Regulation of Enterococcus faecalis Biofilm Formation and Quorum Sensing Related Virulence Factors with Ultra-low Dose Reactive Species Produced by Plasma Activated Water

Abstract

Enterococcus faecalis (E. faecalis) is the species which can cause life-threatening infections in human beings and exhibit highly antibacterial resistance in the nosocomial environment. The purpose of this study was to evaluate the inhibition effect and the mechanisms of ultra-low dose reactive oxygen species produced by plasma activated water (PAW) to the E. faecalis biofilm. The bactericidal effect of E. faecalis planktonic growth was evaluated to find out the maximum processing time. The level of intracellular reactive oxygen species (ROS) and extracellular hydrogen peroxide (H2O2) produced by the PAW without significant killing effect were defined as ultra-low. Then the pre-treated bacteria suspension by the PAW were incubated with brain heart infusion (BHI) broth for 6 h, 12 h, 24 h and 48 h. The biofilm development was evaluated with 96-well polystyrene method (OD570) and confocal laser scanning microscopy (CLSM). Meanwhile, total organic carbon (TOC) was measured with a Shimadzu TOC-L analyzer. The quorum sensing related virulence genes, cylR1, cylA, gelE and sprE, were evaluated with real-time PCR. The PAW did not have significant killing effect within 30 s and the maximum corresponding fluroscence intensity of ROS is under 57.33 ± 0.58 and the concentration of H2O2 is 19.87 ± 2.01 μmol/L. The OD570, CLSM results and TOC results indicated that the biofilm development was effectively inhibited with PAW treatment. Four quorum sensing related virulence genes were all down-regulated. In conclusion, the PAW treatment has potential application to control E. faecalis biofilm development and reduce quorum sensing related virulence genes expression in vitro.