Open AccessQuantitative confocal microscopy and calibration for measuring differences in cyclic-di-GMP signalling by bacteria on biomedical hydrogels
Author(s) -
Jacob Blacutt,
Ziyang Lan,
Elizabeth CosgriffHernandez,
Vernita Gordon
Publication year - 2021
Publication title -
royal society open science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.84
H-Index - 51
ISSN - 2054-5703
DOI - 10.1098/rsos.201453
Subject(s) - biofilm , self healing hydrogels , green fluorescent protein , confocal microscopy , bacteria , confocal , biophysics , fluorescence microscope , intracellular , confocal laser scanning microscope , microbiology and biotechnology , ethylene glycol , pseudomonas aeruginosa , chemistry , confocal laser scanning microscopy , microscopy , fluorescence , plasmid , biochemistry , biology , biomedical engineering , dna , medicine , genetics , geometry , mathematics , physics , organic chemistry , optics , quantum mechanics , gene
The growth of bacterial biofilms on implanted medical devices causes harmful infections and device failure. Biofilm development initiates when bacteria attach to and sense a surface. For the common nosocomial pathogen Pseudomonas aeruginosa and many others, the transition to the biofilm phenotype is controlled by the intracellular signal and second messenger cyclic-di-GMP (c-di-GMP). It is not known how biomedical materials might be adjusted to impede c-di-GMP signalling, and there are few extant methods for conducting such studies. Here, we develop such a method. We allowed P. aeruginosa to attach to the surfaces of poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These bacteria contained a plasmid for a green fluorescent protein (GFP) reporter for c-di-GMP. We used laser-scanning confocal microscopy to measure the dynamics of the GFP reporter for 3 h, beginning 1 h after introducing bacteria to the hydrogel. We controlled for the effects of changes in bacterial metabolism using a promoterless plasmid for GFP, and for the effects of light passing through different hydrogels being differently attenuated by using fluorescent plastic beads as ‘standard candles’ for calibration. We demonstrate that this method can measure statistically significant differences in c-di-GMP signalling associated with different PEGDA gel types and with the surface-exposed protein PilY1.