盐酸小檗碱对耐碳青霉烯类鲍曼不动杆菌生物膜的作用

Effects of berberine hydrochloride on biofilm of carbapenem resistant Acinetobacter baumannii

目的探究盐酸小檗碱对耐碳青霉烯类鲍曼不动杆菌(CRAB)生物膜形成能力的影响。方法收集首都医科大学附属北京友谊医院2021年6—12月临床分离的14株CRAB,采用微量肉汤稀释法测定盐酸小檗碱对CRAB的最低抑菌浓度(MIC),用MTT染色法检测CRAB生物膜形成能力以及不同浓度盐酸小檗碱对生物膜形成的影响。选取生物膜形成能力强的菌株ab216进行中心静脉导管体外生物膜培养并采用扫描电镜分析。采用实时荧光定量PCR法检测用药前后生物膜相关基因abaI、bap、ompA以及外排泵基因adeB、adeG、adeJ表达情况。结果盐酸小檗碱对14株CRAB的MIC均大于512μg/mL;11株(78.6%)CRAB形成生物膜;盐酸小檗碱能抑制CRAB生物膜的形成,并随着药物浓度升高,生物膜的形成显著减少,盐酸小檗碱浓度在256μg/mL(t=2.819,P=0.006)、512μg/mL(t=9.202,P=0.000)、1024μg/mL(t=18.805,P=0.000)可显著降低CRAB生物膜的形成;扫描电镜结果表明,中心静脉导管体外培养72 h形成成熟的生物膜,导管生物膜在盐酸小檗碱作用下细胞之间的交联断裂,网状结构消失;PCR结果显示,外排泵基因adeG表达显著下调,差异有统计学意义(菌株A3:t=5.86,P=0.03;菌株B2:t=6.76,P=0.02;菌株C3:t=13.67,P=0.01;菌株ab216:t=9.21,P=0.02)。结论盐酸小檗碱对CRAB的生物膜形成有抑制作用,并显著下调外排泵基因adeG的表达,为CRAB形成的生物膜的治疗提供新策略。

Objective To investigate the effects of berberine hydrochloride on biofilm formation ability of carbapenem-resistant Acinetobacter baumannii(CRAB).Methods A total of 14 strains of carbapenem-resistant Acinetobacter baumannii were collected from Beijing Friendship Hospital from June 2021 to December 2021.The broth microdilution method was used to detect the minimum inhibitory concentration(MIC)of berberine hydrochloride for CRAB in vitro.The biofilm-forming ability of the 14 isolated CRAB and the effects of different concentrations of berberine hydrochloride on the biofilm formation were determined by MTT dyeing method.The strain ab216 with strong biofilm-forming ability was selected for biofilm culture of central venous catheter in vitro and the formed biofilm was analyzed by scanning electron microscope(SEM).Real-time fluorescent quantitative PCR was used to detect the expressions of biofilm related genes,i.e.,abaI,bap,ompA,and efflux pump genes,i.e.,adeB,adeG and adeJ before and after medication.Results The MICs of berberine hydrochloride on the 14 CRAB strains were all above 512μg/mL.Among them,11 strains(78.6%)formed biofilm.Berberine hydrochloride could inhibit the formation of CRAB biofilm.The biofilm formation significantly decreased with increase of drug concentration.Berberine hydrochloride at the concentrations of 256μg/mL(t=2.819,P=0.006),512μg/mL(t=9.202,P=0.000)and 1024μg/mL(t=18.805,P=0.000)significantly reduced the formation of CRAB biofilm.The results of SEM showed that the central venous catheter formed mature biofilm after 72 hours of incubation in vitro.The cross-links between the cells of catheter biofilm were broken and the reticular structure disappeared under the treatment of berberine hydrochloride.PCR results showed that the expressions of efflux pump gene adeG were significantly down-regulated(strain A3:t=5.86,P=0.03;strain B2:t=6.76,P=0.02;strain C3:t=13.67,P=0.01;strain ab216:t=9.21,P=0.02)with statistically significant differences.Conclusion Berberine hydrochloride can inhibit the biofilm formation of CRAB and significantly downregulate the expression of efflux pump gene adeG.Berberine hydrochloride may provide a new therapeutic strategy for the CRAB-formed biofilm.