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 optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Davey, M. E. & O'Toole, G. A. Microbial biofilms: from ecology to molecular genetics. Microbiol. Mol. Biol. Rev. 64, 847–867 (2000)
Hoyle, B. D. & Costerton, W. J. Bacterial resistance to antibiotics: the role of biofilms. Prog. Drug Res. 37, 91–105 (1991)
Costerton, J. W., Stewart, P. S. & Greenberg, E. P. Bacterial biofilms: a common cause of persistent infections. Science 284, 318–322 (1999)
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)
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)
Hentzer, M. et al. Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function. J. Bacteriol. 183, 5395–5401 (2001)
Heydorn, A. et al. Quantification of biofilm structures by the novel computer program COMSTAT. Microbiol. 146, 2395–2407 (2000)
Stover, C. K. et al. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406, 959–964 (2000)
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)
Breedveld, M. W. & Miller, K. J. Cyclic beta-glucans of members of the family Rhizobiaceae. Microbiol. Rev. 58, 145–161 (1994)
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)
Loewus, F. A. Improvement in the anthrone assay for determination of carbohydrates. Anal. Chem. 24, 219 (1952)
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)
Szejtli, J. The cyclodextrins and their applications in biotechnology. Carbohyd. Polym. 12, 375–392 (1990)
Whiteley, M. et al. Gene expression in Pseudomonas aeruginosa biofilms. Nature 413, 860–864 (2001)
Mah, T.-F. & O'Toole, G. A. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 9, 34–39 (2001)
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)
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)
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)
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)
Caetano-Annoles, G. Amplifying DNA with arbitrary oligonucleotide primers. PCR Methods Appl. 3, 85–92 (1993)
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)
Christensen, B. B. et al. Molecular tools for study of biofilm physiology. Methods Enzymol. 310, 20–42 (1999)
Heydorn, A. et al. Experimental reproducibility in flow-chamber biofilms. Microbiol. 146, 2409–2415 (2000)
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)
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.
G.A.O'T. has stock options for and consults with Microbia, Inc., which provided some funding for this study.
About this article
Population dynamics and transcriptomic responses of Pseudomonas aeruginosa in a complex laboratory microbial community
npj Biofilms and Microbiomes (2019)
Structural and Functional Insights into PpgL, a Metal-Independent β-Propeller Gluconolactonase That Contributes to Pseudomonas aeruginosa Virulence
Infection and Immunity (2019)
Inhibition of biofilm formation, quorum sensing activity and molecular docking study of isolated 3, 5, 7-Trihydroxyflavone from Alstonia scholaris leaf against P.aeruginosa
Bioorganic Chemistry (2019)
Chemical composition, antioxidant, antimicrobial and antibiofilm activities of Vitex gardneriana schauer leaves's essential oil
Microbial Pathogenesis (2019)
Biochemical and Biophysical Research Communications (2019)