中药成分对多药耐药肺炎克雷伯菌的抑菌及耐药逆转作用的体外研究

In vitro study on the antibacterial and resistance-reversing effects of traditional Chinese medicine components against multidrug-resistant Klebsiella pneumoniae

目的研究没食子酸、绿原酸、小檗碱3种中药成分对多药耐药肺炎克雷伯菌(MDR-KP)的抑菌及耐药逆转作用。方法搜集2021年7月—2022年7月,院内分离培养鉴定的23株MDR-KP菌株制备成菌悬液,将没食子酸、绿原酸、小檗碱制备成初始浓度64.0 mg/ml药液,然后用倍比稀释法判断其对各菌株的抑菌效果;采用纸片法(K-B)检测各中药成分对头孢吡肟、氨曲南、哌拉西林、环丙沙星、亚胺培南、左氧氟沙星、美罗培南7种抗菌素均耐药的MDR-KP菌株及原始菌株(阴性对照)的最小抑菌浓度(MIC),计算两者抑菌圈直径。抑菌圈直径为正态分布的计量资料,组间比较采用t检验;各中药成分对MDR-KP耐药逆转效果评价,根据抑菌圈直径与阴性对照抑菌圈直径的差值,差值≥3 mm为具有逆转作用。结果3种中药成分对MDR-KP均有抑菌效果,其中没食子酸对MDR-KP抑菌效果最强,抑菌浓度为1 mg/ml;其次为小檗碱,绿原酸最弱。没食子酸、绿原酸、小檗碱对MDR-KP的逆转实验结果显示:小檗碱对耐头孢吡肟MDR-KP菌株及绿原酸对耐氨曲南、哌拉西林、亚胺培南、左氧氟沙星株、美罗培南的MDR-KP菌株作用后,与阴性对照药物抑菌圈直径比较,差异均无统计学意义(P均>0.05);没食子酸对所有MDR-KP菌株、绿原酸对耐头孢吡肟、环丙沙星MDR-KP菌株,小檗碱对耐氨曲南、哌拉西林、环丙沙星、亚胺培南、左氧氟沙星、美罗培南MDR-KP菌株作用后,与阴性对照药物抑菌圈直径比较,差异均具有统计学意义(P均<0.05),但与阴性对照组抑菌圈直径的差值均<3 mm。结论没食子酸、绿原酸、小檗碱均对MDR-KP具有抑菌作用;但对MDR-KP未达到耐药逆转效果。

Objective To study the antibacterial and resistance-reversing effects of three traditional Chinese medicine components,namely gallic acid,chlorogenic acid,and berberine,against multidrug-resistant Klebsiella pneumoniae(MDR-KP).Methods July 2021 to July 2022,a total of 23 MDR-KP strains isolated and identified in the hospital were prepared as bacterial suspension.Gallic acid,chlorogenic acid,and berberine were prepared as initial concentration of 64.0 mg/ml solutions,and their antibacterial effects on each strain were determined using the serial dilution method.The minimum inhibitory concentration(MIC)of 7 antibiotics,namely cefpirome,amikacin,piperacillin,ciprofloxacin,imipenem,levofloxacin,and meropenem,aginst MDR-KP strains and the original strain(negative control),which were all resistant to all 7 antibiotics,were measured using the paper disk method(K-B).The difference in the diameter of the inhibition zone between the test compounds and negative control was calculated.The diameter of the inhibition zone was normally distributed,and intergroup comparisons were made using the t test.The reversal effect of each Chinese medicine component(TCM)components on MDR-KP resisitance was evaluated based on the the difference in the inhibition zone diameter compared to the negative control,with a difference of≥3 mm indicating a reversal effect.Results All three traditional Chinese medicine components exhibited antibacterial effects against MDR-KP,with gallic acid showing the strongest inhibitory effect at a concentration of 1mg/ml,followed by berberine,and chlorogenic acid being the weakest.The results of the reversal experiments showed that the differences in inhibition zone diameter between berberine and resistant strains of MDR-KP to cefpirome,and between chlorogenic acid and resistant strains to amikacin,piperacillin,imipenem,levofloxacin,and meropenem,were not statistically significant(all P>0.05).However,the differences in inhibition zone diameter between gallic acid and all MDR-KP strains,between chlorogenic acid and resistant strains to cefpirome and ciprofloxacin,and between berberine and resistant strains to amikacin,piperacillin,ciprofloxacin,imipenem,levofloxacin,and meropenem,were statistically significant(all P<0.05).Nevertheless,the differences were<3 mm when compared to the inhibition zone diameter of the negative control group.Conclusion Gallic acid,chlorogenic acid,and berberine all have antibacterial effects against MDR-KP,but they did not achieve resistance-reversing effects on MDR-KP.