Abstract
Bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a global secondary bacterial messenger that controls the formation of drug-resistant multicellular biofilms. Lowering the intracellular c-di-GMP content can disperse biofilms, and it is proposed as a biofilm eradication strategy. However, freshly dispersed biofilm cells exhibit a physiology distinct from biofilm and planktonic cells, and they might have a clinically relevant role in infections. Here we present in vitro and in vivo protocols for the generation and characterization of dispersed cells from Pseudomonas aeruginosa biofilms by reducing the intracellular c-di-GMP content through modulation of phosphodiesterases (PDEs). Unlike conventional protocols that demonstrate biofilm dispersal by biomass quantification, our protocols enable physiological characterization of the dispersed cells. Biomarkers of dispersed cells are identified and quantified, serving as potential targets for treating the dispersed cells. The in vitro protocol can be completed within 4 d, whereas the in vivo protocol requires 7 d.
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Acknowledgements
This research is supported by the National Research Foundation and Ministry of Education Singapore under its Research Centre of Excellence Programme, by a Start-up grant (M4330002.C70) from Nanyang Technological University, and by Academic Research Fund (AcRF) Tier 2 (MOE2014-T2-2-172) from the Ministry of Education, Singapore (to L.Y.). This work was supported by grants from the Danish Council for Strategic Research (M.G.).
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S.L.C., M.Y., T.E.N. and M.R. performed the in vitro dispersal experiments. L.D.H. performed the in vivo dispersal experiments. S.L.C., M.G., T.T.-N. and L.Y. wrote the manuscript. M.G., T.T.-N. and L.Y. designed the experiments.
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Chua, S., Hultqvist, L., Yuan, M. et al. In vitro and in vivo generation and characterization of Pseudomonas aeruginosa biofilm–dispersed cells via c-di-GMP manipulation. Nat Protoc 10, 1165–1180 (2015). https://doi.org/10.1038/nprot.2015.067
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DOI: https://doi.org/10.1038/nprot.2015.067
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