Imaging the internal architecture of pseudomonas aeruginosa biofilms in single- and dual-species communities with E. coli

Pseudomonas aeruginosa and Escherichia coli have been extensively studied in monoculture and are commonly found in polymicrobial biofilms in clinical settings, such as catheters and the airways of cystic fibrosis patients. Biofilm growth is influenced by dynamic biological and physical factors and in natural environments often consist of multiple species, exhibiting collective behaviours that cannot be easily predicted from individual species' biofilms. Previously, intra-colony channels have been identified in E. coli macrocolony biofilms and shown to facilitate nutrient transport from the surrounding environment. This study used conventional and novel imaging techniques, such as confocal microscopy and widefield mesoscopy with the Mesolens and discovered undocumented channel structures within P. aeruginosa macrocolony biofilms. Specimen preparation methods enabled visualisation of internal structures of P. aeruginosa biofilms, revealing, for the first time, that P. aeruginosa forms radial structures capable of transporting fluorescent microspheres into the biomass. Co-cultured E. coli/P. aeruginosa biofilms developed sinuous channel structures with a different morphology to its monoculture, indicating that E. coli may alter the internal architecture of P. aeruginosa biofilms. Demonstrating that these structures form and can uptake exogenously in two phylogenetically distinct bacteria indicates the need to determine how widespread these structures are among other bacterial genera. Along with these novel findings of internal architecture in P. aeruginosa, the current work is focused on expanding this research to clinically relevant isolates to investigate whether these channel structures are conserved across diverse strains and to better understand their potential role in polymicrobial biofilm development.