Use of multiparameter flow cytometry to determine the effects of monoterpenoids and phenylpropanoids on membrane polarity and permeability in staphylococci and enterococci.

Monoterpenoids and phenylpropanoids are major components of many plant essential oils and are relatively simple, low-molecular-weight compounds with antimicrobial activity. This study used multiparameter flow cytometry to examine changes in membrane polarity and permeability in Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis following exposure to the monoterpenoids carvacrol, 1,8-cineole and terpinen-4-ol and the phenylpropanoids eugenol and cinnamaldehyde. Melaleuca alternifolia (tea tree) essential oil was also investigated. The fluorescent dyes DiOC(2)(3) (3,3'-diethyloxacarbocyanine oxide) and TO-PRO(®)-3 were used to assess membrane potential and permeability, respectively, following treatment with the minimum inhibitory concentration (MIC) of each test compound for 5 min and 30 min. Four subpopulations of cells were identified based on polarity and permeability. Eugenol treatment resulted in the greatest depolarisation and permeabilisation at 5 min, followed by carvacrol. Cinnamaldehyde, whilst having the lowest MICs (0.006-0.1%, v/v), did not induce changes in polarity or permeability at the MIC, and substantially higher concentrations were required to induce significant effects. At 30 min, treatment with all six compounds resulted in significant depolarisation (60.9-99.3% of cells), whereas fewer compounds (ranging from two to five per organism) resulted in significant permeabilisation. The extent of permeabilisation was always less than depolarisation, with overall means for all treatments of 46.1% and 89.4% of cells permeabilised and depolarised, respectively, at 30 min. These data demonstrate that several monoterpenoids and phenylpropanoids as well as tea tree oil alter membrane properties by decreasing polarity and increasing permeability in a time- and concentration-dependent manner.