Efflux Pump Inhibition and Resistance Modulation in Mycobacterium smegmatis by Peucedanum ostruthium and Its Coumarins

Abstract

Antibiotic resistance is a growing problem and may become the next major global health crisis if no timely actions are taken. Mycobacterial infections are widespread and, due to antibiotic resistance, also hard to treat and a major cause of mortality. Natural compounds have the potential to increase antibiotic effectiveness due to their resistance modulatory and antimicrobial effects. In this study, Peucedanum ostruthium extracts, fractions, and isolated compounds were investigated regarding their antimicrobial and resistance-modulatory effects as well as efflux pump inhibition in Mycobacterium smegmatis. P. ostruthium extracts were found to have anti-mycobacterial potential and resistance modulating effects on ethidium bromide activity. The major antibacterial effect was attributed to ostruthin, and we found that the more lipophilic the substrate, the greater the antimicrobial effect. Imperatorin caused potent modulatory effects by interfering with the action of the major LfrA efflux pump in M. smegmatis. The plant P. ostruthuim has a complex effect on M. smegmatis, including antibacterial, efflux pump inhibition, resistance modulation, and membrane permeabilization, and its major constituents, ostruthin and imperatorin, have a distinct role in these effects. This makes P. ostruthium and its coumarins promising therapeutics to consider in the fight against drug-resistant mycobacteria.

Keywords: Mycobacterium smegmatis; Peucedanum ostruthium; antibacterial; efflux pump inhibitor; ethidium bromide accumulation; imperatorin; ostruthin; resistance modulation.

Figure 1

UV chromatogram of P. ostruthium ethanol (E) (A) and hexane (H) (B) extracts at 310 nm, molecular structures of main identified coumarins in Peucedanum ostruthium extracts oxypeucedanin hydrate (1), oxypeucedanin (2), ostruthol (3), imperatorin (4), isoimperatorin (5), ostruthin (6) (C) and constituents identified in extracts and subsequent fractions according to LC-PDA-ESI-MSⁿ results with the relative composition of identified coumarins in subfractions (%) (D).

Figure 2

Ethidium bromide (EtBr) accumulation in M. smegmatis mc² 155 untreated culture (control) and treated with (A) the crude hexane (H) and (B) ethanol (E) extract at the minimal inhibitory concentration (MIC; 31.35 mg/L), and sub-inhibitory concentrations of 0.5 × MIC (15.62 mg/L), 0.25 × MIC (7.81 mg/L) and 0.12 × MIC (3.91 mg/L), and the known EPI verapamil in 0.5 × MIC (250 mg/L), presented as relative fluorescent units (RFU). A higher fluorescence indicates stronger efflux pump inhibition.

Figure 3

Fold change in ethidium bromide (EtBr) accumulation in M. smegmatis mc² 155 culture treated with a sub-inhibitory concentration of 0.25 × MIC of P. ostruthium hexane (H) and ethanol (E) extract and their fractions (H1-H5 and E1-E9), and the known EPI verapamil (VP), compared to untreated control at 60 min treatment time point. The dotted line presents no change in EtBr accumulation fold change (baseline). * p < 0.05.

Figure 4

Schematic representation of ethidium bromide (EtBr) accumulation and efflux in M. smegmatis mc² 155 wild type and ΔlfrA mutant without inhibitor (A) and with efflux pumps inhibitor (B); and EtBr accumulation in M. smegmatis mc² 155 (C) and M. smegmatis mc² 155ΔlfrA (D) untreated culture (control) and treated with the isolated compounds imperatorin (4) and ostruthin (6) at the sub-inhibitory concentration of 0.25 × MIC, presented as relative fluorescent units (RFU) with indicated FC in EtBr accumulation in the treated culture vs. the untreated control.

Figure 5

Membrane integrity of M. smegmatis wild type (A) and the ∆lfrA mutant (B) in untreated control culture and culture treated with ethanol extract at concentrations of 0.25 and 0.125 × MIC (E 0.25 × MIC and E 0.125 × MIC), hexane extract at 0.25 and 0.125 × MIC (H 0.25 × MIC and H 0.125 × MIC), imperatorin at 0.25 × MIC, and ostruthin at 0.25 × MIC. Heat-treated culture was used as a control for total membrane disruption. Membrane integrity is presented as relative fluorescent units (RFU) through a 60 min treatment time.