Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance


Biofilms are surface-attached microbial communities with characteristic architecture and phenotypic and biochemical properties distinct from their free-swimming, planktonic counterparts1. One of the best-known of these biofilm-specific properties is the development of antibiotic resistance that can be up to 1,000-fold greater than planktonic cells2. We report a genetic determinant of this high-level resistance in the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa. We have identified a mutant of P. aeruginosa that, while still capable of forming biofilms with the characteristic P. aeruginosa architecture, does not develop high-level biofilm-specific resistance to three different classes of antibiotics. The locus identified in our screen, ndvB, is required for the synthesis of periplasmic glucans. Our discovery that these periplasmic glucans interact physically with tobramycin suggests that these glucose polymers may prevent antibiotics from reaching their sites of action by sequestering these antimicrobial agents in the periplasm. Our results indicate that biofilms themselves are not simply a diffusion barrier to these antibiotics, but rather that bacteria within these microbial communities employ distinct mechanisms to resist the action of antimicrobial agents.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Flow cell assay for antibiotic sensitivity.
Figure 2: Colony biofilm assay for antibiotic sensitivity.
Figure 3: Periplasmic glucans interact with Tb.
Figure 4: ndvB is preferentially expressed in biofilms.


  1. 1

    Davey, M. E. & O'Toole, G. A. Microbial biofilms: from ecology to molecular genetics. Microbiol. Mol. Biol. Rev. 64, 847–867 (2000)

    CAS  Article  Google Scholar 

  2. 2

    Hoyle, B. D. & Costerton, W. J. Bacterial resistance to antibiotics: the role of biofilms. Prog. Drug Res. 37, 91–105 (1991)

    CAS  PubMed  Google Scholar 

  3. 3

    Costerton, J. W., Stewart, P. S. & Greenberg, E. P. Bacterial biofilms: a common cause of persistent infections. Science 284, 318–322 (1999)

    Article  Google Scholar 

  4. 4

    Anderl, J. N., Franklin, M. J. & Stewart, P. S. Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob. Agents Chemother. 44, 1818–1824 (2000)

    CAS  Article  Google Scholar 

  5. 5

    Walters, M. C., Roe, F., Bugnicourt, A., Franklin, M. J. & Stewart, P. S. 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 (2003)

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

    Heydorn, A. et al. Quantification of biofilm structures by the novel computer program COMSTAT. Microbiol. 146, 2395–2407 (2000)

    CAS  Article  Google Scholar 

  8. 8

    Stover, C. K. et al. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406, 959–964 (2000)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Bhagwat, A. A., Gross, K. C., Tully, R. E. & Keister, D. L. β-glucan synthesis in Bradyrhizobium japonicum: characterization of a new locus (ndvC) influencing beta-(1 → 6) linkages. J. Bacteriol. 178, 4635–4642 (1996)

    CAS  Article  Google Scholar 

  10. 10

    Breedveld, M. W. & Miller, K. J. Cyclic beta-glucans of members of the family Rhizobiaceae. Microbiol. Rev. 58, 145–161 (1994)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Chen, R., Bhagwat, A. A., Yaklich, R. & Keister, D. L. Characterization of ndvD, the third gene involved in the synthesis of cyclic β-(1 → 3), (1 → 6)-D-glucans in Bradyrhizobium japonicum. Can. J. Microbiol. 48, 1008–1016 (2002)

    CAS  Article  Google Scholar 

  12. 12

    Loewus, F. A. Improvement in the anthrone assay for determination of carbohydrates. Anal. Chem. 24, 219 (1952)

    CAS  Article  Google Scholar 

  13. 13

    Gonzalez, J. E., Semino, C. E., Wang, L. X., Castellano-Torres, L. E. & Walker, G. C. Biosynthetic control of molecular weight in the polymerization of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti. Proc. Natl Acad. Sci. USA 95, 13477–13482 (1998)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Szejtli, J. The cyclodextrins and their applications in biotechnology. Carbohyd. Polym. 12, 375–392 (1990)

    CAS  Article  Google Scholar 

  15. 15

    Whiteley, M. et al. Gene expression in Pseudomonas aeruginosa biofilms. Nature 413, 860–864 (2001)

    ADS  CAS  Article  Google Scholar 

  16. 16

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

    CAS  Article  Google Scholar 

  17. 17

    Pardee, A. B., Jacob, F. & Monod, J. The genetic control and cytoplasmic expression of “inducibility” in the synthesis of β-galactosidase in E. coli. J. Mol. Biol. 1, 165–178 (1959)

    CAS  Article  Google Scholar 

  18. 18

    Bloemberg, G. V., O'Toole, G. A., Lugtenberg, B. J. 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 

  19. 19

    Simon, R., Quandt, J. & Klipp, W. New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in Gram-negative bacteria. Gene 80, 160–169 (1989)

    Article  Google Scholar 

  20. 20

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

    CAS  Article  Google Scholar 

  21. 21

    Caetano-Annoles, G. Amplifying DNA with arbitrary oligonucleotide primers. PCR Methods Appl. 3, 85–92 (1993)

    Article  Google Scholar 

  22. 22

    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 

  23. 23

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

    CAS  Article  Google Scholar 

  24. 24

    Heydorn, A. et al. Experimental reproducibility in flow-chamber biofilms. Microbiol. 146, 2409–2415 (2000)

    CAS  Article  Google Scholar 

  25. 25

    Wang, L. X., Wang, Y., Pellock, B. & Walker, G. C. Structural characterization of the symbiotically important low-molecular-weight succinoglycan of Sinorhizobium meliloti. J. Bacteriol. 181, 6788–6796 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

Download references


We thank L.-X. Wang for discussions. This work was supported by grants from the NSF to P.S.S., from the NIH to G.C.W, from the Canadian Cystic Fibrosis Foundation to T.-F.M. and from the NIH, Microbia, Inc. and The Pew Charitable Trusts to G.A.O'T., who is a Pew Scholar in the Biomedical Sciences.

Author information



Corresponding author

Correspondence to George A. O'Toole.

Ethics declarations

Competing interests

G.A.O'T. has stock options for and consults with Microbia, Inc., which provided some funding for this study.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Mah, TF., Pitts, B., Pellock, B. et al. A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance. Nature 426, 306–310 (2003).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing