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Phenol-soluble modulins (PSMs) are amphipathic, alpha-helical peptides that are secreted by staphylococci in high amounts in a quorum-sensing-controlled fashion. Studies performed predominantly in Staphylococcus aureus showed that PSMs structure biofilms, which results in reduced biofilm mass, while it has also been reported that S. aureus PSMs stabilize biofilms due to amyloid formation. We here analyzed the roles of PSMs in in vitro and in vivo biofilms of Staphylococcus epidermidis , the leading cause of indwelling device-associated biofilm infection. We produced isogenic deletion mutants for every S. epidermidis psm locus and a sequential deletion mutant in which production of all PSMs was abolished. In vitro analysis substantiated the role of all PSMs in biofilm structuring. PSM-dependent biofilm expansion was not observed, in accordance with our finding that no S. epidermidis PSM produced amyloids. In a mouse model of indwelling device-associated infection, the total psm deletion mutant had a significant defect in dissemination. Notably, the total psm mutant produced a significantly more substantial biofilm on the implanted catheter than the wild-type strain. Our study, which for the first time directly quantified the impact of PSMs on biofilm expansion on an implanted device, shows that the in vivo biofilm infection phenotype in S. epidermidis is in accordance with the PSM biofilm structuring and detachment model, which has important implications for the potential therapeutic application of quorum-sensing blockers. Unlabelled Image [ABSTRACT FROM AUTHOR]