In vitro and in vivo generation and characterization of Pseudomonas aeruginosa biofilm–dispersed cells via c-di-GMP manipulation
Chua, Song Lin
Hultqvist, Louise D.
Nielsen, Thomas E.
Date of Issue2015
School of Biological Sciences
Singapore Centre for Environmental Life Sciences and Engineering
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.
© 2015 Nature America, Inc. This is the author created version of a work that has been peer reviewed and accepted for publication by Nature Protocols, Nature America, Inc. It incorporates referee’s comments but changes resulting from the publishing process, such as copyediting, structural formatting, may not be reflected in this document. The published version is available at: [http://dx.doi.org/10.1038/nprot.2015.067].