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Isolation and characterization of Klebsiella pneumoniae specific bacteriophages from sewage samples

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Abstract

Five bacteriophages (Kpn5, Kpn12, Kpn13, Kpn17 and Kpn22), each having specificity against Klebsiella pneumoniae strain B5055, were isolated from sewage samples and characterized in terms of growth characteristics, genetic material, morphology and structural proteins. Adsorption rate as well as single step growth curve experiments showed variation among phages. Restriction enzyme digestion of DNA confirmed the presence of double stranded DNA as well as the heterogeneous nature of genetic material. RAPD-PCR was performed to further distinguish these closely related phages. Their genome fingerprint confirmed their diversity. Transmission electron microscopy, on the other hand, showed their morphological similarity; they were assigned to family Podoviridae, order Caudovirales on the basis of their head and tail morphology. Structural proteins resolved on SDS-PAGE showed the presence of similar major outer membrane proteins. The bacteriophages, belonging to Podoviridae family with short stumpy tails, were found to be nontoxic to mice. They showed maximum count in various organs at 6 h post inoculation, which persisted till 36 h. These phages thus have the potential to be used for phage therapy.

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Abbreviations

CFU:

colony forming units

MDR:

multi drug resistant (bacteria)

MOI:

multiplicity of infection

PCR:

polymerase chain reaction

PEG:

polyethylene glycol

PFU:

plaque forming units

RAPD:

random amplified polymorphic DNA

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TEM:

transmission electron microscopy

References

  • Ackermann H.W.: Frequency of morphological phage descriptions in the year 2000. Arch.Virol.146, 843–857 (2001).

    Article  CAS  PubMed  Google Scholar 

  • Adam M.H.: Bacteriophages. Interscience Publishers, New York 1959.

    Google Scholar 

  • Atterbury J., Connerton P.L., Dodd C.E.R., Rees C.E.D., Connerton I.F.: Isolation and characterization of Campylobacter bacteriophages from retail poultry. Appl.Environ.Microbiol.69, 4511–4518 (2003).

    Article  CAS  PubMed  Google Scholar 

  • Barrangou R., Yoon S.S., Breidt F., Fleming H.P., Klenhammer T.R.: Characterization of six Leuconostoc fallax bacteriophages isolated from industrial sauerkraut fermentation. Appl.Environ.Microbiol.68, 5452–5458 (2002).

    Article  CAS  PubMed  Google Scholar 

  • Benedict L.R.N., Flamiano R.S.: Use of bacteriophages as therapy for Escherichia coli-induced bacteremia in mouse models. Phil. J.Microbiol.Infect.Dis.33, 47–51 (2004).

    Google Scholar 

  • Bogovazova G.G., Voroshilova N.N., Gorbatkova G.A., Bondarenko V.M., Kazakova T.B., Mamleeva A.G., Sirnov V.D., Glukharev I.A., Erastova E.I., Krylov I.A., Ovcherenko T.M., Baturo A.P., Yalsyk G.V., Arefyeva N.A.: Immunobiological properties and therapeutic effectiveness of preparations from Klebsiella bacteriophages. (In Russian) Zh.Mikrobiol.Epidemiol.Imunobiol.3, 30–33 (1992).

    Google Scholar 

  • Carlton M.: Phage therapy: past history and future prospects. Arch.Immunol.Ther.Exp.(Warsaw)47, 267–274 (1999).

    CAS  Google Scholar 

  • Cerveny K.E., Depaola A., Duckworth D.H., Gulig P.A.: Phage therapy of local and systemic disease caused by Vibrio vulnificus in iron-dextran-treated mice. Infect.Immun.70, 6251–6262 (2002).

    Article  CAS  PubMed  Google Scholar 

  • Chakrabarti A.K., Ghosh A.N., Nair G.B., Niyogi S.K., Bhattacharya S.K., Sarkar B.L.: Development and evaluation of a phage typing scheme for Vibrio cholerae O139. J.Clin.Microbiol.38, 44–49 (2000).

    CAS  PubMed  Google Scholar 

  • Chang H.-C., Chen C.-R., Lin J.-W., Shen G.-H., Chang K.-M., Tseng Y.-H., Weng S.-F.: Isolation and characterization of novel giant Stenotrophomonas maltophilia phage ΦSMA5. Appl.Environ.Microbiol.71, 1387–1393 (2005).

    Article  CAS  PubMed  Google Scholar 

  • Chhibber S., Kaur S., Kumari S.: Therapeutic potential of bacteriophage in treating Klebsiella pneumoniae B5055-mediated lobar pneumonia in mice. J.Med.Microbiol.57, 1508–1513 (2008).

    Article  PubMed  Google Scholar 

  • Dabrowska K., Switala-Jelen K., Opolski A., Weber-Dabrowska B., Gorski A.: Bacteriophage penetration in vertebrates. J.Appl.Microbiol.98, 7–13 (2005).

    Article  CAS  PubMed  Google Scholar 

  • Drake J.W., Hwang C.B.C.: On the mutation rate of herpes simplex virus type 1. Genetics170, 969–970 (2005).

    Article  CAS  PubMed  Google Scholar 

  • Hanlon G.W.: Bacteriophages: an appraisal of their role in the treatment of bacterial infections. Internat.J.Antimicrob.Agents30, 118–128 (2007).

    Article  CAS  Google Scholar 

  • Johansson M.L., Quednau M., Molin G., Ahrne S.: Randomly amplified polymorphic DNA (RAPD) for rapid typing of Lactobacillus plantarum strains. Lett.Appl.Microbiol.21, 155–159 (1999).

    Article  Google Scholar 

  • Jothikumar N., Reddy C.G., Sundari R.B., Saigopal D.V.R.: Isolation of coliphages specific to enterotoxigenic E. coli (ETEC). J.Environ.Monit.2, 372–374 (2000).

    Article  CAS  PubMed  Google Scholar 

  • Kumari S., Harjai K., Chhibber S.: Bacteriophage treatment of burn wound infection caused by Pseudomonas aeruginosa PAO in BALB/c mice. Am.J.BioMed.Sci.1, 385–394 (2009a).

    Article  Google Scholar 

  • Kumari S., Harjai K., Chhibber S.: Efficacy of bacteriophages treatment in murine burn wound infection induced by Klebsiella pneumoniae. J.Microbiol.Biotechnol.19, 622–628 (2009b).

    CAS  PubMed  Google Scholar 

  • Kumari S., Harjai K., Chhibber S.: Characterization of Pseudomonas aeruginosa PAO specific bacteriophages isolated from sewage samples. Am.J.BioMed.Sci.1, 91–102 (2009c).

    Article  CAS  Google Scholar 

  • Kurzępa A., Dąbrowska K., Świtała-Jeleń K., Górski A.: Molecular modification of T4 bacteriophage proteins and its potential application — review. Folia Microbiol.54, 5–15 (2009).

    Article  Google Scholar 

  • Laemmli U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature22, 680–685 (1970).

    Article  Google Scholar 

  • Letkiewicz S., Międzybrodzki R., Fortuna W., Weber-Dąbrowska B., Górski A.: Eradication of Enterococcus faecalis by phage therapy in chronic prostatitis — case report. Folia Microbiol.54, 457–462 (2009).

    Article  CAS  Google Scholar 

  • Malik R., Chhibber S.: Protection with bacteriophages Kϕ1 Klebsiella pneumoniae-induced burn wound infection in mice. J.Microbiol. Immunol.Infect.42, 134–140 (2009).

    PubMed  Google Scholar 

  • Matsuzaki S., Rashel M., Uchiyama J., Sakurai S., Ujihara T., Kuroda M., Ikeuchi M., Tani T., Fujieda M., Wakiguchi H., Imai S.: Bacteriophage therapy: a revitalized therapy against bacterial infectious diseases. J.Infect.Chemother.11, 211–219 (2005).

    Article  PubMed  Google Scholar 

  • Mcvay C., Velasquez S.M., Fralick J.A.: Phage therapy of Pseudomonas aeruginosa infection in a mouse burn wound model. Antimicrob. Agents Chem.51, 1934–1938 (2007).

    Article  CAS  Google Scholar 

  • Mitra K., Ghosh A.N.: Characterization of Vibrio cholerae O1 El Tor typing phage S5. Arch.Virol.152, 1775–1786 (2007).

    Article  CAS  PubMed  Google Scholar 

  • Niemann H., Kwiatkowski B., Westphal U., Stirm S.: Klebsiella serotype 25 capsular polysaccharide: primary structure and depolymerization by a bacteriophage-borne glycanase. J.Bacteriol.130, 366–374 (1977).

    CAS  PubMed  Google Scholar 

  • Nordeen R.O., Morgan M.K., Currier T.C.: Isolation and partial characterization of bacteriophages of the phytopathogen Pseudomonas syringae. Appl.Environ.Microbiol.45, 1890–1898 (1983).

    CAS  PubMed  Google Scholar 

  • Pajunen M., Kiljunen S., Skrunik M.: Bacteriophage YeO3-12, specific for Yersinia enterocolitica serotype O:3, is related to coliphages T3 and T7. J.Bacteriol.182, 5114–5120 (2000).

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Sambrook J., Fritsch E.F., Maniatis T.: Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (NY, USA) 1989.

    Google Scholar 

  • Sen A., Ghosh A.N.: Physicochemical characterization of vibriophage N5. Virol.J.2, 27 (2005).

    Article  PubMed  Google Scholar 

  • Shukla I., Tiwari R., Agrawal M.: Prevalence of extended spectrum β-lactamase producing Klebsiella pneumoniae in a tertiary care hospital. Ind.J.Med.Microbiol.22, 87–91 (2004).

    CAS  Google Scholar 

  • Skurnik M., Pajunen M., Kiljunen S.: Biotechnological challenges of phage therapy. Biotechnol.Lett.29, 995–1003 (2007).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Watnick P., Kolter R.: Biofilm, city of microbes. J.Bacteriol.182, 2675–2679 (2000).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Yamamoto K.R., Alberts B.M., Benzinger R., Lawhorne L., Treiber G.: Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large scale virus purification. Virology40, 734–744 (1970).

    Article  CAS  PubMed  Google Scholar 

  • Yoon S.-S., Barrangou-Poueys R., Breid F. Jr., Fleming H.P.: Detection and characterization of a lytic Pediococcus bacteriophage from the fermenting cucumber brine. J.Microbiol.Biotechnol.17, 262–270 (2007).

    CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Microbiology, Panjab University, Basic Medical Sciences Building, Chandigarh, 160014, India

    S. Kumari, K. Harjai & S. Chhibber

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  1. S. Kumari
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  2. K. Harjai
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  3. S. Chhibber
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Correspondence to S. Chhibber.

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Kumari, S., Harjai, K. & Chhibber, S. Isolation and characterization of Klebsiella pneumoniae specific bacteriophages from sewage samples. Folia Microbiol 55, 221–227 (2010). https://doi.org/10.1007/s12223-010-0032-7

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  • DOI: https://doi.org/10.1007/s12223-010-0032-7

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