Protocol | Published:

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

Nature Protocols volume 10, pages 11651180 (2015) | Download Citation

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.).

Author information

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Liang Yang.

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DOI

https://doi.org/10.1038/nprot.2015.067

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