1887

Abstract

Biofilms represent microbial communities, encased in a self-produced matrix or extracellular polymeric substance. Microbial biofilms are likely responsible for a large proportion of clinically significant infections and the multicellular nature of biofilm existence has been repeatedly associated with antibiotic resistance. Classical antibiotic-susceptibility testing utilizes artificial growth media and planktonic microbes, but this method may not account for the variability inherent in environments subject to biofilm growth . Experiments were designed to test the hypothesis that nutrient concentration can modulate the antibiotic susceptibility of biofilms. Developing biofilms initiated on surgical sutures, and in selected experiments planktonic cultures, were incubated for 16 h in 66 % tryptic soy broth, 0.2 % glucose (1× TSBg), supplemented with bactericidal concentrations of gentamicin, streptomycin, ampicillin or vancomycin. In parallel experiments, antibiotics were added to growth medium diluted one-third (1/3× TSBg) or concentrated threefold (3× TSBg). Following incubation, viable bacteria were enumerated from planktonic cultures or suture sonicates, and biofilm biomass was assayed using spectrophotometry. Interestingly, bactericidal concentrations of gentamicin (5 µg gentamicin ml) and streptomycin (32 µg streptomycin ml) inhibited biofilm formation in samples incubated in 1/3× or 1× TSBg, but not in samples incubated in 3× TSBg. The nutrient dependence of aminoglycoside susceptibility is not only associated with biofilm formation, as planktonic cultures incubated in 3× TSBg in the presence of gentamicin also showed antibiotic resistance. These findings appeared specific for aminoglycosides because biofilm formation was inhibited in all three growth media supplemented with bactericidal concentrations of the cell wall-active antibiotics, ampicillin and vancomycin. Additional experiments showed that the ability of 3× TSBg to overcome the antibacterial effects of gentamicin was associated with decreased uptake of gentamicin by . Uptake is known to be decreased at low pH, and the kinetic change in pH of growth medium from biofilms incubated in 5 µg gentamicin ml in the presence of 3× TSBg was decreased when compared with pH determinations from biofilms formed in 1/3× or 1× TSBg. These studies underscore the importance of environmental factors, including nutrient concentration and pH, on the antibiotic susceptibility of planktonic and biofilm bacteria.

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2014-06-01
2019-10-15
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References

  1. 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][PubMed]
    [Google Scholar]
  2. Chiang W. C., Nilsson M., Jensen P. Ø., Høiby N., Nielsen T. E., Givskov M., Tolker-Nielsen T.. ( 2013;). Extracellular DNA shields against aminoglycosides in Pseudomonas aeruginosa biofilms. . Antimicrob Agents Chemother 57:, 2352–2361. [CrossRef][PubMed]
    [Google Scholar]
  3. CLSI ( 2006;). Performance Standards for Antimicrobial Susceptibility Testing; Approved Standard, 16th edn, M100-S16. . Wayne, PA:: Clinical and Laboratory Standards Institute;.
  4. Cos P., Toté K., Horemans T., Maes L.. ( 2010;). Biofilms: an extra hurdle for effective antimicrobial therapy. . Curr Pharm Des 16:, 2279–2295. [CrossRef][PubMed]
    [Google Scholar]
  5. Dai C. F., Mangiardi D., Cotanche D. A., Steyger P. S.. ( 2006;). Uptake of fluorescent gentamicin by vertebrate sensory cells in vivo. . Hear Res 213:, 64–78. [CrossRef][PubMed]
    [Google Scholar]
  6. Dunne W. M. Jr, Mason E. O. Jr, Kaplan S. L.. ( 1993;). Diffusion of rifampin and vancomycin through a Staphylococcus epidermidis biofilm. . Antimicrob Agents Chemother 37:, 2522–2526. [CrossRef][PubMed]
    [Google Scholar]
  7. Eisenberg E. S., Mandel L. J., Kaback H. R., Miller M. H.. ( 1984;). Quantitative association between electrical potential across the cytoplasmic membrane and early gentamicin uptake and killing in Staphylococcus aureus. . J Bacteriol 157:, 863–867.[PubMed]
    [Google Scholar]
  8. Fux C. A., Costerton J. W., Stewart P. S., Stoodley P.. ( 2005;). Survival strategies of infectious biofilms. . Trends Microbiol 13:, 34–40. [CrossRef][PubMed]
    [Google Scholar]
  9. Gilbert P., Das J., Foley I.. ( 1997;). Biofilm susceptibility to antimicrobials. . Adv Dent Res 11:, 160–167. [CrossRef][PubMed]
    [Google Scholar]
  10. Hall-Stoodley L., Stoodley P.. ( 2009;). Evolving concepts in biofilm infections. . Cell Microbiol 11:, 1034–1043. [CrossRef][PubMed]
    [Google Scholar]
  11. Hall-Stoodley L., Costerton J. W., Stoodley P.. ( 2004;). Bacterial biofilms: from the natural environment to infectious diseases. . Nat Rev Microbiol 2:, 95–108. [CrossRef][PubMed]
    [Google Scholar]
  12. Hess D. J., Henry-Stanley M. J., Wells C. L.. ( 2011;). Gentamicin promotes Staphylococcus aureus biofilms on silk suture. . J Surg Res 170:, 302–308. [CrossRef][PubMed]
    [Google Scholar]
  13. Hong Y., Brown D. G.. ( 2009;). Variation in bacterial ATP level and proton motive force due to adhesion to a solid surface. . Appl Environ Microbiol 75:, 2346–2353. [CrossRef][PubMed]
    [Google Scholar]
  14. Kotra L. P., Haddad J., Mobashery S.. ( 2000;). Aminoglycosides: perspectives on mechanisms of action and resistance and strategies to counter resistance. . Antimicrob Agents Chemother 44:, 3249–3256. [CrossRef][PubMed]
    [Google Scholar]
  15. Lewis K.. ( 2001;). Riddle of biofilm resistance. . Antimicrob Agents Chemother 45:, 999–1007. [CrossRef][PubMed]
    [Google Scholar]
  16. Lewis K.. ( 2007;). Persister cells, dormancy and infectious disease. . Nat Rev Microbiol 5:, 48–56. [CrossRef][PubMed]
    [Google Scholar]
  17. Lewis K.. ( 2010;). Persister cells. . Annu Rev Microbiol 64:, 357–372. [CrossRef][PubMed]
    [Google Scholar]
  18. Mates S. M., Eisenberg E. S., Mandel L. J., Patel L., Kaback H. R., Miller M. H.. ( 1982;). Membrane potential and gentamicin uptake in Staphylococcus aureus. . Proc Natl Acad Sci U S A 79:, 6693–6697. [CrossRef][PubMed]
    [Google Scholar]
  19. Peeters E., Nelis H. J., Coenye T.. ( 2008;). Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. . J Microbiol Methods 72:, 157–165. [CrossRef][PubMed]
    [Google Scholar]
  20. Resch A., Rosenstein R., Nerz C., Götz F.. ( 2005;). Differential gene expression profiling of Staphylococcus aureus cultivated under biofilm and planktonic conditions. . Appl Environ Microbiol 71:, 2663–2676. [CrossRef][PubMed]
    [Google Scholar]
  21. Römling U., Balsalobre C.. ( 2012;). Biofilm infections, their resilience to therapy and innovative treatment strategies. . J Intern Med 272:, 541–561. [CrossRef][PubMed]
    [Google Scholar]
  22. Shanks R. M. Q., Donegan N. P., Graber M. L., Buckingham S. E., Zegans M. E., Cheung A. L., O’Toole G. A.. ( 2005;). Heparin stimulates Staphylococcus aureus biofilm formation. . Infect Immun 73:, 4596–4606. [CrossRef][PubMed]
    [Google Scholar]
  23. Smith P. A., Romesberg F. E.. ( 2007;). Combating bacteria and drug resistance by inhibiting mechanisms of persistence and adaptation. . Nat Chem Biol 3:, 549–556. [CrossRef][PubMed]
    [Google Scholar]
  24. Stewart P. S., Costerton J. W.. ( 2001;). Antibiotic resistance of bacteria in biofilms. . Lancet 358:, 135–138. [CrossRef][PubMed]
    [Google Scholar]
  25. Stewart P. S., Franklin M. J.. ( 2008;). Physiological heterogeneity in biofilms. . Nat Rev Microbiol 6:, 199–210. [CrossRef][PubMed]
    [Google Scholar]
  26. Suci P. A., Mittelman M. W., Yu F. P., Geesey G. G.. ( 1994;). Investigation of ciprofloxacin penetration into Pseudomonas aeruginosa biofilms. . Antimicrob Agents Chemother 38:, 2125–2133. [CrossRef][PubMed]
    [Google Scholar]
  27. Taber H. W., Mueller J. P., Miller P. F., Arrow A. S.. ( 1987;). Bacterial uptake of aminoglycoside antibiotics. . Microbiol Rev 51:, 439–457.[PubMed]
    [Google Scholar]
  28. Tseng B. S., Zhang W., Harrison J. J., Quach T. P., Song J. L., Penterman J., Singh P. K., Chopp D. L., Packman A. I., Parsek M. R.. ( 2013;). The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin. . Environ Microbiol 15:, 2865–2878.[PubMed]
    [Google Scholar]
  29. 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][PubMed]
    [Google Scholar]
  30. Wells C. L., Henry-Stanley M. J., Barnes A. M. T., Dunny G. M., Hess D. J.. ( 2011;). Relation between antibiotic susceptibility and ultrastructure of Staphylococcus aureus biofilms on surgical suture. . Surg Infect (Larchmt) 12:, 297–305. [CrossRef][PubMed]
    [Google Scholar]
  31. Wu J., Xi C.. ( 2009;). Evaluation of different methods for extracting extracellular DNA from the biofilm matrix. . Appl Environ Microbiol 75:, 5390–5395. [CrossRef][PubMed]
    [Google Scholar]
  32. Zhu Y., Weiss E. C., Otto M., Fey P. D., Smeltzer M. S., Somerville G. A.. ( 2007;). Staphylococcus aureus biofilm metabolism and the influence of arginine on polysaccharide intercellular adhesin synthesis, biofilm formation, and pathogenesis. . Infect Immun 75:, 4219–4226. [CrossRef][PubMed]
    [Google Scholar]
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