Unveiling the biofilm architecture of Pseudomonas aeruginosa in single and dual species with Escherichia coli using multi-scale imaging techniques

Biofilms are complex communities of microbes that can pose a significant threat in clinical and industrial settings due to their increased antibiotic resistance, aiding towards recurring chronic infections and contamination of water systems. Previously, intra colony-channels were discovered in mature colony biofilms of E. coli and observed to transport nutrients from the surrounding environment. Additionally, most biofilms in nature are polymicrobial and single species biofilms fail to represent the interactions observed in clinical settings. This study used conventional and novel imaging techniques to analyse the internal architecture of single and dual species biofilms of E. coli and P. aeruginosa. The Mesolens provided a field of view of entire biofilms with a subcellular resolution capturing the differences in macrostructure across the sample area. Innovative specimen preparation methods were designed and implemented to allow the visualisation of the internal structures of P. aeruginosa biofilms. Here, we show that P. aeruginosa formed radial intra-colony channels and can transport fluorescent microspheres into the biomass, like the channels formed by E. coli. Co-cultured E. coli/P. aeruginosa biofilms developed sinuous channel structures with different morphologies, indicating that E. coli may alter the internal architecture of P. aeruginosa biofilms. Demonstrating that these channels form and function similarly in two phylogenetically distinct organisms opens avenues for scientific enquiry into other polymicrobial biofilm architectures. These findings of novel internal architecture and interspecies interactions give new insights into polymicrobial biofilms that may assist with the management of biofilm associated infections and offer fresh perspectives on the fight against antimicrobial resistance.