Ultrasound-mediated plasma-activated microbubbles inhibit mixed biofilms of Enterococcus faecalis and Candida albicans via quorum sensing regulation

Microorganisms represented by Enterococcus faecalis ( E. faecalis ) are key factors in root canal treatment failure. This study aims to explore the antimicrobial effect of ultrasound-mediated plasma-activated microbubbles (PMBs) against mixed infections of E. faecalis and Candida albicans ( C. albicans ), and to investigate the regulation mechanisms of the quorum sensing (QS) system with PMBs. PMBs were prepared through a modified emulsification method and mediated by ultrasound to achieve targeted release of active species (such as NO and H₂O₂). Mixed planktonic microorganisms E. faecalis and C. albicans were treated with different concentrations of PMBs, with OD₆₀₀ measured to evaluate the antimicrobial effect. In order to evaluate the regulation effects and mechanisms of quorum sensing system with PMBs, the following methods were used. The crystal violet staining (OD₅₇₀) method was used to assess the impact of different concentrations of PMBs on mixed biofilms. Scanning electron microscopy (SEM) was employed to observe morphological changes of the E. faecalis biofilms after different treatments. The qRT-PCR was used to analyze the effects of different concentrations of PMBs on the expression of quorum sensing (QS)-related genes (cylR1 and cylA) in E. faecalis single biofilms, and the expression of LuxS in mixed biofilms. The reporter strain Vibrio harveyi BB170 was used to detect the activity of AI-2 expression levels in mixed biofilms. PMBs showed significant antimicrobial effects against both mixed planktonic microorganisms and biofilms, with PMBs-10 showing the best antibacterial effect ( p < 0.05), and the inhibitory effects increased with concentration. SEM analysis revealed that PMBs-10 treatment led to an almost complete disruption of the E. faecalis biofilm structure. Furthermore, qRT-PCR results indicated that PMBs-10 significantly downregulated the expression of cylR1 and cylA in E. faecalis biofilms ( p < 0.05). PMBs effectively inhibited the expression level of LuxS and the activity level of AI-2 in mixed biofilms. Ultrasound-mediated PMBs is a promising strategy that can effectively control both single and mixed infections by regulating the QS system, providing a promising clinical therapy for root canal infection control.