通过测定冬凌草甲素对嗜水气单胞菌的最低抑菌浓度(minimum inhibitory concentration, MIC)、最低杀菌浓度(minimum bactericidal concentration, MBC)和对细菌生长的影响，及其对菌体形态、电导率、乳酸脱氢酶(lactate dehydrogenase, LDH)活性、蛋白质和DNA的影响，探究冬凌草甲素对耐药性嗜水气单胞菌的体外抑菌作用及其机制。结果显示，冬凌草甲素对嗜水气单胞菌具有明显抑制作用，MIC为256 μg/mL，MBC为512 μg/mL。当2MIC浓度的冬凌草甲素作用于嗜水气单胞菌CW株8、16 h后，菌体变形，细胞壁和细胞膜分离，细胞质空化。与对照组相比，药物组菌悬液的电导率显著升高，并且在冬凌草甲素作用6 h后达到5.66%。冬凌草甲素作用8 h后嗜水气单胞菌的LDH活性降低了20.8%，可溶性蛋白含量显著降低。DAPI染色结果显示，药物组荧光密度和强度均较对照组弱。DNA外渗量结果显示，较对照组上升了(29.32±1.02) mg/L。研究表明，冬凌草甲素对嗜水气单胞菌具有较强的抗菌作用，主要通过增加细胞膜的通透性，干扰蛋白质代谢进而抑制细菌的生长繁殖。

Aeromonas hydrophila is known as a common pathogen of human, animal and fish worldwide, which is of great significance to aquaculture and public health. Currently, antibiotics as a kind of high effective and widely used medicine are used in the prevention, control and treatment of A. hydrophlia infections. However, abuse of antibiotics caused the problem of drug-resistant strains. New sustainable drugs to control bacterial infection needed to be studied. In recent years, the active ingredients extracted from traditional Chinese herbal medicine have excellent antibacterial effects in vitro, not easy to develop drug resistance and being able to reverse the properties of bacterial resistance. And these characteristics merit attention. The objective of this study is to analyze the potential inhibitory mechanism of oridonin against A. hydrophila, to provide a theoretical basis for the control of drug-resistant A. hydrophila infection using oridonin as an alternative drug. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and the effect of growth curve were measured in this study. Morphological changes in A. hydrophila following oridonin treatment were determined with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The effects of lactate dehydrogenase (LDH) and soluble proteins were detected by lactic dehydrogenase kits and polyacrylamide gel electrophoresis (SDS-PAGE). Electrical conductivity was analysed to study oridonin effects on cell membrane. Effect of oridonin on A. hydrophila cell membrane was measured based on electrical conductivity. Oridonin had definite inhibitory activity on A. hydrophila, and the MIC and MBC values of oridonin were 256 μg/mL and 512 μg/mL, respectively. After treating A. hydrophila (CW) with 2 MIC oridonin, SEM images showed the surface roughness and collapse of the bacteria, and TEM images showed that the structure of the bacteria was damaged, the cell membrane and cell wall were separated, and the cytoplasm showed vacuolation, while the control group showed no significant changes. 6 h later, the conductivity level of oridonin-treated A. hydrophila CW reached by 5.66% (P<0.05), indicating that oridonin changed cell membrane and wall permeability of CW strain. The results of content of LDH showed that oridonin reduced the formation of LDH by 20.8% (P<0.05). SDS-PAGE results show that the soluble protein content was lower compared with the control group, indicating that oridonin inhibited the protein metabolism of bacteria. It was shown that density and intensity of fluorescence decreased and DNA exosmosis level improved by (29.32±1.02) mg/L(P<0.01). Thus, the decrease of DNA content caused by oridonin affected the cell membrane and cell wall. The results demonstrated that oridonin could inhibit the growth of A. hydrophila significantly by increasing the permeability of cell membrane and affecting the metabolism of protein.

S941.42

农业农村部渔用药物创制重点实验室开放基金(201702)；四川省科技厅应用基础项目(21YYJC2011)