Biological Merits and Antibiofilm Mechanisms of Action of a Specially Designed Double-Shelled Microparticle.

We recently fabricated a double-shelled microparticle (DSMP) incorporating a quaternary ammonium compound (QAC) and quinine, which demonstrated promising anti-infective properties. In this study, we aimed to elucidate biological merits and antibiofilm mechanisms of action of the specifically designed DSMP. Antimicrobial activities of the DSMP against 32 Gram-positive clinical isolates and laboratory strains were assessed by determining the minimum inhibitory concentrations (MICs). Antibiofilm efficacies of the DSMP were evaluated by using a tetrazolium salt reduction assay and confocal laser scanning microscopy. The fractional inhibitory concentration index was determined to clarify drug-drug interactions between QAC and quinine. Evolution of DSMP resistance was experimentally assessed by using an adaptive laboratory evolutionary assay. Molecular mechanisms underlying DSMP resistance was investigated by whole genome sequencing. The DSMP demonstrated strong activity against antibiotic-susceptible clinical isolates of species, and , with MICs of 4-8 μg/mL, and notable antibiofilm capacities. Weakly positively charged DSMP readily penetrated through the negatively charged biofilm matrix in 3 h and accumulated in biofilm water pores by 24 h, thereby killing biofilm-embedded cells. The copresence of the QAC and quinine demonstrated synergistic effects against Gram-positive bacteria while simultaneously mitigating the development of resistance. Whole-genome sequences revealed genetic mutations in efflux pumps that are associated with cross-resistance to antibiotics and the DSMP components. Co-presence of the QAC and quinine in the DSMP lays the foundation for its antimicrobial and antibiofilm properties against Gram-positive bacteria, allowing the DSMP to maintain efficacy while limiting the development of resistance.