Effects of Sub-Inhibitory Rifampicin, Minocycline, and Dalbavancin on Early Biofilm Formation and Transcriptional Responses in Staphylococcus aureus SA113

Abstract

Implant-associated infections are often caused by biofilm-forming pathogens such as Staphylococcus (S.) aureus. While local antibiotic delivery systems aim to prevent adhesion and biofilm establishment, declining drug concentrations may result in sub-inhibitory exposure, which can modulate bacterial adaptation linked to antibiotic resistance, biofilm formation, and regulatory responses. We investigated the effects of sub-inhibitory antibiotic exposure on biofilm formation in S. aureus SA113 and associated transcriptional responses. The biofilm-producing strain S. aureus SA113 was exposed to sub-inhibitory concentrations of rifampicin, minocycline, and dalbavancin during early biofilm formation. Phenotypic effects were assessed by crystal violet staining, enumeration of colony-forming units, and scanning electron microscopy, while transcriptional responses were analyzed by qPCR. Despite stable counts of culturable adherent bacteria, sub-inhibitory antibiotic exposure differentially altered biofilm formation and transcriptional responses. Rifampicin was associated with increased biomass at higher sub-inhibitory concentrations and increased early expression of icaA, icaD (+4.2 log2) and fnbA (+2.7 log2) at 1/2× MIC. Minocycline reduced biomass at lower concentrations with partial recovery toward control levels at 1/2× MIC, while transcriptional analysis at 1/4× MIC after 6 h showed increased expression of icaA, icaD (+2.1 log2) and fnbA (+4.3 log2). Dalbavancin induced a distinct transcriptional response characterized by increased expression of vraS (+1.1 log2) and lrgA (+1.8 log2), without induction of matrix-associated genes, while adherent biofilm biomass was reduced to 42.9% at 1/2× MIC. Morphologically, this was associated with compact aggregates rather than diffuse biofilm structures. Sub-inhibitory antibiotic exposure differentially modulated early biofilm formation in SA113 in a drug-specific manner. Overall, the distinct dalbavancin-associated response may be relevant for the development of preventive local drug-delivery systems.