百里香酚对鱼源耐药性维氏气单胞菌的体外抑菌效果及其机制研究

为探究百里香酚对鱼源耐药性维氏气单胞菌(Aeromonas veronii)的抑菌效果和作用机制,从患病黄颡鱼(Pelteobagrus fulvidraco)体内分离得到一株维氏气单胞菌,药敏试验结果显示,其对氟苯尼考、环丙沙星和恩诺沙星耐药。通过测定最低抑菌浓度(MIC)、最低杀菌浓度(MBC)和生长曲线来评价百里香酚对耐药性维氏气单胞菌的抑菌活性;通过测定百里香酚对其细胞膜通透性、可溶性蛋白、乳酸脱氢酶活性和DNA的影响,结合电镜观察细菌超微结构的变化研究其作用机制。结果显示,百里香酚对耐药性维氏气单胞菌的抑菌效果明显,MIC为256 μg·mL^-1,MBC为512 μg·mL^-1。经512 μg·mL^-1的百里香酚作用1 h后,维氏气单胞菌液电导率极显著(P<0.01)上升,DNA外渗量迅速上升至(115.6±0.5)mg·L^-1。经512 μg·mL^-1的百里香酚作用后,维氏气单胞菌的可溶性蛋白明显变少,细菌乳酸脱氢酶活性在2、4、6、8 h分别极显著(P<0.01)降低了(32.8±0.7)%、(46.2±0.3)%、(46.1±1.6)%、(60.0±1.0)%,DAPI(4',6-二脒基-2-苯基吲哚)染色的荧光强度和密度降低,电镜下可见菌体表面溶解塌陷,皱缩变形,细胞壁和细胞膜分离,细胞质丢失,内部空化。上述结果表明,百里香酚对维氏气单胞菌具有较强的抗菌活性,主要通过增加细胞膜通透性导致胞内物质流失造成细菌死亡。百里香酚可以作为治疗耐药性维氏气单胞菌感染的备选药物进行研究。

Antibacterial effect of thymol against fish-derived drug-resistant Aeromonas veronii in vitro and its mechanism

In order to investigate the antibacterial effect and mechanism of thymol against fish-derived drug-resistant Aeromonas veroniae, a strain of A. veronii was isolated from diseased Pelteobagrus fulvidraco. Drug sensitivity test showed that this strain was resistant to florfenicol, ciprofloxacin and enrofloxacin. The antibacterial activity of thymol against drug-resistant A. veronii was studied by determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and growth curve, and the mechanism was explored by measuring its influence on membrane permeability, soluble protein, lactate dehydrogenase activity, DNA and cell ultrastructural changes. The results showed that thymol had inhibitory effect on A. veronii with MIC of 256 μg·mL^-1 and MBC of 512 μg·mL^-1, respectively. After treatment with 512 μg·mL^-1 thymol for 1 h, the electrical conductivity in strain solution was significantly (P<0.01) increased, and the DNA content in strain solution was increased to (115.6±0.5) mg·L^-1. After treatment with 512 μg·mL^-1 thymol, the soluble protein of this strain decreased, the activity of lactate dehydrogenase significantly (P<0.01) decreased by (32.8±0.7)%, (46.2±0.3)%, (46.1±1.6)%, (60.0±1.0)% at 2, 4, 6, 8 h, respectively, the fluorescence intensity and density of DAPI (4',6-diamidino-2-phenylindole) staining were also decreased, and the surface of this strain seemed to dissolve and collapse, shrink and deform, cell wall and membrane separated, with cytoplasm loss and internal cavitation under electron microscope. All these results suggested that thymol had strong antibacterial activity against A. veronii. The increase of membrane permeability with thymol treatment would cause the loss of intracellular substances, and result in bacterial death. Therefore, thymol could be used as a candidate for the treatment of antibiotics-resistant A. veronii infection.