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

TY - JOUR

T1 - In vitro and in vivo generation and characterization of Pseudomonas aeruginosa biofilm-dispersed cells via c-di-GMP manipulation

AU - Chua, Song Lin

AU - Hultqvist, Louise D

AU - Yuan, Mingjun

AU - Rybtke, Morten

AU - Nielsen, Thomas Eiland

AU - Givskov, Michael

AU - Tolker-Nielsen, Tim

AU - Yang, Liang

PY - 2015/8/25

Y1 - 2015/8/25

N2 - 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.

AB - 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.

U2 - 10.1038/nprot.2015.067

DO - 10.1038/nprot.2015.067

M3 - Journal article

C2 - 26158442

SN - 1754-2189

VL - 10

SP - 1165

EP - 1180

JO - Nature Protocols

JF - Nature Protocols

IS - 8

ER -