小檗碱对耐甲氧西林金黄色葡萄球菌抑菌机制的研究

The mechanism of berberine on Methicillin resistant Staphylococcus aureus in vitro

目的探讨中药单体小檗碱对耐甲氧西林金黄色葡萄球菌(MRSA)细胞膜完整性、通透性的影响以及细菌细胞壁结构的改变,为中药单体成分小檗碱在抗菌中的临床应用奠定基础。方法本研究采用非随机同期对照试验。收集上海市第八人民医院检验科2019—2020年老年科患者下呼吸道样本分离培养的MRSA菌株3株。采用梅里埃VETEK MS全自动快速微生物质谱检测系统及VETEK 2 Compact全自动微生物鉴定仪鉴定细菌药敏实验检测细菌菌种及药敏;采用微量肉汤稀释法测定小檗碱对MRSA的最低抑菌浓度(MIC);采用培养基电导率实验观察不同浓度小檗碱对MRSA细胞膜通透性的改变;透射电子显微镜观察不同浓度小檗碱作用后MRSA细胞壁结构的变化。通过GraphPad Prism5统计软件,对电导率实验结果进行差异性分析。结果小檗碱对MRSA的MIC为64μg/ml;8μg/ml(1/8 MIC)、16μg/ml(1/4 MIC)、32μg/ml(1/2 MIC)、64μg/ml(1 MIC)、128μg/ml(2 MIC)小檗碱作用菌体4 h后,电导率分别增加了24.49%、34.59%、208.92%、196.40%及208.68%。透射电子显微镜观察发现低浓度(8μg/ml;1/8 MIC)小檗碱对细菌无明显破坏作用,MRSA菌体结构完整;中浓度(64μg/ml,1 MIC)小檗碱作用于MRSA后发现MRSA的细胞壁变薄;高浓度(512μg/ml;8 MIC)小檗碱诱导MRSA的细胞壁结构大量破坏溶解,菌体内容物大量外泄,导致细菌裂解死亡。结论小檗碱通过改变MRSA细胞膜通透性及破坏和溶解细胞壁的结构,达到杀灭细菌的作用。

Objective To explore the impact of traditional Chinese medicine berberine(BBR)on membrane integrity and permeability of Methicillin-resistant Staphylococcus aureus(MRSA)and the change of bacterial cell wall structure,laying a foundation for the clinical application of berberine in antibacterial.Methods This study used a non-randomized concurrent controlled trial.The 3 MRSA strains were isolated and cultured from lower respiratory tract samples of geriatric patients from Shanghai Eighth People's Hospital between 2019 and 2020.The Meirier VETEK MS fully automated rapid microbial mass spectrometry detection system and VETEK 2 Compact fully automated microbial identification instrument were used to identify bacterial drug sensitivity experiments to detect bacterial species and drug sensitivity.The minimal inhibitory concentration(MIC)of BBR on MRSA strains was determined by broth microdilution.This study used conductivity tests to assess the changes in membrane permeability in response to different concentration of BBR on MRSA,while also investigating the changes in MRSA morphology by transmission electron microscopy.GraphPad Prism5 was used to analyze the differences in the electrical conductivity experimental results.Results The MIC of BBR on MRSA was 64μg/ml.After co-culturing MRSA with BBR for 4 h at 8μg/ml,16μg/ml,32μg/ml,64μg/ml and 128μg/ml,respectively,the electrical conductivity increased,compared with the control group,by 24.49%,34.59%,208.92%,196.40% and 208.68%,respectively.By transmission electron microscopy,This study found that low concentration of BBR(8μg/ml,1/8 MIC)caused no significant damage to MRSA,and the bacterial structure of MRSA remained intact.The cell wall of MRSA became thinner after treatment with berberine at medium concentration(64μg/ml,1 MIC),while high concentration of BBR(512μg/ml,8 MIC)induced the destruction and dissolution of MRSA cell wall structure and the leakage of bacterial contents,leading to bacterial lysis.Conclusion Berberine can kill bacteria by altering the permeability of MRSA cell membrane and destroying and dissolving the structure of the cell wall.