Biofilme Interkingdom de Enterococcus faecalis e Candida albicans

Abstract

Enterococcus faecalis and Candida albicans are important agent of persistent apical periodontitis. However, the impact that the interaction between them has on these infections is still poorly understood. The present study aimed to (1) optimize and characterize E. faecalis and C. albicans dual-species biofilms; (2) evaluate the antimicrobial potential of promethazine (PMZ) and chlorpromazine (CPZ) against the dual-species biofilm; (3) investigate the outcomes that this interkingdom interaction may have on E. faecalis’ gene expression. Therefore, different physicochemical conditions for biofilm formation were tested. Susceptibility assays to antimicrobials, biochemical composition and ultrastructure analyses were performed. The susceptibility of planktonic cells to PMZ, CPZ chlorhexidine (CHX), and sodium hypochlorite (NaClO) was investigated. The effect of the interaction between phenothiazines and CHX on antimicrobial activity against planktonic cells was examined by chequerboard assay. The effect of NaClO, PMZ, CPZ, CHX, PMZ + CHX, and CPZ + CHX on dual-species biofilms was investigated by susceptibility assays and biochemical and ultra-morphological analyses. RNAseq analyses were performed to compare the gene expression of E. faecalis in mono-species and dual-species biofilms. Further assays to confirm the transcriptomics findings were performed. Results were evaluated through the one-way ANOVA and Tukey’s multiple comparison post-test. Reproducible dual-species biofilms were established in BHI medium, at 35°C for 48 h in a microaerophilic atmosphere. An increase in biomass and chitin content was detected after vancomycin treatment, which might be related to C. albicans’ virulence. Structural analysis revealed that the dual-species biofilm was formed by both microorganisms adhering to the substrate. PMZ, alone or in combination with CHX, was the most efficient phenothiazine against biofilms. Neither PMZ nor CPZ increased the antimicrobial activity of CHX. The presence of C. albicans considerably upregulated E. faecalis’ phosphoenolpyruvate transport system (PTS) gene expression. The increase of PTS activity for mannose uptake in the presence of C. albicans was confirmed experimentally, as well as the significant effect that mannose uptake has on the biofilm formation by E. faecalis. The proposed protocol could be useful for the study of interkingdom relationships and help to find new strategies against periapical infections. Studies investigating the clinical properties of PMZ should be performed to recommend their use in endodontics. No synergism was detected between CHX and the phenothiazines. The present study suggests that the presence of C. albicans induces metabolic changes that may benefit E. faecalis’ virulence as an endodontic pathogen.