Microbe-Derived Indole Metabolite Demonstrates Potent Multidrug Efflux Pump Inhibition in Staphylococcus aureus

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 Ca 2+ 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 Ca 2+ 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.

Deciphering the Antibacterial Role of Peptide From Bacillus subtilis subsp. spizizenii Ba49 Against Staphylococcus aureus

“…The post-antibiotic studies were performed according to Tambat et al (2019) with some modifications. S. aureus cells (∼10 5 CFU/ml) were treated with the two different concentrations, 4 μM (1 × MIC) and 8 μM (2 × MIC), of peptide-Ba49 in MHB medium.…”

Identified Seaweed Compound Diphenylmethane Serves as an Efflux Pump Inhibitor in Drug-Resistant Escherichia coli

“…These data indicate that the addition of DPM does not seem to exert a significant effect on the PAE of erythromycin and clarithromycin. Tambat et al [ 44 ] indicated that an indole of microbial origin, the 2-(2-aminophenyl) indole, could increase the PAE of ciprofloxacin against gram-positive bacterium S. aureus expressing efflux pump NorA by 1.1–1.9 h. For example, the combination of ciprofloxacin (8 μg/mL) and EPI 2-(2-aminophenyl) indoles (32 μg/mL) could extend the PAE for the S. aureus from 1.43 h (8 μg/mL ciprofloxacin alone) to 3.33 h. So far, few studies have reported that EPIs could extend the PAE of antibiotics against gram-negative bacteria.…”

“…The H33342 and EtBr dye accumulation assays were performed according to the method described in a previous report [ 44 ], with some modifications. The E. coli Kam3/pSYC- acrB solution for the dye efflux assay was prepared as described in Section 3.5 .…”

In Vitro Evaluation of Antimicrobial Peptide Tridecaptin M in Combination with Other Antibiotics against Multidrug Resistant Acinetobacter baumannii

“…The synergy of tridecaptin M with other antibiotics was initially identified by checkerboard assay [ 21 , 53 ]. Briefly, in a 96-well plate, the concentration of tridecaptin M was varied vertically, whereas the antibiotics were varied horizontally in a matrix of 8 × 12.…”

“…The inoculum was prepared in a similar way as for MIC determination, and the plates were kept at 37 °C for MIC observation in combination. The fractional inhibitory concentration index (FICI) was calculated for each antibiotic to identify the synergistic effect of tridecaptin M [ 53 ] using following equation: …”