Bacillus subtilismaintains antibiofilm activity againstStaphylococcus aureusfollowing adaptive laboratory evolution

Abstract

AbstractQuorum sensing interference has been touted as an ideal mechanism for the development of new anti-virulence therapies. Recent work has establishedBacillus subtilis6D1, a Gram-positive spore forming bacterium with probiotic qualities, produces metabolites that inhibitStaphylococcus aureusvirulence and biofilm formation via quorum sensing interference. However, it remains unknown how long-term exposure to these molecules driveS. aureusadaptation and evolution.S. aureusplanktonic cells and biofilms were propagated in the presence ofB. subtilis6D1 cell free extracts (CFE) for ∼73 generations. Fitness, virulence, and antibiotic resistance assays of the ancestor and all evolved lineages revealed the emergence of treatment and lifestyle associated ecological traits. Compared to the ancestor and media-evolved lineages,S. aureuslineages evolved in the presence ofB. subtilis6D1 CFE were less competitive in a biofilm and exhibited increased phenotypic sensitivity to multiple antibiotics. Notably,B. subtilis6D1 CFE maintained its ability to inhibitS. aureusbiofilm growth and disassemble mature biofilm in all evolved lineages.S. aureuspopulations propagated in the presence of CFE acquired missense mutations in genes associated with plasmid-borne efflux systems and RNA polymerase. Furthermore, CFE-evolved lineages did not develop mutations in both competence and drug resistance pathways found in similarly evolved control lineages. Our data suggest long-term exposure to biofilm inhibitory molecules, like those produced byB. subtilis6D1, can reduceS. aureus’fitness in a biofilm and increase sensitivity to multiple antibiotics.ImportanceQuorum sensing interference (QSI) has been touted as an ideal mechanism to diminish bacterial virulence and improve antibiotic killing, however few studies investigate the genetic and phenotypic adaptations that occur after long-term exposure to QSI therapies. Recent studies revealedBacillus subtilisreduces biofilm formation and virulence via signaling interference with theS. aureusAgr QS system; however, it remains unclear how long-term exposure to these compounds drivesS. aureusadaptation and evolution. This study helps to address these gaps by investigating whether QSI strategies deployed by probiotic bacteria are viable approaches to increase antibiotic efficacy without increasing antibiotic resistance evolution.