Abstract
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.
Highlights
The growth of bacterial biofilms on implanted medical devices causes harmful infections and device failure
We demonstrate that this method can measure statistically significant differences in c-diGMP signalling associated with different poly(ethylene glycol) diacrylate (PEGDA) gel types and with the surface-exposed protein PilY1
We have demonstrated that quantitative confocal microscopy and image analysis, combined with calibration for both optical attenuation and bacterial metabolism, can measure statistically significant differences in the production of CdrA, which can act as a proxy measure for c-di-GMP
Summary
Poly(ethylene glycol) (PEG, 2 kDa (Mn = 1917) and 10 kDa (Mn = 12 157)), acryloyl chloride, triethylamine, potassium bicarbonate, sodium sulfate and Irgacure 2959 were purchased from Sigma-Aldrich (St Louis, MO). Dichloromethane and deuterated chloroform with 0.03 vol% TMS were purchased from VWR Chemicals (Radnor, PA). Diethyl ether and Dulbecco’s phosphate buffered saline were purchased from Fisher Scientific (Hampton, NH). SecureSeal Imaging Spacers were purchased from Grace Bio-Labs (Bend, OR). All reagents were used as received unless specified otherwise
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