| Summary: | Antifungal agents are widely used to specifically eliminate infections by fungal pathogens. However, the specificity of antifungal agents has been challenged by a few studies demonstrating antibacterial inhibitory effects against <i>Mycobacteria</i> and <i>Streptomyces</i> species. Here, we evaluated for the first time the potential effect of fluconazole, the most clinically used antifungal agent, on a human oral microbiota biofilm model. The results showed that biofilm viability on blood and mitis salivarius agar media was increased over time in the presence of fluconazole at clinically relevant concentrations, despite a reduction in biomass. Targeted PCR revealed a higher abundance of <i>Veillonella atypica</i>, <i>Veillonella dispar,</i> and <i>Lactobacillus</i> spp. in the fluconazole-treated samples compared to the control, while <i>Fusobacterium nucleatum</i> was reduced and <i>Streptococcus</i> spp were not significantly affected. Further, we tested the potential impact of fluconazole using single-species models. Our results, using <i>Streptococcus mutans</i> and <i>Streptococcus mitis</i> luciferase reporters, showed that <i>S. mutans</i> planktonic growth was not significantly affected by fluconazole, whereas for <i>S. mitis</i>, planktonic growth, but not biofilm viability, was inhibited at the highest concentration. Fluconazole’s effects on <i>S. mitis</i> biofilm biomass were concentration and time dependent. Exposure for 48 h to the highest concentration of fluconazole was associated with <i>S. mitis</i> biofilms with the most increased biomass. Potential growth inhibitory effects were further tested using four non-streptococcal species. Among these, the planktonic growth of both <i>Escherichia coli</i> and <i>Granulicatella adiacens</i> was inhibited by fluconazole. The data indicate bacterial responses to fluconazole that extend to a broader range of bacterial species than previously anticipated from the literature, with the potential to disturb biofilm communities.
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