甘草查耳酮A对貂源铜绿假单胞菌的抗菌活性

Antibacterial Activity of Licochalcone A Against Pseudomonas aeruginosa Isolated from Mink

【目的】研究甘草查耳酮A对貂源铜绿假单胞菌的抗菌活性,为临床合理使用甘草查耳酮A抗感染治疗提供依据。【方法】采用微量肉汤稀释法和平板计数法测定甘草查耳酮A对貂源铜绿假单胞菌的最小抑菌浓度(minimal inhibitory concentration,MIC)和最小杀菌浓度(minimum bactericidal concentration,MBC);平板计数法测定1MIC、2MIC、4MIC和8MIC甘草查耳酮A对铜绿假单胞菌的杀菌效果;吸光光度法测定1/16MIC、1/8MIC、1/4MIC和1/2MIC甘草查耳酮A对铜绿假单胞菌生长的影响;结晶紫染色法测定1/8MIC、1/4MIC、1/2MIC和1MIC的甘草查耳酮A对铜绿假单胞菌生物被膜的抑制效果以及1/4MIC、1/2MIC、1MIC和1MBC甘草查耳酮A对生物被膜的清除效果;建立小鼠皮肤刀伤模型和皮肤脓肿模型评价甘草查耳酮A对铜绿假单胞菌感染小鼠的治疗效果。【结果】甘草查耳酮A对貂源铜绿假单胞菌临床分离菌株具有良好的抗菌活性,MIC和MBC分别为3.125和12.5μg/mL。时间-杀菌曲线结果显示,2MIC和1MIC组在6 h内铜绿假单胞菌数量逐渐减少,之后基本维持在1×10^(5) CFU/mL左右;8MIC和4MIC组铜绿假单胞菌的数量一直处于下降趋势,并且分别在14和16 h后均无菌落形成。1/16MIC甘草查耳酮A对铜绿假单胞菌生长影响不明显,而1/8MIC、1/4MIC和1/2MIC甘草查耳酮A可抑制细菌生长。1MIC甘草查耳酮A对铜绿假单胞菌生物被膜形成的抑制率约为70%,1MBC甘草查耳酮A可清除约50%预形成的生物被膜。体内试验结果表明,甘草查耳酮A有助于铜绿假单胞菌感染小鼠刀伤的创口愈合,并减少皮肤脓肿。与对照组相比,甘草查耳酮A和氨苄西林组脓肿中的细菌数量均极显著减少(P<0.01)。【结论】甘草查耳酮A对貂源铜绿假单胞菌具有良好的体外和体内抗菌活性,可进一步用于临床治疗铜绿假单胞菌感染制剂的研发。

【Objective】The aim of this experiment was to study the antibacterial activity of licochalcone A against Pseudomonas aeruginosa isolated from mink,and to provide experimental basis for the rational use of licochalcone A on the infective treatment clinically.【Method】The minimal inhibitory concentration(MIC)and the minimum bactericidal concentration(MBC)of licochalcone A against Pseudomonas aeruginosa were determined by microbroth dilution method and plate counting method,respectively.The germicidal efficacy of 1MIC,2MIC,4MIC and 8MIC of licochalcone A against Pseudomonas aeruginosa was determined by plate counting method.The effect of 1/16MIC,1/8MIC,1/4MIC and 1/2MIC of licochalcone A on the growth of Pseudomonas aeruginosa was determined by absorbance method.The crystal violet staining was used for the determination of the biofilm inhibition with 1/8MIC,1/4MIC,1/2MIC,and 1MIC of licochalcone A and the biofilm clearance with 1/4MIC,1/2MIC,1MIC and 1MBC of licochalcone A.The model of skin wound and skin abscess were established in mice to evaluate the therapeutic effect of licochalcone A on Pseudomonas aeruginosa infected mice.【Result】Licochalcone A showed a good antibacterial activity against Pseudomonas aeruginosa from mink.The MIC and MBC values were 3.125 and 12.5μg/mL,respectively.The results of time-bactericidal curve showed that the number of Pseudomonas aeruginosa was decreased gradually in 2MIC and 1MIC groups within 6 hours,and then remained at about 1×10^(5) CFU/mL,while in 8MIC and 4MIC groups the number of Pseudomonas aeruginosa was in a downward trend,and no colonies were formed after 14 and 16 hours,respectively.Licochalcone A with 1/16MIC had little effect on the growth of Pseudomonas aeruginosa,but 1/8MIC,1/4MIC and 1/2MIC could inhibit the growth of Pseudomonas aeruginosa.The inhibitory rate of licochalcone A with 1MIC on biofilm formation of Pseudomonas aeruginosa was about 70%,and 1MBC could clear about 50%of preformed biofilm.The results of in vivo experiments showed that licochalcone A could promote wound healing and reduce the formation of skin abscess in mice infected with Pseudomonas aeruginosa.Compared with the control group,the number of bacteria in abscess in licochalcone A and ampicillin groups was extremely significantly decreased(P<0.01).【Conclusion】Licochalcone A had good antibacterial activity against Pseudomonas aeruginosa isolated from mink in vitro and in vivo,and could be further used in the development of the preparation for clinical treatment of Pseudomonas aeruginosa infection.