巯乙磺酸钠联合环丙沙星对铜绿假单胞菌生物膜的作用

J4 ›› 2012, Vol. 38 ›› Issue (4): 623-627.

巯乙磺酸钠联合环丙沙星对铜绿假单胞菌生物膜的作用

陈盛¹|余加林²|罗则佳³|何念海¹|孙凤军⁴

（1.第三军医大学西南医院儿科|重庆400038；2.重庆医科大学附属儿童医院新生儿科|重庆400014；3.成都军区总医院医务部|四川成都 610083；4. 第三军医大学西南医院药学部|重庆400038）

收稿日期:2012-02-27 出版日期:2012-07-28 发布日期:2012-07-28
通讯作者: 余加林 E-mail:（Tel：023-63632756，E-mail：yujialin486@sohu.com）
作者简介:陈盛（1974-）|男|重庆市人|主治医师|讲师|在读医学博士|主要从事新生儿感染诊治的研究。
基金资助:
国家自然科学基金资助课题( 30772363)

Effects of mesna combined with ciprofloxacin on biofilm of Pseudomonas aeruginosa

CHEN Sheng¹|YU Jia-lin²|LUO Ze-jia³|HE Nian-hai¹|SUN Feng-jun⁴

（ 1.Department of Pediatric| Southwest Hospital|Third Military Medical University|Chongqing 400038|China； 2.Department of Neonatology|Children’s Hospital of Chongqing,Medical University|Chongqing 400014|China；3. Department of Medical Administration|Chengdu Military General Hospital,Chengdu 610083|China；4.Department of Pharmacy| Southwest Hospital|Third Military Medical University|Chongqing 400038|China）

Received:2012-02-27 Online:2012-07-28 Published:2012-07-28

摘要/Abstract

摘要：

的：探讨巯乙磺酸钠（mesna）对成熟铜绿假单胞菌生物膜(BF)的影响及其联合环丙沙星(CIP)对铜绿假单胞菌BF的作用。方法：微量稀释法检测 mesna和CIP对铜绿假单胞菌PAO1的最低抑菌浓度（MIC）；建立铜绿假单胞菌BF体外模型后分为对照组、低浓度mesna组及高浓度mesna组，用激光共聚焦显微镜(CLSM) 采集并结合BF图像结构分析软件(ISA) 定量分析mesna对铜绿假单胞菌BF作用后的结构参数；铜绿假单胞菌BF再次分为mesna组、CIP组及CIP联合mesna组，用CLSM采集BF图像并结合Amira 5.2三维重建软件定性分析mesna单独及联合CIP对成熟铜绿假单胞菌BF的作用；以棋盘设计，将不同浓度的mesna (0、0.5、1.0、2.0、4.0 g•L^-1)和不同浓度的CIP(浓度依次为（0、1/2、1、2、4和8 MIC）两两组合，平板计数法检测mesna单独及与CIP联用后BF内活菌数。结果： mesna对PAO1的 MIC为10 g•L^-1，CIP对PAO1的MIC为0.125 mg•L^-1； CLSM图像经ISA软件定量分析显示，与对照组比较，mesna能使BF厚度、平均扩散距离和结构熵均减少(P<0.01)，区域孔率增加(P<0.01)，高浓度组较低浓度组效应更明显(P<0.01)；CLSM图像经三维重建后可见，单用mesna（2 g•L^-1）， BF内细菌密集层叠，厚度较厚，可见大量活菌,未见明显死菌;单用CIP（2MIC），BF内仍可见大量活菌，死菌数少; mesna（2 g•L-1）联合CIP（2 MIC）组可见BF细菌稀疏、厚度薄，活菌数少，BF内可见大量死菌；单用2 g•L-1mesna能使BF中的活菌数减少(P<0.05)，单用2MIC CIP可使BF中活菌数降低(P<0.05)， 1 g•L-1 mesna与1 MIC的CIP联合应用可使BF上的活菌数明显减少(P<0.05)。结论：mesna能破坏铜绿假单胞菌BF形态结构，mesna与CIP存在协同作用，可增强其杀菌能力。

关键词: 铜绿假单胞菌；生物膜；巯乙磺酸钠；环丙沙星

Abstract:

Abstract:Objective
To investigate the effects of mesna or/ and in combination with ciprofloxacin on biofilm of Pseudomonas aeruginosa.Methods The broth microdilution method was performed to determine the minimal inhibitory concentration(MIC) of mesna and ciprofloxacin to PAO1,then a biofilm model of Pseudomonas aeruginosa in vitro was established,the biofilms were randomly divided into 3 groups: control group,low-dose mesna group and high-dose mesna group,the parameters　of biofilm structure were analyzed through pictures from CLSM with Image Structure Analyzer(ISA)software after the biofilms were treated with mesna;then the biofilms of Pseudomonas aeruginosa were randomly divided into: mesna group,CIP group,mesna+CIP group,they were treated with mesna,ciprofloxacin (alone and in combination),three dimensional images of the biofilms were obtained with Amira 5.2 software. Through a aboard design,different consistence mesna (0,0.5,1.0，2.0 and 4.0 g•L^-1)were respectively combined with different consistence ciprofloxacin （0，1/2，1，2，4，8 MIC）,the number of bacteria in biofilms was measured by agar plate.Results The MIC values against PAO1 were 10 g•L^-1 for mesna and 0.12 mg•L^-1 for ciprofloxacin.The results of ISA showed that with the treatment of low-dose mesna,the biofilms were decreased in thickness,average difusion distance(ADD)and textual entropy(TE) as compared with control group (P<0.01),however the areal porosity(AP) was increased(P<0.01),the same tendency of these parameters was observed in the high-dose group and the effects were more obvious(P<0.01); The images from CLSM showed that the biofilms in mesna (2 g•L^-1) group were intensive and thicker,the masses of alive bacteria were found in the biofilms but no dead bacteria;ciprofloxacin(2 MIC) decreased the alive bacteria,the dead bacteria were increased relatively slightly;the biofilms were thinner and more scattered,plenty of bacteria were killed in the presence of ciprofloxacin (2 MIC) and mesna (2 g•L^-1).The single use of mesna at the concentration of 2 g•L^-1 could significantly decline the viable cells in biofilms,the individual use of ciprofloxacin at 2 MIC could significantly desease the total bacterial quantity;however,when 1 g•L^-1 mesna was combined with 1 MIC ciprofloxacin,the viable counts in biofilms were less than they were given alone.Conclusion Mesna can destroy the construction of biofilm of Pseudomonas aeruginosa and has the synergistic effect on bactericidal activity with ciprofloxacin.

Key words: Pseudomonas aeruginosa；biofilm；mesna；ciprofloxacin

中图分类号:

Q939.112

陈盛|余加林|罗则佳|何念海|孙凤军. 巯乙磺酸钠联合环丙沙星对铜绿假单胞菌生物膜的作用[J]. J4, 2012, 38(4): 623-627.

CHEN Sheng|YU Jia-lin|LUO Ze-jia|HE Nian-hai|SUN Feng-jun. Effects of mesna combined with ciprofloxacin on biofilm of Pseudomonas aeruginosa[J]. J4, 2012, 38(4): 623-627.