Abstract
Efflux pumps are always at the forefront of bacterial multidrug resistance and account for the failure of antibiotics. The present study explored the potential of 2-(2-Aminophenyl) indole (RP2), an efflux pump inhibitor (EPI) isolated from the soil bacterium, to overcome the efflux-mediated resistance in Staphylococcus aureus. The RP2/antibiotic combination was tested against efflux pump over-expressed S. aureus strains. The compound was further examined for the ethidium bromide (EtBr) uptake and efflux inhibition assay (a hallmark of EPI functionality) and cytoplasmic membrane depolarization. The safety profile of RP2 was investigated using in vitro cytotoxicity assay and Ca2+ channel inhibitory effect. The in vivo efficacy of RP2 was studied in an animal model in combination with ciprofloxacin. RP2 exhibited the synergistic activity with several antibiotics in efflux pump over-expressed strains of S. aureus. In the mechanistic experiments, RP2 increased the accumulation of EtBr, and demonstrated the inhibition of its efflux. The antibiotic-EPI combinations resulted in extended post antibiotic effects as well as a decrease in mutation prevention concentration of antibiotics. Additionally, the in silico docking studies suggested the binding of RP2 to the active site of modeled structure of NorA efflux pump. The compound displayed low mammalian cytotoxicity and had no Ca2+ channel inhibitory effect. In ex vivo experiments, RP2 reduced the intracellular invasion of S. aureus in macrophages. Furthermore, the RP2/ciprofloxacin combination demonstrated remarkable efficacy in a murine thigh infection model. In conclusion, RP2 represents a promising candidate as bacterial EPI, which can be used in the form of a novel therapeutic regimen along with existing and upcoming antibiotics, for the eradication of S. aureus infections.
Highlights
Methicillin-resistant Staphylococcus aureus (MRSA), a high priority pathogen (Tacconelli et al, 2018), is responsible for skin and soft tissue infections, endocarditis, and several bloodstream infections (Tong et al, 2015)
The current study describes the isolation, identification and assessment of efflux pump inhibitory activity of EPI 2(2- Aminophenyl) indole from the terrestrial bacterial isolate, Streptomyces Sp
The initial screening identified 50 microbial isolates (Supplementary Table S1), which potentiated the activity of ethidium bromide (EtBr) in S. aureus by at least four folds
Summary
Methicillin-resistant Staphylococcus aureus (MRSA), a high priority pathogen (Tacconelli et al, 2018), is responsible for skin and soft tissue infections, endocarditis, and several bloodstream infections (Tong et al, 2015). For the last few decades, there is a substantial decline in the development of new antimicrobial compounds (Boucher et al, 2009). With diminished hope on dwindling discovery pipeline, bridging the gap between widespread multi-drug resistance, and development of new antibiotics requires innovative use of available antibiotic arsenal. The over-expression of efflux pumps in S. aureus is one of the major cause of antibiotic resistance (Munita and Arias, 2016). Several efflux pumps have been reported in bacteria, of which major facilitator superfamily (MFS) is predominant (Costa et al, 2013), which utilizes a proton gradient for extruding the drugs. In S. aureus, NorA (MFS) is the most studied efflux pump and is reported to be over-expressed in more than 50% of the clinical isolates (DeMarco et al, 2007). The NorA pump exports a broad range of compounds such as fluoroquinolones, detergents, and several dyes like ethidium bromide (EtBr) (Fontaine et al, 2015; Tintino et al, 2016), whereas TetK and MsrA efflux pumps are specific for tetracycline and erythromycin, respectively (Smith et al, 2007)
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