Biofilms are adherent aggregates of bacterial cells that form on biotic and abiotic surfaces, including human tissues. Biofilms resist antibiotic treatment and contribute to bacterial persistence in chronic infections1,2. Hence, the elucidation of the mechanisms by which biofilms are formed may assist in the treatment of chronic infections, such as Pseudomonas aeruginosa in the airways of patients with cystic fibrosis2. Here we show that subinhibitory concentrations of aminoglycoside antibiotics induce biofilm formation in P. aeruginosa and Escherichia coli. In P. aeruginosa, a gene, which we designated aminoglycoside response regulator (arr), was essential for this induction and contributed to biofilm-specific aminoglycoside resistance. The arr gene is predicted to encode an inner-membrane phosphodiesterase whose substrate is cyclic di-guanosine monophosphate (c-di-GMP)—a bacterial second messenger that regulates cell surface adhesiveness3. We found that membranes from arr mutants had diminished c-di-GMP phosphodiesterase activity, and P. aeruginosa cells with a mutation changing a predicted catalytic residue of Arr were defective in their biofilm response to tobramycin. Furthermore, tobramycin-inducible biofilm formation was inhibited by exogenous GTP, which is known to inhibit c-di-GMP phosphodiesterase activity4. Our results demonstrate that biofilm formation can be a specific, defensive reaction to the presence of antibiotics, and indicate that the molecular basis of this response includes alterations in the level of c-di-GMP.
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Mah, T. F. et al. A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance. Nature 426, 306–310 (2003)
Whiteley, M. et al. Gene expression in Pseudomonas aeruginosa biofilms. Nature 413, 860–864 (2001)
D'Argenio, D. A. & Miller, S. I. Cyclic di-GMP as a bacterial second messenger. Microbiol. 150, 2497–2502 (2004)
Ross, P. et al. The cyclic diguanylic acid regulatory system of cellulose synthesis in Acetobacter xylinum. Chemical synthesis and biological activity of cyclic nucleotide dimer, trimer, and phosphothioate derivatives. J. Biol. Chem. 265, 18933–18943 (1990)
Goh, E. B. et al. Transcriptional modulation of bacterial gene expression by subinhibitory concentrations of antibiotics. Proc. Natl Acad. Sci. USA 99, 17025–17030 (2002)
Goodman, L. S. & Gilman, A. Goodman and Gilman's The Pharmacological Basis of Therapeutics (Macmillan, New York, 1985)
Barclay, M. L. et al. Adaptive resistance to tobramycin in Pseudomonas aeruginosa lung infection in cystic fibrosis. J. Antimicrob. Chemother. 37, 1155–1164 (1996)
Bergey, D. H. & Holt, J. G. Bergey's Manual of Systematic Bacteriology (Williams & Wilkins, Baltimore, 1984)
Mukhopadhyay, S. et al. The quantitative distribution of nebulized antibiotic in the lung in cystic fibrosis. Respir. Med. 88, 203–211 (1994)
Ramsey, B. W. et al. Intermittent administration of inhaled tobramycin in patients with cystic fibrosis. Cystic Fibrosis Inhaled Tobramycin Study Group. N. Engl. J. Med. 340, 23–30 (1999)
Chiang, P. & Burrows, L. L. Biofilm formation by hyperpiliated mutants of Pseudomonas aeruginosa. J. Bacteriol. 185, 2374–2378 (2003)
Rachid, S., Ohlsen, K., Witte, W., Hacker, J. & Ziebuhr, W. Effect of subinhibitory antibiotic concentrations on polysaccharide intercellular adhesin expression in biofilm-forming Staphylococcus epidermidis. Antimicrob. Agents Chemother. 44, 3357–3363 (2000)
Bagge, N. et al. Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and beta-lactamase and alginate production. Antimicrob. Agents Chemother. 48, 1175–1187 (2004)
Paul, R. et al. Cell cycle-dependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain. Genes Dev. 18, 715–727 (2004)
Simm, R., Morr, M., Kader, A., Nimtz, M. & Romling, U. GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility. Mol. Microbiol. 53, 1123–1134 (2004)
Galperin, M. Y., Nikolskaya, A. N. & Koonin, E. V. Novel domains of the prokaryotic two-component signal transduction systems. FEMS Microbiol. Lett. 203, 11–21 (2001)
Jacobs, M. A. et al. Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 100, 14339–14344 (2003)
Bobrov, A. G., Kirillina, O. & Perry, R. D. The phosphodiesterase activity of the HmsP EAL domain is required for negative regulation of biofilm formation in Yersinia pestis. FEMS Microbiol. Lett. 247, 123–130 (2005)
Huang, B., Whitchurch, C. B. & Mattick, J. S. FimX, a multidomain protein connecting environmental signals to twitching motility in Pseudomonas aeruginosa. J. Bacteriol. 185, 7068–7076 (2003)
O'Toole, G. A. & Kolter, R. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 30, 295–304 (1998)
Moskowitz, S. M., Foster, J. M., Emerson, J. & Burns, J. L. Clinically feasible biofilm susceptibility assay for isolates of Pseudomonas aeruginosa from patients with cystic fibrosis. J. Clin. Microbiol. 42, 1915–1922 (2004)
Drenkard, E. & Ausubel, F. M. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 416, 740–743 (2002)
He, J. et al. The broad host range pathogen Pseudomonas aeruginosa strain PA14 carries two pathogenicity islands harboring plant and animal virulence genes. Proc. Natl Acad. Sci. USA 101, 2530–2535 (2004)
Wolfgang, M. C. et al. Conservation of genome content and virulence determinants among clinical and environmental isolates of Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 100, 8484–8489 (2003)
Bader, M. W. et al. Regulation of Salmonella typhimurium virulence gene expression by cationic antimicrobial peptides. Mol. Microbiol. 50, 219–230 (2003)
Rosenfeld, M. et al. Early pulmonary infection, inflammation, and clinical outcomes in infants with cystic fibrosis. Pediatr. Pulmonol. 32, 356–366 (2001)
Hisert, K. M. et al. A glutamate-alanine-leucine (EAL) domain protein of Salmonella controls bacterial survival in mice, antioxidant defence and killing of macrophages: role of cyclic diGMP. Mol. Microbiol. 56, 1234–1245 (2005)
Amikam, D. & Benziman, M. Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens. J. Bacteriol. 171, 6649–6655 (1989)
We thank R. K. Ernst for assistance with preparing the manuscript and laboratory techniques; T. Guina and M. Wu for help in two-dimensional protein electrophoresis analyses; J. Foster and S. Moskowitz for instruction in biofilm growth and measurement; M. Olson and M. Jacobs for providing transposon-insertion mutants; M. Bader for assistance with the cloning and complementation of arr; and J. Burns, B. Ramsey and R. Gibson for discussions. This work was supported by the Cystic Fibrosis Foundation and the National Institutes of Health.
Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
These four panels demonstrate further effects of subinhibitory tobramycin concentrations on P. aeruginosa phenotypes, including growth, flagellar swimming, and biofilm formation. The effects of polymyxin B on flagellar swimming is also shown for comparison, and as well as the effects of mutation in various predicted cyclic diguanylate regulators (including arr). (DOC 211 kb)
The results in these four panels further support the model in Figure 3, in which aminoglycosides induce physiologic changes in P. aeruginosa via c-di-GMP signaling that includes augmented biofilm formation. Such changes, including augmented antibiotic susceptibility, are specific to the biofilm state. Altering c-di-GMP signaling is an attractive therapeutic target. (DOC 96 kb)
This file describes methods and reagents used in this work that were felt to be useful in interpreting the results described in this manuscript, but which were too detailed to include in the main text. (DOC 39 kb)
More detailed text descriptions to accompany the above Figures. (DOC 21 kb)
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Hoffman, L., D'Argenio, D., MacCoss, M. et al. Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 436, 1171–1175 (2005). https://doi.org/10.1038/nature03912
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