{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T13:18:06Z","timestamp":1789132686412,"version":"build-2803163510"},"reference-count":34,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,10,22]],"date-time":"2025-10-22T00:00:00Z","timestamp":1761091200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Key Research","award":["2023YFD1800303"],"award-info":[{"award-number":["2023YFD1800303"]}]},{"name":"Development Program of China","award":["2021YFD1800900"],"award-info":[{"award-number":["2021YFD1800900"]}]},{"name":"Key R&D Program of Henan Province","award":["242102111032"],"award-info":[{"award-number":["242102111032"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibiotics"],"abstract":"<jats:p>Objectives: To explore the genetic basis of phage resistance in sequentially generated capsular mutants of phage-resistant Klebsiella pneumoniae using an established phage library. Methods: Sequential induction strategies were employed to obtain phage-resistant K. pneumoniae capsular mutants by exposing ST11-K64 K. pneumoniae Kp2325 to different single phages. Whole genome sequencing and bioinformatic analysis were used to elucidate the capsular-related genetic changes in phage-resistant mutants. Phenotypic changes were assessed through gene complementation, growth assays, phage cleavage spectrum analysis, TEM for phage morphology, CPS analysis, biofilm formation, and virulence assays. Results: Three sequentially generated phage-resistant K. pneumoniae capsular mutants were obtained, designated R1, R2 and R3. The narrowing of the phage cleavage spectrum and the evolutionary trade-offs of biological phenotypes were observed. Key genetic changes included: (1) ISKpn26 insertion disrupting wcaJ in R1; (2) combined wcaJ insertion and 9-bp deletion in waaH in R2; and (3) CPS gene cluster deletion in R3 were identified as key mechanisms of phage resistance in K. pneumoniae mutants R1, R2 and R3, respectively. Conclusions: Sequential exposure to different single phages led to rapid evolution of phage resistance in K. pneumoniae via genetic mutations that disrupt capsular synthesis. These findings highlight the critical role of bacterial capsule in phage\u2013host interactions and emphasize the need to use phage cocktails targeting different types of receptors to counteract the evolution of bacterial defense mechanisms in phage therapy.<\/jats:p>","DOI":"10.3390\/antibiotics14111056","type":"journal-article","created":{"date-parts":[[2025,10,22]],"date-time":"2025-10-22T07:03:51Z","timestamp":1761116631000},"page":"1056","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Identification of the Genetic Basis of Phage Resistance in Sequentially Generated Phage-Resistant Klebsiella pneumoniae Using an Established Phage Library"],"prefix":"10.3390","volume":"14","author":[{"given":"Wenbo","family":"Zhao","sequence":"first","affiliation":[{"name":"International Joint Research Center of National Animal Immunology, Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Congyang","family":"Du","sequence":"additional","affiliation":[{"name":"International Joint Research Center of National Animal Immunology, Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zheng","family":"Chen","sequence":"additional","affiliation":[{"name":"International Joint Research Center of National Animal Immunology, Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yunze","family":"Zhao","sequence":"additional","affiliation":[{"name":"International Joint Research Center of National Animal Immunology, Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6303-8212","authenticated-orcid":false,"given":"Stefan","family":"Schwarz","sequence":"additional","affiliation":[{"name":"Institute of Microbiology and Epizootics, Centre for Infection Medicine, School of Veterinary Medicine, Freie Universit\u00e4t Berlin, 14195 Berlin, Germany"},{"name":"Veterinary Centre of Resistance Research (TZR), School of Veterinary Medicine, Freie Universit\u00e4t Berlin, 14195 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hong","family":"Yao","sequence":"additional","affiliation":[{"name":"International Joint Research Center of National Animal Immunology, Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3043-7230","authenticated-orcid":false,"given":"Chenglong","family":"Li","sequence":"additional","affiliation":[{"name":"International Joint Research Center of National Animal Immunology, Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunyan","family":"Xu","sequence":"additional","affiliation":[{"name":"International Joint Research Center of National Animal Immunology, Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7431-7607","authenticated-orcid":false,"given":"Xiang-Dang","family":"Du","sequence":"additional","affiliation":[{"name":"International Joint Research Center of National Animal Immunology, Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1128\/MMBR.00078-15","article-title":"Klebsiella pneumoniae: Going on the offense with a strong defense","volume":"80","author":"Paczosa","year":"2016","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e00001-19","DOI":"10.1128\/CMR.00001-19","article-title":"Hypervirulent Klebsiella pneumoniae","volume":"32","author":"Russo","year":"2019","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/S1473-3099(18)30605-4","article-title":"Attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the EU and the European Economic Area in 2015: A population-level modelling analysis","volume":"19","author":"Cassini","year":"2019","journal-title":"Lancet Infect. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2658","DOI":"10.1016\/j.scib.2023.09.040","article-title":"\u201cSuperbugs\u201d with hypervirulence and carbapenem resistance in Klebsiella pneumoniae: The rise of such emerging nosocomial pathogens in China","volume":"68","author":"Pu","year":"2023","journal-title":"Sci. Bull."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"212","DOI":"10.1016\/j.tibtech.2006.03.003","article-title":"Bacteriophages and biotechnology: Vaccines, gene therapy and antibacterials","volume":"24","author":"Clark","year":"2006","journal-title":"Trends Biotechnol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e2041","DOI":"10.1002\/rmv.2041","article-title":"The dawn of phage therapy","volume":"29","author":"Rehman","year":"2019","journal-title":"Rev. Med. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Wang, Z., Cai, R., Wang, G., Guo, Z., Liu, X., Guan, Y., Ji, Y., Zhang, H., Xi, H., and Zhao, R. (2021). Combination therapy of phage vB_KpnM_P-KP2 and gentamicin combats acute pneumonia caused by K47 serotype Klebsiella pneumoniae. Front. Microbiol., 12.","DOI":"10.3389\/fmicb.2021.674068"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1016\/j.jgar.2019.09.018","article-title":"Phage therapy for treatment of virulent Klebsiella pneumoniae infection in a mouse model","volume":"21","author":"Anand","year":"2020","journal-title":"J. Glob. Antimicrob. Resist."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1734","DOI":"10.1093\/infdis\/jiu332","article-title":"Isolation of a bacteriophage and its depolymerase specific for K1 capsule of Klebsiella pneumoniae: Implication in typing and treatment","volume":"210","author":"Lin","year":"2014","journal-title":"J. Infect. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e01281-19","DOI":"10.1128\/AAC.01281-19","article-title":"A dutch case report of successful treatment of chronic relapsing urinary tract infection with bacteriophages in a renal transplant patient","volume":"64","author":"Kuipers","year":"2019","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"771","DOI":"10.1080\/22221751.2020.1747950","article-title":"Non-active antibiotic and bacteriophage synergism to successfully treat recurrent urinary tract infection caused by extensively drug-resistant Klebsiella pneumoniae","volume":"9","author":"Bao","year":"2020","journal-title":"Emerg. Microbes Infect."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e00926-19","DOI":"10.1128\/IAI.00926-19","article-title":"Fitness trade-offs resulting from bacteriophage resistance potentiate synergistic antibacterial strategies","volume":"88","author":"Mangalea","year":"2020","journal-title":"Infect. Immun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1601.e1","DOI":"10.1016\/j.cmi.2023.08.022","article-title":"Development of phage resistance in multidrug-resistant Klebsiella pneumoniae is associated with reduced virulence: A case report of a personalised phage therapy","volume":"29","author":"Li","year":"2023","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3261","DOI":"10.1038\/s41467-023-39029-9","article-title":"Bacteriophage therapy against pathological Klebsiella pneumoniae ameliorates the course of primary sclerosing cholangitis","volume":"14","author":"Ichikawa","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"106218","DOI":"10.1016\/j.micpath.2023.106218","article-title":"Effective of phage cocktail against Klebsiella pneumoniae infection of murine mammary glands","volume":"182","author":"Liang","year":"2023","journal-title":"Microb. Pathog."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1038\/nrmicro2315","article-title":"Bacteriophage resistance mechanisms","volume":"8","author":"Labrie","year":"2010","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e00093-19","DOI":"10.1128\/JB.00093-19","article-title":"Regulation of waaH by PhoB during Pi starvation promotes biofilm formation by Escherichia coli O157:H7","volume":"201","author":"Vogeleer","year":"2019","journal-title":"J. Bacteriol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"891","DOI":"10.1099\/mic.0.000827","article-title":"Absence of the glycosyltransferase WcaJ in Klebsiella pneumoniae ATCC13883 affects biofilm formation, increases polymyxin resistance and reduces murine macrophage activation","volume":"165","author":"Pal","year":"2019","journal-title":"Microbiology"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"289","DOI":"10.3201\/eid2602.190594","article-title":"Novel subclone of carbapenem-resistant Klebsiella pneumoniae sequence type 11 with enhanced virulence and transmissibility, China","volume":"26","author":"Zhou","year":"2020","journal-title":"Emerg. Infect. Dis."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Qin, K., Shi, X., Yang, K., Xu, Q., Wang, F., Chen, S., Xu, T., Liu, J., Wen, W., and Chen, R. (2024). Phage-antibiotic synergy suppresses resistance emergence of Klebsiella pneumoniae by altering the evolutionary fitness. mBio, 15.","DOI":"10.1128\/mbio.01393-24"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Devanga Ragupathi, N.K., Muthuirulandi Sethuvel, D.P., Triplicane Dwarakanathan, H., Murugan, D., Umashankar, Y., Monk, P.N., Karunakaran, E., and Veeraraghavan, B. (2020). The influence of biofilms on carbapenem susceptibility and patient outcome in device associated K. pneumoniae infections: Insights into phenotype vs genome-wide analysis and correlation. Front. Microbiol., 11.","DOI":"10.3389\/fmicb.2020.591679"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Tan, D., Zhang, Y., Qin, J., Le, S., Gu, J., Chen, L.K., Guo, X., and Zhu, T. (2020). A frameshift mutation in wcaJ associated with phage resistance in Klebsiella pneumoniae. Microorganisms, 8.","DOI":"10.3390\/microorganisms8030378"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Geng, H., Song, L., Yang, X., Xing, S., Wang, R., Xu, Y., Jia, X., and Luan, G. (2023). Resistance of Klebsiella pneumoniae to phage hvKpP3 Due to high-molecular weight lipopolysaccharide synthesis failure. Microbiol. Spectr., 11.","DOI":"10.1128\/spectrum.04384-22"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"675","DOI":"10.1038\/nrmicro3096","article-title":"Revenge of the phages: Defeating bacterial defences","volume":"11","author":"Samson","year":"2013","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"760","DOI":"10.1038\/s41579-018-0070-8","article-title":"Targeting mechanisms of tailed bacteriophages","volume":"16","author":"Nobrega","year":"2018","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Thomanek, N., Arends, J., Lindemann, C., Barkovits, K., Meyer, H.E., Marcus, K., and Narberhaus, F. (2019). Intricate crosstalk between lipopolysaccharide, phospholipid and fatty acid metabolism in Escherichia coli modulates proteolysis of lpxC. Front. Microbiol., 9.","DOI":"10.3389\/fmicb.2018.03285"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"8111","DOI":"10.1074\/jbc.M112.445981","article-title":"Molecular and structural basis of inner core lipopolysaccharide alterations in Escherichia coli: Incorporation of glucuronic acid and phosphoethanolamine in the heptose region","volume":"288","author":"Klein","year":"2013","journal-title":"J. Biol. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"e02530-19","DOI":"10.1128\/mBio.02530-19","article-title":"Phage resistance in multidrug-resistant Klebsiella pneumoniae ST258 evolves via diverse mutations that culminate in impaired adsorption","volume":"11","author":"Hesse","year":"2020","journal-title":"mBio"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Cai, R., Wang, G., Le, S., Wu, M., Cheng, M., Guo, Z., Ji, Y., Xi, H., Zhao, C., and Wang, X. (2019). Three capsular polysaccharide synthesis-related glucosyltransferases, GT-1, GT-2 and WcaJ, are associated with virulence and phage sensitivity of Klebsiella pneumoniae. Front. Microbiol., 10.","DOI":"10.3389\/fmicb.2019.01189"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1452.e1","DOI":"10.1016\/j.cmi.2023.07.030","article-title":"Emergence of high-level tigecycline resistance due to the amplification of a tet(A) gene variant in clinical carbapenem-resistant Klebsiella pneumoniae","volume":"29","author":"Yu","year":"2023","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Stachurska, X., Roszak, M., Jab\u0142o\u0144ska, J., Mizieli\u0144ska, M., and Nawrotek, P. (2021). Double-layer agar (DLA) modifications for the first step of the phage-antibiotic synergy (PAS) identification. Antibiotics, 10.","DOI":"10.3390\/antibiotics10111306"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1186\/s12985-021-01655-4","article-title":"Genomic and functional characterization of five novel salmonella-targeting bacteriophages","volume":"18","author":"Ugorski","year":"2021","journal-title":"Virol. J."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Cai, R., Wu, M., Zhang, H., Zhang, Y., Cheng, M., Guo, Z., Ji, Y., Xi, H., Wang, X., and Xue, Y. (2018). A Smooth-Type, Phage-Resistant Klebsiella pneumoniae Mutant Strain Reveals that OmpC Is Indispensable for Infection by Phage GH-K3. Appl. Environ. Microbiol., 84.","DOI":"10.1128\/AEM.01585-18"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Mike, L.A., Stark, A.J., Forsyth, V.S., Vornhagen, J., Smith, S.N., Bachman, M.A., and Mobley, H.L. (2021). A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness. PLoS Pathog., 17.","DOI":"10.1371\/journal.ppat.1009376"}],"container-title":["Antibiotics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6382\/14\/11\/1056\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,22]],"date-time":"2025-10-22T07:18:05Z","timestamp":1761117485000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6382\/14\/11\/1056"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,22]]},"references-count":34,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["antibiotics14111056"],"URL":"https:\/\/doi.org\/10.3390\/antibiotics14111056","relation":{},"ISSN":["2079-6382"],"issn-type":[{"value":"2079-6382","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,22]]}}}