Schilcher, KatrinAlanazi, Maher2025-09-152025https://hdl.handle.net/20.500.14154/76396Abstract Bacterial interspecies interactions are fundamental to microbial community dynamics, involving complex sensing and response mechanisms that mediate co-existence or competitive behaviours. Coinfection with Staphylococcus aureus and Burkholderia cenocepacia is a significant concern in cystic fibrosis, as both pathogens colonise the lung environment in these patients. However, their bidirectional interactions remain poorly characterised, necessitating further investigation. S. aureus produces phenol-soluble modulins (PSMs), surfactant-like cytolytic peptides implicated in modulating the behaviour of neighbouring bacteria, including B. cenocepacia. This study aimed to investigate whether surfactants produced by S. aureus influence the B. cenocepacia surface motility and fitness, aiming to elucidate the molecular mechanisms underlying these interspecies interactions. A motility assay was established using B. cenocepacia H111 wild-type on AB minimal medium with optimised agar concentrations (0.3% and 0.5%). Responses to S. aureus cell-free supernatants (CFS) and synthetic PSM peptides (PSMα2, PSMα3, PSMβ; 0.1 and 1 mM) were assessed. Motility parameters (colony, swarm, and total colony+swarm) areas and directional motility indices were quantified. B. cenocepacia fitness was evaluated by monitoring growth kinetics over 24 hours in the presence of PSMs using a microtiter-based optical density assay. CFS from S. aureus did not induce detectable motility in B. cenocepacia on 1.5% agar. After assay optimisation, synthetic PSMα2 significantly enhanced surface motility in a dose-dependent manner, with local suppression at the peptide site. Although PSMα3 and PSMβ did not significantly enhance B. cenocepacia motility, a trend toward increased motility was observed for both peptides. PSMα2 and PSMβ did not induce directional motility in B. cenocepacia toward the PSM peptides, while PSMα3 induced concentration-dependent directional motility, with 0.1 mM showed strongest response (p < 0.05). Growth kinetics and overall fitness remained unaffected by synthetic PSMs. This study identifies PSMs as interspecies signalling molecules that selectively modulate B. cenocepacia motility. Moreover, it highlights the role of surfactant-mediated interactions in microbial community dynamics and competitive sensing74enBurkholderia cenocepaciaStaphylococcus aureusphenol-soluble modulinssurfactantssurface motilityfitnessinterspecies interactionThesis