Microscopic and mesoscopic optical imaging techniques reveal transport channels and inter-species interactions in pseudomonas aeruginosa and Escherichia coli Biofilms

Biofilms are a complex community of microorganisms that pose a significant threat to clinical and industrial settings due to their increased antibiotic resistance compared to their planktonic counterpart. Previously, intra colony channels were discovered in E. coli mature colony biofilms and were found to support the transportation of nutrients from the surrounding environment. However, most biofilms in nature are polymicrobial and monoculture biofilms fail to represent the interactions observed in clinical settings. This study utilised conventional and novel imaging techniques to analyse the internal architecture of mono- and polymicrobial biofilms of E. coli and P. aeruginosa. Confocal Laser Scanning Microscopy (CLSM) provided high-resolution, three-dimensional reconstructions that highlighted intricate biofilm microstructures. The Mesolens allowed a broader visualization of entire biofilms at subcellular resolution without image artifacts, capturing macrostructural differences across the sample area. Incorporating fluorescent microspheres allowed the visualisation of intra-colony channels within P. aeruginosa biofilms and showed they can also uptake beads into their structure from the external environment. Notably, in co-culture, P. aeruginosa developed unique, sinuous channel structures indicating E. coli may be altering the internal architecture of P. aeruginosa. These findings illustrate the capabilities of advanced imaging in visualizing complex microbial interactions and could inform strategies for disrupting biofilms in clinical and industrial settings.