Abstract
Staphylococcus aureus is an opportunistic pathogen and the major causative agent of life-threatening hospital- and community-acquired infections. A combination of antibiotics could be an opportunity to address the widespread emergence of antibiotic-resistant strains, including Methicillin-Resistant S. aureus (MRSA). We here investigated the potential synergy between ampicillin and plant-derived antibiotics (pentacyclic triterpenes, ursolic acid (UA) and oleanolic acid (OA)) towards MRSA (ATCC33591 and COL) and the mechanisms involved. We calculated the Fractional Inhibitory Concentration Index (FICI) and demonstrated synergy. We monitored fluorescence of Bodipy-TR-Cadaverin, propidium iodide and membrane potential-sensitive probe for determining the ability of UA and OA to bind to lipoteichoic acids (LTA), and to induce membrane permeabilization and depolarization, respectively. Both pentacyclic triterpenes were able to bind to LTA and to induce membrane permeabilization and depolarization in a dose-dependent fashion. These effects were not accompanied by significant changes in cellular concentration of pentacyclic triterpenes and/or ampicillin, suggesting an effect mediated through lipid membranes. We therefore focused on membranous effects induced by UA and OA, and we investigated on models of membranes, the role of specific lipids including phosphatidylglycerol and cardiolipin. The effect induced on membrane fluidity, permeability and ability to fuse were studied by determining changes in fluorescence anisotropy of DPH/generalized polarization of Laurdan, calcein release from liposomes, fluorescence dequenching of octadecyl-rhodamine B and liposome-size, respectively. Both UA and OA showed a dose-dependent effect with membrane rigidification, increase of membrane permeabilization and fusion. Except for the effect on membrane fluidity, the effect of UA was consistently higher compared with that obtained with OA, suggesting the role of methyl group position. All together the data demonstrated the potential role of compounds acting on lipid membranes for enhancing the activity of other antibiotics, like ampicillin and inducing synergy. Such combinations offer an opportunity to explore a larger antibiotic chemical space.
Highlights
In the past few decades, the antibiotic resistance of bacteria has emerged as a serious threat in human and veterinary medicine
These values are lower than the Minimal Inhibitory Concentration (MIC) of ampicillin against MethicillinResistant S. aureus (MRSA) (ATCC33591 and COL)
To gain an insight into the molecular mechanisms involved in the effects induced by ursolic acid (UA) and oleanolic acid (OA) on MRSA membranes, we explored the ability of these two pentacyclic triterpenes to bind to lipoteichoic acids (LTA), and to induce MRSA membrane permeabilization and depolarization
Summary
In the past few decades, the antibiotic resistance of bacteria has emerged as a serious threat in human and veterinary medicine. To tackle this situation, development of new antibiotics or therapeutic strategies against multi-drug resistant bacteria is urgently needed. One approach is to enhance the bacteria susceptibility to antibiotics. Plant-derived chemicals have the potential to be used as therapeutics to enhance the activity of antibiotics against multidrug-resistant pathogens in addition or through their own effect(s) [1,2]. Two main representative compounds are ursolic acid (UA) (3β-hydroxy-urs-12-en-28-oic acid) and oleanolic acid (OA) (3β-hydroxyolean-12-en-28-oic acid). Both show an antimicrobial activity against a large number of pathogens including carbapenem-resistant Klebsiella pneumoniae [3], colistin-resistant
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