| Objective Investigate the synergistic effect of Terpinen-4-ol(T4O)and α-Bisabolol(Bis)on two common skin pathogenic bacteria,Staphylococcus aureus(S.aureus)and Cutibacterium acnes(C.acnes).Provide a theoretical basis for the development of T4O in combination with Bis as a novel pathogenic bacteria inhibitory formulation.Methods 1.The minimum inhibitory concentration(MIC)and fractional inhibitory concentration index(FICI)of T4O and Bis over S.aureus and C.acnes was respectively determined by micro tessellation dilution method.Time-killing curves were used to investigate the in vitro inhibitory effects of MIC and double concentration(2×MIC)of T4O and Bis on S.aureus and C.acnes.2.Zeta potential,intracellular nucleic acid and protein leakage,respiratory chain dehydrogenase activity of S.aureus were measured after T4O and Bis combined treatment.And bacterial morphology was observed by transmission electron microscopy to evaluate the mechanism of inhibiting S.aureus and C.acnes after combined treatment.3.Crystal violet staining was used to evaluate the damage effect of T4O combined with Bis on biofilms of S.aureus and C.acnes.4.Bioinformatics method was used to predict the correlation between T4O and Bis and human skin infection.Results 1.The MIC of T4O alone was 2.50 mg/m L for S.aureus and C.acnes,and the MIC of Bis alone was more than 12.5 mg/m L for the two experimental bacteria alone.The MIC of the combination of the two components,T4O was 0.31 mg/m L for C.acnes and0.63 mg/m L for S.aureus.And the MIC of Bis was 0.39-1.56 mg/m L.T4O combined with Bis inhibited S.aureus and C.acnes with FICI<0.5,showing synergistic effect.2.There were significant differences between the MIC group and the 2×MIC group after 4 h treatment(P<0.01).The inhibition of bacterial growth was dose-dependent,and the CFU of C.acnes decreased to 0 in the 2×MIC group at 24 h.3.Compared with the negative control group that only added solvent and medium,the negative zeta potential on the surface of S.aureus and C.acnes was decreased in MIC and2×MIC groups,and the values of A260 and A280 of the superneant were increased,which reduced the activity of respiratory chain dehydrogenase of S.aureus and damaged the cell integrity of the two experimental strains.4.Residue of S.aureus and C.acnes biofilm on 96-well plates treated with T4O and Bis was lower than that of negative control group.5.Bioinformatics analysis predicted that 14 target proteins of T4O and Bis overlaps with proteins related to skin infection,and the pathways related to skin infection are neural active ligand-receptor interaction signaling pathway and calcium signaling pathway.Conclusion The combination of T4O and Bis has a synergic effect on the inhibition of S.aureus and C.acnes.The mechanism can be explained by the destruction of the cell wall structure of the bacteria,resulting in the leakage of nucleic acid and protein and other intracellular molecules,affecting the function of the respiratory chain and destroying the bacterial biofilm.These mechanisms precisely target the currently discovered bacterial resistance mechanisms,thus reducing the development of bacterial resistance.The results of in vitro bacteriostatic effect and bioinformatics prediction showed that T4O combined with Bis had the potential to be developed as an effective component of bacteriostatic products. |
