Use of carvacrol, thymol, and eugenol for biofilm eradication and resistance modifying susceptibility of Salmonella enterica serovar Typhimurium strains to nalidixic acid

Abstract

The Aims of the study was to evaluate the antibacterial susceptibility and the biofilm eradication of three natural compounds carvacrol (CAR), thymol (TH) and eugenol (EUG), alone or in combination with nalidixic acid (NA) against twelve Salmonella Typhimurium strains. The minimum inhibitory concentration (MIC) and the minimum biofilm eradication concentration (BEC₅₀) of the tested compounds (CAR, TH and EUG) and their combinations with NA were evaluated. In order to assess whether these bacteria had active efflux pumps, ethidium bromide (EtBr) accumulation assays was achieved using spectrophotometric accumulation assays. Moreover, scanning electron microscopy was used to visualize the bacterial biofilm formation on stainless steel surfaces after exposed to NA, CAR, TH and EUG alone and in combination. TH was the most effective essential oil, with the lowest MICs values ranging from 32 to 128 μg/mL followed by EUG and CAR. In addition, the combination of NA with the different compounds enhances antibiotic susceptibility of the tested bacterial strains. These results were confirmed by EtBr accumulation assays. A pronounced effect in decreasing biofilm mass was also noticed. Moreover, SEM revealed that bacterial membrane was disrupted and a complete loss of membrane integrity was also evident. The combination of natural compounds with antibiotic enhances bacterial susceptibility to NA. This combination ameliorates eradication of biofilm formed by S. Typhimurium on polystyrene microtitre plates. Additionally, this synergy induces an alteration of the bacterial cell surface visualized by SEM.

Keywords: Antibacterial; Antibiofilm; Carvacrol; Efflux pumps inhibitors; Eugenol; Membrane disruption; Thymol.