Inhibition of multidrug-resistant foodborne Staphylococcus aureus biofilms by a natural terpenoid (+)-nootkatone and related molecular mechanism

Staphylococcus aureus, a foodbome pathogen, poses serious problems to the food industries owing to biofilm formation, and over 25% of the foodbome illnesses in China have been attributed to S. aureus only. Phytochemicals are widely used as anti-biofilm agents with promising efficacy, and most of them are widely available and safe. This study reported the anti-biofilm efficacy of (+)-nootkatone, a sesquiterpene ketone found in a common fruit grapefruit, against multidrug-resistant S. aureus and its potential molecular mechanism. (+)-Nootkatone exhibited bacteriostatic and bactericidal effects at 200 and 400 mu g/mL, respectively, against S. aureus SJTUF 20758 and S. aureus ATCC 25923. Crystal violet staining indicated that (+)-nootkatone inhibited S. aureus biofilm formation (p < 0.05) at a sub-MIC of 50 mu g/mL and reduced exopolysaccharide production. The thickness of biofilms was significantly reduced by (+)-nootkatone, which was supported by the light microscopy and confocal laser scanning microscopy. Growth curve of bacteria showed that the antibacerial activity of (+)-nootkatone was dose-dependent, and the sub-MIC concentrations did not affect the bacterial growth of planktonic cells. Besides, (+)-nootkatone affected the sliding motility of S. aureus. At 200 mu g/mL, (+)-nootkatone led to the reduction of preformed biofilm mass by 50% and bacterial cell death of 79%, accompanied with a reduction of exopolysaccharide. The expression of biofilm-related genes, including sarA, icaA, agrA, RNAIII, and spa, was suppressed by (+)-nootkatone, as revealed by the transcriptional analysis. Additionally, MTT assay revealed that there was no toxicity of (+)-nootkatone to the human foreskin fibroblasts (HFF) cells. Therefore, (+)-nootkatone is a promising phytochemical against S. aureus biofilms, and has the potential to be used in food industry to fight against S. aureus-induced safety issues.