Aspirin Combined with Antifungal Drugs Suppresses Biofilm by Activating Autophagy.

Aspirin (ASA) induces autophagic death of human tumor cells and autophagy changes the susceptibility of biofilm to antifungal agents. This study investigates whether ASA suppresses biofilm by autophagy regulation and its combination effect with antifungals. Biofilm sensitivity to ASA alone and in combination with antifungals was evaluated using the checkerboard method, and drug interactions were assessed by the fractional inhibition concentration index (FICI) and ΔE models. The effects of ASA on mTOR signaling were examined by western blotting. Alkaline phosphatase activity, acridine orange stain assay, and autophagy-related gene expressions were examined to evaluate autophagic activity. Autophagosomes were observed by transmission electron microscopy. Reactive oxygen species (ROS) were detected by DCFH-DA. Mitochondrial membrane potential (MMP), malondialdehyde (MDA), and ATP levels were determined using commercial kits. ASA inhibited biofilm in a concentration dependent manner and showed synergistic effects against biofilms when combined with amphotericin B or 5-fluorocytosine. ASA treatment induced oxidative stress, evidenced by increased ROS and MDA levels, alongside a reduction in ATP and MMP. ASA inhibited mTOR signaling and induced autophagy in biofilms by increasing oxidative stress and mitochondrial dysfunction, contributing to biofilm inhibition. This study provides valuable insights into the potential of ASA as an adjunct therapy in combination with antifungal agents for managing biofilm-related infections.