Thymus maroccanus essential oil, a membranotropic compound active on Gram-negative bacteria and resistant isolates

AbstractAims: The effects of Thymus maroccanus essential oil (EO) on the integrity ofthe cell membranes and the permeability of the outer membrane (OM) andinner membrane (IM) of Escherichia coli, Enterobacter aerogenes and Salmonellaenterica Typhimurium were investigated.Methods and Results: The bacterial release of intracellular proteins,cytoplasmic b-galactosidase and periplasmic b-lactamase induced byT. maroccanus EO was compared to the membranotropic activity of polymyxinB (PB) known as an effective permeabilizer of the membrane of Gram-negativebacteria. Results showed that T. maroccanus EO increased the permeability ofthe OM and IM of studied bacteria and induced the release of intracellularproteins into the external medium.Conclusions: The effect of T. maroccanus EO on the outer membrane wascomparable to that of PB, and both T. maroccanus EO and PB induce similarlevels of b-lactamase release. In addition, it also promoted the release of thecytoplasmic b-galactosidase. Moreover, the lipopolysaccharide molecules andthe overexpression of efflux pumps seem to play a crucial role in the level ofsusceptibility of studied bacteria to the permeabilizing effect of T. maroccanusEO.Significance and Impact of Study: These results demonstrate thatT. maroccanus EO can restore antibiotic activity by targeting the two bacterialmembranes and would be attractive candidates for developing new adjuvantsfor combating resistant Gram-negative bacteria.IntroductionA noteworthy characteristic of Gram-negative bacteria isthe presence of an outer membrane surrounding the bac-terial cell (Benson 1998; Nikaido 2003). This complexbarrier represents an efficient permeation control (Benson1998; Nikaido 2003), able to impair the uptake of macro-molecules (such as large peptides) and hydrophobic sub-stances (e.g. hydrophobic antibiotics) (Vaara 1992). Thus,the outer membrane (OM) confers a solid protectionagainst molecules that can normally damage the innermembrane (IM), peptidoglycan or cytoplasmic targetsand also controls the uptake of various compoundsincluding nutrients and hydrophilic antibiotics via theproteinaceous channels located through the outer mem-brane (Weiss et al. 1984; Vaara 1992; Page`s et al. 2008).The membrane permeability of Gram-negative bacteria isdirectly associated with the functional assembly of themembrane proteins and the structure of the lipopolysac-charide (LPS). The expression of narrow porin channelsslows down the penetration of hydrophilic solutes (Nikaido1996), while LPS structure controls the rate of trans-membrane diffusion of lipophilic solutes (Nikaido andVaara 1985; Ple´siat and Nikaido 1992). It is important tonote that many of the modifications reported in the outermembrane permeability contribute to the drastic decrease