Proteomic Insights into Antibiotic Resistance and Biofilm Adaptation of Staphylococcus epidermidis to Subinhibitory Levofloxacin, Rifampicin, and Combination Treatment

Staphylococcus epidermidis biofilms are inherently tolerant to antimicrobial treatment, contributing to the persistence and recurrence of biofilm-associated infections. In this study, we examined the phenotypic and proteomic responses of S. epidermidis ATCC 35984 biofilms following exposure to subinhibitory concentrations (1/8× MIC) of levofloxacin, rifampicin, and their combination. Phenotypic analyses revealed clear differences among the treatments. Rifampicin significantly reduced the number of viable biofilm-associated cells, whereas levofloxacin alone had little effect. The combination of rifampicin and levofloxacin produced the most pronounced antibiofilm activity, significantly reducing both biofilm biomass and viability. These findings were supported by confocal laser scanning microscopy, which showed the greatest loss of viable cells and biofilm integrity in the combination-treated biofilms. Label-free quantitative proteomic analysis demonstrated distinct treatment-specific adaptive responses. Rifampicin, either alone or in combination with levofloxacin, triggered broadly increased abundance of proteins involved in translation, energy production, amino acid and nucleotide metabolism, ATP-binding cassette transport, and quorum sensing. In contrast, levofloxacin predominantly suppressed proteins associated with central metabolism, transcription, translation, and biosynthetic pathways. Hierarchical clustering further showed that the proteomic profile of the combination treatment closely resembled that of rifampicin alone, suggesting that rifampicin was the dominant driver of proteomic remodeling. Expression pattern network analysis identified a conserved stress-response signature comprising Fhs, YdaG, PerR, and YcsE across all treatment conditions. Notably, the multidrug efflux pumps NorA and NorC showed decreased protein abundance across all treatment conditions, indicating a potential reduction in efflux-mediated resistance under sub-MIC antibiotic exposure. Taken together, these findings demonstrate that rifampicin retains substantial antibiofilm activity at subinhibitory concentrations and elicits extensive metabolic adaptation, whereas levofloxacin primarily induces a metabolically repressed state. The enhanced activity of the rifampicin–levofloxacin combination appears to arise from complementary effects on biofilm viability and cellular physiology. This study provides new insights into the adaptive responses of S. epidermidis biofilms to sublethal antibiotic stress and highlights the potential of rifampicin-based combination therapies for the management of biofilm-associated infections.