{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T20:07:07Z","timestamp":1791576427740,"version":"4.3.4"},"reference-count":49,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2020,4,30]],"date-time":"2020-04-30T00:00:00Z","timestamp":1588204800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001704","name":"European Society of Clinical Microbiology and Infectious Diseases","doi-asserted-by":"publisher","award":["20200063"],"award-info":[{"award-number":["20200063"]}],"id":[{"id":"10.13039\/501100001704","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000781","name":"European Research Council","doi-asserted-by":"publisher","award":["724519"],"award-info":[{"award-number":["724519"]}],"id":[{"id":"10.13039\/501100000781","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004837","name":"Ministerio de Ciencia e Innovaci\u00f3n","doi-asserted-by":"publisher","award":["BFU2017-89594R"],"award-info":[{"award-number":["BFU2017-89594R"]}],"id":[{"id":"10.13039\/501100004837","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100014440","name":"Ministerio de Ciencia, Innovaci\u00f3n y Universidades","doi-asserted-by":"publisher","award":["FPU16\/02139"],"award-info":[{"award-number":["FPU16\/02139"]}],"id":[{"id":"10.13039\/100014440","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The emergence of multidrug-resistant bacteria is a major global health concern. The search for new therapies has brought bacteriophages into the spotlight, and new phages are being described as possible therapeutic agents. Among the bacteria that are most extensively resistant to current antibiotics is Klebsiella pneumoniae, whose hypervariable extracellular capsule makes treatment particularly difficult. Here, we describe two new K. pneumoniae phages, \u03c0VLC5 and \u03c0VLC6, isolated from environmental samples. These phages belong to the genus Drulisvirus within the family Podoviridae. Both phages encode a similar tail spike protein with putative depolymerase activity, which is shared among other related phages and probably determines their ability to specifically infect K. pneumoniae capsular types K22 and K37. In addition, we found that phage \u03c0VLC6 also infects capsular type K13 and is capable of striping the capsules of K. pneumoniae KL2 and KL3, although the phage was not infectious in these two strains. Genome sequence analysis suggested that the extended tropism of phage \u03c0VLC6 is conferred by a second, divergent depolymerase. Phage \u03c0VLC5 encodes yet another putative depolymerase, but we found no activity of this phage against capsular types other than K22 and K37, after testing a panel of 77 reference strains. Overall, our results confirm that most phages productively infected one or few Klebsiella capsular types. This constitutes an important challenge for clinical applications.<\/jats:p>","DOI":"10.3390\/ijms21093160","type":"journal-article","created":{"date-parts":[[2020,5,4]],"date-time":"2020-05-04T14:00:43Z","timestamp":1588600843000},"page":"3160","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":58,"title":["Isolation and Characterization of Two Klebsiella pneumoniae Phages Encoding Divergent Depolymerases"],"prefix":"10.3390","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2829-8809","authenticated-orcid":false,"given":"Pilar","family":"Domingo-Calap","sequence":"first","affiliation":[{"name":"Institute for Integrative Systems Biology, I<sup>2<\/sup>SysBio, Universitat de Val\u00e8ncia-CSIC, 46980 Paterna, Spain"},{"name":"Department of Genetics, Universitat de Val\u00e8ncia, 46980 Paterna, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0863-3390","authenticated-orcid":false,"given":"Beatriz","family":"Beamud","sequence":"additional","affiliation":[{"name":"FISABIO-Salud P\u00fablica, Generalitat Valenciana, 46020 Valencia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9933-9861","authenticated-orcid":false,"given":"Lucas","family":"Mora-Quilis","sequence":"additional","affiliation":[{"name":"Institute for Integrative Systems Biology, I<sup>2<\/sup>SysBio, Universitat de Val\u00e8ncia-CSIC, 46980 Paterna, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0879-5798","authenticated-orcid":false,"given":"Fernando","family":"Gonz\u00e1lez-Candelas","sequence":"additional","affiliation":[{"name":"Institute for Integrative Systems Biology, I<sup>2<\/sup>SysBio, Universitat de Val\u00e8ncia-CSIC, 46980 Paterna, Spain"},{"name":"FISABIO-Salud P\u00fablica, Generalitat Valenciana, 46020 Valencia, Spain"},{"name":"CIBER in Epidemiology and Public Health, 46020 Valencia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1844-545X","authenticated-orcid":false,"given":"Rafael","family":"Sanju\u00e1n","sequence":"additional","affiliation":[{"name":"Institute for Integrative Systems Biology, I<sup>2<\/sup>SysBio, Universitat de Val\u00e8ncia-CSIC, 46980 Paterna, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,4,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"E3574","DOI":"10.1073\/pnas.1501049112","article-title":"Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health","volume":"112","author":"Holt","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s12941-019-0343-8","article-title":"Klebsiella pneumoniae: An increasing threat to public health","volume":"19","author":"Effah","year":"2020","journal-title":"Ann. Clin. Microbiol. Antimicrob."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Founou, R.C., Founou, L.L., and Essack, S.Y. (2017). Clinical and economic impact of antibiotic resistance in developing countries: A systematic review and meta-analysis. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0189621"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1111\/tan.12742","article-title":"Back to the future: Bacteriophages as promising therapeutic tools","volume":"87","author":"Georgel","year":"2016","journal-title":"HLA"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2517","DOI":"10.3389\/fmicb.2018.02517","article-title":"Phage-Borne Depolymerases Decrease Klebsiella pneumoniae Resistance to Innate Defense Mechanisms","volume":"9","author":"Latka","year":"2018","journal-title":"Front. Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/j.chom.2019.01.014","article-title":"Phage Therapy: A Renewed Approach to Combat Antibiotic-Resistant Bacteria","volume":"25","author":"Kortright","year":"2019","journal-title":"Cell Host Microbe"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"946","DOI":"10.1186\/s12879-019-4498-x","article-title":"Clinical characteristics in blood stream infections caused by Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae: A comparative study, Japan, 2014\u20132017","volume":"19","author":"Imai","year":"2019","journal-title":"BMC Infect. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e01443-18","DOI":"10.1128\/mBio.01443-18","article-title":"Identification of Two Regulators of Virulence That Are Conserved inKlebsiella pneumonia Classical and Hypervirulent Strains","volume":"9","author":"Palacios","year":"2018","journal-title":"mBio"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"719","DOI":"10.1016\/0042-6822(56)90054-X","article-title":"An enzyme produced by a phage-host cell system","volume":"2","author":"Adams","year":"1956","journal-title":"Virology"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1016\/0042-6822(81)90162-8","article-title":"Comparative study of host capsule depolymerases associated with Klebsiella bacteriophages","volume":"113","author":"Stirm","year":"1981","journal-title":"Virology"},{"key":"ref_11","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_12","doi-asserted-by":"crossref","unstructured":"Majkowska-Skrobek, G., Latka, A., Berisio, R., Maciejewska, B., Squeglia, F., Romano, M., Lavigne, R., Struve, C., and Drulis-Kawa, Z. (2016). Capsule-Targeting Depolymerase, Derived from Klebsiella KP36 Phage, as a Tool for the Development of Anti-Virulent Strategy. Viruses, 8.","DOI":"10.3390\/v8120324"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4624","DOI":"10.1038\/s41598-017-04644-2","article-title":"Two T7-like Bacteriophages, K5-2 and K5-4, Each Encodes Two Capsule Depolymerases: Isolation and Functional Characterization","volume":"7","author":"Hsieh","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2768","DOI":"10.3389\/fmicb.2019.02768","article-title":"A Novel Polysaccharide Depolymerase Encoded by the Phage SH-KP152226 Confers Specific Activity Against Multidrug-Resistant Klebsiella pneumoniae via Biofilm Degradation","volume":"10","author":"Wu","year":"2019","journal-title":"Front. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e02457-16","DOI":"10.1128\/JVI.02457-16","article-title":"Klebsiella Phage \u03a6K64-1 Encodes Multiple Depolymerases for Multiple Host Capsular Types","volume":"91","author":"Pan","year":"2017","journal-title":"J. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"472","DOI":"10.1111\/1751-7915.13370","article-title":"Identification of three podoviruses infecting Klebsiella encoding capsule depolymerases that digest specific capsular types","volume":"12","author":"Pan","year":"2019","journal-title":"Microb. Biotechnol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2949","DOI":"10.3389\/fmicb.2019.02949","article-title":"Diversity and Function of Phage Encoded Depolymerases","volume":"10","author":"Knecht","year":"2020","journal-title":"Front. Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Domingo-Calap, P., Beamud, B., Vienne, J., Gonz\u00e1lez-Candelas, F., and Sanju\u00e1n, R. (2020). Isolation of Four Lytic Phages Infecting Klebsiella pneumoniae K22 Clinical Isolates from Spain. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21020425"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e01064-17","DOI":"10.1128\/JVI.01064-17","article-title":"Genomic and Biochemical Characterization of Acinetobacter Podophage Petty Reveals a Novel Lysis Mechanism and Tail-Associated Depolymerase Activity","volume":"92","author":"Morales","year":"2018","journal-title":"J. Virol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"160","DOI":"10.1093\/jac\/dki177","article-title":"Treatment of experimental Escherichia coli infection with recombinant bacteriophage-derived capsule depolymerase","volume":"56","author":"Mushtaq","year":"2005","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1804","DOI":"10.3390\/v7041804","article-title":"A Suggested New Bacteriophage Genus, \u201cKp34likevirus\u201d, within the Autographivirinae Subfamily of Podoviridae","volume":"7","author":"Eriksson","year":"2015","journal-title":"Viruses"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.virusres.2017.09.026","article-title":"Comparative genome analysis of novel Podoviruses lytic for hypermucoviscous Klebsiella pneumoniae of K1, K2, and K57 capsular types","volume":"243","author":"Solovieva","year":"2018","journal-title":"Virus Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2334","DOI":"10.1093\/bioinformatics\/btq413","article-title":"genoPlotR: Comparative gene and genome visualization in R","volume":"26","author":"Guy","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1016\/0008-6215(88)84126-0","article-title":"Primary structure of Klebsiella serotype K22 capsular polysaccharide: Another glycan containing 4-O-[(S-1-carboxyethyl]-d-glucuronic acid","volume":"179","author":"Parolis","year":"1988","journal-title":"Carbohydr. Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1099\/00221287-70-2-331","article-title":"The Exopolysaccharides of Klebsiella Serotype 2 Strains as Substrates for Phage-induced Polysaccharide Depolymerases","volume":"70","author":"Sutherland","year":"1972","journal-title":"J. Gen. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1016\/S0008-6215(00)83303-0","article-title":"Klebsiella serotype-13 capsular polysaccharide: Primary structure and depolymerization by a bacteriophage-borne glycanase","volume":"59","author":"Niemann","year":"1977","journal-title":"Carbohydr. Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1016\/S0008-6215(00)90061-2","article-title":"The use of bacteriophage depolymerization in the structural investigation of the capsular polysaccharide from Klebsiella serotype K3","volume":"149","author":"Dutton","year":"1986","journal-title":"Carbohydr. Res."},{"key":"ref_28","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_29","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S0378-1097(04)00041-2","article-title":"The interaction of phage and biofilms","volume":"232","author":"Sutherland","year":"2004","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Pan, Y.-J., Lin, T.-L., Chen, Y.-H., Hsu, C.-R., Hsieh, P.-F., Wu, M.-C., and Wang, J.-T. (2013). Capsular Types of Klebsiella pneumoniae Revisited by wzc Sequencing. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0080670"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1099\/00222615-41-6-423","article-title":"The use of bacteriophages to differentiate serologically cross-reactive isolates of Klebsiella pneumoniae","volume":"41","author":"Pieroni","year":"1994","journal-title":"J. Med. Microbiol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1016\/j.ijbiomac.2020.03.196","article-title":"Determination of the capsular polysaccharide structure of the Klebsiella pneumoniae ST512 representative strain KPB-1 and assignments of the glycosyltransferases functions","volume":"155","author":"Bellich","year":"2020","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1054","DOI":"10.1111\/j.1365-2958.2005.04918.x","article-title":"Identification of Klebsiella pneumonia virulence determinants using an intranasal infection model","volume":"58","author":"Lawlor","year":"2005","journal-title":"Mol. Microbiol."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Domingo-Calap, P., and Delgado-Mart\u00ednez, J. (2018). Bacteriophages: Protagonists of a Post-Antibiotic Era. Antibiotics, 7.","DOI":"10.3390\/antibiotics7030066"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Bansal, S., Harjai, K., and Chhibber, S. (2014). Depolymerase improves gentamicin efficacy during Klebsiella pneumoniae induced murine infection. BMC Infect. Dis., 14.","DOI":"10.1186\/1471-2334-14-456"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1186\/s13059-019-1891-0","article-title":"Improved metagenomic analysis with Kraken 2","volume":"20","author":"Wood","year":"2019","journal-title":"Genome Biol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1089\/cmb.2012.0021","article-title":"SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing","volume":"19","author":"Bankevich","year":"2012","journal-title":"J. Comput. Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"W16","DOI":"10.1093\/nar\/gkw387","article-title":"PHASTER: A better, faster version of the PHAST phage search tool","volume":"44","author":"Arndt","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Darling, A.E., Mau, B., and Perna, N.T. (2010). progressiveMauve: Multiple Genome Alignment with Gene Gain, Loss and Rearrangement. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0011147"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"4537","DOI":"10.1093\/bioinformatics\/btz265","article-title":"PHANOTATE: A novel approach to gene identification in phage genomes","volume":"35","author":"McNair","year":"2019","journal-title":"Bioinformatics"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"4636","DOI":"10.1093\/nar\/27.23.4636","article-title":"Improved microbial gene identification with GLIMMER","volume":"27","author":"Delcher","year":"1999","journal-title":"Nucleic Acids Res."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Hyatt, D., Chen, G., Locascio, P.F., Land, M., Larimer, F.W., and Hauser, L. (2010). Prodigal: Prokaryotic gene recognition and translation initiation site identification. BMC Bioinform., 11.","DOI":"10.1186\/1471-2105-11-119"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2141","DOI":"10.1007\/s00253-015-7247-0","article-title":"Bacteriophage-encoded depolymerases: Their diversity and biotechnological applications","volume":"100","author":"Pires","year":"2016","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1236","DOI":"10.1093\/bioinformatics\/btu031","article-title":"InterProScan 5: Genome-scale protein function classification","volume":"30","author":"Jones","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"2237","DOI":"10.1016\/j.jmb.2017.12.007","article-title":"A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core","volume":"430","author":"Zimmermann","year":"2018","journal-title":"J. Mol. Biol."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Lechner, M., Findeiss, S., M\u00fcller, L., Marz, M., Stadler, P.F., and Prohaska, S. (2011). Proteinortho: Detection of (Co-)orthologs in large-scale analysis. BMC Bioinform., 12.","DOI":"10.1186\/1471-2105-12-124"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1093\/molbev\/msu300","article-title":"IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies","volume":"32","author":"Nguyen","year":"2014","journal-title":"Mol. Biol. Evol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1002\/jemt.1060110212","article-title":"Improved preservation of bacterila capsule for electron microscopy","volume":"11","author":"Jacques","year":"1989","journal-title":"J. Electron Microsc. Tech."},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Patro, L.P.P., Sudhakar, K.U., and Rathinavelan, T. (2019). K-PAM: A unified platform to distinguish Klebsiella species K- and O-antigen types, model antigen structures and identify hypervirulent strains. bioRxiv.","DOI":"10.1038\/s41598-020-73360-1"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/9\/3160\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T13:24:10Z","timestamp":1760361850000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/9\/3160"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,4,30]]},"references-count":49,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2020,5]]}},"alternative-id":["ijms21093160"],"URL":"https:\/\/doi.org\/10.3390\/ijms21093160","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,4,30]]}}}