Natural product inhibitors of bacterial type-IV secretion systems and efflux pumps

Multidrug-resistance is a global health concern and in bacteria it may be conferred by the acquisition of multiple-drug resistance genes and/or by the action of multidrug-efflux pumps. The current study targeted these processes as a means to combat the spread of multidrug resistance genes among bacteria, and reinstate the efficacy of antibiotics against efflux-mediated drug-resistant strains. Our aim was therefore to isolate and characterise natural products that function by either inhibiting bacterial conjugation and/or by potentiating antibiotic activity against efflux-related multidrug-resistant (MDR) strains. Selected medicinal plants, some of which have antibacterial properties (Amoracia rusticana, Borago officinalis, Brassica oleracea, Lepidium sativum, Myristica Iowiana, Sinapis alba, Uncaria tomentosa and Zingiber officinale) were extracted with solvents of increasing polarity. The extracts were then screened against Escherichia coli conjugation pairs of donors (pKM101, IncN; TP114, IncI2; pUB307, IncP; and R7K, IncW), and recipients (ER1793 and JM109), and Staphylococcus aureus strains expressing distinct efflux-related multidrug-resistance pumps; SA-1199B (NorA) and XU212 (TetK). The active extracts were further fractionated using various chromatographic techniques (Thin Layer Chromatography, Solid Phase Extraction, Vacuum Liquid Chromatography, Column Chromatography and High Performance Liquid Chromatography). The compounds, which were isolated from the bioactive fractions, were then characterized by the use of spectroscopic techniques (NMR, MS, IR and UV) and re-assessed for anti-conjugation and antibiotic potentiation activity. The isolated glucosinolates from the Brassica plants showed moderate activity (10 - 50% reduction) against the conjugal transfer of the tested plasmids while the isothiocyanates, which are degradation products of the glucosinolates, showed better broad-range anti-conjugal activity. An amide, isolated from M. lowiana, showed significant anti-conjugal inhibitory activity (16.7 ± 2.0%) against the R7K plasmid. Its anti-conjugal activity was plasmid specific and non-toxic to human dermal fibroblasts, adult cells. In addition, a gingerol compound isolated from Z. officinale, the isolated amide from M. lowiana, and benzyl isothiocyanate, significantly potentiated the activity of norfloxacin and tetracycline against SA-1199B (NorA) and XU212 (TetK), respectively. Their potentiation activity ranged from 2 to 512-fold. In conclusion, the study identified natural product inhibitors of the type-IV secretion-related processes and efflux pump systems. Compounds with such anti-conjugative and antibiotic potentiation activity could help decrease the spread of multidrug resistance genes via conjugation and prolonging the efficacy of existing antibiotics