Anandamide alters the membrane properties, halts the cell division and prevents drug efflux in multidrug resistant Staphylococcus aureus

Abstract

Antibiotic resistance is a serious public health problem throughout the world. Overcoming methicillin and multidrug-resistant Staphylococcus aureus (MRSA/MDRSA) infections has become a challenge and there is an urgent need for new therapeutic approaches. We have previously demonstrated that the endocannabinoid Anandamide (AEA) can sensitize MRSA to antibiotics. Here we have studied the mechanism of action using a MDRSA clinical isolate that are sensitized by AEA to methicillin and norfloxacin. We found that AEA treatment halts the growth of both antibiotic-sensitive and antibiotic-resistant S. aureus. The AEA-treated bacteria become elongated and the membranes become ruffled with many protrusions. AEA treatment also leads to an increase in the percentage of bacteria having a complete septum, suggesting that the cell division is halted at this stage. The latter is supported by cell cycle analysis that shows an accumulation of bacteria in the G2/M phase after AEA treatment. We further observed that AEA causes a dose-dependent membrane depolarization that is partly relieved upon time. Nile red staining of the bacterial membranes indicates that AEA alters the membrane structures. Importantly, 4′-6-diamidino-2-phenylindole (DAPI) accumulation assay and ethidium bromide efflux (EtBr) assay unveiled that AEA leads to a dose-dependent drug accumulation by inhibiting drug efflux. In conclusion, our study demonstrates that AEA interferes with cell division, alters the membrane properties of MDRSA, and leads to increased intracellular drug retention, which can contribute to the sensitization of MDRSA to antibiotics.

Anti-Microbial Activity of Phytocannabinoids and Endocannabinoids in the Light of Their Physiological and Pathophysiological Roles

“…The anti-microbial effect of endocannabinoids depends on the strain studied and the endocannabinoid used [16][17][18]389,390] (Table 2). Among the tested organisms, Streptococcus salivarius, Bacteroides fragilis, and Enterococcus faecalis were the most susceptible bacteria to AEA and N-Linoleoylethanolamine (LEA) [390].…”

“…Among the tested organisms, Streptococcus salivarius, Bacteroides fragilis, and Enterococcus faecalis were the most susceptible bacteria to AEA and N-Linoleoylethanolamine (LEA) [390]. MSSA and MDRSA become immediately growth arrested by AEA, an effect that was transient and relieved upon time [16]. On the other hand, the growth of Lactobacillus gasseri species becomes enhanced by LEA and OEA [390].…”

“…Cannabis sativa L. subspecies are plants that contain a large variety of secondary metabolites, including phytocannabinoids, terpenoids and flavonoids, which have profound anti-microbial activities, in addition to possessing anti-inflammatory, anti-oxidative and neuromodulatory properties [12][13][14]. In mammalians, the phytocannabinoids interact with the same receptors (e.g., cannabinoid receptors CB1 and CB2) as the endocannabinoids [15], which are endogenous substances with anti-microbial, anti-inflammatory and neuromodulatory activities [16][17][18][19][20][21][22][23][24]. While much is known about the cannabinoid targets in mammalians, so far, little is known about the microbial targets of these compounds.…”

“…AraS had a MIC of 16 and 128 µg/mL on MRSA ATCC 33592 and MRSA ATCC 43300, respectively, and a MIC above 256 µg/mL for the clinical MRSA isolate [18]. A kinetic study of AEA on a multidrug-resistant Staphylococcus aureus (MDRSA) clinical isolate and the MSSA ATCC 25923 strain showed that AEA caused a transient bacteriostatic effect that was overcome with time [16]. The bacteriostatic effect of AEA was independent of the drug-resistant phenotype [16].…”

Effect of epigallocatechin gallate on dental biofilm of Streptococcus mutans: An in vitro study

“…The bacterial suspension was divided into 1 ml aliquots with different concentrations of EGCG and stained with 10 µl of 3 mM DiOC 2 (3) for 30 min in the dark. The samples were analyzed by flow cytometry (LSR-Fortessa flow cytometer, BD Biosciences), using the 488 nm excitation laser and collecting the data using the green (530nm) and red (610/620 nm) filters [ 40 ]. In addition, we measured the forward scatter (FSC) and the side scatter (SSC) of the untreated and EGCG-treated bacteria.…”