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Enterococcus faecalis  is the most important agent of persistent apical periodontitis, and recently,  Candida albicans  has also been implicated in periapical infections. This study aimed to optimize an  in vitro E. faecalis  and  C. albicans  dual-species biofilm protocol for endodontic research. Different physicochemical conditions for biofilm formation were tested. Susceptibility assays to antimicrobials, biochemical composition and an ultra-morphological structure analyses were performed. Reproducible dual-species biofilms were established in BHI medium at 35 °C, for 48 h and in a microaerophilic atmosphere. An increase in biomass and chitin content was detected after vancomycin treatment. Structural analysis revealed that the dual-species biofilm was formed by both microorganisms adhered to the substrate. The proposed protocol could be useful for the study of interkingdom relationships and help to find new strategies against periapical infections.

Enterococcus faecalis and Candida albicans dual-species biofilm: establishment of an in vitro protocol and characterization