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In vitro and in vivo generation and characterization of Pseudomonas aeruginosa biofilm–dispersed cells via c-di-GMP manipulation

Nature Protocols volume 10, pages 1165–1180 (2015)Cite this article

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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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Figure 1: Schematic workflow of biofilm dispersal and evaluation of dispersed cells in vitro.
Figure 2: Schematic workflow of biofilm dispersal and evaluation of dispersed cells in vivo.
Figure 3: Quantification of dispersed cells and biofilm after 5 h of dispersal treatment in vitro.
Figure 4: c-di-GMP as a universal biomarker for PAO1/pBAD-yhjH in vitro–dispersed cells with induction by L-arabinose in comparison with planktonic cells.
Figure 5: Biomarkers specific to P. aeruginosa in vitro–dispersed cells post biofilm dispersal in comparison with planktonic cells.
Figure 6: Pyoverdine production of dispersed cells and planktonic cells measured in PAO1/pBAD-yhjH (planktonic or 0.25% arabinose-dispersed) in vitro cultures by means of fluorescence (excitation 400 nm/emission 460 nm) by a Tecan microplate reader (Step 44).
Figure 7: In vivo dispersal of P. aeruginosa biofilms.

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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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Authors and Affiliations

  1. Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore

    Song Lin Chua, Mingjun Yuan, Michael Givskov & Liang Yang

  2. National University of Singapore (NUS) Graduate School of Integrative Sciences and Engineering, National University of Singapore, Singapore

    Song Lin Chua

  3. Department of International Health, Immunology and Microbiology, Costerton Biofilm Center, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.,

    Louise D Hultqvist, Morten Rybtke, Thomas E Nielsen, Michael Givskov & Tim Tolker-Nielsen

  4. School of Biological Sciences, Nanyang Technological University, Singapore

    Liang Yang

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Contributions

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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Correspondence to Liang Yang.

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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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