Biological characterization and complete genome analysis of the newly isolated Serratia liquefaciens  phage vB_SlqS_ZDD2

Biological characterization and complete genome analysis of the newly isolated Serratia liquefaciens phage vB_SlqS_ZDD2 | 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 Biological characterization and complete genome analysis of the newly isolated Serratia liquefaciens phage vB_SlqS_ZDD2 Qian Zhao, Linzi Han, Chen Chu, Lili Wang, Likun Zhuang, Rongtao Yuan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3786849/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Apr, 2024 Read the published version in Archives of Virology → Version 1 posted 5 You are reading this latest preprint version Abstract A novel lytic phage named vB_SlqS_ZDD2 infecting Serratia liquefaciens was isolated from hospital sewage. BLASTn analysis showed that the genome sequence of phage vB_SlqS_ZDD2 shared 0% query coverage with other phages in the NCBI database. Phenotype and phylogeny analysis indicated that this phage might be a new member of the order Caudoviricetes . Genome sequence displayed that phage vB_SlqS_ZDD2 has a 49,178 bp dsDNA with 55% GC content and has 74 open reading frames. Besides, the phage exhibited strong lytic activity and a wide range of pH (3-12) and temperature tolerance (80℃, 80 min). Figures Figure 1 Figure 2 Main Text Serratia liquefaciens is one of the opportunistic pathogens that widely exited in the environment and it is the major cause of Serratia infections which can lead bacteremia, respiratory tract infection, pneumonia and a series of other diseases [1, 2]. In recent years, Serratia liquefaciens has developed high resistance to currently available antibiotics, such as Penicillin, and more and more multidrug-resistant strainswere also found [3, 4]. Hence, it is necessary to develop efficient methods to combat Serratia liquefaciens infections. As the most abundant microorganisms on the earth, bacteriophages could invade bacteria and have the advantage of host specificity and non-toxicity to animals, which are proposed to be the alternatives to conventional antibiotic treatments [5-7]. In this study, a Serratia liquefaciens phage, named vB_SlqS_ZDD2, was isolated from hospital sewage and its biological characterizations and complete genome sequence were determined. vB_SlqS_ZDD2 showed strong lytic properties and formed clear plaques on the plates (approximately 0.5-1.0 mm in diameter) (Fig. 1A). Transmission electron microscope was used to explore the morphology of phage, and the result showed that vB_SlqS_ZDD2 has a head with a diameter of 64-69 nm and a long tail of 143-150 nm in length (Fig. 1B). The morphological analysis indicated that phage vB_SlqS_ZDD2 may be a member of the order Caudoviricetes , showing Siphovirus characteristic features. To evaluate the biological characterizations of phage vB_SlqS_ZDD2, the optimal multiplicity of infection (MOI), one-step growth curve, pH and temperature stability of this phage were measured. MOI of phage was determined according to the procedures described by Xi et al. with modification [8]. Briefly, phage was added into the host bacteria culture according to different MOI ratios, and the mixture was incubated at 37°C, 180 rpm for 8 h. Double-layer agar method was used to measure the phage titer [9]. The results (Fig. 1C) showed that the phage titer reached 1.9 × 10 10 PFU/ml at an MOI of 0.001, which was significantly higher ( p < 0.01) than other groups. To test the life cycle of phage, one-step growth curve measurement was conducted [10]. Firstly, vB_SlqS_ZDD2 was mixed with host bacteria at the optimal MOI of 0.001 and incubated at 37°C for 15 min. Then, the culture was centrifuged (12,000 × g, 2 min) to remove the unabsorbed phage, and the pellet was resuspended and transferred into 50 ml LB medium and incubated at 37°C (140 rpm). Next, three samples were taken at 0, 5, 10 min, and then every 10 min for the first 120 min and every 30 min from 120 to 270 min of incubation. The phage titer was determined immediately by double-layer method. As shown in Fig 1D, the latent period of vB_SlqS_ZDD2 was approximately 5 min, and the lysis period was approximately 140 min, as well as the burst size of phage was 104 PFU per cell. The pH stability of phage vB_SlqS_ZDD2 was examined as Ding et al. described with minor modification [11]. The phage was incubated in LB broth at various pH values for 1 h and then the titer was measured. The results (Fig. 1E) showed that phage vB_SlqS_ZDD2 exhibited a strong tolerance from pH 3-11, and it was inactivated when the pH below 2 or above 13. To investigate its temperature stability [12], phage samples were incubated at different temperatures (40, 50, 60, 70 and 80℃). Samples were collected every 20 min from 0 to 80 min of incubation and the titer was determined. As displayed in Fig. 1F, the titer of phage vB_SlqS_ZDD2 were remaining basically unchanged from 0 to 80 min when the temperature below 60℃. When the temperature rose to 70℃, phage titer decreased from 2.5 × 10 9 to 6.7 × 10 6 PFU/ml (0 to 80 min). However, phage vB_SlqS_ZDD2 maintained a titer of 2.3 × 10 3 PFU/ml even after incubation at 80 °C for 80 min, indicating that it has excellent temperature stability. Phage genomic DNA was extracted using phage DNA isolation kit (Tiangen Biotek, Beijing) according to the manufacturer's instructions. High-throughput sequencing of the genomic DNA of phage vB_SlqS_ZDD2 was performed by BGI-Shenzhen using a MGISEQ-2000 platform. Whole genome sequence of phage was submitted to the NCBI GenBank database with the accession number OR881364. The genome map was shown in Fig. 2A&B. Genome sequence results revealed that phage vB_SlqS_ZDD2 was 49,178 bp with 55% GC content. Open reading frames (ORFs) were predicted using RAST software (https://rast.nmpdr.org/rast.cgi) and subsequently verified using the ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). There were 74 ORFs in the genome of phage vB_SlqS_ZDD2, and 20 of which were functionally annotated by searching the non-redundant protein database using BLASTp (http://blast.ncbi.nlm.nih.gov/). These major functional proteins could be classified into five categories according to NCBI and UniProt databases (Fig. 2B): structural proteins (gp01, gp02, gp03, gp05, gp06, gp07, gp72, gp74), DNA replication and nucleotide metabolism (gp10, gp25, gp60, gp66), DNA packaging (gp16), lysis proteins (gp19, gp20) and other functions proteins (gp04, gp08, gp15, gp71, gp73). Among all open reading frames, there are three different initial codons, with ATG having the most, accounting for 79.73% (59/74). All open reading frames can be divided into two encoding directions, with ORF22, ORF57, ORF58, ORF59, ORF62, ORF63, ORF64, ORF65, ORF66 and ORF67 being forward encoding, and the remaining 64 open reading frames being reverse encoding (Fig. 2B). In addition, no tRNA genes were found using tRNAscan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/). No genes related to drug resistance, virulence, or toxins were found in the genome of phage vB_SlqS_ZDD2, which indicated that vB_SlqS_ZDD2 might be a potential biotherapeutic agent against Serratia liquefaciens . Phylogenetic relationship of phage vB_SlqS_ZDD2 to other reported prokaryotic double-stranded DNA viruses was analyzed using the ViPTree version 2.1 (https://www.genome.jp/viptree/) based on the whole genome (Fig. 2C). A total of 3178 sequences were selected by system into comparing with phage vB_SlqS_ZDD2 (Fig. 2C showed section data), and the results showed that phage vB_SlqS_ZDD2 was grouped into one new group. Further BLASTn alignment of phage vB_SlqS_ZDD2 showed that it shared 0% query coverage with other phages in the NCBI database, which demonstrated that it is a newly discovered phage. Therefore, based on its lower similarity to other phages at the nucleotide level and its morphological analysis, phage vB_SlqS_ZDD2 was considered to represent a new member of the order Caudoviricetes . In conclusion, a new Serratia liquefaciens phage, vB_SlqS_ZDD2, was isolated and characterized in this study. It showed strong lytic activity, a wide range of pH and temperature tolerance, which might be useful for treating Serratia liquefaciens infections. Based on electron microscopy and complete genome analysis, vB_SlqS_ZDD2 might be a new member of the order Caudoviricetes . This study provides a theoretical basis and reference for further research on treatment of Serratia liquefaciens infections. Declarations Data availability The complete genome sequence of phage vB_SlqS_ZDD2 is available in the GenBank database, with the accession number OR881364 (The record will be released to the public database once they are processed). Compliance with Ethical Standards Funding: This project was supported by Shandong Provincial Natural Science Foundation (ZR2022QH132, ZR2016HM34). Conflicts of interest: No conflict of interests. Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors. References Momose T, Masutani S, Oshima A, Kawasaki H, Tanaka R, Iwamoto Y, Ishido H, Senzaki H (2018) First pediatric case of infective endocarditis caused by Serratia Liquefaciens . Int Heart J 59:1485–1487. https://doi.org/10.1536/ihj.17-595 Mahlen SD (2011) Serratia infections: from military experiments to current practice. Clin Microbiol Rev 24:755–791. https://doi.org/10.1128/CMR.00017-11 Gaetti-Jardim E, Nakano V, Wahasugui TC, Cabral FC, Gamba R, Avila-Campos MJ (2008) Occurrence of yeasts, enterococci and other enteric bacteria in subgingival biofilm of HIV-positive patients with chronic gingivitis and necrotizing periodontitis. Braz J Microbiol 39:257–261. https://doi.org/10.1590/S1517-83822008000200011 Jepsen K, Falk W, Brune F, Cosgarea R, Fimmers R, Bekeredjian-Ding I, Jepsen S (2022) Prevalence and antibiotic susceptibility trends of selected enterobacteriaceae , enterococci , and candida albicans in the Subgingival Microbiota of German Periodontitis Patients: A Retrospective Surveillance Study. Antibiotics (Basel) 11:385. https://doi.org/10.3390/antibiotics11030385 Hibstu Z, Belew H, Akelew Y, Mengist HM (2022) Phage Therapy: A different approach to fight bacterial infections. Biol-Targets Ther 16:173–186. https://doi.org/10.2147/BTT.S381237 Laanto E (2024) Overcoming Bacteriophage Resistance in Phage Therapy. Method Protoc 2739:401–410. https://doi.org/10.1007/978-1-0716-3549-0_23 Krylov VN, Bourkaltseva MV, Pleteneva EA (2019) Bacteriophage's Dualism in Therapy. Trends Microbiol 27:566–567. https://doi.org/10.1016/j.tim.2019.05.001 Xi HY, Dai JX, Tong YJ, Cheng MJ, Zhao FY, Fan H, Li XW, Cai RP, Ji YL, Sun CJ, Feng X, Lei LC, Rahman SU, Han WY, Gu JM (2019) The Characteristics and Genome Analysis of vB_AviM_AVP, the First Phage Infecting Aerococcus viridans . Viruses 11:104. https://doi.org/10.3390/v11020104 Li M, Li MZ, Lin H, Wang JX, Jin YQ, Han F (2016) Characterization of the novel T4-like Salmonella enterica bacteriophage STP4-a and its endolysin. Arch Virol 161:377–384. https://doi.org/10.1007/s00705-015-2647-0 Zhang L, Shahin K, Soleimani-Delfan A, Ding H, Wang H, Sun L, Wang R (2022) Phage JS02, a putative temperate phage, a novel biofilm-degrading agent for Staphylococcus aureus . Lett Appl Microbiol 75:643–654. https://doi.org/10.1111/lam.13663 Ding TY, Sun HZ, Pan Q, Zhao FY, Zhang ZZ, Ren HY (2020) Isolation and characterization of Vibrio parahaemolyticus bacteriophage vB_VpaS_PG07. Virus Res 286:198080. https://doi.org/10.1016/j.virusres.2020.198080 Svab D, Falgenhauer L, Rohde M, Chakraborty T, Toth I (2018) Identification and characterization of new broad host-range rV5-like coliphages C203 and P206 directed against enterobacteria . Infect Genet Evol 64:254–261. https://doi.org/10.1016/j.meegid.2018.07.004 Supplementary Files SupplementaryTableS1.docx Supplementaryfile.docx Cite Share Download PDF Status: Published Journal Publication published 18 Apr, 2024 Read the published version in Archives of Virology → Version 1 posted Reviewers agreed at journal 04 Jan, 2024 Reviewers invited by journal 03 Jan, 2024 First submitted to journal 21 Dec, 2023 Editor assigned by journal 21 Dec, 2023 Editorial decision: Minor Revision 20 Dec, 2023 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-3786849","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":265208948,"identity":"d416ea88-7ab9-4dea-8f14-0a77083549cd","order_by":0,"name":"Qian Zhao","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Zhao","suffix":""},{"id":265208949,"identity":"95004ff0-7054-4822-a449-53b47f506fbc","order_by":1,"name":"Linzi Han","email":"","orcid":"","institution":"Qingdao stomatological hospital affiliated to qingdao university","correspondingAuthor":false,"prefix":"","firstName":"Linzi","middleName":"","lastName":"Han","suffix":""},{"id":265208950,"identity":"0087d26f-9542-4707-a527-128784b6e776","order_by":2,"name":"Chen Chu","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Chu","suffix":""},{"id":265208951,"identity":"fd30e499-7203-4487-9ae5-178e22bc6bfb","order_by":3,"name":"Lili Wang","email":"","orcid":"","institution":"Qingdao municipal hospital affiliated to university of health and rehabilitation sciences","correspondingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Wang","suffix":""},{"id":265208952,"identity":"4ce43b8b-0e81-489e-98a4-afbdef6d1c25","order_by":4,"name":"Likun Zhuang","email":"","orcid":"","institution":"Qingdao municipal hospital affiliated to university of health and rehabilitation sciences","correspondingAuthor":false,"prefix":"","firstName":"Likun","middleName":"","lastName":"Zhuang","suffix":""},{"id":265208953,"identity":"dcb5c580-d204-4536-842c-f3cc79d40a49","order_by":5,"name":"Rongtao Yuan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYDACCTBihvIqJOT4SdRyxsJYsoEkLYxtFYkbCGmRn91jeOPnDmt5c4ncY9KF8yQYNzAwP3x0A48WxjlnjC17z6Qb7pyRlyY9c5sEszkDm7FxDh4tzBI5ZhK8bYcZN9zIMZPm3SbBZtnAwyaNTwsbUIvk37bD9hAtcyR4DA4Q0MIjAVLZdjgRoqVBQoKgFgmJtGJr2bb05A1n3iVbzzgmYSDZTMAv8jOSN95822Ztu+F47sHbBTV19f3szQ8f49OC7EZo7DATUIdFyygYBaNgFIwCNAAAa2JEmPES2MsAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0007-8457-5387","institution":"Qingdao municipal hospital affiliated to university of health and rehabilitation sciences","correspondingAuthor":true,"prefix":"","firstName":"Rongtao","middleName":"","lastName":"Yuan","suffix":""}],"badges":[],"createdAt":"2023-12-21 12:19:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3786849/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3786849/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00705-024-06026-2","type":"published","date":"2024-04-18T23:30:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49299609,"identity":"7a03fddd-b3b8-425e-9fcc-ef363f500cef","added_by":"auto","created_at":"2024-01-08 09:11:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":810160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological characteristics and biological characterizations of phage vB_SlqS_ZDD2. \u003c/strong\u003e(A) Plaque morphology. (B) TEM photograph. (C) The MOI test of phage. (D) One-step growth curve. (E) pH stability. (F) Temperature stability.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3786849/v1/b4326f3ec29decb2bd482e2d.png"},{"id":49300208,"identity":"7f95dc62-8485-4eec-abed-916f8ddd15f3","added_by":"auto","created_at":"2024-01-08 09:19:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1006953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComplete genome and phylogenetic analysis of phage vB_SlqS_ZDD2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Circular representation depicting the whole genome of phage vB_SlqS_ZDD2. The outside ring showed the physical map of the genome scaled in kbp. The inner rings showed the histogram represent GC content (black) and GC skew (green/purple). (B) Graphical representation of the whole genome of vB_SlqS_ZDD2. Different colors indicated the different functional categories of the genes, and arrows indicated the orientation of each ORF. (C) ViPTree analysis of phage vB_SlqS_ZDD2. A total of 3179 phage sequences were used to construct phylogenetic trees. Phages were identified and classified by ICTV, the outer and inner rings represented the host group and virus family, respectively. In addition, an expanded view of the region of the tree containing the most closely related phages were shown, vB_SlqS_ZDD2 is shown in red and with star.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3786849/v1/b8c0e224f174c073b0499b7f.png"},{"id":55696610,"identity":"599b7389-9884-4889-bb8f-e18c1b67accc","added_by":"auto","created_at":"2024-05-02 01:47:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1565168,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3786849/v1/9df6d2fa-b6df-4742-8c4b-101af1887983.pdf"},{"id":49299615,"identity":"a439a761-4388-4c01-9c90-76dc78b977a7","added_by":"auto","created_at":"2024-01-08 09:11:52","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":42215,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3786849/v1/445dcb802e20e1c206cbef8b.docx"},{"id":49299612,"identity":"cbbaa546-4619-4221-868f-1d1e6866f6d1","added_by":"auto","created_at":"2024-01-08 09:11:51","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":40739,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-3786849/v1/17cc60e342b91dd0710dbe40.docx"}],"financialInterests":"","formattedTitle":"Biological characterization and complete genome analysis of the newly isolated Serratia liquefaciens phage vB_SlqS_ZDD2","fulltext":[{"header":"Main Text","content":"\u003cp\u003e\u003cem\u003eSerratia liquefaciens\u0026nbsp;\u003c/em\u003eis one of the opportunistic pathogens that widely exited in the environment and it is the major cause of \u003cem\u003eSerratia\u003c/em\u003e infections which can lead bacteremia, respiratory tract infection, pneumonia and a series of other diseases [1, 2]. In recent years, \u003cem\u003eSerratia liquefaciens\u0026nbsp;\u003c/em\u003ehas developed high resistance to currently available antibiotics, such as Penicillin, and more and more\u0026nbsp;multidrug-resistant strainswere also found\u0026nbsp;[3, 4]. Hence, it is necessary to develop efficient methods to combat \u003cem\u003eSerratia liquefaciens\u003c/em\u003e infections.\u003c/p\u003e\n\u003cp\u003eAs the most abundant microorganisms on the earth, bacteriophages could invade bacteria and have the advantage of host specificity and non-toxicity to animals, which are proposed to be the alternatives to conventional antibiotic treatments [5-7]. In this study, a \u003cem\u003eSerratia liquefaciens\u003c/em\u003e phage, named vB_SlqS_ZDD2, was isolated from hospital sewage and its biological characterizations and complete genome sequence were determined.\u003c/p\u003e\n\u003cp\u003evB_SlqS_ZDD2 showed strong lytic properties and formed clear plaques on the plates (approximately 0.5-1.0 mm in diameter) (Fig. 1A). Transmission electron microscope was used to explore the morphology of phage, and the result showed that vB_SlqS_ZDD2 has a head with a diameter of 64-69 nm and a long tail of 143-150 nm in length (Fig. 1B). The morphological analysis indicated that phage vB_SlqS_ZDD2 may be a member of the order \u003cem\u003eCaudoviricetes\u003c/em\u003e, showing Siphovirus characteristic features.\u003c/p\u003e\n\u003cp\u003eTo evaluate the biological characterizations of phage vB_SlqS_ZDD2, the optimal multiplicity of infection (MOI), one-step growth curve, pH and temperature stability of this phage were measured. MOI of phage was determined according to the procedures described by Xi et al. with modification [8]. Briefly, phage was added into the host bacteria culture according to different MOI ratios, and the mixture was incubated\u0026nbsp;at 37\u0026deg;C, 180 rpm for 8 h. Double-layer agar method was used to measure the phage titer\u0026nbsp;[9]. The results (Fig. 1C) showed that the phage titer reached 1.9\u0026nbsp;\u0026times; 10\u003csup\u003e10\u003c/sup\u003e PFU/ml at an MOI of 0.001, which was significantly higher (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01) than other groups. To test the life cycle of phage, one-step growth curve measurement was conducted [10]. Firstly, vB_SlqS_ZDD2 was mixed with host bacteria at the optimal MOI of 0.001 and incubated at 37\u0026deg;C for 15 min. Then, the culture was centrifuged (12,000 \u0026times; g, 2 min) to remove the unabsorbed phage, and the pellet was resuspended and transferred into 50 ml\u0026nbsp;LB medium and incubated at 37\u0026deg;C (140 rpm). Next, three samples were taken at 0, 5, 10 min, and then every 10 min for the first 120 min and every 30 min from 120 to 270 min of incubation. The phage titer\u0026nbsp;was determined immediately by double-layer method. As shown in Fig 1D, the latent period of vB_SlqS_ZDD2 was approximately 5 min, and the lysis period was approximately 140 min, as well as the burst size of phage was 104 PFU per cell.\u003c/p\u003e\n\u003cp\u003eThe pH stability of phage vB_SlqS_ZDD2 was examined as Ding et al. described with minor modification [11]. The phage was incubated in LB broth at various pH values for 1 h and then the titer was measured. The results (Fig. 1E) showed that phage vB_SlqS_ZDD2 exhibited a strong tolerance from pH 3-11, and it was inactivated when the pH below 2 or above 13. To investigate its temperature stability [12], phage samples were incubated at different temperatures (40, 50, 60, 70 and 80℃). Samples were collected every 20 min from 0 to 80 min of incubation and the titer was determined. As displayed in Fig. 1F, the titer of phage vB_SlqS_ZDD2 were remaining basically unchanged from 0 to 80 min when the temperature below 60℃. When the temperature rose to 70℃, phage titer decreased from 2.5 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e to 6.7 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e PFU/ml (0 to 80 min). However, phage vB_SlqS_ZDD2 maintained a titer of 2.3 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e PFU/ml even after incubation at\u0026nbsp;80\u0026thinsp;\u0026deg;C for 80\u0026thinsp;min, indicating that it has excellent temperature stability.\u003c/p\u003e\n\u003cp\u003ePhage genomic DNA was extracted using phage DNA isolation kit (Tiangen Biotek, Beijing) according to the manufacturer\u0026apos;s instructions. High-throughput sequencing of the genomic DNA of phage vB_SlqS_ZDD2 was performed by BGI-Shenzhen\u0026nbsp;using a MGISEQ-2000 platform.\u0026nbsp;Whole genome sequence of phage was submitted to the NCBI GenBank database with the accession number OR881364. The genome map was shown in Fig. 2A\u0026amp;B. Genome sequence results revealed that phage vB_SlqS_ZDD2 was 49,178 bp with 55% GC content. Open reading frames (ORFs) were predicted using RAST software (https://rast.nmpdr.org/rast.cgi) and subsequently verified using the ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). There were 74 ORFs in the genome of phage vB_SlqS_ZDD2, and 20 of which were functionally annotated by searching the non-redundant protein database using BLASTp (http://blast.ncbi.nlm.nih.gov/). These major functional proteins could be classified into five categories according to NCBI and UniProt databases (Fig. 2B): structural proteins (gp01, gp02, gp03, gp05, gp06, gp07, gp72, gp74), DNA replication and nucleotide metabolism (gp10, gp25, gp60, gp66), DNA packaging (gp16), lysis proteins (gp19, gp20) and other functions proteins (gp04, gp08, gp15, gp71, gp73). Among all open reading frames, there are three different initial codons, with ATG having the most, accounting for 79.73% (59/74). All open reading frames can be divided into two encoding directions, with ORF22, ORF57, ORF58, ORF59, ORF62, ORF63, ORF64, ORF65, ORF66 and ORF67 being forward encoding, and the remaining 64 open reading frames being reverse encoding (Fig. 2B). In addition, no\u0026nbsp;tRNA genes were found using tRNAscan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/). No genes related to drug resistance, virulence, or toxins were found in the genome of phage vB_SlqS_ZDD2, which indicated that vB_SlqS_ZDD2 might be a potential biotherapeutic agent against \u003cem\u003eSerratia liquefaciens\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003ePhylogenetic relationship of phage vB_SlqS_ZDD2 to other reported prokaryotic double-stranded DNA viruses was analyzed using\u0026nbsp;the ViPTree version 2.1 (https://www.genome.jp/viptree/) based on the whole genome (Fig. 2C). A total of 3178 sequences were selected by system into comparing with phage vB_SlqS_ZDD2\u0026nbsp;(Fig. 2C showed section data), and the results showed that phage vB_SlqS_ZDD2 was grouped into one new group.\u0026nbsp;Further BLASTn alignment of phage vB_SlqS_ZDD2\u0026nbsp;showed that it shared 0% query coverage with other phages in the NCBI database, which demonstrated that it is a newly discovered phage. Therefore, based on its lower similarity to other phages at the nucleotide level and its morphological analysis, phage vB_SlqS_ZDD2 was considered to\u0026nbsp;represent a new member of the order \u003cem\u003eCaudoviricetes\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn conclusion, a new \u003cem\u003eSerratia liquefaciens\u003c/em\u003e phage, vB_SlqS_ZDD2, was isolated and characterized in this study. It showed strong lytic activity, a wide range of pH and temperature tolerance, which might be useful for treating \u003cem\u003eSerratia liquefaciens\u003c/em\u003e infections. Based on electron microscopy and complete genome analysis, vB_SlqS_ZDD2 might be a new member\u0026nbsp;of the order \u003cem\u003eCaudoviricetes\u003c/em\u003e. This study provides a theoretical basis and reference for further research on treatment of \u003cem\u003eSerratia liquefaciens\u003c/em\u003e infections.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe complete genome sequence of phage\u0026nbsp;vB_SlqS_ZDD2\u0026nbsp;is available in the GenBank database, with the accession number\u0026nbsp;OR881364 (The record will be released to the public database once they are processed).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding: \u003c/strong\u003eThis project was supported by Shandong Provincial Natural Science Foundation (ZR2022QH132, ZR2016HM34).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest: \u003c/strong\u003eNo conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval: \u003c/strong\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMomose T, Masutani S, Oshima A, Kawasaki H, Tanaka R, Iwamoto Y, Ishido H, Senzaki H (2018) First pediatric case of infective endocarditis caused by \u003cem\u003eSerratia Liquefaciens\u003c/em\u003e. Int Heart J 59:1485\u0026ndash;1487. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1536/ihj.17-595\u003c/span\u003e\u003cspan address=\"10.1536/ihj.17-595\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahlen SD (2011) \u003cem\u003eSerratia\u003c/em\u003e infections: from military experiments to current practice. Clin Microbiol Rev 24:755\u0026ndash;791. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/CMR.00017-11\u003c/span\u003e\u003cspan address=\"10.1128/CMR.00017-11\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGaetti-Jardim E, Nakano V, Wahasugui TC, Cabral FC, Gamba R, Avila-Campos MJ (2008) Occurrence of yeasts, \u003cem\u003eenterococci\u003c/em\u003e and other enteric bacteria in subgingival biofilm of HIV-positive patients with chronic gingivitis and necrotizing periodontitis. Braz J Microbiol 39:257\u0026ndash;261. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/S1517-83822008000200011\u003c/span\u003e\u003cspan address=\"10.1590/S1517-83822008000200011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJepsen K, Falk W, Brune F, Cosgarea R, Fimmers R, Bekeredjian-Ding I, Jepsen S (2022) Prevalence and antibiotic susceptibility trends of selected \u003cem\u003eenterobacteriaceae\u003c/em\u003e, \u003cem\u003eenterococci\u003c/em\u003e, and \u003cem\u003ecandida albicans\u003c/em\u003e in the Subgingival Microbiota of German Periodontitis Patients: A Retrospective Surveillance Study. Antibiotics (Basel) 11:385. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/antibiotics11030385\u003c/span\u003e\u003cspan address=\"10.3390/antibiotics11030385\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHibstu Z, Belew H, Akelew Y, Mengist HM (2022) Phage Therapy: A different approach to fight bacterial infections. Biol-Targets Ther 16:173\u0026ndash;186. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/BTT.S381237\u003c/span\u003e\u003cspan address=\"10.2147/BTT.S381237\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaanto E (2024) Overcoming Bacteriophage Resistance in Phage Therapy. Method Protoc 2739:401\u0026ndash;410. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-1-0716-3549-0_23\u003c/span\u003e\u003cspan address=\"10.1007/978-1-0716-3549-0_23\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrylov VN, Bourkaltseva MV, Pleteneva EA (2019) Bacteriophage's Dualism in Therapy. Trends Microbiol 27:566\u0026ndash;567. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tim.2019.05.001\u003c/span\u003e\u003cspan address=\"10.1016/j.tim.2019.05.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXi HY, Dai JX, Tong YJ, Cheng MJ, Zhao FY, Fan H, Li XW, Cai RP, Ji YL, Sun CJ, Feng X, Lei LC, Rahman SU, Han WY, Gu JM (2019) The Characteristics and Genome Analysis of vB_AviM_AVP, the First Phage Infecting \u003cem\u003eAerococcus viridans\u003c/em\u003e. Viruses 11:104. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/v11020104\u003c/span\u003e\u003cspan address=\"10.3390/v11020104\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi M, Li MZ, Lin H, Wang JX, Jin YQ, Han F (2016) Characterization of the novel T4-like \u003cem\u003eSalmonella enterica\u003c/em\u003e bacteriophage STP4-a and its endolysin. Arch Virol 161:377\u0026ndash;384. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00705-015-2647-0\u003c/span\u003e\u003cspan address=\"10.1007/s00705-015-2647-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Shahin K, Soleimani-Delfan A, Ding H, Wang H, Sun L, Wang R (2022) Phage JS02, a putative temperate phage, a novel biofilm-degrading agent for \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. Lett Appl Microbiol 75:643\u0026ndash;654. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/lam.13663\u003c/span\u003e\u003cspan address=\"10.1111/lam.13663\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing TY, Sun HZ, Pan Q, Zhao FY, Zhang ZZ, Ren HY (2020) Isolation and characterization of \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e bacteriophage vB_VpaS_PG07. Virus Res 286:198080. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.virusres.2020.198080\u003c/span\u003e\u003cspan address=\"10.1016/j.virusres.2020.198080\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSvab D, Falgenhauer L, Rohde M, Chakraborty T, Toth I (2018) Identification and characterization of new broad host-range rV5-like coliphages C203 and P206 directed against \u003cem\u003eenterobacteria\u003c/em\u003e. Infect Genet Evol 64:254\u0026ndash;261. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.meegid.2018.07.004\u003c/span\u003e\u003cspan address=\"10.1016/j.meegid.2018.07.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3786849/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3786849/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA novel lytic phage named vB_SlqS_ZDD2 infecting \u003cem\u003eSerratia liquefaciens\u003c/em\u003e was isolated from hospital sewage. BLASTn analysis showed that the genome sequence of phage vB_SlqS_ZDD2 shared 0% query coverage with other phages in the NCBI database. Phenotype and phylogeny analysis indicated that this phage might be a new member of the order \u003cem\u003eCaudoviricetes\u003c/em\u003e. Genome sequence displayed that phage vB_SlqS_ZDD2 has a 49,178 bp dsDNA with 55% GC content and has 74 open reading frames. Besides, the phage exhibited strong lytic activity and a wide range of pH (3-12) and temperature tolerance (80℃, 80 min).\u003c/p\u003e","manuscriptTitle":"Biological characterization and complete genome analysis of the newly isolated Serratia liquefaciens phage vB_SlqS_ZDD2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-08 09:11:46","doi":"10.21203/rs.3.rs-3786849/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-01-04T16:22:42+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-03T18:44:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2023-12-21T05:36:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-12-21T05:28:27+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor Revision","date":"2023-12-21T03:00:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b6fe8aa2-d7fc-4e6f-a470-8e7865734161","owner":[],"postedDate":"January 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-01T23:30:56+00:00","versionOfRecord":{"articleIdentity":"rs-3786849","link":"https://doi.org/10.1007/s00705-024-06026-2","journal":{"identity":"archives-of-virology","isVorOnly":false,"title":"Archives of Virology"},"publishedOn":"2024-04-18 23:30:56","publishedOnDateReadable":"April 18th, 2024"},"versionCreatedAt":"2024-01-08 09:11:46","video":"","vorDoi":"10.1007/s00705-024-06026-2","vorDoiUrl":"https://doi.org/10.1007/s00705-024-06026-2","workflowStages":[]},"version":"v1","identity":"rs-3786849","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3786849","identity":"rs-3786849","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}