Characterization and genome analysis of novel bacteriophages infecting the opportunistic human pathogens Klebsiella oxytoca and K. pneumoniae


Klebsiella is a genus of well-known opportunistic human pathogens that are associated with diabetes mellitus and chronic pulmonary obstruction; however, this pathogen is often resistant to multiple drugs. To control this pathogen, two Klebsiella-infecting phages, K. oxytoca phage PKO111 and K. pneumoniae phage PKP126, were isolated from a sewage sample. Analysis of their host range revealed that they infect K. pneumoniae and K. oxytoca, suggesting host specificity for members of the genus Klebsiella. Stability tests confirmed that the phages are stable under various temperature (4 to 60 °C) and pH (3 to 11) conditions. A challenge assay showed that PKO111 and PKP126 inhibit growth of their host strains by 2 log and 4 log, respectively. Complete genome sequencing of the phages revealed that their genome sizes are quite different (168,758 bp for PKO111 and 50,934 bp for PKP126). Their genome annotation results showed that they have no human virulence-related genes, an important safety consideration. In addition, no lysogen-formation gene cluster was detected in either phage genome, suggesting that they are both virulent phages in their bacterial hosts. Based on these results, PKO111 and PKP126 may be good candidates for development of biocontrol agents against members of the genus Klebsiella for therapeutic purposes. A comparative analysis of tail-associated gene clusters of PKO111 and PKP126 revealed relatively low homology, suggesting that they might differ in the way they recognize and infect their specific hosts.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. 1.

    Abbasifar R, Kropinski AM, Sabour PM, Ackermann HW, Lingohr EJ, Griffiths MW (2012) Complete genome sequence of Cronobacter sakazakii bacteriophage vB_CsaM_GAP161. J Virol 86:13806–13807

    Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Adams MH (1959) Enumeration of bacteriophage particles. Bacteriophages. Interscience Publishers, New York, pp 27–30

    Google Scholar 

  3. 3.

    Altermann E, Klaenhammer TR (2003) GAMOLA: a new local solution for sequence annotation and analyzing draft and finished prokaryotic genomes. OMICS 7:161–169

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Besemer J, Lomsadze A, Borodovsky M (2001) GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res 29:2607–2618

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Bouza E, Cercenado E (2002) Klebsiella and Enterobacter: antibiotic resistance and treatment implications. Semin Resp Infect 17:215–230

    Article  Google Scholar 

  7. 7.

    Cao F, Wang X, Wang L, Li Z, Che J, Wang L, Li X, Cao Z, Zhang J, Jin L (2015) Evaluation of the efficacy of a bacteriophage in the treatment of pneumonia induced by multidrug resistance Klebsiella pneumoniae in mice. Biomed Res Int 2015:752930

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Carver TJ, Rutherford KM, Berriman M, Rajandream M-A, Barrell BG, Parkhill J (2005) ACT: the Artemis comparison tool. Bioinformatics 21:3422–3423

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Demirdag K, Hosoglu S (2010) Epidemiology and risk factors for ESBL-producing Klebsiella pneumoniae: a case control study. J Infect Dev Ctries 4:717–722

    PubMed  Google Scholar 

  10. 10.

    Deresinski S (2009) Bacteriophage therapy: exploiting smaller fleas. Clin Infect Dis 48:1096–1101

    Article  PubMed  Google Scholar 

  11. 11.

    Duckworth DH, Gulig PA (2002) Bacteriophages: potential treatment for bacterial infections. BioDrugs 16:57–62

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Failla ML, Benedict C, Weinberg E (1975) Bacterial and fungal growth in total parenteral nutrition solutions. Antonie Van Leeuwenhoek 41:319–328

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Gyles C (2007) Shiga toxin-producing Escherichia coli: an overview. J Anim Sci 85:E45–E62

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Hayashi K, Morooka N, Yamamoto Y, Fujita K, Isono K, Choi S, Ohtsubo E, Baba T, Wanner BL, Mori H (2006) Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110. Mol Syst Biol 2(2006):0007

    PubMed  Google Scholar 

  15. 15.

    Hendrix RW (2003) Bacteriophage genomics. Curr Opin Microbiol 6:506–511

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Hirsch EB, Tam VH (2010) Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrug-resistant infection. J Antimicrobe Chemother 65:1119–1125

    CAS  Article  Google Scholar 

  17. 17.

    Kumari S, Harjai K, Chhirbber S (2010) Topical treatment of Klebsiella pneumoniae B5055 induced burn wound infection in mice using natural products. J Infect Dev Ctries 4:367–377

    Article  PubMed  Google Scholar 

  18. 18.

    Lang LH (2006) FDA approves use of bacteriophages to be added to meat and poultry product. Gastroenterology 131:1370

    PubMed  Google Scholar 

  19. 19.

    Maal KB, Delfan AS, Salmanizadeh S (2014) Isolation and identification of Klebsiella pneumonia and Klebsiella oxytoca bacteriophages and their application in wastewater treatment and coliforrn’s phage therapy. Res J Environ Sci 8:123–138

    CAS  Article  Google Scholar 

  20. 20.

    Maki DG (1976) Growth properties of microorganisms in infusion fluid and methods of detection. In: Phillips I, Meers PD, D’Arcy PF (eds) Microbiological hazards of infusion therapy. Microbiological hazards of infusion therapy. MTP Press, Lancaster, pp 13–47

    Google Scholar 

  21. 21.

    McClelland M, Sanderson KE, Spieth J, Clifton SW, Latreille P, Courtney L, Porwollik S, Ali J, Dante M, Du F (2001) Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413:852–856

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    McNair K, Bailey BA, Edwards RA (2012) PHACTS, a computational approach to classifying the lifestyle of phages. Bioinformatics 28:614–618

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Park K-H, Kurokawa K, Zheng L, Jung D-J, Tateishi K, Jin J-O, Ha N-C, Kang HJ, Matsushita M, Kwak J-Y (2010) Human serum mannose-binding lectin senses wall teichoic acid Glycopolymer of Staphylococcus aureus, which is restricted in infancy. J Biol Chem 285:27167–27175

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Park M, Lee J-H, Shin H, Kim M, Choi J, Kang D-H, Heu S, Ryu S (2012) Characterization and comparative genomic analysis of a novel bacteriophage, SFP10, simultaneously inhibiting both Salmonella enterica and Escherichia coli O157:H7. Appl Environ Microbiol 78:58–69

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Podschun R, Ullmann U (1998) Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 11:589–603

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Pomakova D, Hsiao C, Beanan J, Olson R, MacDonald U, Keynan Y, Russo T (2012) Clinical and phenotypic differences between classic and hypervirulent Klebsiella pneumonia: an emerging and under-recognized pathogenic variant. Eur J Clin Microbiol Infect Dis 31:981–989

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, Apweiler R, Lopez R (2005) InterProScan: protein domains identifier. Nucleic Acids Res 33:W116–W120

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Ribot EM, Wierzba RK, Angulo FJ, Barrett TJ (2002) Salmonella enterica serotype Typhimurium DT104 isolated from humans, United States, 1985, 1990, and 1995. Emerg Infect Dis 8:387–391

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream M-A, Barrell B (2000) Artemis: sequence visualization and annotation. Bioinformatics 16:944–945

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Shen Y-J, Jiang H, Jin J-P, Zhang Z-B, Xi B, He Y-Y, Wang G, Wang C, Qian L, Li X (2004) Development of genome-wide DNA polymorphism database for map-based cloning of rice genes. Plant Physiol 135:1198–1205

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Shoma S, Kamruzzaman M, Ginn AN, Iredell JR, Partridge SR (2014) Characterization of multidrug-resistant Klebsiella pneumoniae from Australia carrying blaNDM-1. Diagn Microbiol Infect Dis 78:93–97

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539

    Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Struve C, Krogfelt KA (2004) Pathogenic potential of environmental Klebsiella pneumoniae isolates. Environ Microbiol 6:584–590

    Article  PubMed  Google Scholar 

  34. 34.

    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Tatusova TA, Madden TL (1999) BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences. FEMS Microbiol Lett 174:247–250

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Unerwood AP, Mulder A, Gharbia S, Green J (2005) Virulence Searcher: a tool for searching raw genome sequences from bacterial genomes for putative virulence factors. Clin Microbiol Infect 11:770–772

    Article  Google Scholar 

  37. 37.

    Verma V, Harjai K, Chhibber S (2009) Characterization of a T7-like lytic bacteriophage of Klebsiella pneumoniae B5055: a potential therapeutic agent. Curr Microbiol 59:274–281

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Wilcox S, Toder R, Foster J (1996) Rapid isolation of recombinant lambda phage DNA for use in fluorescence in situ hybridization. Chromosome Res 4:397–404

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Wu L-T, Chang S-Y, Yen M-R, Yang T-C, Tseng Y-H (2007) Characterization of extended-host-range pseudo-T-even bacteriophage Kpp95 isolated on Klebsiella pneumoniae. Appl Environ Microbiol 73:2532–2540

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Zhu J, Jiang R, Kong H, Zhang R, Lü H, Sun C, Huang Z (2013) Emergence of novel variants of gyrA, parC, qnrS genes in multi-drug resistant Klebsiella caused pneumonia. Zhonghua Liu Xing Bing Xue Za Zhi 34:61–66

    PubMed  Google Scholar 

Download references


This research was supported by the Public Welfare & Safety research program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning (NRF-2012M3A2A1051684).

Author information



Corresponding author

Correspondence to Ju-Hoon Lee.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 671 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Park, EA., Kim, YT., Cho, JH. et al. Characterization and genome analysis of novel bacteriophages infecting the opportunistic human pathogens Klebsiella oxytoca and K. pneumoniae . Arch Virol 162, 1129–1139 (2017).

Download citation


  • Biocontrol Agent
  • Indicator Strain
  • Phage Genome
  • Endolysin
  • Phage Therapy