In vitro Antibiofilm Activity of Cold Atmospheric Plasma on Enterococcus Faecalis

Abstract

Apical periodontitis is an infection primarily caused by bacteria that invade the dental pulp and periapical tissues, necessitating effective management to preserve tooth function and aesthetics through endodontic procedures. E. faecalis is one of the most significant bacteria implicated in endodontic treatment failure. Effective disinfection of the root canal system is crucial for reducing the bacterial load and ensuring long-term treatment success. Traditionally, NaOCl has been the gold standard for root canal disinfection due to its broad-spectrum antimicrobial properties. However, NaOCl presents several significant drawbacks, including potential safety concerns, adverse effects on dentine mechanical properties, and limited penetration into the dentinal tubules. These limitations necessitate the exploration of alternative or adjunctive disinfection methods that can offer enhanced efficacy and safety. Atmospheric plasma jet (APJ) technology has emerged as a promising alternative in various fields due to its potent antimicrobial properties. APJ has been investigated in areas such as food safety, industrial sanitation, medical sterilisation, and more recently, dentistry. The APJ generates reactive oxygen species (ROS), which are believed to play a critical role in its antimicrobial activity. Although the precise mode of action of APJ is not yet fully understood, its broad-spectrum antimicrobial effectiveness highlights its potential as a significant area of interest for researchers aiming to enhance endodontic treatment disinfection. The primary aim of this research was to investigate the antimicrobial efficacy of APJ against E. faecalis biofilms on hydroxyapatite (HA) discs. A secondary aim was to determine whether adding NaOCl 0.5% to APJ treatment would enhance its antimicrobial efficacy, thereby testing the synergistic potential of the combined treatment. In addition, phosphate-buffered saline (PBS) was used as the liquid medium and tested in two ways to evaluate its antimicrobial efficacy. The first method involved applying APJ to PBS without the HA disc sample for a determined period. The sample was then inserted into the activated PBS without further application of APJ to observe any antimicrobial activities generated by the APJ. This group was referred to as Plasma-Activated PBS (PAPBS). The second method involved exposing the sample to continuous APJ application while it was in the PBS medium. This approach aimed to enhance the disinfection process by utilising both APJ and the ROS generated in the PBS. The PBS media treated the sample with APJ during its time in PBS, resulting in a group termed Plasma-Enhanced PBS (PEPBS). The objective of comparing these two methods was to determine which technique would result in more significant bacterial reduction, thus guiding future clinical applications. The methodology involved culturing E. faecalis biofilms on HA discs in10 BHI broth under anaerobic conditions at 37°C. Biofilms were grown for different periods: 24 hours, 1 week, and 3 weeks, to represent varying stages of biofilm maturity. Treatment groups comprised a negative control PBS, a positive control (1% NaOCl), and various combinations involving APJ alone, PEPBS, PAPBS and APJ with 0.5% NaOCl. Biofilms were exposed to the antimicrobial agents for 1, 5, and 10 min. Bacterial reduction was assessed using CFU counting. For statistical analysis, a three-way ANOVA was employed to evaluate the significance of the results. The results demonstrated that while 1% NaOCl exhibited the highest reduction rate for immature biofilms, the mature biofilm showed higher resistance. In contrast, APJ showed superior performance in killing mature biofilms. APJ alone, PEPBS, and PAPBS provided significant differences compared to the control group. Notably, the combination of APJ and 0.5% NaOCl achieved 100% eradication of E. faecalis biofilms regardless of their age, highlighting a synergistic effect that significantly enhances antimicrobial efficacy, indicating the potential of APJ to improve the performance of various irrigants.