Inhibition of biofilm formation of foodborne pathogens by selected South African medicinal plants

Abstract

Microbial biofilm and quorum sensing are related traits employed by microorganisms to improve their survival and virulence. They have been increasingly implicated in the food processing and the medical industries where they cause surface and food surface contamination. In this thesis, the available literature regarding the value of South African plants as potential sources of anti-biofilm and quorum quenching bioactive secondary metabolites was surveyed. The literature survey also covered antimicrobial activity investigations of medicinal plants against foodborne pathogens. The survey revealed that a total of thirty plant species belonging to nineteen families have anti-biofilm and quorum quenching capacity against foodborne pathogens. The survey served to summarize present knowledge and to provide a basis for further investigation of South African medicinal plants with known anti-biofilm and quorum quenching potential. In subsequent research, the antimicrobial, anti-biofilm, antioxidant and cytotoxicity activities of nine South African medicinal plants was evaluated. The plants (Combretum elaeagnoides, Combretum molle, Combretum oxystachyum, Carpobrotus edulis, Vachellia rehmanniana, Vachellia xanthophloea, Kigelia africana, Elephantorrhiza elephantina and Ochna pretoriensis) were investigated. The selection of plant species was based on their known antimicrobial activity, chemotaxonomic relationships to plant species with antibacterial activity, availability and/or the existence of traditional uses against foodborne diseases. The serial microdilution technique and the crystal violet assay were used to assess the antimicrobial and anti-biofilm potential of the acetone and methanol extracts, fractions and isolated compound. The antioxidant activity of the extracts, fractions and isolated compound was determined using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assays. The tetrazolium dye cell viability assay was used to ascertain the cytotoxicity of the samples. The methanol extract of C. elaeagnoides was fractionated into five fractions (hexane, dichloromethane, ethyl acetate, butanol and water) via solvent-solvent fractionation. Column chromatography making use of silica gel as stationary phase combined with gradient elution of increasing polarity using hexane and ethyl acetate was used to isolate a bioactive compound via bioassay-guided fractionation. The isolated compound was identified as quercetin 3-O-rhamnoside using mass spectrometry (MS) and nuclear magnetic resonance (NMR). The extracts of the nine plants were active against all selected test organisms with minimum inhibitory concentration (MIC) values ranging from <0.02 to 2.5 mg/ml. The acetone extracts of Vachellia xanthophloea, Combretum molle, and Carpobrotus edulis had excellent MIC activity of <0.02 mg/mL against Enterobacter cloacae. The dichloromethane fraction of Combretum elaeagnoides had the best MIC value of 0.03 mg/mL against Escherichia coli (ATCC 25922). The result of the minimum bactericidal concentration (MBC) investigation showed that the acetone extracts of C. elaeagnoides and C. molle had the best activity against E. coli (ATCC 25922) with no growth at 0.31 mg/mL, and acetone extracts of C. elaeagnoides and V. xanthophloea against Staphylococcus aureus at 0.63 mg/mL respectively. The acetone extracts of C. molle and V. xanthophloea had the best MBC activity against the clinical isolate of E. coli (clinical isolate), Campylobacter jejuni, and E. cloacae with 100% inhibition at 0.08, 0.31 and 0.02 mg/mL respectively. The acetone extract of C. edulis also inhibited (100%) E. cloacae at 0.02 mg/mL. The acetone extract of C. molle exhibited 100% inhibition against Salmonella Typhimurium, Stenotrophomonas maltophilia and Klebsiella pneumoniae at 0.04, 0.16 and 0.08 mg/mL respectively. The acetone extract of V. xanthophloea also exhibited 100% inhibition against Salmonella Typhimurium and S. Enteriditis at 0.08 mg/mL. Of all the extracts, fractions and the isolated compound, the acetone extract of C. molle had the best MBC against the tested pathogens. Most of the extracts, fractions and the isolated compound, quercetin 3-O-rhamnoside, selectively reduced biofilm growth by at least 50% against the foodborne pathogens. The acetone extract of C. molle had the greatest anti-biofilm activity against S. Typhimurium. The antioxidant assay revealed that the methanol extract of V. xanthophloea had very good activity against the radical scavenging DPPH while the acetone extract of C. edulis had excellent activity against the electron reducing ABTS with IC50 values of 0.14±0.11μg/mL and 0.01±0.02 μg/mL respectively. Most of the fractions had good antioxidant activity against DPPH (IC50= 0.01 to >100 μg/mL) and ABTS (IC50= 0.01±0.007 to 20.00±1.79 μg/mL) radicals. The butanol fraction of C. elaeagnoides and quercetin 3-O-rhamnoside had excellent antioxidant activity with the same IC50 value of 0.01 μg/mL. The cytotoxicity assay revealed that most of the extracts were relatively safe to cells except for the acetone extract of C. molle (0.01 mg/mL) and the methanol extract of O. pretoriensis (0.02 mg/ml). Therefore the good antimicrobial activity of C. molle may be largely owing to non-specific toxicity. In summary, the study has established that the leaf extracts of the nine South African medicinal plants as well as the fractions and isolated compound from C. elaeagnoides have antimicrobial, antibiofilm and antioxidant potential and are generally relatively non-cytotoxic. Further work is needed to explore the quorum quenching, synergistic effects and possible mechanisms of action of the plants.