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

doi:10.1038/nprot.2015.067

. 2015 Aug;10(8):1165-80.

doi: 10.1038/nprot.2015.067. Epub 2015 Jul 9.

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

Song Lin Chua¹, Louise D Hultqvist², Mingjun Yuan³, Morten Rybtke², Thomas E Nielsen², Michael Givskov⁴, Tim Tolker-Nielsen², Liang Yang⁵

Affiliations

¹ 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.
² Department of International Health, Immunology and Microbiology, Costerton Biofilm Center, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
³ Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore.
⁴ 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.
⁵ 1] Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore. [2] School of Biological Sciences, Nanyang Technological University, Singapore.

PMID: 26158442
DOI: 10.1038/nprot.2015.067

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

Song Lin Chua et al. Nat Protoc. 2015 Aug.

. 2015 Aug;10(8):1165-80.

doi: 10.1038/nprot.2015.067. Epub 2015 Jul 9.

Authors

Song Lin Chua¹, Louise D Hultqvist², Mingjun Yuan³, Morten Rybtke², Thomas E Nielsen², Michael Givskov⁴, Tim Tolker-Nielsen², Liang Yang⁵

Affiliations

¹ 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.
² Department of International Health, Immunology and Microbiology, Costerton Biofilm Center, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
³ Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore.
⁴ 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.
⁵ 1] Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore. [2] School of Biological Sciences, Nanyang Technological University, Singapore.

PMID: 26158442
DOI: 10.1038/nprot.2015.067

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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[1] Crit Rev Eukaryot Gene Expr. 2014;24(3):269-79 - PubMed

[2] Crit Rev Eukaryot Gene Expr. 2014;24(3):269-79 - PubMed

[3] Acta Pathol Microbiol Scand B. 1980 Jun;88(3):125-31 - PubMed

[4] Acta Pathol Microbiol Scand B. 1980 Jun;88(3):125-31 - PubMed

[5] Clin Infect Dis. 2001 Oct 15;33(8):1387-92 - PubMed

[6] Clin Infect Dis. 2001 Oct 15;33(8):1387-92 - PubMed

[7] Infect Immun. 2013 Aug;81(8):2705-13 - PubMed

[8] Infect Immun. 2013 Aug;81(8):2705-13 - PubMed

[9] J Bacteriol. 2006 Nov;188(21):7335-43 - PubMed

[10] J Bacteriol. 2006 Nov;188(21):7335-43 - PubMed

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

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

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