Current Microbiology

, Volume 66, Issue 3, pp 251–258 | Cite as

Isolation and Characterisation of Lytic Bacteriophages of Klebsiella pneumoniae and Klebsiella oxytoca

  • Natia Karumidze
  • Ia Kusradze
  • Sophio Rigvava
  • Marine Goderdzishvili
  • Kumar RajakumarEmail author
  • Zemphira AlavidzeEmail author


Klebsiella bacteria have emerged as an increasingly important cause of community-acquired nosocomial infections. Extensive use of broad-spectrum antibiotics in hospitalised patients has led to both increased carriage of Klebsiella and the development of multidrug-resistant strains that frequently produce extended-spectrum β-lactamases and/or other defences against antibiotics. Many of these strains are highly virulent and exhibit a strong propensity to spread. In this study, six lytic Klebsiella bacteriophages were isolated from sewage-contaminated river water in Georgia and characterised as phage therapy candidates. Two of the phages were investigated in greater detail. Biological properties, including phage morphology, nucleic acid composition, host range, growth phenotype, and thermal and pH stability were studied for all six phages. Limited sample sequencing was performed to define the phylogeny of the K. pneumoniae- and K. oxytoca-specific bacteriophages vB_Klp_5 and vB_Klox_2, respectively. Both of the latter phages had large burst sizes, efficient rates of adsorption and were stable under different adverse conditions. Phages reported in this study are double-stranded DNA bacterial viruses belonging to the families Podoviridae and Siphoviridae. One or more of the six phages was capable of efficiently lysing ~63 % of Klebsiella strains comprising a collection of 123 clinical isolates from Georgia and the United Kingdom. These phages exhibit a number of properties indicative of potential utility in phage therapy cocktails.


Klebsiella Brain Heart Infusion Broth Phage Therapy Lytic Bacteriophage Phage Lysate 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank Seydina M. Diene, PhD, Pathologie Humaine/Maladies infectieuses, Faculté de Médecine et de Pharmacie, Université de la Méditerranée Aix Marseille II, for help with bioinformatics analyses. This study was supported by a GNSF (Georgian National Science Foundation) grant N 04/01 to NK. Work in KR’s laboratory was partly funded by a Medisearch grant.


  1. 1.
    Adams M (1959) Bacteriophages. Interscience, New York, pp 137–150Google Scholar
  2. 2.
    Drulis-Kawa Z, Mackiewicz P, Kesik-Szeloch A, Maciaszczyk-Dziubinska E, Weber-Dabrowska B, Dorotkiewicz-Jach A, Augustyniak D, Majkowska-Skrobek G, Bocer T, Empel J, Kropinski AM (2011) Isolation and characterisation of KP34-a novel phiKMV-like bacteriophage for Klebsiella pneumoniae. Appl Microbiol Biotechnol 90:1333–1345. doi: 10.1007/s00253-011-3149-y PubMedCrossRefGoogle Scholar
  3. 3.
    Gatedee J, Kritsiriwuthinan K, Galyov EE, Shan J, Dubinina E, Intarak N, Clokie MR, Korbsrisate S (2011) Isolation and characterization of a novel podovirus which infects Burkholderia pseudomallei. Virol J 8:366. doi: 10.1186/1743-422X-8-366 PubMedCrossRefGoogle Scholar
  4. 4.
    Gu J, Liu X, Li Y, Han W, Lei L, Yang Y, Zhao H, Gao Y, Song J, Lu R, Sun C, Feng X (2012) A method for generation phage cocktail with great therapeutic potential. PLoS One 7:e31698. doi: 10.1371/journal.pone.0031698 PubMedCrossRefGoogle Scholar
  5. 5.
    Hyman P, Abedon ST (2009) Practical methods for determining phage growth parameters. Methods Mol Biol 501:175–202. doi: 10.1007/978-1-60327-164-6_18 PubMedCrossRefGoogle Scholar
  6. 6.
    Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP (2009) Enumeration of bacteriophages by double agar overlay plaque assay. Methods Mol Biol 501:69–76. doi: 10.1007/978-1-60327-164-6_7 PubMedCrossRefGoogle Scholar
  7. 7.
    Kropinski AM, Prangishvili D, Lavigne R (2009) Position paper: the creation of a rational scheme for the nomenclature of viruses of Bacteria and Archaea. Environ Microbiol 11:2775–2777. doi: 10.1111/j.1462-2920.2009.01970.x PubMedCrossRefGoogle Scholar
  8. 8.
    Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, Chaudhary U, Doumith M, Giske CG, Irfan S, Krishnan P, Kumar AV, Maharjan S, Mushtaq S, Noorie T, Paterson DL, Pearson A, Perry C, Pike R, Rao B, Ray U, Sarma JB, Sharma M, Sheridan E, Thirunarayan MA, Turton J, Upadhyay S, Warner M, Welfare W, Livermore DM, Woodford N (2010) Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis 10:597–602. doi: 10.1016/S1473-3099(10)70143-2 PubMedCrossRefGoogle Scholar
  9. 9.
    Kumari S, Harjai K, Chhibber S (2010) Evidence to support the therapeutic potential of bacteriophage Kpn5 in burn wound infection caused by Klebsiella pneumoniae in BALB/c mice. J Microbiol Biotechnol 20:935–941PubMedCrossRefGoogle Scholar
  10. 10.
    Kutter E (2009) Phage host range and efficiency of plating. Methods Mol Biol 501:141–149. doi: 10.1007/978-1-60327-164-6_14 PubMedCrossRefGoogle Scholar
  11. 11.
    Lee M, Miller RC Jr (1974) T7 exonuclease (gene 6) is necessary for molecular recombination of bacteriophage T7. J Virol 14:1040–1048PubMedGoogle Scholar
  12. 12.
    Lingohr E, Frost S, Johnson RP (2009) Determination of bacteriophage genome size by pulsed-field gel electrophoresis. Methods Mol Biol 502:19–25. doi: 10.1007/978-1-60327-565-1_3 PubMedCrossRefGoogle Scholar
  13. 13.
    Mathers AJ, Cox HL, Kitchel B, Bonatti H, Brassinga AK, Carroll J, Scheld WM, Hazen KC, Sifri CD (2011) Molecular dissection of an outbreak of carbapenem-resistant enterobacteriaceae reveals intergenus KPC carbapenemase transmission through a promiscuous plasmid. MBio 2:e00204–e00211. doi: 10.1128/mBio.00204-11 PubMedCrossRefGoogle Scholar
  14. 14.
    Nagaraj S, Chandran SP, Shamanna P, Macaden R (2012) Carbapenem resistance among Escherichia coli and Klebsiella pneumoniae in a tertiary care hospital in south India. Indian J Med Microbiol 30:93–95. doi: 10.4103/0255-0857.93054 PubMedCrossRefGoogle Scholar
  15. 15.
    Nale JY, Shan J, Hickenbotham PT, Fawley WN, Wilcox MH, Clokie MR (2012) Diverse temperate bacteriophage carriage in Clostridium difficile 027 strains. PLoS One 7:e37263. doi: 10.1371/journal.pone.0037263 PubMedCrossRefGoogle Scholar
  16. 16.
    Podschun R, Ullmann U (1998) Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 11:589–603PubMedGoogle Scholar
  17. 17.
    Ramphal R, Ambrose PG (2006) Extended-spectrum beta-lactamases and clinical outcomes: current data. Clin Infect Dis 42(Suppl 4):S164–S172. doi: 10.1086/500663 PubMedCrossRefGoogle Scholar
  18. 18.
    Serwer P, Watson RH, Son M (1990) Role of gene 6 exonuclease in the replication and packaging of bacteriophage T7 DNA. J Mol Biol 215:287–299. doi: 10.1016/S0022-2836(05)80347-X PubMedCrossRefGoogle Scholar
  19. 19.
    Son M, Serwer P (1992) Role of exonuclease in the specificity of bacteriophage T7 DNA packaging. Virology 190:824–833PubMedCrossRefGoogle Scholar
  20. 20.
    Stahlhut SG, Struve C, Krogfelt KA, Reisner A (2012) Biofilm formation of Klebsiella pneumoniae on urethral catheters requires either type 1 or type 3 fimbriae. FEMS Immunol Med Microbiol 65:350–359. doi: 10.1111/j.1574-695X.2012.00965.x PubMedCrossRefGoogle Scholar
  21. 21.
    Sulakvelidze A, Kutter E (2005) Bacteriophage therapy in humans. In: Kutter E, Sulakvelidze A (eds) Bacteriophages: biology and applications. CRC, New York, pp 377–428 Google Scholar
  22. 22.
    Sulakvelidze A, Alavidze Z, Morris JG Jr (2001) Bacteriophage therapy. Antimicrob Agents Chemother 45:649–659. doi: 10.1128/AAC.45.3.649-659.2001 PubMedCrossRefGoogle Scholar
  23. 23.
    Summers WC(1999) Felix d’Herelle and the origins of molecular biology. Yale University Press, New Haven, pp 108–124Google Scholar
  24. 24.
    Tsai SS, Huang JC, Chen ST, Sun JH, Wang CC, Lin SF, Hsu BR, Lin JD, Huang SY, Huang YY (2010) Characteristics of Klebsiella pneumoniae bacteremia in community-acquired and nosocomial infections in diabetic patients. Chang Gung Med J 33:532–539PubMedGoogle Scholar
  25. 25.
    Tsay RW, Siu LK, Fung CP, Chang FY (2002) Characteristics of bacteremia between community-acquired and nosocomial Klebsiella pneumoniae infection: risk factor for mortality and the impact of capsular serotypes as a herald for community-acquired infection. Arch Intern Med 162:1021–1027PubMedCrossRefGoogle Scholar
  26. 26.
    Verma V, Harjai K, Chhibber S (2009) Restricting ciprofloxacin-induced resistant variant formation in biofilm of Klebsiella pneumoniae B5055 by complementary bacteriophage treatment. J Antimicrob Chemother 64:1212–1218. doi: 10.1093/jac/dkp360 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Natia Karumidze
    • 1
    • 2
    • 3
  • Ia Kusradze
    • 1
    • 2
  • Sophio Rigvava
    • 1
    • 2
  • Marine Goderdzishvili
    • 1
  • Kumar Rajakumar
    • 3
    • 4
    Email author
  • Zemphira Alavidze
    • 1
    Email author
  1. 1.Eliava Institute of Bacteriophages, Microbiology and VirologyTbilisiGeorgia
  2. 2.Ilia State UniversityTbilisiGeorgia
  3. 3.Department of Infection, Immunity and InflammationUniversity of LeicesterLeicesterUK
  4. 4.Department of Clinical MicrobiologyUniversity Hospitals of Leicester National Health Service TrustLeicesterUK

Personalised recommendations