黄藤素对动物源耐药沙门氏菌抑菌作用的初步研究

A preliminary study on the bacteriostatic actions of palmatine on animal-derived resistant Salmonella

初步研究黄藤素对动物源耐药沙门氏菌的抑菌作用。用微量肉汤稀释法检测黄藤素和12种抗生素对耐药沙门氏菌的最小抑菌浓度(Minimal inhibitory concentration,MIC);用微量棋盘法测定黄藤素联合抗生素使用对耐药沙门氏菌的抑菌效果;用PCR法检测13个耐药基因;用细菌浓度OD值的变化和透射电镜检测黄藤素对耐药沙门氏菌生长曲线和形态的影响;通过碱性磷酸酶含量、电导率、可溶性蛋白浓度、胞内蛋白浓度、acrA表达等的变化来研究黄藤素对耐药沙门氏菌的抑菌作用。结果表明,黄藤素对编号为8、10、15的3株沙门氏菌的MIC分别为64μg/mL、256μg/mL和128μg/mL。黄藤素与恩诺沙星等7种抗生素联用表现为协同作用或相加作用,黄藤素与多粘菌素B等4种抗生素联用表现为拮抗作用。3株沙门氏菌均携带了多种耐药基因。0.5 MIC(32μg/mL)和1 MIC(64μg/mL)的黄藤素能抑制沙门氏菌的生长,使细菌细胞壁变薄,空腔增多,使碱性磷酸酶增多,电导率变大,可溶性蛋白与胞内蛋白含量均增加,降低acrA的表达。结果提示,黄藤素引起耐药菌形态、蛋白表达和外排泵等的变化。

To study the bacteriostatic actions of palmatine on animal-derived resistant Salmonella,MICs of palmatine and 12 antibiotics were tested by the broth dilution method,and the bacteriostatic actions of palmatine combining with antibiotics were determined by micro-checkerboard method.13 Resistant genes were detected by PCR.The effect of palmatine on the growth and morphology of Salmonella was detected by the changes of OD value of bacterial concentration and transmission electron microscopy.The bacteriostatic actions of palmatine on resistant Salmonella were studied by alkaline phosphatase content,electrical conductivity,soluble protein concentration,intracellular protein concentration and acrA expression.The results showed that MICs of palmatine to 3 Salmonella strains which were named 8,10 and 15 were 64μg/mL,256μg/mL and 128μg/mL respectively.Combinations of palmatine with seven antibiotics such as enoxacin showed synergistic additive effects,and antagonistic effects while when combined with 4 antibiotics such as polymyxin B.All three strains carried multiple resistance genes.The different concentrations of palmatine at 0.5 MIC and 1 MIC(32μg/mL and 64μg/mL)could lead to changes in Salmonella such as inhibiting growth,making the bacterial cell wall thin,increasing the number of cavities,the content of the alkaline phosphatase,the electrical conductivity and the soluble protein and intracellular protein content,and reducing the expression of acrA.The results demonstrated that palmatine could change the morphology,protein expression and efflux pump of the resistant bacteria.