pCF10 reprograms Enterococcus faecalis biofilms to promote nonattached aggregate biofilms, oxidative stress adaptation, and spatial antibiotic protection

Mobile genetic elements are major drivers of microbial niche development. The pheromone-responsive plasmid pCF10 enhances virulence of Enterococcus faecalis . Previously, we showed that pCF10 induces organized complex biofilm structures containing aggregated bacteria in the commensal strain OG1RF. Exposure to 100× the MIC of erythromycin (100× Erm) rapidly doubled both bacterial numbers and structure size. Here, we show that these structures form under flow and in chemostats, conditions relevant to infection, and nonattached biofilm aggregates were formed. Aggregation substance was required for complex structure formation and resistance to 100× Erm. Redox staining revealed elevated hydrogen peroxide (H 2 O 2 ) within the structures. Catalase inhibited complex structure formation, consistent with our published mathematical model predicting a stress signal for spatially organized growth. pCF10 increased tolerance to H 2 O 2 . Exogenous H 2 O 2 increased pCF10 copy number from ~3 to ~10 per cell, whereas cysteine-to-alanine substitutions in conserved cysteine residues of the replication initiation protein PrgW abolished this response. Erythromycin-induced population expansion persisted for at least 15 h, and clinically relevant subinhibitory and 1× Erm increased population and structure size. Mechanically disrupted biofilms were antibiotic sensitive, revealing some persisters. Mathematical simulations revealed spatial arrangements of aggregated structures capable of generating protected microenvironments enabling continued survival during antibiotic exposure.