| Summary: | The study of bacterial interaction between <i>Streptococcus mutans</i> and <i>Actinomyces naeslundii</i> may disclose important features of biofilm interspecies relationships. The aim of this study was to characterize—with an emphasis on biofilm formation and composition and metabolic activity—single- and dual-species biofilms of <i>S. mutans</i> or <i>A. naeslundii,</i> and to use a drip flow reactor (DFR) to evaluate biofilm stress responses to 0.2% chlorhexidine diacetate (CHX). Single- and dual-species biofilms were grown for 24 h. The following factors were evaluated: cell viability, biomass and total proteins in the extracellular matrix, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide—“XTT”—reduction and lactic acid production. To evaluate stress response, biofilms were grown in DFR. Biofilms were treated with CHX or 0.9% sodium chloride (NaCl; control). Biofilms were plated for viability assessment. Confocal laser-scanning microscopy (CLSM) was also performed. Data analysis was carried out at 5% significance level. <i>S. mutans</i> viability and lactic acid production in dual-species biofilms were significantly reduced. <i>S. mutans</i> showed a higher resistance to CHX in dual-species biofilms. Total protein content, biomass and XTT reduction showed no significant differences between single- and dual-species biofilms. CLSM images showed the formation of large clusters in dual-species biofilms. In conclusion, dual-species biofilms reduced <i>S. mutans</i> viability and lactic acid production and increased <i>S. mutans’</i> resistance to chlorhexidine.
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