Evaluation of the antibacterial effect of (R)-(+)-Limonene against Enterococcus faecalis and Enterobacter cloacae strains isolated from food

Abstract

The aim of this study was to evaluate the potential antibacterial and anti-adherent activities of the monoterpene (R)-(+)-limonene, as well as its synergistic potential with synthetic antimicrobials against strains of Enterococcus faecalis and Enterobacter cloacae. The antibacterial properties of (R)-(+)-limonene were assessed using the broth microdilution technique to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Additionally, the infusion disc method was employed to explore the association of the compound with antimicrobials, and the test tube method was used to determine the minimum inhibitory concentration of adherence (MICA). It was observed that the MIC for (R)-(+)-limonene was 1000 μg mL-1 for five of the six E. faecalis strains tested, while for E. cloacae, the MIC exceeded 1000 μg mL-1 for all strains tested. Identical values were recorded for the MBC in E. faecalis. In terms of its combination with synthetic antimicrobials, (R)-(+)-limonene demonstrated a synergistic effect with gentamicin and ciprofloxacin for most strains. Regarding the MICA, both (R)-(+)-limonene and 0.12% chlorhexidine digluconate failed to inhibit biofilm formation at the tested concentrations. Given the need for new therapeutic alternatives for treating bacterial infections, this study revealed that the tested monoterpene exhibited moderate bactericidal effects against E. faecalis strains and no antibacterial effect against E. cloacae strains. However, when combined with various classes of antimicrobials, (R)-(+)-limonene showed synergistic effects with gentamicin and ciprofloxacin for most strains. This suggests that (R)-(+)-limonene holds promise for enhancing the treatment of bacterial infections and could support conventional therapies. Nonetheless, further in vitro, ex vivo, and in vivo studies are necessary to confirm and elucidate its efficacy and mechanisms.