Antipathogenic properties of selected South African medicinal plants to combat multidrug-resistant bacteria

Abstract

South African native plants have been predominantly explored for their antibacterial properties, overlooking their antipathogenic and antivirulence (also known as antiquorum sensing) potential. The bacterial quorum sensing (QS) system controls the virulence, biofilm formation and pathogenesis and collectively promotes diseases caused by multi-drug resistant (MDR) bacteria. Therefore, blocking the QS system may aid the management of various infectious diseases caused by human pathogens. Plant secondary metabolites can thwart bacterial colonization and virulence. Hence, this research was undertaken to explore the phytochemical, antivirulence or antipathogenic properties, molecular modeling and safety use of bioactive South African medicinal plants against selected MDR bacteria. Ten medicinal plants namely Acokanthera oppositifolia (Lam), Aloe arborescens (Mill), Artemesia afra (Jacq. ex Willd), Lippia javanica (Burm. f. Spreng), Melianthus comosus (Vahl), Pelargonium sidoides (DC), Plectranthus ecklonii (Benth), Tetradenia riparia ((Hochst.) Codd), Trichilia emetica (Vahl) and Vachellia karroo (Hayne. Banfi and Glasso) were selected based on their therapeutic use for various treatments such as skin infections, respiratory diseases and urinary tract infections (UTI). Plants were collected at Manie van der Schijff Botanical Garden, extracted using solvents with varying polarities then determined their minimum inhibitory concentrations (MIC) using the microdilution method against five MDR bacteria (Streptococcus pyogenes, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli). From the 50 plant extracts, Melianthus comosus (dichloromethane, aqueous and methanol), Pelargonium sidoides (aqueous) and Vachellia karroo (aqueous and methanol) extracts showed potent MIC values between 0.19 - 0.78 mg/mL against the five tested bacterial pathogens. A selected bio-monitor strain of Chromobacterium violaceum (ATCC 12472) with a violacein phenotype was used to assess plant extracts for their antiquorum sensing activities. The violacein production in C. violaceum was reduced up to 38.34% by M. comosus, Plectranthus ecklonii and P. sidoides extracts. The plant extracts that exhibited promising activities (antibacterial and antiquorum sensing), warranted further investigation for the identification of the compounds by using gas chromatography-mass spectrophotometry (GC-MS). Chemical vii characterization of six active extracts of M. comosus (dichloromethane, aqueous and methanol), P. sidoides (aqueous) and V. karroo (aqueous and methanol) revealed compounds such as α-D-glucopyranoside, methyl, guanosine, neophytadiene, phytol, hexadecanoic acid and linalool, octadecanoic acid. An in-silico approach was then conducted to validate the antiquorum sensing properties of the compounds through the QS regulator protein (CviR´) of C. violaceum. Sucrose, guanosine, α-D-glucopyranoside, methyl, phytol and 1,2,3-benzenetriol exhibited good docking scores between -7.59 and -5.57 kcal/mol for the CviR´ protein of C. violaceum. The active plant extracts and phytochemicals were assessed on their effect to decrease the biofilm formation of selected MDR bacteria and their cytotoxic effect on the African monkey kidney Vero (epithelial). Treatment of five MDR bacterial pathogens with plant extracts and compounds for anti-adhesion and biofilm development showed up to 78.88% and 31.82% inhibitory effects, respectively. In-vitro cytotoxicity activities of plant extracts were shown to reduce the Vero cell’s viability in a dose-dependent manner, showing a cytostatic effect, particularly at higher concentrations. Whilst both compounds of guanosine and phytol caused no cytotoxic effect on Vero cells. Pseudomonas aeruginosa was used as an MDR model pathogen to ascertain the molecular QS mechanism of the compounds. Two P. aeruginosa QS regulator proteins of LasR (2UV0) and PqsR (4JVD) were investigated through in-silico studies. Phytochemical compounds of sucrose, guanosine, α-D-glucopyranoside, methyl, phytol and 1,2,3-benzenetriol showed varying docking scores between 5.66 - 8.37 kcal/mol and 7.79 - 9.13 kcal/mol for 2UV0 and 4JVD proteins, respectively. Further, molecular dynamics simulation (MDS) findings showed phytol was the most stable compound that bound to the 2UV0 protein with an average root-mean-square deviation (RMSD) value of 1.47Å indicating a good solution whereas guanosine was the most stable for the 4JVD protein with an RMSD value of 2.10Å. The binding of all compounds resulted in a more flexible 4JVD conformation than the 2UV0. The in-silico findings were validated by targeting several P. aeruginosa virulence properties. M. comosus extracts and guanosine showed a significant reduction of pyocyanin production and inhibited swarming and swimming motility at a higher concentration. Overall, guanosine was the ideal potential QS compound that could be a candidate in search of antivirulent or antipathogenic drugs to combat bacterial infections caused by MDR bacteria.