O-antigen polysaccharides confer higher antibiotic tolerance to High-Risk Pseudomonas aeruginosa biofilms

1. AbstractBiofilm-associated persistent infections caused by epidemic high-risk clones (HiRiCs) of Pseudomonas aeruginosa have raised major clinical concerns due to their multidrug resistance and global prevalence. For some HiRiCs, such as ST111, global success has been linked to a shift in expression of lipopolysaccharide (LPS) O-specific antigens from serotype O4 to O12, which affects surface adhesion and virulence. However, whether this serotype switch also alters biofilm formation remains unknown.A key challenge in studying bacterial biofilms is the lack of experimental systems that support long-term, physiologically relevant biofilm formation while enabling real-time monitoring. Here, we employed a compact centrifugal microfluidic system, Bacterial Culture on Disc (BCoD), to monitor biofilm development and antibiotic responses in P. aeruginosa strains expressing different O-antigen types.Using the BCoD platform, we observed distinct differences in biofilm formation between ST111 strains lacking the O-specific antigen (ΔOSA mutants) and those expressing either O4 or O12. Although O-antigen production was not essential for biofilm formation, its absence reduced biofilm robustness and cell viability. Importantly, we also observed that the variation between O4 and O12 further modulated biofilm productivity, with O12 forming more biomass than O4. Despite these differences, ΔOSA and O12 biofilms showed similar susceptibility to colistin treatment.These findings demonstrate that the chemical composition of the O-antigen influences P. aeruginosa biofilm formation and may therefore help to explain the selective advantages of driving serotype switching and the global emergence of O12 ST111. In addition, this study highlights the utility of the BCoD platform for investigating biofilm behavior in clinically relevant isolates with subtle phenotypic differences. In future work, the BCoD will be further developed for fundamental studies on biofilm dynamics, heterogeneity, and stress responses, as well as for systematic screening of biofilm eradication strategies under physiologically relevant flow conditions.