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Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation

Abstract

Colonization of the lungs of cystic fibrosis (CF) patients by the opportunistic bacterial pathogen Pseudomonas aeruginosa is the principal cause of mortality in CF populations1,2. Pseudomonas aeruginosa infections generally persist despite the use of long-term antibiotic therapy1,3. This has been explained by postulating that P. aeruginosa forms an antibiotic-resistant biofilm4,5 consisting of bacterial communities embedded in an exopolysaccharide matrix. Alternatively, it has been proposed that resistant P. aeruginosa variants may be selected in the CF respiratory tract by antimicrobial therapy itself1,6. Here we report that both explanations are correct, and are interrelated. We found that antibiotic-resistant phenotypic variants of P. aeruginosa with enhanced ability to form biofilms arise at high frequency both in vitro and in the lungs of CF patients. We also identified a regulatory protein (PvrR) that controls the conversion between antibiotic-resistant and antibiotic-susceptible forms. Compounds that affect PvrR function could have an important role in the treatment of CF infections.

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Figure 1: Characterization of P. aeruginosa antibiotic-resistant RSCV.
Figure 2: Appearance of phenotypic variants resistant to kanamycin depends on environmental factors.
Figure 3: Identification and characterization of the P. aeruginosa PvrR response regulator.

References

  1. Govan, J. R. & Deretic, V. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol. Rev. 60, 539–574 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Smith, J. J., Travis, S. M., Greenberg, E. P. & Welsh, M. J. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85, 229–236 (1996).

    CAS  Article  PubMed  Google Scholar 

  3. Hoiby, N. Antibiotic therapy for chronic infection of pseudomonas in the lung. Annu. Rev. Med. 44, 1–10 (1993).

    CAS  Article  PubMed  Google Scholar 

  4. Singh, P. K. et al. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407, 762–764 (2000).

    ADS  CAS  Article  PubMed  Google Scholar 

  5. Costerton, J. W., Stewart, P. S. & Greenberg, E. P. Bacterial biofilms: a common cause of persistent infections. Science 284, 1318–1322 (1999).

    ADS  CAS  Article  PubMed  Google Scholar 

  6. Speert, D. P. et al. Conversion of Pseudomonas aeruginosa to the phenotype characteristic of strains from patients with cystic fibrosis. J. Clin. Microbiol. 28, 188–194 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Rahme, L. G. et al. Common virulence factors for bacterial pathogenicity in plants and animals. Science 268, 1899–1902 (1995).

    ADS  CAS  Article  PubMed  Google Scholar 

  8. Haussler, S., Tummler, B., Weissbrodt, H., Rohde, M. & Steinmetz, I. Small-colony variants of Pseudomonas aeruginosa in cystic fibrosis. Clin. Infect. Dis. 29, 621–625 (1999).

    CAS  Article  PubMed  Google Scholar 

  9. Drumm, B., Neumann, A. W., Policova, Z. & Sherman, P. M. Bacterial cell surface hydrophobicity properties in the mediation of in vitro adhesion by the rabbit enteric pathogen Escherichia coli strain RDEC-1. J. Clin. Invest. 84, 1588–1594 (1989).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Hentzer, M. et al. Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function. J. Bacteriol. 183, 5395–5401 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Henderson, I. R., Owen, P. & Nataro, J. P. Molecular switches—the ON and OFF of bacterial phase variation. Mol. Microbiol. 33, 919–932 (1999).

    CAS  Article  PubMed  Google Scholar 

  12. Grewal, S. I., Han, B. & Johnstone, K. Identification and characterization of a locus which regulates multiple functions in Pseudomonas tolaasii, the cause of brown blotch disease of Agaricus bisporus. J. Bacteriol. 177, 4658–4668 (1995).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Holloway, B. W., Krishnapillai, V. & Morgan, A. F. Chromosomal genetics of Pseudomonas. Microbiol. Rev. 43, 73–102 (1979).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Oliver, A., Canton, R., Campo, P., Baquero, F. & Blazquez, J. High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 288, 1251–1254 (2000).

    ADS  CAS  Article  PubMed  Google Scholar 

  15. Han, B., Pain, A. & Johnstone, K. Spontaneous duplication of a 661 bp element within a two-component sensor regulator gene causes phenotypic switching in colonies of Pseudomonas tolaasii, cause of brown blotch disease of mushrooms. Mol. Microbiol. 25, 211–218 (1997).

    CAS  Article  PubMed  Google Scholar 

  16. Lewis, K. Riddle of biofilm resistance. Antimicrob. Agents Chemother. 45, 999–1007 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Mah, T. F. & O'Toole, G. A. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 9, 34–39 (2001).

    CAS  Article  PubMed  Google Scholar 

  18. McNamara, P. J. & Proctor, R. A. Staphylococcus aureus small colony variants, electron transport and persistent infections. Int. J. Antimicrob. Agents 14, 117–122 (2000).

    CAS  Article  PubMed  Google Scholar 

  19. Ausubel, F. M. et al. Short Protocols in Molecular Biology (Greene Publishing and Wiley, New York, 1992).

    Google Scholar 

  20. O'Toole, G. A. & Kolter, R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol. Microbiol. 28, 449–461 (1998).

    CAS  Article  PubMed  Google Scholar 

  21. Sherman, P. M., Houston, W. L. & Boedeker, E. C. Functional heterogeneity of intestinal Escherichia coli strains expressing type 1 somatic pili (fimbriae): assessment of bacterial adherence to intestinal membranes and surface hydrophobicity. Infect. Immun. 49, 797–804 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Christensen, B. B. et al. Molecular tools for study of biofilm physiology. Methods Enzymol. 310, 20–42 (1999).

    CAS  Article  PubMed  Google Scholar 

  23. Bloemberg, G. V., O'Toole, G. A., Lugtenberg, B. J. & Kolter, R. Green fluorescent protein as a marker for Pseudomonas spp. Appl. Environ. Microbiol. 63, 4543–4551 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Samadpour, M., Moseley, S. L. & Lory, S. Biotinylated DNA probes for exotoxin A and pilin genes in the differentiation of Pseudomonas aeruginosa strains. J. Clin. Microbiol. 26, 2319–2323 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Andrews, J. M. Determination of minimum inhibitory concentrations. J. Antimicrob. Chemother. 48, (Suppl. A) 5–16 (2001).

    CAS  Article  PubMed  Google Scholar 

  26. Figurski, D. H. & Helinski, D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl Acad. Sci. USA 76, 1648–1652 (1979).

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Schweizer, H. P. Escherichia-Pseudomonas shuttle vectors derived from pUC18/19. Gene 97, 109–121 (1991).

    CAS  Article  PubMed  Google Scholar 

  28. Donnenberg, M. S. & Kaper, J. B. Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect. Immun. 59, 4310–4317 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank J. Lai and R. A. Rogers for image analysis of biofilm structures; T. Mylonakis for help in obtaining the samples and clinical data from CF patients; M. J. Ferraro and K. Henry for providing the CF sputum samples; and M. Wildermuth for help analysing protein sequences. This work was supported by grants from Aventis SA and the National Institutes of Health.

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Correspondence to Frederick M. Ausubel.

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The work described in this manuscript was supported by Aventis SA, a pharmaceutical compnay. Although the data as described has no direct commercial application, it could be perceived that Aventis SA has a financial stake in the outcome of the experiments described. The authors have filed a patent application describing the work presented in this manuscript.

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Drenkard, E., Ausubel, F. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 416, 740–743 (2002). https://doi.org/10.1038/416740a

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