Bacillus subtilis maintains antibiofilm activity against Staphylococcus aureus following adaptive laboratory evolution

Quorum sensing interference has been touted as an ideal mechanism for the development of new anti-virulence therapies. Recent work has established Bacillus subtilis 6D1, a Gram-positive spore forming bacterium with probiotic qualities, produces metabolites that inhibit Staphylococcus aureus virulence and biofilm formation via quorum sensing interference. However, it remains unknown how long-term exposure to these molecules drive S. aureus adaptation and evolution. S. aureus planktonic cells and biofilms were propagated in the presence of B. subtilis 6D1 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. aureus lineages evolved in the presence of B. subtilis 6D1 CFE were less competitive in a biofilm and exhibited increased phenotypic sensitivity to multiple antibiotics. Notably, B. subtilis 6D1 CFE maintained its ability to inhibit S. aureus biofilm growth and disassemble mature biofilm in all evolved lineages. S. aureus populations 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 by B. subtilis 6D1, can reduce S. 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 revealed Bacillus subtilis reduces biofilm formation and virulence via signaling interference with the S. aureus Agr QS system; however, it remains unclear how long-term exposure to these compounds drives S. aureus adaptation 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.