1887

Abstract

In patients with diabetes mellitus, foot infections pose a significant risk. These are complex infections commonly caused by , and , all of which are potentially susceptible to bacteriophages. Here, we characterized five bacteriophages that we had determined previously to have antimicrobial and wound-healing potential in chronic , and infections. Morphological and genetic features indicated that the bacteriophages were lytic members of the family or and did not harbour any known bacterial virulence genes. Combinations of the bacteriophages had broad host ranges for the different target bacterial species. The activity of the bacteriophages against planktonic cells revealed effective, early killing at 4 h, followed by bacterial regrowth to pre-treatment levels by 24 h. Using metabolic activity as a measure of cell viability within established biofilms, we found significant cell impairment following bacteriophage exposure. Repeated treatment every 4 h caused a further decrease in cell activity. The greatest effects on both planktonic and biofilm cells occurred at a bacteriophage : bacterium input multiplicity of 10. These studies on both planktonic cells and established biofilms allowed us to better evaluate the effects of a high input multiplicity and a multiple-dose treatment protocol, and the findings support further clinical development of bacteriophage therapy.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.071753-0
2014-08-01
2019-12-08
Loading full text...

Full text loading...

/deliver/fulltext/jmm/63/8/1055.html?itemId=/content/journal/jmm/10.1099/jmm.0.071753-0&mimeType=html&fmt=ahah

References

  1. Abedon S. T. . ( 2009; ). Kinetics of phage-mediated biocontrol of bacteria. . Foodborne Pathog Dis 6:, 807–815. [CrossRef] [PubMed]
    [Google Scholar]
  2. Abedon S. T. . ( 2010; ). The ‘nuts and bolts’ of phage therapy. . Curr Pharm Biotechnol 11:, 1. [CrossRef] [PubMed]
    [Google Scholar]
  3. Abedon S. T. , Thomas-Abedon C. . ( 2010; ). Phage therapy pharmacology. . Curr Pharm Biotechnol 11:, 28–47. [CrossRef] [PubMed]
    [Google Scholar]
  4. Abedon S. T. , Kuhl S. J. , Blasdel B. G. , Kutter E. M. . ( 2011; ). Phage treatment of human infections. . Bacteriophage 1:, 66–85. [CrossRef] [PubMed]
    [Google Scholar]
  5. Ackermann H.-W. . ( 2009; ). Phage classification and characterization. . In Bacteriophages Methods and Protocols, pp. 127–140. Edited by Clokie M. R. J. , Kropinski A. M. . . Clifton, NJ:: Humana Press;. [CrossRef]
    [Google Scholar]
  6. Adams M. H. . ( 1959; ). Methods of study of bacterial viruses: isolation of bacterial viruses. . In Bacteriophages, pp. 447–449. New York:: Interscience;.
    [Google Scholar]
  7. Ansaldi M. . ( 2012; ). Cell biology perspectives in phage biology. . Front Biosci (Elite Ed) 4:, 1823–1829. . [CrossRef] [PubMed]
    [Google Scholar]
  8. Armon R. , Kott Y. . ( 1993; ). A simple, rapid and sensitive presence/absence detection test for bacteriophage in drinking water. . J Appl Bacteriol 74:, 490–496. [CrossRef] [PubMed]
    [Google Scholar]
  9. Bruynoghe R. , Maisin J. . ( 1921; ). Essais de thérapeutique au moyen du bacteriophage. C R . Soc Biol 85:, 1120–1121 (in French).
    [Google Scholar]
  10. Capparelli R. , Parlato M. , Borriello G. , Salvatore P. , Iannelli D. . ( 2007; ). Experimental phage therapy against Staphylococcus aureus in mice. . Antimicrob Agents Chemother 51:, 2765–2773. [CrossRef] [PubMed]
    [Google Scholar]
  11. Carlton R. M. . ( 1999; ). Phage therapy: past history and future prospects. . Arch Immunol Ther Exp (Warsz) 47:, 267–274.[PubMed]
    [Google Scholar]
  12. Cerca N. , Martins S. , Cerca F. , Jefferson K. K. , Pier G. B. , Oliveira R. , Azeredo J. . ( 2005; ). Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry. . J Antimicrob Chemother 56:, 331–336. [CrossRef] [PubMed]
    [Google Scholar]
  13. Ceyssens P. J. , Lavigne R. , Mattheus W. , Chibeu A. , Hertveldt K. , Mast J. , Robben J. , Volckaert G. . ( 2006; ). Genomic analysis of Pseudomonas aeruginosa phages LKD16 and LKA1: establishment of the phiKMV subgroup within the T7 supergroup. . J Bacteriol 188:, 6924–6931. [CrossRef] [PubMed]
    [Google Scholar]
  14. Ceyssens P. J. , Brabban A. , Rogge L. , Lewis M. S. , Pickard D. , Goulding D. , Dougan G. , Noben J. P. , Kropinski A. et al. ( 2010; ). Molecular and physiological analysis of three Pseudomonas aeruginosa phages belonging to the “N4-like viruses”. . Virology 405:, 26–30. [CrossRef] [PubMed]
    [Google Scholar]
  15. Chan B. K. , Abedon S. T. . ( 2012; ). Phage therapy pharmacology phage cocktails. . Adv Appl Microbiol 78:, 1–23. [CrossRef] [PubMed]
    [Google Scholar]
  16. Chang K. C. , Lin N. T. , Hu A. , Lin Y. S. , Chen L. K. , Lai M. J. . ( 2011; ). Genomic analysis of bacteriophage ϕAB1, a ϕKMV-like virus infecting multidrug-resistant Acinetobacter baumannii . . Genomics 97:, 249–255. [CrossRef] [PubMed]
    [Google Scholar]
  17. Chao L. , Levin B. R. , Stewart F. M. . ( 1977; ). A complex community in a simple habitat: an experimental study with bacteria and phage. . Ecology 58:, 369–378. [CrossRef]
    [Google Scholar]
  18. Chibeu A. , Lingohr E. J. , Masson L. , Manges A. , Harel J. , Ackermann H. W. , Kropinski A. M. , Boerlin P. . ( 2012; ). Bacteriophages with the ability to degrade uropathogenic Escherichia coli biofilms. . Viruses 4:, 471–487. [CrossRef] [PubMed]
    [Google Scholar]
  19. Chopra I. , Hodgson J. , Metcalf B. , Poste G. . ( 1997; ). The search for antimicrobial agents effective against bacteria resistant to multiple antibiotics. . Antimicrob Agents Chemother 41:, 497–503.[PubMed]
    [Google Scholar]
  20. Cornelissen A. , Ceyssens P. J. , T’Syen J. , Van Praet H. , Noben J. P. , Shaburova O. V. , Krylov V. N. , Volckaert G. , Lavigne R. . ( 2011; ). The T7-related Pseudomonas putida phage ϕ15 displays virion-associated biofilm degradation properties. . PLoS ONE 6:, e18597. [CrossRef] [PubMed]
    [Google Scholar]
  21. Gill J. J. , Hyman P. . ( 2010; ). Phage choice, isolation, and preparation for phage therapy. . Curr Pharm Biotechnol 11:, 2–14. [CrossRef] [PubMed]
    [Google Scholar]
  22. Goode D. , Allen V. M. , Barrow P. A. . ( 2003; ). Reduction of experimental Salmonella and Campylobacter contamination of chicken skin by application of lytic bacteriophages. . Appl Environ Microbiol 69:, 5032–5036. [CrossRef] [PubMed]
    [Google Scholar]
  23. Gu J. , Liu X. , Lu R. , Li Y. , Song J. , Lei L. , Sun C. , Feng X. , Du C. et al. ( 2012; ). Complete genome sequence of Staphylococcus aureus bacteriophage GH15. . J Virol 86:, 8914–8915. [CrossRef] [PubMed]
    [Google Scholar]
  24. Hughes K. A. , Sutherland I. W. , Jones M. V. . ( 1998; ). Biofilm susceptibility to bacteriophage attack: the role of phage-borne polysaccharide depolymerase. . Microbiology 144:, 3039–3047. [CrossRef] [PubMed]
    [Google Scholar]
  25. Kasman L. M. , Kasman A. , Westwater C. , Dolan J. , Schmidt M. G. , Norris J. S. . ( 2002; ). Overcoming the phage replication threshold: a mathematical model with implications for phage therapy. . J Virol 76:, 5557–5564. [CrossRef] [PubMed]
    [Google Scholar]
  26. Kropinski A. , Mazzocco A. , Waddell T. , Lingohr E. , Johnson R. . ( 2009; ). Enumeration of bacteriophages by double agar overlay plaque assay. . In Bacteriophages Methods and Protocols, vol. 1. Isolation, Characterization, and Interactions (Methods in Molecular Biology) series, vol. 501, pp. 69–76. Edited by Clokie M. , Kropinski A. . . New York:: Humana Press, Springer Science + Business Media;. [CrossRef]
    [Google Scholar]
  27. Kumari S. , Harjai K. , Chhibber S. . ( 2010; ). Topical treatment of Klebsiella pneumoniae B5055 induced burn wound infection in mice using natural products. . J Infect Dev Ctries 4:, 367–377.[PubMed] [CrossRef]
    [Google Scholar]
  28. Kussell E. , Kishony R. , Balaban N. Q. , Leibler S. . ( 2005; ). Bacterial persistence: a model of survival in changing environments. . Genetics 169:, 1807–1814. [CrossRef] [PubMed]
    [Google Scholar]
  29. Kutter E. . ( 2009; ). Phage host range and efficiency of plating. . Methods Mol Biol 501:, 141–149. [CrossRef] [PubMed]
    [Google Scholar]
  30. Kutter E. , De Vos D. , Gvasalia G. , Alavidze Z. , Gogokhia L. , Kuhl S. , Abedon S. T. . ( 2010; ). Phage therapy in clinical practice: treatment of human infections. . Curr Pharm Biotechnol 11:, 69–86. [CrossRef] [PubMed]
    [Google Scholar]
  31. Lammens E. , Ceyssens P. J. , Voet M. , Hertveldt K. , Lavigne R. , Volckaert G. . ( 2009; ). Representational difference analysis (RDA) of bacteriophage genomes. . J Microbiol Methods 77:, 207–213. [CrossRef] [PubMed]
    [Google Scholar]
  32. Lavery L. A. , Armstrong D. G. , Wunderlich R. P. , Mohler M. J. , Wendel C. S. , Lipsky B. A. . ( 2006; ). Risk factors for foot infections in individuals with diabetes. . Diabetes Care 29:, 1288–1293. [CrossRef] [PubMed]
    [Google Scholar]
  33. Lavigne R. , Burkal’tseva M. V. , Robben J. , Sykilinda N. N. , Kurochkina L. P. , Grymonprez B. , Jonckx B. , Krylov V. N. , Mesyanzhinov V. V. , Volckaert G. . ( 2003; ). The genome of bacteriophage ϕKMV, a T7-like virus infecting Pseudomonas aeruginosa . . Virology 312:, 49–59. [CrossRef] [PubMed]
    [Google Scholar]
  34. Lavigne R. , Darius P. , Summer E. J. , Seto D. , Mahadevan P. , Nilsson A. S. , Ackermann H. W. , Kropinski A. M. . ( 2009; ). Classification of Myoviridae bacteriophages using protein sequence similarity. . BMC Microbiol 9:, 224. [CrossRef] [PubMed]
    [Google Scholar]
  35. Lenski R. E. , Levin B. R. . ( 1985; ). Constraints on the coevolution of bacteria and virulent phage: a model, some experiments and predictions for natural communities. . Am Nat 125:, 585–602. [CrossRef]
    [Google Scholar]
  36. Lipsky B. A. , Berendt A. R. , Deery H. G. , Embil J. M. , Joseph W. S. , Karchmer A. W. , LeFrock J. L. , Lew D. P. , Mader J. T. et al. ( 2004; ). Diagnosis and treatment of diabetic foot infections. . Clin Infect Dis 39:, 885–910. [CrossRef] [PubMed]
    [Google Scholar]
  37. Loc-Carrillo C. , Wu S. , Beck J. P. . ( 2012; ). Phage therapy of wounds and related purulent infections. . In Bacteriophages in Health and Disease. pp. 185–202. Edited by Hyman P. . . Cambridge, USA:: CAB International;. [CrossRef]
    [Google Scholar]
  38. Mendes J. J. , Marques-Costa A. , Vilela C. , Neves J. , Candeias N. , Cavaco-Silva P. , Melo-Cristino J. . ( 2012; ). Clinical and bacteriological survey of diabetic foot infections in Lisbon. . Diabetes Res Clin Pract 95:, 153–161. [CrossRef] [PubMed]
    [Google Scholar]
  39. Mendes J. J. , Leandro C. , Corte-Real S. , Barbosa R. , Cavaco-Silva P. , Melo-Cristino J. , Górski A. , Garcia M. . ( 2013; ). Wound healing potential of topical bacteriophage therapy on diabetic cutaneous wounds. . Wound Repair Regen 21:, 595–603. [CrossRef] [PubMed]
    [Google Scholar]
  40. Merabishvili M. , Pirnay J. P. , Verbeken G. , Chanishvili N. , Tediashvili M. , Lashkhi N. , Glonti T. , Krylov V. , Mast J. et al. ( 2009; ). Quality-controlled small-scale production of a well-defined bacteriophage cocktail for use in human clinical trials. . PLoS ONE 4:, e4944. [CrossRef] [PubMed]
    [Google Scholar]
  41. Międzybrodzki R. , Borysowski J. , Weber-Dąbrowska B. , Fortuna W. , Letkiewicz S. , Szufnarowski K. , Pawełczyk Z. , Rogóż P. , Kłak M. et al. ( 2012; ). Clinical aspects of phage therapy. . Adv Virus Res 83:, 73–121. [CrossRef] [PubMed]
    [Google Scholar]
  42. Miller H. . ( 1987; ). Practical aspects of preparing phage and plasmid DNA: growth, maintenance, and storage of bacteria and bacteriophage. . Methods Enzymol 152:, 145–170. [CrossRef] [PubMed]
    [Google Scholar]
  43. Mottola C. , Mendes J. , Cavaco-Silva P. , Melo-Cristino J. , Oliveira M. . ( 2013; ). Relevance of inoculum size on biofilm formation by diabetic foot bacterial isolates. . In Portuguese Congress of Microbiology and Biotechnology 2013. 6–8 December, Aveiro Portugal.
    [Google Scholar]
  44. Murray P. , Baron E. , Jorgensen J. , Pfaller M. , Yolken R. . ( 2003; ). Manual of Clinical Microbiology, , 8th edn.. Washington, DC:: American Society for Microbiology;.
    [Google Scholar]
  45. NCCLS ( 1999; ). Methods for Determining Bactericidal Activity of Antimicrobial Agents: approved guideline M26-A. . Wayne, PA:: National Committee for Clinical Laboratory Standards;.
  46. O’Flaherty S. , Coffey A. , Edwards R. , Meaney W. , Fitzgerald G. F. , Ross R. P. . ( 2004; ). Genome of staphylococcal phage K: a new lineage of Myoviridae infecting Gram-positive bacteria with a low G+C content. . J Bacteriol 186:, 2862–2871. [CrossRef] [PubMed]
    [Google Scholar]
  47. O’Flaherty S. , Ross R. P. , Meaney W. , Fitzgerald G. F. , Elbreki M. F. , Coffey A. . ( 2005; ). Potential of the polyvalent anti-Staphylococcus bacteriophage K for control of antibiotic-resistant staphylococci from hospitals. . Appl Environ Microbiol 71:, 1836–1842. [CrossRef] [PubMed]
    [Google Scholar]
  48. O’Flynn G. , Ross R. P. , Fitzgerald G. F. , Coffey A. . ( 2004; ). Evaluation of a cocktail of three bacteriophages for biocontrol of Escherichia coli O157 : H7. . Appl Environ Microbiol 70:, 3417–3424. [CrossRef] [PubMed]
    [Google Scholar]
  49. Percival S. L. , Hill K. E. , Williams D. W. , Hooper S. J. , Thomas D. W. , Costerton J. W. . ( 2012; ). A review of the scientific evidence for biofilms in wounds. . Wound Repair Regen 20:, 647–657. [CrossRef] [PubMed]
    [Google Scholar]
  50. Pettit R. K. , Weber C. A. , Kean M. J. , Hoffmann H. , Pettit G. R. , Tan R. , Franks K. S. , Horton M. L. . ( 2005; ). Microplate Alamar blue assay for Staphylococcus epidermidis biofilm susceptibility testing. . Antimicrob Agents Chemother 49:, 2612–2617. [CrossRef] [PubMed]
    [Google Scholar]
  51. Pettit R. K. , Weber C. A. , Pettit G. R. . ( 2009; ). Application of a high throughput Alamar blue biofilm susceptibility assay to Staphylococcus aureus biofilms. . Ann Clin Microbiol Antimicrob 8:, 28. [CrossRef] [PubMed]
    [Google Scholar]
  52. Popova A. V. , Zhilenkov E. L. , Myakinina V. P. , Krasilnikova V. M. , Volozhantsev N. V. . ( 2012; ). Isolation and characterization of wide host range lytic bacteriophage AP22 infecting Acinetobacter baumannii . . FEMS Microbiol Lett 332:, 40–46. [CrossRef] [PubMed]
    [Google Scholar]
  53. Rabinovitch A. , Aviram I. , Zaritsky A. . ( 2003; ). Bacterial debris – an ecological mechanism for coexistence of bacteria and their viruses. . J Theor Biol 224:, 377–383. [CrossRef] [PubMed]
    [Google Scholar]
  54. Sahm D. F. , Deane J. , Pillar C. M. , Fernandes P. . ( 2013; ). In vitro activity of CEM-102 (fusidic acid) against prevalent clones and resistant phenotypes of Staphylococcus aureus. . Antimicrob Agents Chemother 57:, 4535–4536. [CrossRef] [PubMed]
    [Google Scholar]
  55. Sambrook J. , Fritsch E. , Maniatis T. . ( 1989; ). Molecular Cloning: a Laboratory Manual, , 2nd edn.. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory Press;.
    [Google Scholar]
  56. Turnidge J. , Collignon P. . ( 1999; ). Resistance to fusidic acid. . Int J Antimicrob Agents 12: (Suppl. 2), S35–S44. [CrossRef] [PubMed]
    [Google Scholar]
  57. Wild S. , Roglic G. , Green A. , Sicree R. , King H. . ( 2004; ). Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. . Diabetes Care 27:, 1047–1053. [CrossRef] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.071753-0
Loading
/content/journal/jmm/10.1099/jmm.0.071753-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error