Combating Antimicrobial Resistance with Botanicals: Screening and Evaluation of New Efflux Pump and Bacterial Conjugation Inhibitors

Abstract

The global health challenge of multidrug-resistant (MDR) bacteria stems from their acquisition of drug-resistance genes or from multidrug efflux pump activity. This study examined these mechanisms with the aim of limiting the spread of MDR genes and restoring antibiotic efficacy. The objective was to identify natural compounds that inhibit bacterial conjugation or enhance antibiotic activity against efflux-related resistant strains. Using sequential solvent extraction, crude extracts from several ethnomedicinal plants were screened in vitro against resistant bacterial strains, including Staphylococcus aureus ATCC 25923, MRSA, S. aureus XU212 Tet(K), S. aureus SA1199B NorA, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. In addition to screening plant extracts, selected commercial phytochemicals were also evaluated for antibacterial potency, efflux pump inhibition, and inhibition of bacterial conjugation. Bioactivity-guided fractionation of promising plant extracts led to the isolation of asiatic acid and corosolic acid from Symplocos fasciculata Zoll., which showed minimum inhibitory concentrations (MICs) of 16 μg/mL against S. aureus strains. In Salvia miltiorrhiza, miltirone, dihydrotanshinone I, cryptotanshinone and rosmarinic acid exhibited potency, with minimum inhibitory concentration (MIC) values of 0.5, 4, 8 and 16 μg/mL, respectively. From Tabebuia impetiginosa, plumbagin demonstrated potent activity against E. coli (MIC = 8 μg/mL) and against S. aureus strains (MIC = 0.125 μg/mL). Juglone also showed activity against S. aureus, with an MIC of 8 μg/mL. Furthermore, oleanolic acid enhanced the activity of tetracycline 512-fold against S. aureus XU212 Tet(K). Other compounds from different plant species, including liquiritigenin, bixin, echinocystic acid and rehmannioside D, increased the activity of tetracycline by 256-fold. Rehmannioside D also enhanced the activity of norfloxacin by 256-fold. Plumbagin significantly reduced plasmid conjugal transfer frequency by 69.6 ± 3.6% for plasmid pKM101 (IncN) and 81±5.7% for plasmid R388 (IncW). Similarly, bixin reduced plasmid conjugal transfer frequency by 69.6 ± 3.6% for plasmid R388 (IncW). These findings indicate that plumbagin exerts a greater inhibitory effect on plasmid conjugal

transfer for plasmid pKM101 (IncN) than for plasmid R388 (IncW). Bixin showed a comparable inhibitory effect on plasmid R388 (IncW). This study identifies botanicals as promising candidates for resistance-modifying agents. By interfering with efflux mechanisms and horizontal gene transfer, these natural compounds present innovative strategies to restore antibiotic efficacy and counter the growing threat of antimicrobial resistance