, minimizing safety concerns (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Second, it has a relatively broad action against diverse \u003cem\u003eK. pneumoniae\u003c/em\u003e strains, including MDR and hypervirulent types, which is essential given the genetic diversity of \u003cem\u003eK. pneumoniae\u003c/em\u003e clinical isolates (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Third, the bacteriophage genome encodes several enzymes such as holins, spanins, MazG-like pyrophosphatase, RNA polymerase, and ribonucleotide reductase that collectively enhance its ability to infect and lyse bacterial cells. These enzymes enable efficient membrane disruption, suppress bacterial stress responses, and replication of bacteriophage under nutrient-limited or stressful conditions (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Their presence suggests the bacteriophage is well-suited for therapeutic applications, potentially maintaining stability and activity across various infection environments and delivery routes such as inhalation or topical use.\u003c/p\u003e \u003cp\u003eFourth, its stability in a range of conditions implies it could be formulated for various routes of administration (inhalation for pneumonia, topical for wound infections, etc.) without rapid inactivation.\u003c/p\u003e \u003cp\u003eRecent \u003cem\u003ein vivo\u003c/em\u003e studies give optimism that bacteriophages like Halo KS-7 could be effective. For example, Gan et al. (2022) (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e) demonstrated that two lytic bacteriophages could rescue mice from fatal \u003cem\u003eK. pneumoniae\u003c/em\u003e pneumonia, significantly reducing bacterial load and inflammation. Similarly, Duarte et al. (2022) (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) reported successful compassionate use of bacteriophages in a human patient with recurrent \u003cem\u003eKlebsiella\u003c/em\u003e urinary infection. These cases, alongside rigorous laboratory evaluations, suggest that bacteriophages are moving from bench to bedside for \u003cem\u003eK. pneumoniae\u003c/em\u003e. Halo KS-7\u0026rsquo;s broad host range would be advantageous for treating infections where the specific capsular type is unknown or mixed \u003cem\u003eKlebsiella\u003c/em\u003e populations are present. In a practical scenario, Halo KS-7 could be used as part of a bacteriophage cocktail to preempt resistance \u0026ndash; combining it with other bacteriophages targeting complementary receptors. Indeed, bacteriophage cocktails have broadened the overall killing spectrum and delayed the emergence of bacteriophage-resistant mutants (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile this study provides a comprehensive analysis of Halo KS-7, there are some limitations. We did not perform \u003cem\u003ein vivo\u003c/em\u003e experiments, so the bacteriophage\u0026rsquo;s efficacy and immunogenicity in animal infection models remain to be tested. Given its promising \u003cem\u003ein vitro\u003c/em\u003e profile, testing Halo KS-7 in a mouse pneumonia model, similar to Gan et al. and Li et al., (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e) would be a logical step to confirm therapeutic potential and safety (e.g., absence of immunopathology).\u003c/p\u003e \u003cp\u003eFrom a genomic standpoint, Halo KS-7 underscores how much bacteriophage genetic diversity remains undiscovered. The AI pipeline flagged a number of unique ORFs; characterizing these (via knockout mutagenesis or structural studies) could reveal novel functions. One speculative but intriguing ORF encodes a small protein of 90 amino acids with a repeated motif; such proteins sometimes act as inhibitors of host processes (e.g., protease inhibitors or nucleotide sequestration proteins). Uncovering any anti-host mechanisms could augment our understanding of how bacteriophages subvert \u003cem\u003eKlebsiella\u003c/em\u003e during infection. Furthermore, exploring the relationship of Halo KS-7 with the rare bacteriophages it is related to (like the \u0026ldquo;vB_KpnM_Saline\u0026rdquo;) could help define a new bacteriophage genus. Given the mosaic nature of bacteriophage genomes, the presence or absence of specific functional modules such as lysis-related or host takeover genes, shows the importance of analyzing bacteriophage genes individually and in the context of the complete genome.\u003c/p\u003e \u003cp\u003eComprehensive characterization of Halo KS-7 reveals a potent lytic bacteriophage with distinctive halophilic adaptation and a broad host range. Isolated from a clinical setting and thoroughly examined through laboratory assays and an AI-assisted genomic annotation pipeline, Halo KS-7 represents a novel addition to the growing repertoire of bacteriophages with translational promise. Genomic analysis confirmed the absence of antibioticresistance, integrase, or recombinase gene, underscoring its strictly lytic lifecycle and lack of lysogenic potential. Intriguingly, the genome also harbors three tRNA genes (tRNA\u003csup\u003eTyr\u003c/sup\u003e, tRNA\u003csup\u003ePro\u003c/sup\u003e, and tRNA\u003csup\u003eAsn\u003c/sup\u003e), which likely optimize translational efficiency when host tRNA pools are limited, sustaining rapid protein synthesis and enhancing lytic activity. The presence of a toxin gene and auxiliary factors such as MazG, like pyrophosphatase and HNH endonucleases, further suggest built-in enhancements to bacterial killing without promoting HGT or antimicrobial resistance (AMR), reinforcing its therapeutic safety. Finally, the application of AI in genomic annotation was instrumental in rapidly distinguishing both beneficial and undesirable elements, showcasing the power of computational tools in modern bacteriophage research.\u003c/p\u003e \u003cp\u003eAs AMR continues to escalate, bacteriophages like Halo KS-7 offer a timely and targeted alternative for infection control. Future directions will focus on assessing its efficacy in vivo infection models, exploring formulation strategies, and evaluating its role within bacteriophage cocktails or as a source of recombinant enzymes. Incorporating such bacteriophages into therapeutic pipelines and clinical trials will be crucial to realizing their clinical impact. Our findings contribute to the broader understanding of bacteriophage diversity and function, laying the groundwork for further development of Halo KS-7 in combating critical \u003cem\u003eK. pneumoniae\u003c/em\u003e infections.\u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Bacteriophage Isolation and Host Bacteria\u003c/h2\u003e \u003cp\u003eHalo KS-7 was isolated from wastewater at Shahriar Hospital in Tehran, Iran. The sewage sample was centrifugated (10,000 \u0026times; g, 10 min) and filtered through a 0.22 \u0026micro;m membrane. The host bacterium, \u003cem\u003eK. pneumoniae\u003c/em\u003e, was cultured in Luria-Bertani (LB) agar at 37˚C until the logarithmic growth phase was reached. The wastewater sample (50 mL) was enriched with an equal volume of exponential-phase \u003cem\u003eK. pneumoniae\u003c/em\u003e culture and incubated at 37\u0026deg;C for 24 hours with gentle agitation. The host \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates were carbapenem-resistant clinical strains previously isolated from diabetic foot ulcers of hospital patients.\u003c/p\u003e \u003cp\u003eThe resulting bacteriophage lysate underwent purification via three successive cycles of single-plaque selection and co-culturing. Bacteriophage isolation was verified using the double-layer agar (DLA) method (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). The plate was incubated at 37˚C for 24 h, and clear plaques indicated a successful bacteriophage infection. Bacteriophage titration involved tenfold serial dilutions in SM buffer (100 mM NaCl, 8 mM MgSO4, 50 mM Tris (pH\u0026thinsp;=\u0026thinsp;7.5), and 0.002% gelatin (w/v)). Bacteriophage titers were determined by the double agar overlay plaque assay and reported as plaque-forming units per milliliter (PFU/mL) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Morphological Characterization by Transmission Electron Microscopy (TEM)\u003c/h2\u003e \u003cp\u003eFollowing standard protocols, isolated bacteriophage particles were analyzed using transmission electron microscopy (TEM) (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Briefly, 10 \u0026micro;L of purified bacteriophage suspension was applied to a carbon-coated copper grid and allowed to adsorb for 3\u0026ndash;5 minutes. The sample was then negatively stained with 1% (w/v) uranyl acetate (pH\u0026thinsp;=\u0026thinsp;7). Imaging was performed using a Zeiss LEO 906 TEM (Carl Zeiss LEO EM 906 E, Germany) operating at an accelerating voltage of 100 kV (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Host Range Determination\u003c/h2\u003e \u003cp\u003eThe host range of Halo KS-7 was evaluated on a panel of bacterial strains to assess its lytic spectrum. This panel included 30 CRKP strains (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Host range spot tests were conducted by spotting 10 \u0026micro;L of high-titer bacteriophage (~\u0026thinsp;10^9 PFU/mL) onto lawns of each bacterial strain on appropriate agar. Plates were incubated overnight at 37\u0026deg;C, and lysis was assessed qualitatively based on plaque morphology. Transparent or opaque plaques indicated susceptibility to Halo KS-7, reflecting lytic activity. In contrast, the absence of plaques or only faint clearing suggested resistance to the bacteriophage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Physical and Stability Tests\u003c/h2\u003e \u003cp\u003eThe stability of Halo KS-7 was evaluated under a range of conditions representing physiological and extreme environments. To assess thermal stability, bacteriophage lysates (~\u0026thinsp;10⁸ PFU/mL in SM buffer) were incubated for 1 hour at (-20\u0026deg;C, 4\u0026deg;C, 37\u0026deg;C, 50\u0026deg;C, 60\u0026deg;C, and 70\u0026deg;C), followed by titration using plaque assays on LB agar (Wang et al., 2016). For pH stability, aliquots were adjusted to pH values (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, and \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) using SM buffer modified with HCl or NaOH and incubated at room temperature for 1 hour before titration. Halotolerance was assessed by incubating bacteriophage lysates in SM buffer supplemented with varying NaCl concentrations (5%, 10%, and 15%) at 25\u0026deg;C for 24 hours, followed by titer determination using the double agar layer (DLA) method (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). All experiments were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.5. Bacteriophage DNA Extraction and Genome Sequencing\u003c/h2\u003e \u003cp\u003eAccording to the manufacturer's instructions, the bacteriophage genome was extracted using a commercial DNA extraction kit (DNA Pure, FAVOR-GEN, Iran). Quality and purity of the extracted DNA were verified through optical density measurements, agarose gel electrophoresis, and a PCR assay using bacterial 16S rRNA primers to confirm the absence of bacterial genomic contamination (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAn Illumina library was constructed from 100 ng of purified Halo KS-7 DNA for whole-genome sequencing (WGS) using a Nextera XT Library Preparation Kit. Paired-end sequencing (2 \u0026times; 150 bp) was carried out on the Illumina HiSeq platform. Raw sequencing reads were subjected to quality assessment using FastQC software, followed by adapter removal and quality filtering with Trimmomatic. High-quality reads were de novo assembled using SPAdes v3.15.2 with the \"careful\" mode enabled to reduce mismatches and short indels. Assembly quality was evaluated, resulting in a single contiguous bacteriophage genome. Any remaining gaps or ambiguous regions were resolved via Sanger sequencing of PCR-amplified fragments. The finalized genome was confirmed to be a linear double-stranded DNA molecule with defined termini, as determined by read coverage analysis and identification of putative terminal repeats (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.6. Bioinformatics Analysis\u003c/h2\u003e \u003cp\u003eThis study used two complementary approaches to analyze bacteriophage sequencing data. The first approach utilized an AI-enabled pipeline for viral detection and validation from next-generation sequencing data (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). This pipeline integrates advanced bioinformatics tools with AI to efficiently identify viral sequences and de novo genome assembly, offering a comprehensive view of bacteriophage genetic diversity. The second approach involved using CLC Genomics Workbench 22, a widely adopted platform that supports read processing, mapping, variant calling, and genome assembly through an intuitive graphical interface and robust analytical capabilities.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e4.6.1. AI-Driven Genome Annotation Pipeline\u003c/h2\u003e \u003cp\u003eIn this study, the WGS data of bacteriophage Halo KS-7 was analyzed using a bioinformatics pipeline based on methods described by Ghorbani et al. (2024) (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Raw sequencing reads were initially assessed for quality using FastQC, then trimming with Trimmomatic to remove low-quality bases and adapter sequences. High-quality reads were then assembled de novo using SPAdes, generating a complete genome sequence. The assembly was refined and validated through multiple alignment checks and coverage assessments.\u003c/p\u003e \u003cp\u003eGenome annotation was performed using Prokka, which identified open reading frames (ORFs), tRNAs, and other genomic elements. Functional annotation of predicted ORFs was conducted through comparative analysis against NCBI nr, Pfam, and InterProScan databases. Comparative genomic tools, including Blastn and VICTOR, were employed to evaluate taxonomic classification and genomic similarity to other bacteriophages. Conserved domains, virulence factors, and potential antibiotic resistance genes were screened using PHASTER and the Comprehensive Antibiotic Resistance Database.\u003c/p\u003e \u003cp\u003eAll computational analyses were executed using Python scripts provided in the referenced study, ensuring reproducibility and alignment with established methodologies. This automated workflow streamlined the read processing, genome assembly, annotation, and functional characterization processes. Overall, the results offered in-depth insights into the genetic architecture, structural features, and evolutionary lineage of Halo KS-7, confirming its potential applications (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e4.6.2. Analysis Using CLC Genomics Workbench 22\u003c/h2\u003e \u003cp\u003eSequencing reads were trimmed using CLC Genomics Workbench 22 (QIAGEN) with standard parameters to remove adapter sequences, ambiguous nucleotides, and low-quality bases. Specifically, bases with quality scores\u0026thinsp;\u0026le;\u0026thinsp;5, reads shorter than 15 nucleotides and reads containing more than two ambiguous nucleotides were removed (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). The cleaned reads were assembled de novo using default parameters (word size: 15 nt).\u003c/p\u003e \u003cp\u003eAssembled contigs were subjected to nucleotide BLAST analysis using Geneious version 22 (Biomatters, New Zealand). The contig with the highest similarity to viral sequences in the GenBank database was selected for further study to identify putative bacteriophages. Confirmation was performed using the VirusDetect tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://virusdetect.feilab.net/cgi-bin/virusdetect/vdo_home.cgi\u003c/span\u003e\u003cspan address=\"http://virusdetect.feilab.net/cgi-bin/virusdetect/vdo_home.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, and results were independently validated by mapping cleaned reads back to the viral genome identified in the BLAST results.\u003c/p\u003e \u003cp\u003eGenome annotation was carried out using the CLC Microbial Genomics Module, leveraging a reference genome and integrated BLAST tools. Annotation parameters included a default similarity threshold of 95% and an E-value cutoff of 0.0001 (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). The finalized genome sequence was then aligned with selected bacteriophage genomes from the NCBI database using the Whole Genome Alignment plugin in CLC Genomics Workbench (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e), providing insights into phylogenetic relationships and genomic conservation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSA:\u003c/strong\u003e conceptualization, data curation, formal analysis, investigation, methodology, software, visualization, writing \u0026ndash; original draft, writing \u0026ndash; review \u0026amp; editing, validation, project administration.\u0026nbsp;\u003cstrong\u003eMB:\u003c/strong\u003e conceptualization, formal analysis, validation, software, data curation, writing \u0026ndash; original draft, writing\u0026ndash;review \u0026amp; editing, visualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSS:\u003c/strong\u003e conceptualization, visualization, investigation, data curation, writing \u0026ndash; original draft.\u0026nbsp;\u003cstrong\u003eMSH:\u003c/strong\u003e conceptualization, methodology, project administration, supervision, validation, writing \u0026ndash; review and editing. \u003cstrong\u003ePS:\u003c/strong\u003e validation, writing \u0026ndash; review \u0026amp; editing. \u0026nbsp; \u003cstrong\u003eMP:\u003c/strong\u003e\u0026nbsp; visualization, investigation. \u003cstrong\u003eAHS:\u003c/strong\u003e validation, writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available in the \u003cstrong\u003eGenBank\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003erepository (accession number PV02655, https://www.ncbi.nlm.nih.gov/search/all/?term=PV026551).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interests exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted without any commercial or financial relationships that could potentially create a conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Considerations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe collection of wastewater samples for this study was conducted in accordance with the ethical guidelines set by the Pasteur Institute of Iran under the ethical code IR.PII.AEC.1403.008. Permission was obtained for the collection of wastewater samples by Shahriar Hospital, with no identifiable patient data collected during the research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYuan Y, Wang J, Yao Z, Ma B, Li Y, Yan W, et al. Risk Factors for Carbapenem-Resistant Klebsiella pneumoniae Bloodstream Infections and Outcomes. Infect Drug Resist. 2020;13:207-15.\u003c/li\u003e\n\u003cli\u003eBeig M, Aghamohammad S, Majidzadeh N, Asforooshani MK, Rezaie N, Abed S, et al. Antibiotic resistance rates in hypervirulent Klebsiella pneumoniae strains: A systematic review and meta-analysis. J Glob Antimicrob Resist. 2024;38:376-88.\u003c/li\u003e\n\u003cli\u003eBeig M, Arabestani MR. Investigation of MexAB-OprM efflux pump gene expression in clinical isolates of pseudomonas aeruginosa isolated from Intensive Care Unit. Iranian Journal of Medical Microbiology. 2019;13(2):142-50.\u003c/li\u003e\n\u003cli\u003eMohammadpour D, Memar MY, Leylabadlo HE, Ghotaslou A, Ghotaslou R. Carbapenem-Resistant Klebsiella pneumoniae: A comprehensive review of phenotypic and genotypic methods for detection. The Microbe. 2025;6:100246.\u003c/li\u003e\n\u003cli\u003eSadeqi S, Michniewski S, Nikkhahi F, Jameson E, Marashi SMA. Isolation of new Klebsiella pneumoniae phage PSKP16. Iran J Microbiol. 2023;15(1):62-8.\u003c/li\u003e\n\u003cli\u003eChinemerem Nwobodo D, Ugwu MC, Oliseloke Anie C, Al-Ouqaili MTS, Chinedu Ikem J, Victor Chigozie U, et al. Antibiotic resistance: The challenges and some emerging strategies for tackling a global menace. J Clin Lab Anal. 2022;36(9):e24655.\u003c/li\u003e\n\u003cli\u003eSadeqi S, Hashemi Shahraki A, Nikkhahi F, Javadi A, Amin Marashi SM. Application of bacteriophage cocktails for reducing the bacterial load of nosocomial pathogens in hospital wastewater. Iran J Microbiol. 2022;14(3):395-401.\u003c/li\u003e\n\u003cli\u003eGoodarzi F, Hallajzadeh M, Sholeh M, Talebi M, Mahabadi VP, Amirmozafari N. Biological characteristics and anti-biofilm activity of a lytic phage against vancomycin-resistant Enterococcus faecium. Iran J Microbiol. 2021;13(5):691-702.\u003c/li\u003e\n\u003cli\u003eGoodarzi F, Hallajzadeh M, Sholeh M, Talebi M, Pirhajati Mahabadi V, Amirmozafari N. Anti-biofilm Activity of a Lytic Phage Against Vancomycin-Resistant Enterococcus faecalis. Iran J Pathol. 2022;17(3):285-93.\u003c/li\u003e\n\u003cli\u003eZurabov F, Glazunov E, Kochetova T, Uskevich V, Popova V. Bacteriophages with depolymerase activity in the control of antibiotic resistant Klebsiella pneumoniae biofilms. Sci Rep. 2023;13(1):15188.\u003c/li\u003e\n\u003cli\u003eMohammadi M, Mehrisadat M, Masoumeh B, Somaieh N, Mina Y, and Monfared FN. The Role of Nanotechnology in Overcoming Barriers to Phage Therapy: an Overview. Future Virology. 2023;18(6):387-98.\u003c/li\u003e\n\u003cli\u003eChen C, Tao Z, Li T, Chen H, Zhao Y, Sun X. Isolation and characterization of novel bacteriophage vB_KpP_HS106 for Klebsiella pneumonia K2 and applications in foods. Front Microbiol. 2023;14:1227147.\u003c/li\u003e\n\u003cli\u003ePeng X, Chang J, Zhang H, Li X, Zhang C, Jiao S, et al. Isolation, characterization, and genomic analysis of a novel bacteriophage vB_Kp_XP4 targeting hypervirulent and multidrug-resistant Klebsiella pneumoniae. Front Microbiol. 2025;16:1491961.\u003c/li\u003e\n\u003cli\u003eChen H, Liu H, Gong Y, Dunstan RA, Ma Z, Zhou C, et al. A Klebsiella-phage cocktail to broaden the host range and delay bacteriophage resistance both in vitro and in vivo. NPJ Biofilms Microbiomes. 2024;10(1):127.\u003c/li\u003e\n\u003cli\u003eManuel RD, Snyder JC. The Expanding Diversity of Viruses from Extreme Environments. Int J Mol Sci. 2024;25(6).\u003c/li\u003e\n\u003cli\u003eMotlagh AM, Bhattacharjee AS, Coutinho FH, Dutilh BE, Casjens SR, Goel RK. Insights of Phage-Host Interaction in Hypersaline Ecosystem through Metagenomics Analyses. Front Microbiol. 2017;8:352.\u003c/li\u003e\n\u003cli\u003eFlamholz ZN, Li C, Kelly L. Improving viral annotation with artificial intelligence. mBio. 2024;15(10):e0320623.\u003c/li\u003e\n\u003cli\u003eHo P, Chen Y, Biswas S, Canfield E, Abdolvahabi A, Feldman DE. Bacteriophage antidefense genes that neutralize TIR and STING immune responses. Cell Rep. 2023;42(4):112305.\u003c/li\u003e\n\u003cli\u003eGholizadeh O, Ghaleh HEG, Tat M, Ranjbar R, Dorostkar R. The potential use of bacteriophages as antibacterial agents against Klebsiella pneumoniae. Virol J. 2024;21(1):191.\u003c/li\u003e\n\u003cli\u003eChen Q, Zhang F, Bai J, Che Q, Xiang L, Zhang Z, et al. Bacteriophage-resistant carbapenem-resistant Klebsiella pneumoniae shows reduced antibiotic resistance and virulence. Int J Antimicrob Agents. 2024;64(2):107221.\u003c/li\u003e\n\u003cli\u003eWang Q, Chen R, Liu H, Liu Y, Li J, Wang Y, et al. Isolation and characterization of lytic bacteriophage vB_KpnP_23: A promising antimicrobial candidate against carbapenem-resistant Klebsiella pneumoniae. Virus Res. 2024;350:199473.\u003c/li\u003e\n\u003cli\u003eParmar KM, Gaikwad SL, Dhakephalkar PK, Kothari R, Singh RP. Intriguing Interaction of Bacteriophage-Host Association: An Understanding in the Era of Omics. Front Microbiol. 2017;8:559.\u003c/li\u003e\n\u003cli\u003eNarayanan MP, Kumar A, Kumar Verma G, Bairwa A, Mirza AA, Goyal B. Efficacy of Bacteriophages in Wound Healing: An Updated Review. Cureus. 2024;16(10):e71542.\u003c/li\u003e\n\u003cli\u003eAbdi G, Jain M, Barwant M, Tendulkar R, Tendulkar M, Tariq M, et al. Unveiling the dynamic role of bioinformatics in automation for efficient and accurate data processing and interpretation. Advances in Bioinformatics: Springer; 2024. p. 279-319.\u003c/li\u003e\n\u003cli\u003eRost HL, Sachsenberg T, Aiche S, Bielow C, Weisser H, Aicheler F, et al. OpenMS: a flexible open-source software platform for mass spectrometry data analysis. Nat Methods. 2016;13(9):741-8.\u003c/li\u003e\n\u003cli\u003eSundaram J, Gowri K, Devaraju S, Gokuldev S, Jayaprakash S, Anandaram H, et al. An Exploration of Python Libraries in Machine Learning Models for Data Science. Advanced Interdisciplinary Applications of Machine Learning Python Libraries for Data Science: IGI Global; 2023. p. 1-31.\u003c/li\u003e\n\u003cli\u003eDjaffardjy M, Marchment G, Sebe C, Blanchet R, Bellajhame K, Gaignard A, et al. Developing and reusing bioinformatics data analysis pipelines using scientific workflow systems. Comput Struct Biotechnol J. 2023;21:2075-85.\u003c/li\u003e\n\u003cli\u003eZhai Z, Zhang Z, Zhao G, Liu X, Qin F, Zhao Y. Genomic Characterization of Two Novel RCA Phages Reveals New Insights into the Diversity and Evolution of Marine Viruses. Microbiol Spectr. 2021;9(2):e0123921.\u003c/li\u003e\n\u003cli\u003eCortes-Martin A, Denise R, Guerin E, Stockdale SR, Draper LA, Ross RP, et al. Isolation and characterization of a novel lytic Parabacteroides distasonis bacteriophage phiPDS1 from the human gut. Gut Microbes. 2024;16(1):2298254.\u003c/li\u003e\n\u003cli\u003eSutton TDS, Hill C. Gut Bacteriophage: Current Understanding and Challenges. Front Endocrinol (Lausanne). 2019;10:784.\u003c/li\u003e\n\u003cli\u003eShkoporov AN, Khokhlova EV, Fitzgerald CB, Stockdale SR, Draper LA, Ross RP, et al. PhiCrAss001 represents the most abundant bacteriophage family in the human gut and infects Bacteroides intestinalis. Nat Commun. 2018;9(1):4781.\u003c/li\u003e\n\u003cli\u003eGuerin E, Hill C. Shining Light on Human Gut Bacteriophages. Front Cell Infect Microbiol. 2020;10:481.\u003c/li\u003e\n\u003cli\u003eWahida A, Tang F, Barr JJ. Rethinking phage-bacteria-eukaryotic relationships and their influence on human health. Cell Host Microbe. 2021;29(5):681-8.\u003c/li\u003e\n\u003cli\u003eMirza KA, Tchatchiashvili T, Marquet M, Nietzsche S, Pletz MW, Makarewicz O. Characterization and genome analysis of novel Klebsiella pneumoniae phage vbKpUKJ_2 isolated from hospital sewage water. BMC Microbiol. 2025;25(1):96.\u003c/li\u003e\n\u003cli\u003eYoung R. Phage lysis: do we have the hole story yet? Curr Opin Microbiol. 2013;16(6):790-7.\u003c/li\u003e\n\u003cli\u003eSummer EJ, Berry J, Tran TA, Niu L, Struck DK, Young R. Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts. J Mol Biol. 2007;373(5):1098-112.\u003c/li\u003e\n\u003cli\u003eMiller ES, Kutter E, Mosig G, Arisaka F, Kunisawa T, Ruger W. Bacteriophage T4 genome. Microbiol Mol Biol Rev. 2003;67(1):86-156, table of contents.\u003c/li\u003e\n\u003cli\u003eCheng W, Chen Q, Ren Y, Zhang Y, Lu L, Gui L, et al. The identification of viral ribonucleotide reductase encoded by ORF23 and ORF141 genes and effect on CyHV-2 replication. Front Microbiol. 2023;14:1154840.\u003c/li\u003e\n\u003cli\u003eLobocka M, Dabrowska K, Gorski A. Engineered Bacteriophage Therapeutics: Rationale, Challenges and Future. BioDrugs. 2021;35(3):255-80.\u003c/li\u003e\n\u003cli\u003eWang I-N, Smith DL, Young R. Holins: The Protein Clocks of Bacteriophage Infections. Annual Review of Microbiology. 2000;54(Volume 54, 2000):799-825.\u003c/li\u003e\n\u003cli\u003eBerry J, Summer EJ, Struck DK, Young R. The final step in the phage infection cycle: the Rz and Rz1 lysis proteins link the inner and outer membranes. Mol Microbiol. 2008;70(2):341-51.\u003c/li\u003e\n\u003cli\u003eYang H, Ma Y, Wang Y, Yang H, Shen W, Chen X. Transcription regulation mechanisms of bacteriophages: recent advances and future prospects. Bioengineered. 2014;5(5):300-4.\u003c/li\u003e\n\u003cli\u003eGan L, Fu H, Tian Z, Cui J, Yan C, Xue G, et al. Bacteriophage Effectively Rescues Pneumonia Caused by Prevalent Multidrug-Resistant Klebsiella pneumoniae in the Early Stage. Microbiol Spectr. 2022;10(5):e0235822.\u003c/li\u003e\n\u003cli\u003eDuarte J, Maximo C, Costa P, Oliveira V, Gomes NCM, Romalde JL, et al. Potential of an Isolated Bacteriophage to Inactivate Klebsiella pneumoniae: Preliminary Studies to Control Urinary Tract Infections. Antibiotics (Basel). 2024;13(2):195.\u003c/li\u003e\n\u003cli\u003eLi P, Guo G, Zheng X, Xu S, Zhou Y, Qin X, et al. Therapeutic efficacy of a K5-specific phage and depolymerase against Klebsiella pneumoniae in a mouse model of infection. Vet Res. 2024;55(1):59.\u003c/li\u003e\n\u003cli\u003eZadeh RG, Zadeh MH, Sholeh M, Asgharzadeh S, Darbandi A, Fashami P. Bacteriophage and probiotic therapy in the treatment of Pseudomonas aeruginosa burn infection and their synergistic effect. Egypt J Exp Biol. 2023;19:21-9.\u003c/li\u003e\n\u003cli\u003eAlduhaidhawi AHM, AlHuchaimi SN, Al-Mayah TA, Al-Ouqaili MTS, Alkafaas SS, Muthupandian S, et al. Prevalence of CRISPR-Cas Systems and Their Possible Association with Antibiotic Resistance in Enterococcus faecalis and Enterococcus faecium Collected from Hospital Wastewater. Infect Drug Resist. 2022;15:1143-54.\u003c/li\u003e\n\u003cli\u003eAbed S, Sholeh M, Khazani Asforooshani M, Shafiei M, Hashemi Shahraki A, Nasr S. Insights into the novel Enterococcus faecalis phage: A comprehensive genome analysis. PLoS One. 2024;19(5):e0301292.\u003c/li\u003e\n\u003cli\u003eKhazani Asforooshani M, Elikaei A, Abed S, Shafiei M, Barzi SM, Solgi H, et al. A novel Enterococcus faecium phage EF-M80: unveiling the effects of hydrogel-encapsulated phage on wound infection healing. Front Microbiol. 2024;15:1416971.\u003c/li\u003e\n\u003cli\u003eYu P, Mathieu J, Li M, Dai Z, Alvarez PJ. Isolation of Polyvalent Bacteriophages by Sequential Multiple-Host Approaches. Appl Environ Microbiol. 2016;82(3):808-15.\u003c/li\u003e\n\u003cli\u003eKomijani M, Bouzari M, Rahimi F. Detection and characterization of a novel lytic bacteriophage (vB-KpneM-Isf48) against Klebsiella pneumoniae isolates from infected wounds carrying antibiotic-resistance genes (TEM, SHV, and CTX-M). 2017.\u003c/li\u003e\n\u003cli\u003eGygli SM, Keller PM, Ballif M, Blochliger N, Homke R, Reinhard M, et al. Whole-Genome Sequencing for Drug Resistance Profile Prediction in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2019;63(4):10.1128/aac. 02175-18.\u003c/li\u003e\n\u003cli\u003eBosworth A, Robson J, Lawrence B, Casey AL, Fair A, Khanam S, et al. Deployment of whole genome next-generation sequencing of SARS-CoV-2 in a military maritime setting. BMJ Mil Health. 2024;170(e2):e144-e9.\u003c/li\u003e\n\u003cli\u003eGhorbani A, Rostami M, Guzzi PH. AI-enabled pipeline for virus detection, validation, and SNP discovery from next-generation sequencing data. Front Genet. 2024;15:1492752.\u003c/li\u003e\n\u003cli\u003eCaporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 2012;6(8):1621-4.\u003c/li\u003e\n\u003cli\u003eMisu M, Yoshikawa T, Sugimoto S, Takamatsu Y, Kurosu T, Ouji Y, et al. Rapid whole genome sequencing methods for RNA viruses. Front Microbiol. 2023;14:1137086.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Key Functional Genes in Halo KS-7 Bacteriophage and Their Potential Roles Therapy.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"662\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene Name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFunction\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 234px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBenefit to Bacteriophage Therapy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHolin CDS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eCreates pores in the bacterial membrane, allowing endolysins to access the peptidoglycan.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 234px;\"\u003e\n \u003cp\u003eEnhances bacterial lysis efficiency, accelerating infection.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e(40)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRz-like spanin CDS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eFacilitates the final step of cell lysis by disrupting the outer membrane.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 234px;\"\u003e\n \u003cp\u003eEnsures complete bacterial lysis, preventing persistence.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e(41)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMazG-like pyrophosphatase CDS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eInhibits bacterial stress response by degrading nucleotide alarmones.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 234px;\"\u003e\n \u003cp\u003eReduces bacterial resistance mechanisms. Ensures survival of bacteriophages within the bacterial cell.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e(18)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRNA polymerase CDS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eTranscribes bacteriophage genes independently of host machinery.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 234px;\"\u003e\n \u003cp\u003eEnables efficient viral gene expression even in stressed hosts.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e(42)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eribonucleotide reductase CDS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 217px;\"\u003e\n \u003cp\u003eConverts ribonucleotides to deoxyribonucleotides for DNA synthesis.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 234px;\"\u003e\n \u003cp\u003eSupports rapid bacteriophage replication in nutrient-limited conditions.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e(38)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"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":"Carbapenem-resistant Klebsiella pneumoniae (CRKP), bacteriophage therapy, Halo KS-7, artificial intelligence","lastPublishedDoi":"10.21203/rs.3.rs-6534345/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6534345/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCarbapenem-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e(CRKP) is a multidrug-resistant (MDR) pathogen causing severe infections in immunocompromised patients, prompting the exploration of alternative therapies like bacteriophage therapy. In this study, we isolated and characterized a novel halophilic lytic bacteriophage, Halo KS-7, targeting \u003cem\u003eK. pneumoniae\u003c/em\u003e, and used an AI-driven annotation pipeline in Python to analyze its genome and therapeutic potential.\u003c/p\u003e\n\u003cp\u003eBacteriophages were isolated from Hospital wastewater, purified through plaque isolation, and confirmed using the double-layer agar method. Morphological analysis via transmission electron microscopy (TEM) and plaque assays assessed lytic activity. DNA sequencing was done using Illumina HiSeq 2000, followed by genome assembly, AI-guided annotation, gene prediction, protein function classification, and comparative genomics using CLC Genomics Workbench. We also evaluated host range, temperature stability, pH sensitivity, and salt stress tolerance to assess therapeutic potential.\u003c/p\u003e\n\u003cp\u003eHalo KS-7 exhibited strong lytic activity against CRKP and was classified as a Myoviridae bacteriophage by TEM. Phenotypic assays demonstrated optimal activity at 37 °C and neutral pH, effective activity from pH 4–10, and enhanced performance in high-salinity conditions. Its 58.716 kb linear dsDNA genome (44.4% G+C) contains 49 predicted ORFs, lacks integrase, lysogeny, or antibiotic-resistance genes, and includes three tRNA genes (tRNATyr, tRNAPro, and tRNAAsn). It also includes a toxin gene and auxiliary factors like MazG, pyrophosphatase, and HNH endonucleases that enhance bacterial killing without promoting horizontal gene transfer or resistance. Functional annotation assigned ~65% of ORFs to structural, replication, and packaging roles. Comparative genomics showed moderate similarity to other Myoviridae but with distinct accessory features, emphasizing its novelty and therapeutic value.\u003c/p\u003e\n\u003cp\u003eHalo KS-7 is a novel, strictly lytic bacteriophage with strong antibacterial activity and stress resilience, supporting its use as a promising biocontrol agent against CRKP and its potential for clinical development in managing MDR infections.\u003c/p\u003e","manuscriptTitle":"Genomic Insights into Halo KS-7: An AI-Driven Characterization of a Novel Halophilic Bacteriophage Targeting Carbapenem-Resistant Klebsiella pneumoniae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 09:51:39","doi":"10.21203/rs.3.rs-6534345/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":"faec220f-84a0-4b2b-b21b-2db405c97df8","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48262710,"name":"Biological sciences/Microbiology/Bacteria"},{"id":48262711,"name":"Biological sciences/Microbiology/Bacteriology"},{"id":48262712,"name":"Biological sciences/Microbiology/Phage biology"}],"tags":[],"updatedAt":"2025-06-16T09:24:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 09:51:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6534345","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6534345","identity":"rs-6534345","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}