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Abstract

Bacteriophages (phages) are viruses that target bacteria, with the ability to lyse and kill host bacterial cells. Due to this, they have been of some interest as a therapeutic since their discovery in the early 1900s, but with the recent increase in antibiotic resistance, phages have seen a resurgence in attention. Current methods of isolation and purification of phages can be long and tedious, with caesium chloride concentration gradients the gold standard for purifying a phage fraction. Isolation of novel phages requires centrifugation and ultrafiltration of mixed samples, such as water sources, effluent or faecal samples etc, to prepare phage filtrates for further testing. We propose countercurrent chromatography as a novel and alternative approach to use when studying phages, as a scalable and high-yield method for obtaining phage fractions. However, the full extent of the usefulness and resolution of separation with this technique has not been researched; it requires optimization and ample testing before this can be revealed. Here we present an initial study to determine survivability of two phages, T4 and ϕX174, using only water as a mobile phase in a Spectrum Series 20 HPCCC. Both phages were found to remain active once eluted from the column. Phages do not fully elute from the column and sodium hydroxide is necessary to flush the column between runs to deactivate remaining phages.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2022-02-24
2024-10-10
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References

  1. Clokie MRJ, Millard AD, Letarov AV, Heaphy S. Phages in nature. Bacteriophage 2011; 1:31–45 [View Article] [PubMed]
    [Google Scholar]
  2. Gordillo Altamirano FL, Barr JJ. Phage therapy in the postantibiotic era. Clin Microbiol Rev 2019; 32:1–25 [View Article] [PubMed]
    [Google Scholar]
  3. Schmidt C. Phage therapy’s latest makeover. Nat Biotechnol 2019; 37:581–586 [View Article] [PubMed]
    [Google Scholar]
  4. Gill JJ, Hyman P. Phage choice, isolation, and preparation for phage therapy. Curr Pharm Biotechnol 2010; 11:2–14 [View Article] [PubMed]
    [Google Scholar]
  5. Adriaenssens EM, Lehman SM, Vandersteegen K, Vandenheuvel D, Philippe DL et al. CIM(®) monolithic anion-exchange chromatography as a useful alternative to CsCl gradient purification of bacteriophage particles. Virology 2012; 434:265–270 [View Article] [PubMed]
    [Google Scholar]
  6. Vandenheuvel D, Rombouts S, Adriaenssens EM. Purification of Bacteriophages Using Anion-Exchange Chromatography. In Clokie MRJ, Kropinski AM, Lavigne R. eds Bacteriophages Methods Protoc Vol. 3 New York: Springer; 2018 pp 59–69 [View Article]
    [Google Scholar]
  7. Wommack KE, Sime-Ngando T, Winget DM, Jamindar S, Helton RR. Filtration-based methods for the collection of viral concentrates from large water samples, in: man. Aquat Viral Ecol, American Society of Limnology and Oceanography 2010110–117 [View Article]
    [Google Scholar]
  8. Berthod A, Faure K. Separations with a liquid stationary phase: countercurrent chromatography or centrifugal partition chromatography. Anal Sep Sci 20151177–1206
    [Google Scholar]
  9. Williams SKR, Runyon JR, Ashames AA. Field-flow fractionation: addressing the nano challenge. Anal Chem 2011; 83:634–642 [View Article] [PubMed]
    [Google Scholar]
  10. Fedotov PS, Ermolin MS, Katasonova ON. Field-flow fractionation of nano- and microparticles in rotating coiled columns. J Chromatogr A 2015; 1381:202–209 [View Article] [PubMed]
    [Google Scholar]
  11. Caldwell KD, Karaiskakis G, Calvin Giddings J. Characterization of T4D virus by sedimentation field-flow fractionation. J Chromatogr A 1981; 215:323–332 [View Article]
    [Google Scholar]
  12. Giddings JC, Yang FJF, Myers MN. Application of sedimentation field-flow fractionation to biological particles: molecular weights and separation. Separation Science 1975; 10:133–149 [View Article]
    [Google Scholar]
  13. Yazaki K. Electron microscopic studies of bacteriophage phi X174 intact and "eclipsing’ particles, and the genome by the staining, and shadowing method. J Virol Methods 1981; 2:159–167 [View Article] [PubMed]
    [Google Scholar]
  14. Ackermann HW, Krisch HM. A catalogue of T4-type bacteriophages. Arch Virol 1997; 142:2329–2345 [View Article] [PubMed]
    [Google Scholar]
  15. Clokie MRJ, Kropinski AM. Bacteriophages. In Bacteriophages: Methods and Protocols Totowa, NJ: Humana Press; 2009 [View Article]
    [Google Scholar]
  16. Fortier L-C, Moineau S. Phage production and maintenance of stocks, including expected stock lifetimes. In Clokie MRJ, Kropinski AM. eds Bacteriophages Methods Protoc Vol. 1 New York: Humana Press; 2009 pp 203–219 [View Article]
    [Google Scholar]
  17. Oślizło A, Miernikiewicz P, Piotrowicz A, Owczarek B, Kopciuch A et al. Purification of phage display-modified bacteriophage T4 by affinity chromatography. BMC Biotechnol 2011; 11:1–9 [View Article] [PubMed]
    [Google Scholar]
  18. Sutherland IA. Recent progress on the industrial scale-up of counter-current chromatography. J Chromatogr A 2007; 1151:6–13 [View Article] [PubMed]
    [Google Scholar]
  19. Battu S, Roux A, Delebasee S, Bosgiraud C, Cardot PJP. Sedimentation field-flow fractionation device cleaning, decontamination and sterilization procedures for cellular analysis. J Chromatogr B Biomed Sci Appl 2001; 751:131–141 [View Article] [PubMed]
    [Google Scholar]
  20. Jue E, Yamanishi CD, Chiu RYT, Wu BM, Kamei DT. Using an aqueous two-phase polymer-salt system to rapidly concentrate viruses for improving the detection limit of the lateral-flow immunoassay. Biotechnol Bioeng 2014; 111:2499–2507 [View Article] [PubMed]
    [Google Scholar]
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