ABSTRACT
The objectives of this study were to evaluate tetrahydropyridine derivatives as efflux inhibitors and to understand the mechanism of action of the compounds by in silico studies. Minimum inhibitory concentration (MIC) determination, fluorometric methods and docking simulations were performed. The compounds NUNL02, NUNL09 and NUNL10 inhibited efflux, and NUNL02 is very likely a substrate of the transporter protein AcrB. Docking studies suggested that the mechanism of action could be by competition with substrate for binding sites and protein residues. We showed for the first time the potential of tetrahydropyridines as efflux inhibitors and highlighted compound NUNL02 as an AcrB-specific inhibitor. Docking studies suggested that competition is the putative mechanism of action of these compounds.
Subject(s)
Anti-Bacterial Agents/metabolism , Biological Transport, Active/drug effects , Enzyme Inhibitors/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/drug effects , Multidrug Resistance-Associated Proteins/metabolism , Pyridines/metabolism , Anti-Bacterial Agents/chemistry , Enzyme Inhibitors/chemistry , Escherichia coli Proteins/chemistry , Microbial Sensitivity Tests , Molecular Docking Simulation , Multidrug Resistance-Associated Proteins/chemistry , Protein Binding , Pyridines/chemistryABSTRACT
Six compounds (1-6), isolated from the methanol extract of the roots of the African medicinal plant Zanthoxylum capense Thunb. (Rutaceae), and seven ester derivatives (7-13) were evaluated for their antibacterial activities and modulatory effects on the MIC of antibiotics (erythromycin, oxacillin, and tetracycline) and ethidium bromide (EtBr) against a Staphylococcus aureus reference strain (ATCC 6538). Using the same model, compounds 1-13 were also assessed for their potential as efflux pump inhibitors by a fluorometric assay that measures the accumulation of the broad range efflux pump substrate EtBr. Compounds 8 and 11 were further evaluated for their antibacterial, modulatory and EtBr accumulation effects against four additional S. aureus strains, which included two clinical methicillin-resistant S. aureus (MRSA) strains. Compounds (1-13) have not shown antibacterial activity at the concentration ranges tested. When evaluated against S. aureus ATCC 6538, oxychelerythrine (1) a benzophenanthridine alkaloid, showed the highest modulatory activity enhancing the susceptibility of this strain to all the tested antibiotics from two to four-fold. Ailanthoidiol diacetate (8) and ailanthoidiol di-2-ethylbutanoate (11) were also good modulators when combined with EtBr, increasing the bacteria susceptibility by four and two-fold, respectively. In the EtBr accumulation assay, using ATCC 6538 strain, the phenylpropanoid (+)-ailanthoidiol (6) and most of its ester derivatives (8-11) exhibited higher activity than the positive control verapamil. The highest effects were found for compounds 8 and 11 that also increased the accumulation of EtBr, using S. aureus ATCC 25923 as model. Furthermore, both compounds (8, 11) were able to enhance the ciprofloxacin activity against the MRSA clinical strains tested, causing a reduction of the antibiotic MIC values from two to four-fold. The EtBr accumulation assay revealed that this modulation activity was not due to an inhibition of efflux pumps mechanism. These results suggested that Z. capense constituents may be valuable as leads for restoring antibiotic activity against MRSA strains.
Subject(s)
Anti-Bacterial Agents/pharmacology , Plant Extracts/pharmacology , Staphylococcus aureus/drug effects , Zanthoxylum/chemistry , Anti-Bacterial Agents/isolation & purification , Methicillin-Resistant Staphylococcus aureus/drug effects , Microbial Sensitivity Tests , Molecular Structure , Plant Extracts/chemistry , Plant Roots/chemistryABSTRACT
Resistance mediated by efflux has been recognized in Staphylococcus aureus in the last few decades, although its clinical relevance has only been recognized recently. The existence of only a few studies on the individual and overall contribution of efflux to resistance phenotypes associated with the need of well-established methods to assess efflux activity in clinical isolates contributes greatly to the lack of solid knowledge of this mechanism in S. aureus. This study aims to provide information on approaches useful to the assessment and characterization of efflux activity, as well as contributing to our understanding of the role of efflux to phenotypes of antibiotic resistance and biocide tolerance in S. aureus clinical isolates. The results described show that efflux is an important contributor to fluoroquinolone resistance in S. aureus and suggest it as a major mechanism in the early stages of resistance development. We also show that efflux plays an important role on the reduced susceptibility to biocides in S. aureus, strengthening the importance of this long neglected resistance mechanism to the persistence and proliferation of antibiotic/biocide-resistant S. aureus in the hospital environment.
