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Aminoglycoside antibiotics induce bacterial biofilm formation

Abstract

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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Figure 1: Phenotypic effects of tobramycin on P. aeruginosa and E. coli.
Figure 2: The role of P. aeruginosa arr in tobramycin induction of biofilm formation and biofilm antibiotic resistance.
Figure 3: The role of c-di-GMP in the biofilm response to aminoglycosides.

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Acknowledgements

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.

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Correspondence to Samuel I. Miller.

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Supplementary information

Supplementary Figure S1

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)

Supplementary Figure S2

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)

Supplementary Methods

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)

Supplementary Figure Legends

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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