关键词: 抗菌肽brevilaterin, ε-聚赖氨酸, 协同抑菌机理, 金黄色葡萄球菌

Abstract: Staphylococcus aureus is a common foodborne pathogenic bacterium, and controlling S. aureus is of great importance for the improvement of food safety. Thus, using S. aureus as an indicator microorganism, the synergistic antibacterial mechanism of the antimicrobial peptide brevilaterin combined with ε-polylysine was investigated. Time-kill kinetics showed that brevilaterin and ε-polylysine had a synergistic antibacterial effect on S. aureus. Transmembrane proton potential kinetics indicated that ε-polylysine could disrupt the transmembrane pH gradient, but brevilaterin had no obvious effect. The combination of brevilaterin and ε-polylysine each at 1/4 minimal inhibitory concentration (MIC) synergistically damaged the transmembrane pH gradient. Flow cytometry combined with fluorescence microscopy was used to investigate the cell membrane integrity, and the results showed that 36.3% of the cells were damaged by brevilaterin at 1/4 MIC, while only 10.4% of the cells were destroyed by ε-polylysine at 1/4 MIC. Their combined use could synergistically damage the cell membrane integrity, resulting in 51.3% cell membrane damage. The ultrastructure of cells observed by transmission electron microscopy showed that the combined use of brevilaterin and ε-polylysine had a synergistic effect in disrupting cell morphology and causing leakage of cellular contents when compared with either alone. Sodium dodecylsulfate-polyacrylamide gel?electrophoresis suggested that ε-polylysine could inhibit bacterial protein synthesis or result in the degradation of proteins, while brevilaterin had no effect on protein synthesis. Agarose gel retardation electrophoresis showed that brevilaterin had no effect on bacterial DNA, and ε-polylysine and its combination with brevilaterin could lead to the retention of DNA, indicating that ε-polylysine could inhibit bacteria by binding with DNA. Therefore, the combination of brevilaterin and ε-polylysine could synergistically disrupt the membrane integrity, and exert a synergistic antibacterial effect through inhibition of respiratory chain dehydrogenase activity by brevilaterin, and destruction of transmembrane pH gradient by ε-polylysine as well as its binding with DNA.

Key words: antimicrobial peptide brevilaterin, ε-polylysine, synergistic antibacterial mechanism, Staphylococcus aureus