黄连解毒汤联合阿奇霉素对铜绿假单胞菌的体外抑菌效果

In Vitro Antibacterial Effect of Huanglian Jiedu Decoction Combined with Azithromycin on Pseudomonas Aeruginosa

目的探讨黄连解毒汤联合阿奇霉素对铜绿假单胞菌的体外抑菌效果。方法以铜绿假单胞菌标准质控菌株ATCC27853及开封市第二中医院临床分离的98株铜绿假单胞菌(包括非黏液型32例、黏液型66例)为研究对象,采用肉汤稀释法测量黄连解毒汤、阿奇霉素的最小抑菌浓度(MIC),并以棋盘法作联合测试,通过计算部分抑菌浓度指数(FICI)明确相互作用关系,应用结晶紫染色法评估生物膜抑制的效果,再采用实时荧光定量PCR测定相关密度感应基因的表达。结果铜绿假单胞菌标准质控菌株ATCC 27853、临床黏液型及非黏液型经阿奇霉素、黄连解毒汤单一及联合处理后,整体MIC值呈下降趋势,且FICI值以0~1之间居多;铜绿假单胞菌标准质控菌株ATCC 27853、临床黏液型及非黏液型经阿奇霉素、黄连解毒汤单一及联合处理后,经F分析显示,4组OD值比较,差异有统计学意义(P<0.05),且阿奇霉素组、黄连解毒汤组及联合组OD值均低于空白对照组,不同处理组别内铜绿假单胞菌非黏液型OD值均高于黏液型(P<0.05);铜绿假单胞菌ATCC 27853临床黏液型及非黏液型于阿奇霉素、黄连解毒汤在抑制浓度下单一及联合处理下,LasB基因的mRNA相对表达水平随浓度升高呈下降趋势,但LasR基因、rhIR基因的mRNA相对表达水平不随浓度升高呈线性变化。结论黄连解毒汤可通过抑制生物膜生长用以增强铜绿假单胞菌体外活性,与阿奇霉素发挥协同或相加作用,且非黏液型强铜绿假单胞菌耐药性高于黏液型。此外,黄连解毒汤联合阿奇霉素可能对铜绿假单胞菌密度感应基因表达产生影响。

Objective To investigate the in vitro antibacterial effect of Huanglian Jiedu decoction combined with azithromycin on Pseudomonas aeruginosa.Methods The standard quality control strain ATCC27853 of Pseudomonas aeruginosa and 98 clinical isolates of Pseudomonas aeruginosa(including 32 cases of non-mucus type and 66 cases of mucus type)were used as the research objects.The minimum inhibitory concentration(MIC)was tested in combination with the checkerboard method.The interaction relationship was clarified by calculating the fractionalal inhibitory concentration index(FICI).Crystal violet staining was used to evaluate the biofilm inhibitory effect to measure the relative density-sensing gene expression.Results After single or combined treatment with azithromycin and Huanglian Jiedu decoction,the standard quality control strain ATCC 27853 of Pseudomonas aeruginosa,clinical mucinous and non mucinous types,showed a decreasing trend in overall MIC values,with FICI values mostly ranging from 0 to 1.The standard quality control strain ATCC 27853 of Pseudomonas aeruginosa,clinical mucinous and non mucinous types,were treated with azithromycin and Huanglian Jiedu decoction alone or in combination.F-analysis showed that there was a statistically significant difference in OD values among the four groups(P<0.05),and the OD values of the azithromycin group,Huanglian Jiedu decoction group,and combination group were lower than the blank control group.The OD values of non mucinous Pseudomonas aeruginosa in different treatment groups were higher than those of mucinous type(P<0.05).Pseudomonas aeruginosa ATCC 27853 clinical mucinous and non mucinous types were treated with azithromycin and Huanglian Jiedu decoction at inhibitory concentrations.The relative mRNA expression levels of LasB gene decreased with increasing concentration,but the relative mRNA expression levels of LasR gene and rhIR gene did not show linear changes with increasing concentration.Conclusion Huanglian Jiedu decoction can enhance the in vitro activity of Pseudomonas aeruginosa by inhibiting biofilm growth,and has synergistic or additive effects with azithromycin.Moreover,non mucinous strong Pseudomonas aeruginosa has higher drug resistance than mucinous Pseudomonas aeruginosa.In addition,the combination of Huanglian Jiedu decoction and azithromycin may have an impact on the expression of density sensing genes in Pseudomonas aeruginosa.