Disinfection efficiencies of sage and spearmint essential oils against planktonic and biofilm Staphylococcus aureus cells in comparison with sodium hypochlorite

Staphylococcus aureus causes human infections and foodborne intoxications. This study explored the potential antibacterial actions of sage and spearmint essential oils (EOs) against both its planktonic and biofilm cells, in comparison with sodium hypochlorite (NaOCl), a commonly applied chemical sanitizer. Initially, the minimum inhibitory and bactericidal concentrations (MICs, MBCs) of each plant mixture were determined against planktonic cultures, following growth at 30 °C for 24 h. Stationary phase planktonic bacteria were then individually exposed for 6 min to either each EO (applied at 1-2 × MBC; 2.5-5%), or NaOCl (250-450 ppm). These were also left to form biofilms on 96-well polystyrene microplates, at 30 °C for 96 h, with medium renewal at 48 h, in the presence of 10 different concentrations of each EO, expanding from sub- to super-inhibitory for planktonic growth, and the minimum biofilm inhibitory concentrations (MBICs; > 90% inhibition) of each plant mixture were calculated. Formed biofilms were finally exposed for 6 min to either each EO (applied at 2-6 × MBC; 5-15%), or NaOCl (7500-25,000 ppm; applied either alone or in combination with each EO at 5%). Results showed that both EOs presented MIC and MBC equal to 1.25 and 2.5%, respectively. As expected, their application at their MIC and above significantly inhibited biofilm formation, while spearmint EO was still able to cause this at ½ of its MIC, with MBICs equal to 1.25 and 0.63% for sage and spearmint EOs, respectively. Alarmingly, the application of both EOs at 1/8 to 1/16 of their MIC further increased biofilm formation. Regarding biofilm disinfection experiments, the individual application of each EO against the pre-established sessile communities resulted in log decrease ranges of 0.8-3 log CFU/cm2, while in the case of NaOCl application (either alone or combined with each EO), the observed reductions never exceeded 1.7 log CFU/cm2. These last results highlight the great antimicrobial recalcitrance of biofilm communities, found here to be ca. 100 times more resistant to NaOCl compared to planktonic ones, and stress the urgent need for further research on alternative, adequate and safe disinfection strategies to control them in food processing and other environments.