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On the basis of a primary screening scheme by using plant seed methanol extract against a collection of human pathogenic bacteria, Medicago sativa L. seeds were chosen as the best potential resource against tested Gram positive bacteria. Then an agar-overlay method using fully separated proteins on sodium dodecyl sulphate-polyacryliamide gel electrophoresis (SDS-PAGE) gels was used for initial determination and primary characterization of active putative defensins in the plant seeds. Clear and remarkable zones of inhibition in a region corresponding to peptides slightly larger than 6 kDa were recorded for Methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecium (VRE) strains and yeast but a smaller inhibition zone with several colonies for tested Gram negative strain of Escherichia coli. Further characterization experiments using two-dimensional gel electrophoresis and subsequent agar-overlay assay against both strains confirmed the peptide nature of the active substance. Also gel filtration separation using Sephadex G-25 superfine and subsequent antibacterial assays could confirm the presence of a low molecular weight anti-MRSA and anti-VRE peptide in the total water soluble proteins obtained from M. sativa L. seeds which also showed high level of thermo stability.
Key words: Medicago sativa L., methicillin resistant bacteria, vancomycin resistant bacteria, plant defensins.