Effect of cell-free spent media prepared from Aggregatibacter actinomycetemcomitans on the growth of Candida albicans and Streptococcus mutans in co-species biofilms

This study explored the influence of cell-free spent media prepared from Aggregatibacter actinomycetemcomitans LuxS mutant (Aa-LuxS), its wild type strain (Aa-WT), and the laboratory strain (Aa-Y4), on the interaction between Candida albicans and Streptococcus mutans while growing in co-species biofilm for 48 h. By analyzing the results of crystal violet staining, [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assays, and quantitative real-time polymerase chain reaction (qPCR), we found that the presence of Aa-LuxS in treated biofilms did not affect biofilm development, while added Aa-WT or Aa-Y4 resulted in a significant decrease in both biofilm mass and the number of cells. The inhibitory effect of Aa-WT or Aa-Y4 was not dependent on the protein concentration in the spent media tested (1 and 10%). Gene transcription analyses indicated that Aa-WT/Aa-Y4 exhibits comparable inhibitory effects on the expression of hyphal-associated genes (ALS3 and HWP1), but not on the expression of YWP1, which encodes a yeast form of C. albicans. In contrast, except for gtfD, the expression of S. mutans gtfB/C genes encoding glucosyltransferase was not affected in Aa-WT and Aa-Y4 treated biofilms compared to the levels found in Aa-LuxS treated biofilms. Our results indicate that AI-2-containing spent media derived from Aa can reduce biofilm biomass without significantly inhibiting the survival rate of S. mutans.