1887

Abstract

The presence of persister cells and small-colony variants (SCVs) has been associated with enhanced antibiotic resistance of many organisms in biofilms. This study investigated whether persisters and/or SCVs contribute to the antibiotic resistance of biofilms. A detailed dose-dependent killing of biofilms and planktonic cells with five antibiotics (oxacillin, cefotaxime, amikacin, ciprofloxacin and vancomycin) was analysed by treating them with each antibiotic at a concentration of 0–100 μg ml at 37 °C for 48 h. The killing of biofilm cells by all of the antibiotics showed the presence of persister cells – most cells in the population died, leaving a fraction that persisted, even at higher concentrations of the antibiotics. These persisters represented a transient resistant phenotype and reverted to a killing curve resembling that of the wild-type parent upon re-exposure to the antibiotics. SCVs were observed in biofilms only after treatment with ciprofloxacin, and these SCVs were of a transient nature. The treatment of planktonic cells with oxacillin, cefotaxime, ciprofloxacin and vancomycin killed the entire population and no persisters were detected. Transient SCVs, observed in planktonic cells following exposure to these antibiotics, were killed at higher antibiotic concentrations. The treatment of planktonic cells with amikacin yielded a small subpopulation of survivors that included persisters (at numbers significantly lower than for the biofilms) and highly resistant, stable SCVs with an increased biofilm-forming capacity in comparison with the wild-type parent. Thus the high resistance of biofilms to multiple unrelated antibiotics is largely dependent on the presence of persister cells. Biofilms harbour a large number of persisters in comparison with planktonic cultures, which either do not harbour persisters or harbour only a small number. SCVs, although not specifically associated with biofilms, have an increased biofilm-forming capacity and this may explain the frequent isolation of SCVs from biofilm-associated infections. The intrinsic resistance of these variants may in turn contribute to the enhanced antibiotic resistance of the biofilms thus formed.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.009720-0
2009-08-01
2019-08-18
Loading full text...

Full text loading...

/deliver/fulltext/jmm/58/8/1067.html?itemId=/content/journal/jmm/10.1099/jmm.0.009720-0&mimeType=html&fmt=ahah

References

  1. Acar, J. F., Goldstein, F. W. & Lagrange, P. ( 1978; ). Human infections caused by thiamine- or menadione-requiring Staphylococcus aureus. J Clin Microbiol 8, 142–147.
    [Google Scholar]
  2. Allegrucci, M. & Sauer, K. ( 2007; ). Characterization of colony morphology variants isolated from Streptococcus pneumoniae biofilms. J Bacteriol 189, 2030–2038.[CrossRef]
    [Google Scholar]
  3. Amorena, B., Gracia, E., Monzón, M., Leiva, J., Oteiza, C., Pérez, M., Alabart, J. L. & Hernández-Yago, J. ( 1999; ). Antibiotic susceptibility assay for Staphylococcus aureus in biofilms developed in vitro. J Antimicrob Chemother 44, 43–55.[CrossRef]
    [Google Scholar]
  4. Anderl, J. N., Franklin, M. J. & Stewart, P. S. ( 2000; ). Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob Agents Chemother 44, 1818–1824.[CrossRef]
    [Google Scholar]
  5. Ashby, M. J., Neale, J. E., Knott, S. J. & Critchley, I. A. ( 1994; ). Effect of antibiotics on non-growing planktonic cells and biofilms of Escherichia coli. J Antimicrob Chemother 33, 443–452.[CrossRef]
    [Google Scholar]
  6. Balaban, N. Q., Merrin, J., Chait, R., Kowalik, L. & Leibler, S. ( 2004; ). Bacterial persistence as a phenotypic switch. Science 305, 1622–1625.[CrossRef]
    [Google Scholar]
  7. Bayston, R., Ashraf, W. & Smith, T. ( 2007; ). Triclosan resistance in methicillin-resistant Staphylococcus aureus expressed as small colony variants: a novel mode of evasion of susceptibility to antiseptics. J Antimicrob Chemother 59, 848–853.[CrossRef]
    [Google Scholar]
  8. Bendouah, Z., Barbeau, J., Hamad, W. A. & Desrosiers, M. ( 2006; ). Biofilm formation by Staphylococcus aureus and Pseudomonas aeruginosa is associated with an unfavorable evolution after surgery for chronic sinusitis and nasal polyposis. Otolaryngol Head Neck Surg 134, 991–996.[CrossRef]
    [Google Scholar]
  9. Besier, S., Smaczny, C., von Mallinckrodt, C., Krahl, A., Ackermann, H., Brade, V. & Wichelhaus, T. A. ( 2007; ). Prevalence and clinical significance of Staphylococcus aureus small-colony variants in cystic fibrosis lung disease. J Clin Microbiol 45, 168–172.[CrossRef]
    [Google Scholar]
  10. Bigger, J. W. ( 1944; ). Treatment of staphylococcal infections with penicillin. Lancet 244, 497–500.[CrossRef]
    [Google Scholar]
  11. Brooun, A., Liu, S. & Lewis, K. ( 2000; ). A dose–response study of antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 44, 640–646.[CrossRef]
    [Google Scholar]
  12. Ceri, H., Olson, M. E., Stremick, C., Read, R. R., Morck, D. & Buret, A. ( 1999; ). The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol 37, 1771–1776.
    [Google Scholar]
  13. Chuard, C., Vaudaux, P. E., Proctor, R. A. & Lew, D. P. ( 1997; ). Decreased susceptibility to antibiotic killing of a stable small colony variant of Staphylococcus aureus in fluid phase and on fibronectin-coated surfaces. J Antimicrob Chemother 39, 603–608.[CrossRef]
    [Google Scholar]
  14. CLSI ( 2006; ). Performance Standards for Antimicrobial Susceptibility Testing; 16th Informational Supplement. CLSI document M100-S16. Wayne, PA: Clinical and Laboratory Standards Institute.
  15. Costerton, J. W., Stewart, P. S. & Greenberg, E. P. ( 1999; ). Bacterial biofilms: a common cause of persistent infections. Science 284, 1318–1322.[CrossRef]
    [Google Scholar]
  16. Drenkard, E. & Ausubel, F. M. ( 2002; ). Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 416, 740–743.[CrossRef]
    [Google Scholar]
  17. Fux, C. A., Costerton, J. W., Stewart, P. S. & Stoodley, P. ( 2005; ). Survival strategies of infectious biofilms. Trends Microbiol 13, 34–40.[CrossRef]
    [Google Scholar]
  18. Gilbert, P., Allison, D. G. & McBain, A. J. ( 2002; ). Biofilms in vitro and in vivo: do singular mechanisms imply cross-resistance? Symp Ser Soc Appl Microbiol 92, 98S–110S.[CrossRef]
    [Google Scholar]
  19. Haussler, S., Ziegler, I., Lottel, A., von Gotz, F., Rohde, M., Wehmhohner, D., Saravanamuthu, S., Tummler, B. & Steinmetz, I. ( 2003; ). Highly adherent small-colony variants of Pseudomonas aeruginosa in cystic fibrosis lung infection. J Med Microbiol 52, 295–301.[CrossRef]
    [Google Scholar]
  20. Higashi, J. M. & Sullam, P. M. ( 2006; ). Staphylococcus aureus biofilms. In Biofilms, Infection, and Antimicrobial Therapy, pp. 81–108. Edited by J. L. Pace, M. E. Rupp & R. G. Finch. Boca Raton, FL: Taylor & Francis.
  21. Keren, I., Kaldalu, N., Spoering, A., Wang, Y. & Lewis, K. ( 2004a; ). Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett 230, 13–18.[CrossRef]
    [Google Scholar]
  22. Keren, I., Shah, D., Spoering, A., Kaldalu, N. & Lewis, K. ( 2004b; ). Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli. J Bacteriol 186, 8172–8180.[CrossRef]
    [Google Scholar]
  23. Lewis, K. ( 2007; ). Persister cells, dormancy and infectious disease. Nat Rev Microbiol 5, 48–56.[CrossRef]
    [Google Scholar]
  24. Lewis, K., Spoering, A. L., Kaldalu, N., Keren, I. & Shah, D. ( 2006; ). Persisters: specialized cells responsible for biofilm tolerance to antimicrobial agents. In Biofilms, Infection, and Antimicrobial Therapy, pp. 241–256. Edited by J. L. Pace, M. E. Rupp & R. G. Finch. Boca Raton, FL: Taylor & Francis.
  25. Mah, T. F. & O'Toole, G. A. ( 2001; ). Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 9, 34–39.[CrossRef]
    [Google Scholar]
  26. Miles, A. A., Misra, S. S. & Irwin, J. O. ( 1938; ). The estimation of the bactericidal power of the blood. J Hyg (Lond) 38, 732–749.[CrossRef]
    [Google Scholar]
  27. Muli, F. W. & Struthers, J. K. ( 1998; ). The growth of Gardnerella vaginalis and Lactobacillus acidophilus in Sorbarod biofilms. J Med Microbiol 47, 401–405.[CrossRef]
    [Google Scholar]
  28. Proctor, R. A. & Peters, G. ( 1998; ). Small colony variants in staphylococcal infections: diagnostic and therapeutic implications. Clin Infect Dis 27, 419–422.[CrossRef]
    [Google Scholar]
  29. Proctor, R. A., van Langevelde, P., Kristjansson, M., Maslow, J. N. & Arbeit, R. D. ( 1995; ). Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus. Clin Infect Dis 20, 95–102.[CrossRef]
    [Google Scholar]
  30. Proctor, R. A., Kahl, B., von Eiff, C., Vaudaux, P. E., Lew, D. P. & Peters, G. ( 1998; ). Staphylococcal small colony variants have novel mechanisms for antibiotic resistance. Clin Infect Dis 27 (Suppl. 1), S68–S74.[CrossRef]
    [Google Scholar]
  31. Proctor, R. A., von Eiff, C., Kahl, B. C., Becker, K., McNamara, P., Herrmann, M. & Peters, G. ( 2006; ). Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat Rev Microbiol 4, 295–305.[CrossRef]
    [Google Scholar]
  32. Roberts, M. E. & Stewart, P. S. ( 2005; ). Modelling protection from antimicrobial agents in biofilms through the formation of persister cells. Microbiology 151, 75–80.[CrossRef]
    [Google Scholar]
  33. Spoering, A. L. & Lewis, K. ( 2001; ). Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J Bacteriol 183, 6746–6751.[CrossRef]
    [Google Scholar]
  34. Stewart, P. S. & Costerton, J. W. ( 2001; ). Antibiotic resistance of bacteria in biofilms. Lancet 358, 135–138.[CrossRef]
    [Google Scholar]
  35. Vuong, C., Saenz, H. L., Gotz, F. & Otto, M. ( 2000; ). Impact of the agr quorum-sensing system on adherence to polystyrene in Staphylococcus aureus. J Infect Dis 182, 1688–1693.[CrossRef]
    [Google Scholar]
  36. Walters, M. C., III, Roe, F., Bugnicourt, A., Franklin, M. J. & Stewart, P. S. ( 2003; ). Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. Antimicrob Agents Chemother 47, 317–323.[CrossRef]
    [Google Scholar]
  37. Williams, I., Venables, W. A., Lloyd, D., Paul, F. & Critchley, I. ( 1997; ). The effects of adherence to silicone surfaces on antibiotic susceptibility in Staphylococcus aureus. Microbiology 143, 2407–2413.[CrossRef]
    [Google Scholar]
  38. Yao, J. D. C. & Moellering, R. C., Jr ( 2003; ). Antibacterial agents. In Manual of Clinical Microbiology, 8th edn, pp. 1039–1073. Edited by P. R. Murray, E. J. Baron, M. A. Pfaller, J. H. Jorgensen & R. H. Yolken. Washington, DC: American Society for Microbiology.
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.009720-0
Loading
/content/journal/jmm/10.1099/jmm.0.009720-0
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