Use of confocal laser scanning microscopy to evaluate the metal ion removal and destabilization of Enterococcus faecalis biofilms by EDTA and etidronic acid

Abstract

Chelating substances bind to metals, forming stable complexes, rendering these essential ions unavailable for microbial metabolism and community stability in biofilms. This action can contribute to the disinfection in endodontic treatments. Through confocal laser scanning microscopy (CLSM), this study quantified the metal ion removal by chelating agents in Enterococcus faecalis biofilms and assessed the impact on community stability. E. faecalis biofilms were grown for 21 days on acrylic coverslips, which the following were immersed in (n = 10): G1) saline solution (control, 5 min); G2) 17% ethylenediaminetetraacetic acid (EDTA, 3 min); and G3) 9% etidronic acid (Dual Rinse HEDP, 5 min). Next, they were stained with Phen Green FL to identify metals, 4’,6-diamidino-2-phenylindole (DAPI) to highlight bacterial DNA, and propidium iodide (PI) to mark nucleic acid in cells with damaged membranes. Images were captured (4/sample) by CLSM and analyzed by the software Leica Application Suite X. The individual and total volume of the biofilm-stained components (μm3) and their individual percentages in the biofilms were analyzed using one-way ANOVA with Tukey tests (α < 0.05). EDTA caused a higher metal removal (P < 0.05) that potentially destabilized biofilms, causing detachment of bacterial cells. Consequently, EDTA significantly reduced the total cubic volume of biofilms compared to other irrigants (P < 0.05), while control and etidronic acid groups exhibited similarity (P > 0.05). However, the percentages of nucleic acid and metals remained constant in all treatments (P > 0.05). In conclusion, strong chelating solutions, such as EDTA, can remove substantial amounts of metals from biofilms and affect the community stability.