Exploring the Physics of Surface-Bacteria Interactions and Developing Novel Techniques for Biofilm Characterisation

Abstract

The increasing prevalence of biofilms in medical and industrial contexts poses significant global challenges, necessitating the emergence of innovative solutions in antibiofilm surface design. Central to these efforts is a requirement for an in-depth understanding of how material properties, such as surface wetting, and the behaviour of fouling cells influence cell-surface interactions, such as bacterial motility, surface attachment, surface detachment, and biofilm formation. Unfortunately, existing methodologies for assessing the impact of these properties and behaviours on antifouling performance often fail to provide a clear and comprehensive evaluation of these factors, limiting our understanding of their collective impact on antifouling efficacy. In this project, we have performed various surface-wetting tests — including contact angle, contact angle hysteresis, droplet sliding, and droplet impact measurements — to characterise the static and kinetic frictions of several novel antifouling surfaces. From these tests, we have revealed that antifouling surfaces like SLIPS and SOCAL tend to have low static and kinetic frictions, and, as a result, droplets are far more likely to slide and bounce on these surfaces, thereby reducing droplet contact time and promoting self-cleaning. Theoretically, this behaviour will also correlate to poor bacterial attachment and heightened cell motility, as these slippery surfaces can also potentially restrict cell interaction time and inhibit stable, static bacterial adhesion. Since bacteria-material interactions depend not only on surface properties but also on the specific behaviours of the bacterial cells, we needed to subsequently perform bacterial motility, attachment, and detachment tests on these slippery surfaces to adequately test this hypothesis. To this end, cells of wild-type Pseudomonas aeruginosa, as well as those of the mutants lacking either pili or flagella, were cultured atop these antifouling surfaces to test the combined effect of different cell motility mechanisms and surfacewetting properties on cell adhesion. Here, we demonstrated that flagella-deficient cells demonstrated a higher tendency for static adhesion atop these surfaces, while those strains with flagella displayed enhanced motility resulting from greater programmed detachment. Interestingly, the pili-deficient mutant exhibited unique spiralling swimming behaviour when entrapped atop SLIPS. Among our materials, despite its slightly higher kinetic friction, SOCAL emerged as the most promising of the antifouling surfaces, given a low number of strongly adhered cells. In contrast, while effective at preventing static adhesion, SLIPS proved a poor anti-attachment stratagem due to cells being entrapped in its surface, underscoring the need for design refinements to enhance its antifouling capabilities. In practice, biofilms will form on most surfaces (including those reported to be antifouling) given enough time. While these antifouling strategies do not prevent biofilm formation entirely, they can significantly delay the process and influence the biofilms’ structural integrity, making them mechanically weaker than those formed on surfaces without such antifouling measures. Understanding these surface-induced variations in the mechanical properties of biofilms is critical to developing more effective management and removal strategies. Unfortunately, current mechanics characterisation techniques are often slow, hindering practical, large-scale biofilm assessment. To address this, we developed a groundbreaking Optical Coherence Tomography (OCT)-based air jet indenter to allow us to rapidly characterise the mechanical properties of bacterial biofilms. Using this tool, we discovered that biofilms of Pseudomonas deficient in pili were significantly less stiff than those expressing this surface appendage, indicating that pili are key structural components in biofilm resilience. The development of this tool is crucial for enhancing our understanding of biofilm mechanics, paving the way for more targeted and effective anti-biofilm strategies.