title: In vitro and in vivo generation and characterization of Pseudomonas aeruginosa biofilm-dispersed cells via c-di-GMP manipulation.
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.
authors.affiliation: 1] Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore. [2] National University of Singapore (NUS) Graduate School of Integrative Sciences and Engineering, National University of Singapore, Singapore.
authors.forename: Song Lin
authors.identifier: 0000000170477451
authors.initials: SL
authors.lastname: Chua
authors.affiliation: Department of International Health, Immunology and Microbiology, Costerton Biofilm Center, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Louise D
authors.initials: LD
authors.lastname: Hultqvist
authors.affiliation: Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore.
authors.forename: Mingjun
authors.initials: M
authors.lastname: Yuan
authors.affiliation: Department of International Health, Immunology and Microbiology, Costerton Biofilm Center, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Morten
authors.initials: M
authors.lastname: Rybtke
authors.affiliation: Department of International Health, Immunology and Microbiology, Costerton Biofilm Center, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Nielsen
authors.affiliation: 1] Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore. [2] Department of International Health, Immunology and Microbiology, Costerton Biofilm Center, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Michael
authors.initials: M
authors.lastname: Givskov
authors.affiliation: Department of International Health, Immunology and Microbiology, Costerton Biofilm Center, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Tim
authors.initials: T
authors.lastname: Tolker-Nielsen
authors.affiliation: 1] Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore. [2] School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Liang
authors.identifier: 0000000223620128
authors.initials: L
authors.lastname: Yang
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques; D018441:Biofilms; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D005260:Female; D008807:Mice, Inbred BALB C; D011550:Pseudomonas aeruginosa / Q000502:physiology*
chemical_list: C062025:bis(3',5')-cyclic diguanylic acid; D006152:Cyclic GMP