Characterization and Genomic Analysis of a New Bacteriophage Klebsiella pneumoniae CTF-1 from Turkey

Characterization and Genomic Analysis of a New Bacteriophage Klebsiella pneumoniae CTF-1 from Turkey | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Characterization and Genomic Analysis of a New Bacteriophage Klebsiella pneumoniae CTF-1 from Turkey Kübra Can Kurt, Edip Tokuç, Halil Kurt, Duygu Nur Arabacı, Ahmet Sait, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6506509/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 Background Klebsiella pneumoniae is a clinically important pathogen causing respiratory tract infections, pneumonia, wound infections, urinary tract infections and sepsis. It is on the priority pathogen list of the World Health Organization (WHO) due to the resistant infections it causes. The K. pneumoniae CTF1 phage is a novel lytic phage with the highest overall similarity of 82.19% against K. pneumoniae isolated from clinical wound infections. In this study, the whole genome analysis of The K. pneumoniae CTF1 phage was conducted. Results The K. pneumoniae CTF1 phage has a linear double-stranded DNA genome 40,841 bp long with 53.1% GC content. Totally 44 protein coding genes were identified in phage genome. 31 of which were assigned functions as genome replication, phage packaging, structural proteins or phage lysis. K. pneumoniae CTF-1 is a member of the Przondovirus genus within the Caudovirales order. The objective of the research was to explore the effectiveness of lytic K. pneumoniae CTF-I bacteriophage isolate on panresistant K. pneumoniae isolates. The K. pneumoniae CTF1 phage was effective against 22 (88%) of 25 K. pneumoniae strains. The optimum efficacy of phage K. pneumoniae CTF-1 was 37–45°C, and the optimal effectiveness of the phage was pH between 4–9. The latent time and lytic cycle of the phage were around 40 min and burst size was around 92 PFU/mL. Conclusions The K. pneumoniae CTF-1 phage is an excellent candidate for phage therapy due to its high lytic activity against panresistant Klebsiella and lack of antibiotic resistance genes, toxins, virulence factors or integrase genes. Klebsiella pneumoniae Wound infection Phage therapy Antimicrobial resistance Whole-genome sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Background K. pneumoniae is a Gram-negative opportunistic pathogen belonging to the Enterobacteriaceae family. It can be found everywhere in the environment, in water, soil, animals, plants. It is a causative agent of many infections such as pneumonia, sepsis, wound infections, bacteremia, liver abscess, urinary tract infections [ 1 – 4 ]. K. pneumoniae are also pathogens or opportunistic pathogens that cause pneumonia and sepsis, especially in immunosuppressives. Newborns, immunocompromised individuals, and the elderly are at greatest risk for K. pneumoniae infections [ 5 , 6 ]. Wound infections are cause many deaths worldwide every year. Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa are the most prevalent wound infection agents [ 7 – 9 ]. The emergence and the global rapid spread of multidrug-resistant (MDR) and panresistant K. pneumoniae strains has reached alarming levels due to increased mortality. The WHO recognizes ESBL- and carbapenem-resistant K. pneumoniae as a global health threat. due to it has become resistant to nearly all available antibiotics and are classified within ESKAPE pathogens [ 10 – 14 ]. Colistin is used as an antimicrobial of last resort in the treatment of multidrug-resistant K. pneumoniae [ 15 , 16 ]. Colistin-resistant K. pneumoniae strains are also rapidly increasing day by day [ 17 ]. Those strains were responsible for more than 90,000 infections and more than 7000 deaths per year in Europe alone [ 18 ]. The spread of multidrug-resistant pathogens is indeed a major global problem and needs to be addressed. Bacteriophages alone or combination with antibiotics can be used to complement or replace antibiotics in various infections [ 19 , 20 ]. Using bacteriophages to treat infections caused by resistant pathogens is becoming widespread. Phage therapy is promising for the treatment of multidrug-resistant gram-negative bacilli. Bacteriophages have many advantages such as having low side effects and not causing resistance and destroying the biofilm layer [ 21 – 23 ]. Bacteriophages are a natural entity that infect bacteria and kill them by lysis after proliferation. Because of their high specificity against bacteria, bacteriophages can kill target bacterial strains without harming human cells and any other beneficial bacteria [ 24 – 26 ]. Phage therapy is effective against both antibiotic-resistant and antibiotic-sensitive pathogens. Phage therapy also restores sensitivity to various antibiotics by disrupting antibiotic resistance [ 27 – 30 ]. Thus, Bacteriophages could be a potential treatment in the clinic especially against antibiotic-resistant bacterial pathogens. The aim of this study is to characterize genome structure of K. pneumoniae CTF-I and investigate the effectiveness of the lytic bacteriophage K. pneumoniae CTF-I on panresistant K. pneumoniae isolates. Methods 2.1.Bacterial strains and culture conditions The 25 K. pneumoniae clinical strains, resistant to all antimicrobials, including colistin (panresistant), were originally isolated from clinical wound specimens. The bacteria culturing was performed in Tryptic Soy Broth (TSB), with the supplement of 5 mM CaCl2 and MgCl2. Twenty-five clinical KP strains were isolated Istanbul University- Cerrahpaşa, Cerrahpaşa Medical Faculty, Microbiology Laboratory. The samples collected were inoculated on blood agar, chocolate agar MacConkey agar. Bacterial colonies characterized using MALDI-TOF (MS, Bruker, Germany). The disk diffusion method was performed consistent with EUCAST (European Committee on Antimicrobial Susceptibility Testing) guidelines and the results were evaluated according to EUCAST criteria. 2.2. Isolation and purification of bacteriophage K. pneumoniae -CTF1 The isolation and purification of bacteriophages were carried out using the double-layer plate method. Phages were isolated from wastewater samples. Wastewater samples were filtered through 0.2 µm syringe filters to exclude bacteria. 50 µL Klebsiella was seeded in 2.5 mL medium supplemented with 5 mM MgCl2 and CaCl2 and incubated overnight at 37°C. The cultures were centrifuged and the supernatant was filtered with a 0.22 µm filter to exclude bacterial cells. The filtrate was diluted serially with TSB from 10 − 1 to 10–12 and utilized in an overlay agar plaque assay [ 19 ]. One-Step Growth Curve Analysis Mixture of 0.1 ml phage with 9.9 ml of K. pneumoniae in TSB was incubated for 5 min at room temperature. The mixture was centrifuged for 5 minutes to remove free phages, the residue was then resuspended in 10 ml TSB. 100 µL of samples were taken every 5 minutes to determine phage titters through 60 min. The number of phage progeny/number of latent infected cells determines the burst size. Host range testing The phage host range is determined by spot test method. 5 µL dilutions of phage were pipetted onto bacterial plates in soft agar. Petri dishes were left to dry upside down for 1 hour. Then at the end of the incubation period, the formation of phage plaques was investigated at 37°C overnight. The cell lysis was recorded like those visible plaques or had no effect. This phage may be a good candidate for use in phage therapy due to the advantages of having a wide host range, not containing antibiotic resistance genes, and having a short lysis period. Physical Stability of the Phage The temperature stability of phage KP CTF-1was determined at 4, 28, 37, 40, 45, 50, 55, 60 and 65°C) for 1 hour in TSB, respectively, phage suspension at each temperature for 60 min. The pH tolerance of the known concentration of phage KP-CTF-1 was tested for 1 h in TSB adjusted to different pH values (2.0, 3.0, 4.0, 5.0, 6.0 7.0, 8.0, 9.0, 9.0, 10.0, 11.0 and 12.0).For each assay, the phage titter was tested three times, and the averages were used. 2.3 K. pneumoniae -CTF1 genome sequencing and analysis Bacteriophage genomic DNA isolation The PEG 6000 method was used to concentrate on Klebsiella phage. The phage solution was mixed with PEG 6000 (10% w/v) solution and stirred overnight at + 4°C with gentle agitation (100 rpm).and then centrifuged at 12000g for 4 hours and the supernatant was removed. The obtained pellet was dispersed with 200 µl DNase RNase free water. Phage genomic DNA was isolated in the Roche MagNA pure LC system (Penzberg, Germany) using the Roche MagNA pure LC total nucleic acid isolation kit in accordance with the manufacturer's protocol. The quantity and quality of the phage DNA was measured with Thermo NanoDrop 2000c (Penzberg, Germany). Phage genome sequencing with MinION™ and bioinformatic analysis The phage genome was sequenced using the ONT ligation sequencing kit (SQK-LSK109) and the ONT native barcode kit (EXP-NBD104-114). The prepared library was loaded into an ONT MinION Flowcell v. 9.4.1 (FLO-MIN106D). Raw readings in Fast4 format were obtained using the MinKNOW program (v. 22.03.5). High quality readings were obtained after barcodes and adapters were removed using ONT-guppy (v. 6.0.6). The quality of raw fastq read was controlled by FastQC v0.11.9 ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). Contigs were obtained using flye de novo assembler (v. 2.8) [ 31 ]. BLAST was performed against the NCBI nt reference database for taxonomic analysis of the contigs. The overall identity % of bacteriophage was calculated using the formula of: overall identity % = identity % x coverage. The annotation of protein-coding genes was conducted by BLASTp against the NCBI nr database after identifying the protein-coding genes using the prokka program. The existence of antibiotic resistance genes was checked using the deepARG program [ 32 ]. rho-independent terminators were estimated using ARNold [ 33 ]. Results The phage KP-CTF-1 was isolated from wastewater sample. The phage plaques observed clear, transparent on the lawn of K. pneumoniae . Based on phage genome analysis, phage KP-CTF-1 can be classified as Przondovirus, which belong to Studiervirinae in Caudovirales, according to the latest criteria established by the International Committee on Taxonomy of Viruses. 3.1. Physiological characterization of the bacteriophage K. pneumoniae- CTF-1 The lytic cycle and latent time of Bacteriophage K. pneumoniae- CTF-1 was around 40 min and burst size was around 92 PFU/mL (Fig. 1 A). The optimum efficacy of phage K. pneumoniae- CTF-1 was 36–43°C (Fig. 1 B), and the optimal effectiveness of the phage was pH between 4–8 (Fig. 1 C). Of 25 K. pneumoniae strains isolated from wound samples, 22 (88%) were sensitive to K. pneumoniae CTF-1 phages and three (12%) were resistant. 3.2. Annotations and Characteristics of the Complete Genome of Klebsiella pneumoniae CTF1 Thir generation long read sequencing using The Oxford Nanopore MinION used to decipher Klebsiella pneumoniae CTF1 genome. K. pneumoniae -CTF-1 phage has a linear double-stranded DNA genome 40,841 bp long with 53.1% GC content. Circular map of the whole genome of K. pneumoniae -CTF-1. and comparative genome maps of closely related bacteriophages were shown in Fig. 2 . The similarity of K. pneumoniae -CTF-1 phage to the closest Klebsiella phage cp46 (OX335440.1) in NCBI refseq data is 94.48. The highest overall identity of closest Klebsiella phage cp46 to K. pneumoniae -CTF-1 was 82.19% (Table 1 ). Therefore, K. pneumoniae -CTF-1 is considered as a novel bacteriophage. The phylogenetic tree for K. pneumoniae -CTF-1 phage based on both terminase large subunit sequences and whole genome is given in Fig. 3 . Table 1 The whole genome sequence alignment of K. pneumoniae -CTF-1 against NCBI refseq database. Scientific Name Overall identity % Max Score Total Score Query Cover E value Per. Ident Acc. Len Accession Klebsiella phage cp46 82.197600% 26294 54083 87% 0 94.48% 39694 OX335440.1 Klebsiella phage VLCpiA3b 79.739200% 25398 50989 86% 0 92.72% 40231 ON602742.1 Klebsiella phage K5-4 78.204000% 25357 50533 84% 0 93.10% 40163 NC_047799.1 Klebsiella phage cp28 79.330500% 25236 51261 85% 0 93.33% 39661 OX335408.1 Klebsiella phage cp29 79.330500% 25236 51261 85% 0 93.33% 39662 OX335401.1 The genome structure and anatomy of K. pneumoniae -CTF-1 phage are shown in Fig. 4 . K. pneumoniae -CTF-1 phage is like other Klebsiella phages. A total of 49 ORFs were identified. According to the protein BLAST result, 44 of these ORFs were identified as structural proteins, proteins with functions in replication or phage lysis (Fig. 4 , Table S1 ). The function of 13 remaining ORFs identified as hypothetical protein. Gp13 functions DNA polymerases and Gp45 encodes DNA dependent RNA polymerase. Gp6 encodes a single-stranded DNA-binding protein. Phage Gp07 has been identified as an endonuclease. Gp17 gene encodes exonuclease. Gp1, Gp3, Gp5 and Gp7 are other transcription and replication related genes. Gp35 is annotated as a holin protein and Gp37 as Rz, a spanin protein (Fig. 4 ). Holin and RZ proteins are involved in bacterial lysing. Gp35 and Gp37 share 100% and 98% identity, respectively. Genes range from Gp21 to Gp34 responsible structural genes, head and tail morphogenesis. Gp36 and Gp38, annotated as small and large terminase subunits, respectively. Large terminase subunits are conserved protein and therefore used in the phylogenetic tree construction (Fig. 3 B) No antibiotic resistance genes were found in the K. pneumoniae -CTF-1 genome. Therefore, the newly isolated K. pneumoniae -CTF-1 phage may be a safe therapeutic phage therapy agent. Six rho-independent terminators were found in the K. pneumoniae -CTF-1 genome (S. table2). Discussion The ever-increasing number of carbapenem-resistant and even colistin-resistant K. pneumoniae isolates is a major problem worldwide. For this reason, research and development of new phage-based therapies may help in the treatment of infections caused by resistant bacteria. In this study, a novel K. pneumoniae -CTF-1 phage was isolated against K. pneumoniae strains obtained from clinical wound samples. The research aimed to characterize the K. pneumoniae -CTF-1 phage genome sequence and explore the potential for bacteriophage therapy. The findings provide valuable information for the development of phage-based therapies against K. pneumoniae strains isolated from clinical wound samples. According to phage genome analysis, K. pneumoniae -CTF-1 can be classified as Przondovirus, which belongs to Studiervirinae in Caudovirales. This study showed that K. pneumoniae -CTF-1 had 88% effectiveness against 25 K. pneumoniae strains. Since K. pneumoniae -CTF-1 phage does not contain antibiotic resistance and toxin genes, it can be safely used in treatment. 44 structural protein genes have been found in the phage genome, which are responsible for functions such as genome replication and phage lysis. Wound infections are responsible for one-third of hospital-acquired infections and 70–80% of deaths are caused by these infections. They are major agents in the development of mortality and morbidity in patients, mainly in developing countries [ 34 , 35 ]. Wound infections are difficult to diagnose and treat, are particularly common in hospitalized patients, and are likely to be infected with multiple pathogenic bacteria. The control of wound infections has become difficult due to the rising prevalence of infections caused by polymicrobial flora and common bacterial resistance to antibiotics. E. coli, Klebsiella spp, S. aureus, P. aeruginosa ,. and Acinetobacter spp. are the most common bacterial pathogens causing wound infections [ 36 ], Bacteriophages are used in some countries to treat various bacterial infections, but a standardized medical procedure has not yet been established. In order to utilize the enormous potential of phage therapy, it is necessary to choose the most effective bacteriophage [ 37 , 38 ]. Finally, phage therapy may not be accepted by some people due to a lack of phage awareness among medical staff and the public. With such problems, many researchers and clinicians are also drawing attention to phage therapy. The success of phage therapy relies on determining strategies to treat infections and decrease the outbreak of phage-resistant bacteria [ 39 ]. Today, phages have wide clinical application possibilities for the cure of infections caused by resistant bacteria; however, phages still have restrictions in clinical application. As phage therapy research develops further over time, clinical applications of phage appear to promise a brilliant future. In Vivo pharmacodynamics and pharmacokinetics of various phage products are different compared to antibiotics. There are many differences in the clinical application of phages, because preparations that include various phages have a different biological profile. Purification and manufacturing new phages costs less than antibiotics. There are phage therapy applications against K. pneumoniae strains isolated from the wound [ 40 , 41 ] and sepsis [ 42 ]. KpJH46Φ2 phage was successfully used in combination with antibiotics in a patient with prosthetic joint infection caused by K. pneumoniae [ 40 ]. L Yang, C Wang, Y Zeng, Y Song, G Zhang, D Wei, Y Li and J Feng [ 41 ] isolated K. pneumoniae RCIP0100 phage and demonstrated broad lytic activity against 15 of 27 MDR-KP strains. Like our isolated K. pneumoniae -CTF-1 phage, RCIP0100 phage was a promising candidate for phage therapy. ɸKpBHU7, ɸKpBHU4, and ɸKpBHU14 phages,, effective against K. pneumoniae were isolated [ 42 ]. The in vivo efficacy of these bacteriophages has been successfully tested in a mouse model of septicemia. They were killed 71.42%, 77.14% and 71.14% of clinical isolates, respectively. Compared to this phage, the new phage K. pneumoniae -CTF1 has higher activity against K. pneumoniae strains. Conclusions The K. pneumoniae CTF-1 phage can be considered as a bacteriophage therapeutic candidate for wound infections caused by multidrug-resistant K. pneumoniae strains because it does not contain toxins, integrase genes or virulence factors. Declarations Acknowledgments The author would like to Istanbul University-Cerrahpasa, Scientific Research Projects Coordination Unit (IUC-BAP) Funding This study was supported by the IUC-BAP. Project number 34517. Ethics approval The study was approved by İstanbul University-Cerrahpasa, Cerrahpasa Medical Faculty, Clinical Research Ethics Committee (83045809-604.01.02). Informed consent was obtained from all the subjects and/or their legal guardian(s). All methods were conducted in compliance with relevant guidelines and regulations. Research were conducted in compliance with the Helsinki Declaration. Consent for Publication Not applicable. Consent to participate Not applicable. Competing interests The author declare no conflict of interest. Data availability The data underlying this article are available in GenBank accession number of PV550976 . Author contributions K.C. conceived and designed the study. E.T. and D.N.A. performed the research under the guidance of K.C. S.A. provided technical support and analyzed the data. H.K. and A.S. analyzed sequencing data. K.C. and H.K. wore the manuscript. M.S. and H.B.T. provided the critical support and helped in drafting of the paper. All the authors revised the manuscript. References Effah CY, Sun T, Liu S, Wu Y. Klebsiella pneumoniae: an increasing threat to public health. Annals of clinical microbiology and antimicrobials. 2020;19(1):1-9. Lee C-R, Lee JH, Park KS, Jeon JH, Kim YB, Cha C-J, et al. Antimicrobial resistance of hypervirulent Klebsiella pneumoniae: epidemiology, hypervirulence-associated determinants, and resistance mechanisms. Frontiers in cellular and infection microbiology. 2017;7:483. Dai P, Hu D. The making of hypervirulent Klebsiella pneumoniae. 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Phage cocktails and the future of phage therapy. Future microbiology. 2013;8(6):769-83. Cano EJ, Caflisch KM, Bollyky PL, Van Belleghem JD, Patel R, Fackler J, et al. Phage therapy for limb-threatening prosthetic knee Klebsiella pneumoniae infection: case report and in vitro characterization of anti-biofilm activity. Clinical Infectious Diseases. 2021;73(1):e144-e51. Yang L, Wang C, Zeng Y, Song Y, Zhang G, Wei D, et al. Characterization of a novel phage against multidrug-resistant Klebsiella pneumoniae. Archives of Microbiology. 2024;206(9):379. Singh A, Singh AN, Rathor N, Chaudhry R, Singh SK, Nath G. Evaluation of bacteriophage cocktail on septicemia caused by colistin-resistant Klebsiella pneumoniae in mice model. Frontiers in Pharmacology. 2022;13:778676. Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx TableS2.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6506509","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":455716423,"identity":"ff048e84-e350-4dc4-b234-c48c7a31b51f","order_by":0,"name":"Kübra Can Kurt","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYBACCQYeKAtMVzAkQESJ1nLgDFyLAZFaDrYRoUVyRu6xDz8Y7OTNec6Yff44ry7P4ADzwds8DH/ycWmRlshLntnDkGy4s7fHeMbBbYeLDQ6wJVvzMBhYNuDQIieRYwx00wHGDed5jBkObjuQuOEAj5k0UAtOl4G0MP5hOGAP0TKnDqiF/xteLdJALcxAWxI3nO0BamlgBtnChleLZM+7ZGYZg+TkDWeOFTOcOXa4WPIwm7HlHANjnFokjuceZnxTYWe74UzyZoaKmro8vuPND2+8qZDDHcpggCLNjCEyCkbBKBgFo4BUAABlM0+GNwlVwwAAAABJRU5ErkJggg==","orcid":"","institution":"University of Health Sciences, Hamidiye Faculty of Dentistry, Department of Basic Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Kübra","middleName":"Can","lastName":"Kurt","suffix":""},{"id":455716424,"identity":"5c0ebe0a-76fb-4b4f-9282-00355da1e3e6","order_by":1,"name":"Edip Tokuç","email":"","orcid":"","institution":"İstanbul University-Cerrahpasa, Cerrahpasa Medical Faculty, Medical Microbiology Department","correspondingAuthor":false,"prefix":"","firstName":"Edip","middleName":"","lastName":"Tokuç","suffix":""},{"id":455716425,"identity":"430a32a3-ccd5-41f0-9770-33dd1a34016d","order_by":2,"name":"Halil Kurt","email":"","orcid":"","institution":"University of Health Sciences, Hamidiye International School of Medicine, Medical Biology Department","correspondingAuthor":false,"prefix":"","firstName":"Halil","middleName":"","lastName":"Kurt","suffix":""},{"id":455716426,"identity":"6c7d5d10-271c-4eb7-9ce0-68b27c02c438","order_by":3,"name":"Duygu Nur Arabacı","email":"","orcid":"","institution":"Nişantaşı University, Department of Genetics and Bioengineering","correspondingAuthor":false,"prefix":"","firstName":"Duygu","middleName":"Nur","lastName":"Arabacı","suffix":""},{"id":455716427,"identity":"942d2b58-2f52-433f-a4e7-16c9cefd39df","order_by":4,"name":"Ahmet Sait","email":"","orcid":"","institution":"Virology Laboratory, Pendik Veterinary Control Institute","correspondingAuthor":false,"prefix":"","firstName":"Ahmet","middleName":"","lastName":"Sait","suffix":""},{"id":455716428,"identity":"34fd3888-d41e-4e1b-994c-4347b8e15037","order_by":5,"name":"Sevcan Aydın","email":"","orcid":"","institution":"Istanbul University, Faculty of Science, Department of Biology, Biotechnology Section","correspondingAuthor":false,"prefix":"","firstName":"Sevcan","middleName":"","lastName":"Aydın","suffix":""},{"id":455716429,"identity":"5c67e1af-0d78-4c86-b16b-a464252db7d3","order_by":6,"name":"Mikael Skurnik","email":"","orcid":"","institution":"Department of Bacteriology and Immunology, Human Microbiome Research Program, Faculty of Medicine, University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Mikael","middleName":"","lastName":"Skurnik","suffix":""},{"id":455716430,"identity":"15799403-cbd1-4ee7-9b17-80d50a470e2c","order_by":7,"name":"Hrisi Bahar Tokman","email":"","orcid":"","institution":"İstanbul University-Cerrahpasa, Cerrahpasa Medical Faculty, Medical Microbiology Department","correspondingAuthor":false,"prefix":"","firstName":"Hrisi","middleName":"Bahar","lastName":"Tokman","suffix":""}],"badges":[],"createdAt":"2025-04-22 17:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6506509/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6506509/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82699309,"identity":"d5012cf2-e7bd-411e-adcf-146870597ede","added_by":"auto","created_at":"2025-05-14 09:21:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":87169,"visible":true,"origin":"","legend":"\u003cp\u003eBiological characterization of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e CTF1. (A) One-step growth curve analysis. (B) Thermal stability. (C) pH stability.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6506509/v1/5626acf25003b969baf087cd.png"},{"id":82698659,"identity":"7d071941-5e92-4c4a-92b4-e8d9d6d254b3","added_by":"auto","created_at":"2025-05-14 09:13:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":529959,"visible":true,"origin":"","legend":"\u003cp\u003eCircular map of the whole genome of \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1. and comparative genome maps of genome sequences of closely related bacteriophages. Gaps in the circles represent regions of low or no similarity.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6506509/v1/1bac5b643e5e906cb619c987.png"},{"id":82698657,"identity":"7930b156-c587-447f-a7c4-4a2253b47244","added_by":"auto","created_at":"2025-05-14 09:13:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":826117,"visible":true,"origin":"","legend":"\u003cp\u003eThe evolutionary association of phage with closely related bacteriophages. (A) Phylogenetic trees of \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 constructed from the whole genome alignment generated by VipTree. (B) Terminase large subunit Phylogenetic trees of phage \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 and closely related phages.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6506509/v1/87579b8782877fb01479fc50.png"},{"id":82697488,"identity":"2f9ce462-f12f-48d9-a038-afc24869fb56","added_by":"auto","created_at":"2025-05-14 09:05:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":268916,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic map of \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 and it is genetic characteristics.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6506509/v1/bb8b03f941e243faf777c4b1.png"},{"id":89836750,"identity":"20193436-d587-45de-8d92-db85a06480fe","added_by":"auto","created_at":"2025-08-25 14:47:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2393630,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6506509/v1/3c7c53be-d21c-4be3-852a-cf30b11f6eb7.pdf"},{"id":82697485,"identity":"9d5a641d-9ad8-47fa-acb3-174fe9db1ce5","added_by":"auto","created_at":"2025-05-14 09:05:48","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14152,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6506509/v1/2952dc5c1c10a2b8b0473b68.xlsx"},{"id":82697489,"identity":"5e35e3e2-cc83-4642-b255-f81bb7c113fb","added_by":"auto","created_at":"2025-05-14 09:05:48","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9567,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6506509/v1/13dec339b1e582081381f523.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eCharacterization and Genomic Analysis of a New Bacteriophage \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e CTF-1 from Turkey\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eK. pneumoniae\u003c/em\u003e is a Gram-negative opportunistic pathogen belonging to the Enterobacteriaceae family. It can be found everywhere in the environment, in water, soil, animals, plants. It is a causative agent of many infections such as pneumonia, sepsis, wound infections, bacteremia, liver abscess, urinary tract infections [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. \u003cem\u003eK. pneumoniae\u003c/em\u003e are also pathogens or opportunistic pathogens that cause pneumonia and sepsis, especially in immunosuppressives. Newborns, immunocompromised individuals, and the elderly are at greatest risk for \u003cem\u003eK. pneumoniae\u003c/em\u003e infections [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWound infections are cause many deaths worldwide every year. \u003cem\u003eKlebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa\u003c/em\u003e are the most prevalent wound infection agents [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The emergence and the global rapid spread of multidrug-resistant (MDR) and panresistant \u003cem\u003eK. pneumoniae\u003c/em\u003e strains has reached alarming levels due to increased mortality. The WHO recognizes ESBL- and carbapenem-resistant K. pneumoniae as a global health threat. due to it has become resistant to nearly all available antibiotics and are classified within ESKAPE pathogens [\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Colistin is used as an antimicrobial of last resort in the treatment of multidrug-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Colistin-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e strains are also rapidly increasing day by day [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Those strains were responsible for more than 90,000 infections and more than 7000 deaths per year in Europe alone [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe spread of multidrug-resistant pathogens is indeed a major global problem and needs to be addressed. Bacteriophages alone or combination with antibiotics can be used to complement or replace antibiotics in various infections [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Using bacteriophages to treat infections caused by resistant pathogens is becoming widespread. Phage therapy is promising for the treatment of multidrug-resistant gram-negative bacilli. Bacteriophages have many advantages such as having low side effects and not causing resistance and destroying the biofilm layer [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Bacteriophages are a natural entity that infect bacteria and kill them by lysis after proliferation. Because of their high specificity against bacteria, bacteriophages can kill target bacterial strains without harming human cells and any other beneficial bacteria [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Phage therapy is effective against both antibiotic-resistant and antibiotic-sensitive pathogens. Phage therapy also restores sensitivity to various antibiotics by disrupting antibiotic resistance [\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Thus, Bacteriophages could be a potential treatment in the clinic especially against antibiotic-resistant bacterial pathogens.\u003c/p\u003e \u003cp\u003eThe aim of this study is to characterize genome structure of \u003cem\u003eK. pneumoniae\u003c/em\u003e CTF-I and investigate the effectiveness of the lytic bacteriophage \u003cem\u003eK. pneumoniae\u003c/em\u003e CTF-I on panresistant \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1.Bacterial strains and culture conditions\u003c/h2\u003e \u003cp\u003eThe 25 \u003cem\u003eK. pneumoniae\u003c/em\u003e clinical strains, resistant to all antimicrobials, including colistin (panresistant), were originally isolated from clinical wound specimens. The bacteria culturing was performed in Tryptic Soy Broth (TSB), with the supplement of 5 mM CaCl2 and MgCl2. Twenty-five clinical KP strains were isolated Istanbul University- Cerrahpaşa, Cerrahpaşa Medical Faculty, Microbiology Laboratory. The samples collected were inoculated on blood agar, chocolate agar MacConkey agar. Bacterial colonies characterized using MALDI-TOF (MS, Bruker, Germany). The disk diffusion method was performed consistent with EUCAST (European Committee on Antimicrobial Susceptibility Testing) guidelines and the results were evaluated according to EUCAST criteria.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Isolation and purification of bacteriophage \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF1\u003c/h2\u003e \u003cp\u003eThe isolation and purification of bacteriophages were carried out using the double-layer plate method. Phages were isolated from wastewater samples. Wastewater samples were filtered through 0.2 \u0026micro;m syringe filters to exclude bacteria. 50 \u0026micro;L Klebsiella was seeded in 2.5 mL medium supplemented with 5 mM MgCl2 and CaCl2 and incubated overnight at 37\u0026deg;C. The cultures were centrifuged and the supernatant was filtered with a 0.22 \u0026micro;m filter to exclude bacterial cells. The filtrate was diluted serially with TSB from 10\u0026thinsp;\u0026minus;\u0026thinsp;1 to 10\u0026ndash;12 and utilized in an overlay agar plaque assay [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eOne-Step Growth Curve Analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eMixture of 0.1 ml phage with 9.9 ml of \u003cem\u003eK. pneumoniae\u003c/em\u003e in TSB was incubated for 5 min at room temperature. The mixture was centrifuged for 5 minutes to remove free phages, the residue was then resuspended in 10 ml TSB. 100 \u0026micro;L of samples were taken every 5 minutes to determine phage titters through 60 min. The number of phage progeny/number of latent infected cells determines the burst size.\u003c/p\u003e \u003cp\u003e \u003cem\u003eHost range testing\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe phage host range is determined by spot test method. 5 \u0026micro;L dilutions of phage were pipetted onto bacterial plates in soft agar. Petri dishes were left to dry upside down for 1 hour. Then at the end of the incubation period, the formation of phage plaques was investigated at 37\u0026deg;C overnight. The cell lysis was recorded like those visible plaques or had no effect. This phage may be a good candidate for use in phage therapy due to the advantages of having a wide host range, not containing antibiotic resistance genes, and having a short lysis period.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePhysical Stability of the Phage\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe temperature stability of phage KP CTF-1was determined at 4, 28, 37, 40, 45, 50, 55, 60 and 65\u0026deg;C) for 1 hour in TSB, respectively, phage suspension at each temperature for 60 min. The pH tolerance of the known concentration of phage KP-CTF-1 was tested for 1 h in TSB adjusted to different pH values (2.0, 3.0, 4.0, 5.0, 6.0 7.0, 8.0, 9.0, 9.0, 10.0, 11.0 and 12.0).For each assay, the phage titter was tested three times, and the averages were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF1 genome sequencing and analysis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eBacteriophage genomic DNA isolation\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe PEG 6000 method was used to concentrate on \u003cem\u003eKlebsiella\u003c/em\u003e phage. The phage solution was mixed with PEG 6000 (10% w/v) solution and stirred overnight at +\u0026thinsp;4\u0026deg;C with gentle agitation (100 rpm).and then centrifuged at 12000g for 4 hours and the supernatant was removed. The obtained pellet was dispersed with 200 \u0026micro;l DNase RNase free water. Phage genomic DNA was isolated in the Roche MagNA pure LC system (Penzberg, Germany) using the Roche MagNA pure LC total nucleic acid isolation kit in accordance with the manufacturer's protocol. The quantity and quality of the phage DNA was measured with Thermo NanoDrop 2000c (Penzberg, Germany).\u003c/p\u003e \u003cp\u003ePhage genome sequencing with MinION\u0026trade; and bioinformatic analysis\u003c/p\u003e \u003cp\u003eThe phage genome was sequenced using the ONT ligation sequencing kit (SQK-LSK109) and the ONT native barcode kit (EXP-NBD104-114). The prepared library was loaded into an ONT MinION Flowcell v. 9.4.1 (FLO-MIN106D). Raw readings in Fast4 format were obtained using the MinKNOW program (v. 22.03.5). High quality readings were obtained after barcodes and adapters were removed using ONT-guppy (v. 6.0.6). The quality of raw fastq read was controlled by FastQC v0.11.9 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/fastqc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Contigs were obtained using flye de novo assembler (v. 2.8) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. BLAST was performed against the NCBI nt reference database for taxonomic analysis of the contigs. The overall identity % of bacteriophage was calculated using the formula of: overall identity % = identity % x coverage. The annotation of protein-coding genes was conducted by BLASTp against the NCBI nr database after identifying the protein-coding genes using the prokka program. The existence of antibiotic resistance genes was checked using the deepARG program [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. rho-independent terminators were estimated using ARNold [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe phage KP-CTF-1 was isolated from wastewater sample. The phage plaques observed clear, transparent on the lawn of \u003cem\u003eK. pneumoniae\u003c/em\u003e. Based on phage genome analysis, phage KP-CTF-1 can be classified as Przondovirus, which belong to Studiervirinae in Caudovirales, according to the latest criteria established by the International Committee on Taxonomy of Viruses.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.1. Physiological characterization of the bacteriophage\u003c/b\u003e \u003cb\u003eK. pneumoniae-\u003c/b\u003e \u003cb\u003eCTF-1\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe lytic cycle and latent time of Bacteriophage \u003cem\u003eK. pneumoniae-\u003c/em\u003eCTF-1 was around 40 min and burst size was around 92 PFU/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The optimum efficacy of phage \u003cem\u003eK. pneumoniae-\u003c/em\u003eCTF-1 was 36\u0026ndash;43\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and the optimal effectiveness of the phage was pH between 4\u0026ndash;8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Of 25 K. pneumoniae strains isolated from wound samples, 22 (88%) were sensitive to K. pneumoniae CTF-1 phages and three (12%) were resistant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Annotations and Characteristics of the Complete Genome of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e CTF1\u003c/h2\u003e \u003cp\u003eThir generation long read sequencing using The Oxford Nanopore MinION used to decipher \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e CTF1 genome. \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 phage has a linear double-stranded DNA genome 40,841 bp long with 53.1% GC content. Circular map of the whole genome of \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1. and comparative genome maps of closely related bacteriophages were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The similarity of \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 phage to the closest Klebsiella phage cp46 (OX335440.1) in NCBI refseq data is 94.48. The highest overall identity of closest Klebsiella phage cp46 to \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 was 82.19% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 is considered as a novel bacteriophage. The phylogenetic tree for \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 phage based on both terminase large subunit sequences and whole genome is given in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe whole genome sequence alignment of \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 against NCBI refseq database.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eScientific Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOverall identity %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMax Score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal Score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eQuery Cover\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eE value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePer. Ident\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAcc. Len\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKlebsiella phage cp46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e82.197600%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e54083\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e87%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e94.48%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e39694\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eOX335440.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKlebsiella phage VLCpiA3b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e79.739200%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25398\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50989\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e86%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e92.72%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e40231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eON602742.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKlebsiella phage K5-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e78.204000%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25357\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50533\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e84%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e93.10%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e40163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNC_047799.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKlebsiella phage cp28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e79.330500%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e51261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e93.33%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e39661\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eOX335408.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKlebsiella phage cp29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e79.330500%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e51261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e93.33%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e39662\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eOX335401.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe genome structure and anatomy of \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 phage are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 phage is like other Klebsiella phages. A total of 49 ORFs were identified. According to the protein BLAST result, 44 of these ORFs were identified as structural proteins, proteins with functions in replication or phage lysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The function of 13 remaining ORFs identified as hypothetical protein.\u003c/p\u003e \u003cp\u003eGp13 functions DNA polymerases and Gp45 encodes DNA dependent RNA polymerase. Gp6 encodes a single-stranded DNA-binding protein. Phage Gp07 has been identified as an endonuclease. Gp17 gene encodes exonuclease. Gp1, Gp3, Gp5 and Gp7 are other transcription and replication related genes. Gp35 is annotated as a holin protein and Gp37 as Rz, a spanin protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Holin and RZ proteins are involved in bacterial lysing. Gp35 and Gp37 share 100% and 98% identity, respectively. Genes range from Gp21 to Gp34 responsible structural genes, head and tail morphogenesis. Gp36 and Gp38, annotated as small and large terminase subunits, respectively. Large terminase subunits are conserved protein and therefore used in the phylogenetic tree construction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB)\u003c/p\u003e \u003cp\u003eNo antibiotic resistance genes were found in the \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 genome. Therefore, the newly isolated \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 phage may be a safe therapeutic phage therapy agent. Six rho-independent terminators were found in the \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 genome (S. table2).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe ever-increasing number of carbapenem-resistant and even colistin-resistant \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates is a major problem worldwide. For this reason, research and development of new phage-based therapies may help in the treatment of infections caused by resistant bacteria. In this study, a novel \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 phage was isolated against \u003cem\u003eK. pneumoniae\u003c/em\u003e strains obtained from clinical wound samples. The research aimed to characterize the \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 phage genome sequence and explore the potential for bacteriophage therapy. The findings provide valuable information for the development of phage-based therapies against \u003cem\u003eK. pneumoniae\u003c/em\u003e strains isolated from clinical wound samples. According to phage genome analysis, \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 can be classified as Przondovirus, which belongs to Studiervirinae in Caudovirales. This study showed that \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 had 88% effectiveness against 25 \u003cem\u003eK. pneumoniae\u003c/em\u003e strains. Since \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 phage does not contain antibiotic resistance and toxin genes, it can be safely used in treatment. 44 structural protein genes have been found in the phage genome, which are responsible for functions such as genome replication and phage lysis.\u003c/p\u003e \u003cp\u003eWound infections are responsible for one-third of hospital-acquired infections and 70–80% of deaths are caused by these infections. They are major agents in the development of mortality and morbidity in patients, mainly in developing countries [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Wound infections are difficult to diagnose and treat, are particularly common in hospitalized patients, and are likely to be infected with multiple pathogenic bacteria. The control of wound infections has become difficult due to the rising prevalence of infections caused by polymicrobial flora and common bacterial resistance to antibiotics. \u003cem\u003eE. coli, Klebsiella spp, S. aureus, P. aeruginosa\u003c/em\u003e,. and \u003cem\u003eAcinetobacter spp.\u003c/em\u003e are the most common bacterial pathogens causing wound infections [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e],\u003c/p\u003e \u003cp\u003eBacteriophages are used in some countries to treat various bacterial infections, but a standardized medical procedure has not yet been established. In order to utilize the enormous potential of phage therapy, it is necessary to choose the most effective bacteriophage [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Finally, phage therapy may not be accepted by some people due to a lack of phage awareness among medical staff and the public. With such problems, many researchers and clinicians are also drawing attention to phage therapy. The success of phage therapy relies on determining strategies to treat infections and decrease the outbreak of phage-resistant bacteria [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Today, phages have wide clinical application possibilities for the cure of infections caused by resistant bacteria; however, phages still have restrictions in clinical application. As phage therapy research develops further over time, clinical applications of phage appear to promise a brilliant future.\u003c/p\u003e \u003cp\u003eIn Vivo pharmacodynamics and pharmacokinetics of various phage products are different compared to antibiotics. There are many differences in the clinical application of phages, because preparations that include various phages have a different biological profile. Purification and manufacturing new phages costs less than antibiotics.\u003c/p\u003e \u003cp\u003eThere are phage therapy applications against \u003cem\u003eK. pneumoniae\u003c/em\u003e strains isolated from the wound [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and sepsis [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. KpJH46Φ2 phage was successfully used in combination with antibiotics in a patient with prosthetic joint infection caused by \u003cem\u003eK. pneumoniae\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. L Yang, C Wang, Y Zeng, Y Song, G Zhang, D Wei, Y Li and J Feng [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] isolated \u003cem\u003eK. pneumoniae\u003c/em\u003e RCIP0100 phage and demonstrated broad lytic activity against 15 of 27 MDR-KP strains. Like our isolated \u003cem\u003eK. pneumoniae\u003c/em\u003e-CTF-1 phage, RCIP0100 phage was a promising candidate for phage therapy. ɸKpBHU7, ɸKpBHU4, and ɸKpBHU14 phages,, effective against \u003cem\u003eK. pneumoniae\u003c/em\u003e were isolated [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The in vivo efficacy of these bacteriophages has been successfully tested in a mouse model of septicemia. They were killed 71.42%, 77.14% and 71.14% of clinical isolates, respectively. Compared to this phage, the new phage \u003cem\u003eK. pneumoniae\u003c/em\u003e -CTF1 has higher activity against \u003cem\u003eK. pneumoniae\u003c/em\u003e strains.\u003c/p\u003e "},{"header":"Conclusions","content":"\u003cp\u003eThe K. pneumoniae CTF-1 phage can be considered as a bacteriophage therapeutic candidate for wound infections caused by multidrug-resistant K. pneumoniae strains because it does not contain toxins, integrase genes or virulence factors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003eThe author would like to Istanbul University-Cerrahpasa, Scientific Research Projects Coordination Unit (IUC-BAP)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the IUC-BAP. Project number 34517.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study was approved by İstanbul University-Cerrahpasa, Cerrahpasa Medical Faculty, Clinical Research Ethics Committee (83045809-604.01.02).\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all the subjects and/or their legal guardian(s).\u003c/p\u003e\n\u003cp\u003eAll methods were conducted in compliance with relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003eResearch were conducted in compliance with the Helsinki Declaration.\u003c/p\u003e\n\u003cp\u003eConsent for Publication Not applicable.\u003c/p\u003e\n\u003cp\u003eConsent to participate Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data underlying this article are available in GenBank accession number of\u0026nbsp;\u003cstrong\u003ePV550976\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.C. conceived and designed the study. E.T. and D.N.A. performed the research under the guidance of K.C. S.A. provided technical support and analyzed the data. H.K. and A.S. analyzed sequencing data. K.C. and H.K. wore the manuscript. M.S. and H.B.T. provided the critical support and helped in drafting of the paper. All the authors revised the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEffah CY, Sun T, Liu S, Wu Y. Klebsiella pneumoniae: an increasing threat to public health. Annals of clinical microbiology and antimicrobials. 2020;19(1):1-9.\u003c/li\u003e\n\u003cli\u003eLee C-R, Lee JH, Park KS, Jeon JH, Kim YB, Cha C-J, et al. Antimicrobial resistance of hypervirulent Klebsiella pneumoniae: epidemiology, hypervirulence-associated determinants, and resistance mechanisms. Frontiers in cellular and infection microbiology. 2017;7:483.\u003c/li\u003e\n\u003cli\u003eDai P, Hu D. The making of hypervirulent Klebsiella pneumoniae. 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Frontiers in Pharmacology. 2022;13:778676.\u003c/li\u003e\n\u003c/ol\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":"Klebsiella pneumoniae, Wound infection, Phage therapy, Antimicrobial resistance, Whole-genome sequencing","lastPublishedDoi":"10.21203/rs.3.rs-6506509/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6506509/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e is a clinically important pathogen causing respiratory tract infections, pneumonia, wound infections, urinary tract infections and sepsis. It is on the priority pathogen list of the World Health Organization (WHO) due to the resistant infections it causes. The \u003cem\u003eK. pneumoniae\u003c/em\u003e CTF1 phage is a novel lytic phage with the highest overall similarity of 82.19% against \u003cem\u003eK. pneumoniae\u003c/em\u003e isolated from clinical wound infections. In this study, the whole genome analysis of The \u003cem\u003eK. pneumoniae\u003c/em\u003e CTF1 phage was conducted.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eK. pneumoniae\u003c/em\u003e CTF1 phage has a linear double-stranded DNA genome 40,841 bp long with 53.1% GC content. Totally 44 protein coding genes were identified in phage genome. 31 of which were assigned functions as genome replication, phage packaging, structural proteins or phage lysis. \u003cem\u003eK. pneumoniae\u003c/em\u003e CTF-1 is a member of the \u003cem\u003ePrzondovirus\u003c/em\u003e genus within the Caudovirales order. The objective of the research was to explore the effectiveness of lytic \u003cem\u003eK. pneumoniae\u003c/em\u003e CTF-I bacteriophage isolate on panresistant K. pneumoniae isolates. The \u003cem\u003eK. pneumoniae\u003c/em\u003e CTF1 phage was effective against 22 (88%) of 25 \u003cem\u003eK. pneumoniae\u003c/em\u003e strains. The optimum efficacy of phage \u003cem\u003eK. pneumoniae\u003c/em\u003e CTF-1 was 37\u0026ndash;45\u0026deg;C, and the optimal effectiveness of the phage was pH between 4\u0026ndash;9. The latent time and lytic cycle of the phage were around 40 min and burst size was around 92 PFU/mL.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eK. pneumoniae\u003c/em\u003e CTF-1 phage is an excellent candidate for phage therapy due to its high lytic activity against panresistant \u003cem\u003eKlebsiella\u003c/em\u003e and lack of antibiotic resistance genes, toxins, virulence factors or integrase genes.\u003c/p\u003e","manuscriptTitle":"Characterization and Genomic Analysis of a New Bacteriophage Klebsiella pneumoniae CTF-1 from Turkey","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-14 09:05:43","doi":"10.21203/rs.3.rs-6506509/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":"e3d810ac-2e7f-4b8e-b271-49201f5a9492","owner":[],"postedDate":"May 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-25T14:39:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-14 09:05:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6506509","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6506509","identity":"rs-6506509","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}