Biofilm boundaries and breaches: Contrasting pseudomonas interactions with E. coli

Pseudomonas aeruginosa, Pseudomonas fluorescens, and Escherichia coli are well-studied in monoculture yet often coexist in polymicrobial biofilms in clinical and environmental settings, including catheters, the airways of cystic-fibrosis patients, and wastewater systems. Biofilm growth is an emergent property and frequently unpredictable, particularly in multi-species consortia. Intra-colony channels, first described in E. coli macrocolony biofilms, can transport exogenous materials and distribute nutrients around the biomass but whether such channels are conserved across diverse bacteria has remained unclear. To address this, we combined laser-scanning and spinning-disk confocal microscopy with cross-scale confocal and wide-field mesoscopy using the Mesolens, and we further applied light-sheet imaging to capture channel dynamics with increased depth, speed, and sensitivity. This multi-modal approach resolves transport channel architecture and the dynamics, enabling a comparison across phylogenetically distinct species. When co-cultured with E. coli, P. aeruginosa biofilms formed different channel structures to the respective monoculture biofilms and displayed spatially separate colonies and different channel architectures between species. By contrast, when co-cultured with E. coli, P. fluorescens formed distinct colonies at the E. coli biofilm periphery, but P. fluorescens was also present within the channel networks of the Gram-negative biomass. Along with these novel findings of internal architecture in Pseudomonas, and undocumented biofilm organisation in co-culture environments, current work focuses on a quantitative means of comparing biofilm architecture in single species and polymicrobial biofilms and investigating clinically relevant isolates to determine if biofilm transport channels are conserved across diverse phylotypes and to better understand their potential role in polymicrobial biofilm development.