痰热清注射液与亚胺培南西司他丁联合对广泛耐药铜绿假单胞菌的抑菌作用
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摘要
目的:观察痰热清注射液(痰热清)与亚胺培南西司他丁联合对广泛耐药铜绿假单胞菌的抑菌作用,并研究其作用机制,为临床治疗耐药菌提供参考。方法:将医院临床分离到广泛耐药铜绿假单胞菌采用微量稀释法检测痰热清与亚胺培南西司他丁最小抑菌浓度(MIC);棋盘法检测联合用药抑菌效果;微孔板法检测对成熟生物膜内细菌代谢活性的影响;Bio Flux动态观察药物对生物膜的影响并用激光共聚焦扫描显微镜观察活死细菌的分布;扫描电子显微镜(SEM)观察药物作用后细菌的形态变化。结果:亚胺培南西司他丁和痰热清的MIC分别为512 mg·L~(-1)和>16 500 mg·L~(-1)。棋盘法结果显示痰热清可增强亚胺培南西司他丁的敏感性,二者协同抑菌,并确定联合用药浓度。与空白组或单用亚胺培南西司他丁组比较,联合用药各组显著减少生物膜内活菌量(P<0.05,P<0.01)。Bio Flux结果表明与空白组或单用药物组比较,联合用药各组可破坏生物膜结构,减小生物膜面积(P<0.05);荧光染色结果显示联合用药对动态生物膜内细菌的代谢活性有明显抑制。SEM显示痰热清可抑制细菌分裂。结论:痰热清与亚胺培南西司他丁联合对浮游和生物膜状态的细菌生长有抑制作用,还可对生物膜产生破坏作用。
Objective:To observe the effect of combination of Tanreqing injection(Tanreqing)and imipenem-cilastatin on extensively-drug resistant Pseudomonas aeruginosa(XDPA),and study the mechanism of the combination.Method:The minimum inhibitory concentrations(MICs)of Tanreqing and imipenem-cilastatin against planktonic XDPA strain isolated in clinic were determined by the broth microdilution method.The checkerboard method was used to evaluate the combination effect.The bacterial metabolic activity in mature biofilm was studied by microtiter-plate test.The destructive effect of combination drugs on dynamic biofilm was observed by using BioFlux system,and viable cells were examined by confocal laser scanning microscope(CLSM)after treatment.The scanning electron microscopy(SEM)was used for observing Pseudomonas aeruginosa and length measurement.Result:The MIC values of imipenem-cilastatin and Tanreqing were 512 mg·L~(-1)and more than16 500 mg·L~(-1).The checkerboard analysis showed that Tanreqing could enhance the sensitivity of imipenemcilastatin,while the combination drugs synergistically inhibited the growth of bacteria.Compared with the control group or the imipenem-cilastatin individual group,the combined drugs significantly reduced the amount of living bacteria in the biofilm(P<0.05,P<0.01).BioFlux results showed that the combination drugs destructed the biofilm structure and reduced the area coverage(P<0.05)by comparing with the control group or the single drug group.The results of fluorescent staining showed that the combination drug significantly inhibited the metabolic activity of bacteria in dynamic biofilm.Tanreqing inhibited bacterial division to achieve the antibacterial effect.Conclusion:Tanreqing and imipenem-cilastatin synergistically inhibit the bacterial growth in planktonic and biofilm states,and destruct biofilms.
Objective:To observe the effect of combination of Tanreqing injection(Tanreqing)and imipenem-cilastatin on extensively-drug resistant Pseudomonas aeruginosa(XDPA),and study the mechanism of the combination.Method:The minimum inhibitory concentrations(MICs)of Tanreqing and imipenem-cilastatin against planktonic XDPA strain isolated in clinic were determined by the broth microdilution method.The checkerboard method was used to evaluate the combination effect.The bacterial metabolic activity in mature biofilm was studied by microtiter-plate test.The destructive effect of combination drugs on dynamic biofilm was observed by using BioFlux system,and viable cells were examined by confocal laser scanning microscope(CLSM)after treatment.The scanning electron microscopy(SEM)was used for observing Pseudomonas aeruginosa and length measurement.Result:The MIC values of imipenem-cilastatin and Tanreqing were 512 mg·L~(-1)and more than16 500 mg·L~(-1).The checkerboard analysis showed that Tanreqing could enhance the sensitivity of imipenemcilastatin,while the combination drugs synergistically inhibited the growth of bacteria.Compared with the control group or the imipenem-cilastatin individual group,the combined drugs significantly reduced the amount of living bacteria in the biofilm(P<0.05,P<0.01).BioFlux results showed that the combination drugs destructed the biofilm structure and reduced the area coverage(P<0.05)by comparing with the control group or the single drug group.The results of fluorescent staining showed that the combination drug significantly inhibited the metabolic activity of bacteria in dynamic biofilm.Tanreqing inhibited bacterial division to achieve the antibacterial effect.Conclusion:Tanreqing and imipenem-cilastatin synergistically inhibit the bacterial growth in planktonic and biofilm states,and destruct biofilms.
引文
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[14]任英莉,孟宪卿,王珊珊,等.耐药铜绿假单胞菌肺炎中医证型演变规律研究[J].内蒙古中医药,2016,35(12):5-6.
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[16]宋志香,李刘坤,李兴广.中药痰热清注射液治疗MRSA感染性肺炎的临床观察研究[J].中华医院感染学杂志,2010,20(11):1596-1598.
[17]龙爽,刘绍勇,徐英.痰热清注射液及5种中间体对四氯化碳致急性肝损伤大鼠的保护作用探讨[J].中国实验方剂学杂志,2018,24(11):73-80.
[18]刘珏玲,杨伟峰,孙娅楠等.痰热清注射液对耐甲氧西林金黄色葡萄球菌生物膜三维结构的影响[J].中国病原生物学杂志,2017,12(12):1152-1156.
[19]Seth A K,Geringer M R,Hong S J,et al. In vivo modeling of biofilm-infected wounds:a review[J]. J Surg Res,2012,178(1):330-338.
[20]黄梅,谭余庆,罗俊,等.植物类中药抗细菌耐药性的研究进展[J].中国实验方剂学杂志,2018,24(23):218-224.
[21]周至品,杨兴秀,叶晓雪.传统药物对铜绿假单胞菌生物被膜作用研究进展[J].中药药理与临床,2015,31(6):230-235.
[2]Lebeaux D, Ghigo J M, Beloin C. Biofilm-related infections:bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics[J]. Microbiol Mol Biol Rev,2014,78(3):510-543.
[3]袁莉莉,丁百兴,沈震,等.碳青霉烯类抗生素耐药铜绿假单胞菌感染的临床研究[J].中国感染与化疗杂志,2017,17(2):121-126.
[4]施卫荣,周建英.痰热清注射液联合亚胺培南治疗高龄耐药性铜绿假单胞菌肺部感染患者的临床疗效[J].实用心脑肺血管病杂志,2017,25(10):103-106.
[5]孙广信,常瑞,李珊,等.痰热清注射液治疗下呼吸道多药耐药铜绿假单胞菌感染临床研究[J].中医学报,2012,27(10):1348-1349.
[6]WANG Y,WANG T,HU J J,et al. Anti-biofilm activity of TanRe Qing,a traditional Chinese medicine used for the treatment of acute pneumonia[J]. J Ethnopharmacol,2011,134(1):165-170.
[7]YANG W F,LIU J L,Bla6ekovic'B,et al. In vitro antibacterial effects of Tanreqing injection combined with vancomycin or linezolid against methicillin-resistant Staphylococcus aureus[J]. BMC Complement Altern Med,2018,18(1):169.
[8]ZHU W,ZHANG Y H,Sinko W,et al. Antibacterial drug leads targeting isoprenoid biosynthesis[J]. Proc Natl Acad Sci USA,2013,110(1):123-128.
[9]Benoit M R,Conant C G,Ionescu-Zanetti C,et al.New device for high-throughput viability screening of flow biofilms[J]. Appl Environ Microbiol,2010,76(13):4136-4142.
[10]雷洪涛,刘敏彦,欧阳竞锋,等.连花清瘟胶囊抗金黄色葡萄球菌生物膜研究[J].中国实验方剂学杂志,2013,19(22):161-164.
[11]Kalil A C,Metersky M L,Klompas M,et al. Executive summary:management of adults with hospital-acquired and ventilator-associated pneumonia:2016 clinical practice guidelines by the infectious diseases society of America and the American thoracic society[J]. Clin Infect Dis,2016,63(5):575-582.
[12]张祎博,孙景勇,倪语星,等. 2005—2014年CHINET铜绿假单胞菌耐药性监测[J].中国感染与化疗杂志,2016,16(2):141-145.
[13]胡付品,郭燕,朱德妹,等. 2017年CHINET中国细菌耐药性监测[J].中国感染与化疗杂志,2018,18(3):241-251.
[14]任英莉,孟宪卿,王珊珊,等.耐药铜绿假单胞菌肺炎中医证型演变规律研究[J].内蒙古中医药,2016,35(12):5-6.
[15]徐丽丽.难治性风温肺热病中医证候学规律研究[D].北京:北京中医药大学,2013.
[16]宋志香,李刘坤,李兴广.中药痰热清注射液治疗MRSA感染性肺炎的临床观察研究[J].中华医院感染学杂志,2010,20(11):1596-1598.
[17]龙爽,刘绍勇,徐英.痰热清注射液及5种中间体对四氯化碳致急性肝损伤大鼠的保护作用探讨[J].中国实验方剂学杂志,2018,24(11):73-80.
[18]刘珏玲,杨伟峰,孙娅楠等.痰热清注射液对耐甲氧西林金黄色葡萄球菌生物膜三维结构的影响[J].中国病原生物学杂志,2017,12(12):1152-1156.
[19]Seth A K,Geringer M R,Hong S J,et al. In vivo modeling of biofilm-infected wounds:a review[J]. J Surg Res,2012,178(1):330-338.
[20]黄梅,谭余庆,罗俊,等.植物类中药抗细菌耐药性的研究进展[J].中国实验方剂学杂志,2018,24(23):218-224.
[21]周至品,杨兴秀,叶晓雪.传统药物对铜绿假单胞菌生物被膜作用研究进展[J].中药药理与临床,2015,31(6):230-235.