Bulletin of Experimental Biology and Medicine

, Volume 164, Issue 3, pp 344–346 | Cite as

Evaluation of the Efficiency of Lytic Mycobacteriophage D29 on the Model of M. tuberculosis-Infected Macrophage RAW 264 Cell Line

  • M. B. Lapenkova
  • N. S. Smirnova
  • P. N. Rutkevich
  • M. A. Vladimirsky

Culture of mouse macrophages (RAW 264.7 ATCC strain) in wells of a 6-well plate was infected with M. tuberculosis in proportion of 15 mycobacteria per one macrophage and then treated with a lytic strain of mycobacteriophage D29. Antibacterial efficacy of mycobacteriophages was studied using D29 phage (activity 108 plaque-forming units/ml) previously purified by ion exchange chromatography. After single and double 24-h treatment, the lysed cultures of macrophages were inoculated onto Middlebrook 7H10 agar medium. The number of mycobacterial colonies in control and test wells (at least 3 wells in each group) was 300.178±12.500 and 36.0±5.4, respectively (p<0.01).

Key Words

mycobacterium tuberculosis mycobacteriophages macrophages cell culture phage DNA 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Adriaenssens EM, Lehman SM, Vandersteegen K, Vandenheuvel D, Philippe DL, Cornelissen A, Clokie MR, García AJ, De Proft M, Maes M, Lavigne R.CIM(®) monolithic anion-exchange chromatography as a useful alternative to CsCl gradient purification of bacteriophage particles. Virology. 2012;434(2):265-270.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Eltringham IJ, Wilson SM, Drobniewski FA. Evaluation of a bacteriophage-based assay (phage amplified biologically assay) as a rapid screen for resistance to isoniazid, ethambutol, streptomycin, pyrazinamide, and ciprofloxacin among clinical isolates of Mycobacterium tuberculosis. J. Clin. Microbiol. 1999;37(11):3528-3532.PubMedPubMedCentralGoogle Scholar
  3. 3.
    Ford ME, Sarkis GJ, Belanger AE, Hendrix RW, Hatfull GF. Genome structure of mycobacteriophage D29: implications for phage evolution. J. Mol. Biol. 1998;279(1):143-164.CrossRefPubMedGoogle Scholar
  4. 4.
    Gan Y, Yao Y, Guo S. The dormant cells of Mycobacterium tuberculosis may be resuscitated by targeting-expression system of recombinant mycobacteriophage-Rpf: implication of shorter course of TB chemotherapy in the future. Med. Hypotheses. 2015;84(5):477-480.CrossRefPubMedGoogle Scholar
  5. 5.
    Hatfull GF. Molecular genetics of mycobacteriophages. Microbiol. Spectr. 2014;2(2):1-36.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Kamilla S, Jain V. Mycobacteriophage D29 holin C-terminal region functionally assists in holin aggregation and bacterial cell death. FEBS J. 2016;283(1):173-190.CrossRefPubMedGoogle Scholar
  7. 7.
    Liu K, Wen Z, Li N, Yang W, Wang J, Hu L, Dong X, Lu J, Li J. Impact of relative humidity and collection media on mycobacteriophage D29 aerosol. Appl. Environ. Microbiol. 2012;78(5):1466-1472.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Nieth A, Verseux C, Barnert S, Süss R, Römer W. A first step toward liposome mediated intracellular bacteriophage therapy. Expert Opin. Drug Deliv. 2015;12(9):1411-1424.CrossRefPubMedGoogle Scholar
  9. 9.
    O’Flaherty S, Ross RP, Coffey A. Bacteriophage and their lysins for elimination of infectious bacteria. FEMS Microbiol. Rev. 2009;33(4):801-819.CrossRefPubMedGoogle Scholar
  10. 10.
    Payne KM, Hatfull GF. Mycobacteriophage endolysins: diverse and modular enzymes withmultiple catalytic activities. PLoS One. 2012;7(3):e34052. doi: Scholar
  11. 11.
    Peng L, Chen BW, Luo YA, Wang GZ. Effect of mycobacteriophage to intracellular mycobacteria in vitro. Chin. Med. J. (Engl.) 2006;119(8):692-695.Google Scholar
  12. 12.
    Pholwat S, Ehdaie B, Foongladda S, Kelly K, Houpt E. Real-time PCR using mycobacteriophage DNA for rapid phenotypic drug susceptibility results for Mycobacterium tuberculosis. J. Clin. Microbiol. 2012;50(3):754-761.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Rybniker J, Kramme S, Small PL. Host range of 14 mycobacteriophages in Mycobacterium ulcerans and seven other mycobacteria including Mycobacterium tuberculosis — application for identification and susceptibility testing. J. Med. Microbiol. 2006;55(Pt 1):37-42.CrossRefPubMedGoogle Scholar
  14. 14.
    Xiong X, Zhang HM, Wu TT, Xu L, Gan YL, Jiang LS, Zhang L, Guo SL. Titer dynamic analysis of D29 within MTB-infected macrophages and effect on immune function of macrophages. Exp. Lung Res. 2014;40(2):86-98.CrossRefPubMedGoogle Scholar
  15. 15.
    Yosef I, Kiro R, Molshanski-Mor S, Edgar R, Qimron U. Different approaches for using bacteriophages against antibiotic resistant bacteria. Bacteriophage. 2014;4(1):e28491.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • M. B. Lapenkova
    • 1
  • N. S. Smirnova
    • 1
  • P. N. Rutkevich
    • 2
  • M. A. Vladimirsky
    • 1
  1. 1.Research Institute of PhthisiopulmonologyI. M. Sechenov First Moscow State Medical University, Ministry of Health of the Russian FederationMoscowRussia
  2. 2.Research Institute of Experimental Cardiology, Russian Cardiology Research and Production ComplexMinistry of Health of the Russian FederationMoscowRussia

Personalised recommendations