In Situ Investigation of Pseudomonas aeruginosa Biofilm Development: Interplay between Flow, Growth Medium, and Mechanical Properties of Substrate.

To better understand the impact of biomaterial mechanical properties and growth medium on bacterial adhesion and biofilm formation under flow, we investigated the biofilm formation ability of Pseudomonas aeruginosa in different media on polydimethylsiloxane (PDMS) of different stiffness in real time using a microfluidic platform. P. aeruginosa colonization was recorded with optical microscopy and automated image analysis. The bacterial intracellular level of cyclic diguanylate (c-di-GMP), which regulates biofilm formation, was monitored using the transcription of the putative adhesin gene (cdrA) as a proxy. Contrary to the previous supposition, we revealed that PDMS material stiffness within the tested range has negligible impact on biofilm development and biofilm structures, whereas culture media not only influence the kinetics of biofilm development but also affect the biofilm morphology and structure dramatically. Interestingly, magnesium rather than previously reported calcium was identified here to play a decisive role in the formation of dense P. aeruginosa aggregates and high levels of c-di-GMP. These results demonstrate that although short-term adhesion assays bring valuable insight into bacterial and material interactions, long-term evaluations are essential to better predict overall biofilm outcome. The microfluidic system developed here presents a valuable application potential for studying biofilm development in situ. .