The antibacterial mode of action and properties of Ib-AMP1, a plant-derived antimicrobial peptide, against Escherichia coli O157:H7

The continual occurrence of foodborne outbreaks along with the consumer demand for use of fewer traditional antimicrobial agents in foods has driven research interests in development of plant-derived antimicrobial agents (pAMPs) for use in food and food processing. Ib-AMP1 is a pAMP isolated from seeds of Impatiens balsamina. Previous studies indicated that it is a broad spectrum pAMP and the therapeutic index against eight human pathogens was 23.5; however, for future utilization, other antibacterial properties and mode of action must be elucidated. The purpose of this dissertation was to investigate the antibacterial properties and mode of action of Ib-AMP1 against Escherichia coli O157:H7, a foodborne pathogen that has been continually associated with foodborne outbreaks. The study design provided insight on the implantation and potential application of Ib-AMP1; a specific docking site or ligand-receptor relationship was not studied. The results demonstrated that Ib-AMP1 exhibited bactericidal activity against E. coli O157:H7, Salmonella enterica serovar Newport, Pseudomonas aeruginosa and Staphylococcus aureus. Ib-AMP1 at lethal concentrations (1X and 2X MIC) resulted in 1.46 to 2.69 log reduction of viable cells and prevented outgrowth when tested against low (103 CFU/mL) and medium (106 CFU/mL) E. coli O157:H7 populations. Ib-AMP1 at 2X MIC failed to inhibit and prevent outgrowth when cell numbers were 109 CFU/mL. No residual activity of Ib-AMP1 was apparent following interaction of the peptide with bacteria or the medium. Ib-AMP1 concentration less than 100 µg/mL showed little or no inhibition of human cell proliferation including human small intestine, colon and liver cells, which are associated with oral consumption of an AMP. The mode of action study demonstrated that a concentration dependent effect of Ib-AMP1 on the E. coli O157:H7 cell membrane occurred. Ib-AMP1 treatments resulted in efflux of K+ and ATP, suggesting pores of sufficient size to allow efflux of large molecules. The efflux of intracellular components may be associated with damage to the outer membrane and dissipation of cytoplasmic membrane potential. Results of this study suggest Ib-AMP1 is bactericidal interfering within outer and inner membrane integrity permitting efflux of ATP and interfering with intracellular biosynthesis of DNA, RNA, and protein.