【作者】 何涛；

【导师】 吴聪明； 汪洋；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2015， 博士

【摘要】 抗菌药物敏感性判定标准(折点)是开展病原菌耐药性检测,确定其耐药率、耐药水平的重要技术标准,目前世界上比较权威的折点制定组织有美国临床实验室标准化研究所(Clinical and Laboratory Standards Institute, CLSI)和欧洲抗菌药物敏感性试验委员会(European Committee on Antimicrobial Susceptibility Testing, EUCAST),而我国没有设立制定折点的机构,因此参考的主要是国外的标准。国外标准对一些中国自主研制的新兽药比如乙酰甲喹没有建立敏感性折点；同时国内兽医临床的某些常用剂型,比如氟苯尼考粉剂针对大肠杆菌感染,国外的标准也没有涉及。乙酰甲喹和氟苯尼考是国内兽医临床使用较为普遍的抗菌药物,在控制动物的大肠杆菌感染性疾病发面发挥了巨大作用,而敏感性折点的缺乏严重影响了这两种药物的耐药性监测工作的开展。针对以上问题,本研究将参照国外建立折点的标准流程,系统调查国内动物源大肠杆菌对乙酰甲喹和氟苯尼考的敏感性表型及分子耐药机制,在此基础上设定两种药物针对大肠杆菌的野生型折点,通过动物模型初步探讨与临床效力最为相关的PK-PD参数,以期为PK-PD折点的制定提供数据基础。本研究收集了70年代、80年代、90年代、2001年-2013年国内不同地区不同动物来源的1123株大肠杆菌,通过微量肉汤稀释法测试了乙酰甲喹和氟苯尼考对这些菌株的最小抑菌浓度(Minimum Inhibitory Concentration, MIC),通过体外药效学试验研究了这两种药物对致病性大肠杆菌的最小杀菌浓度(Minimum Bactericidal Concentration, MBC)、防突变浓度(Mutant Prevention Concentration, MPC)、抗菌后效应(Post-antimicrobial effect, PAE)和体外杀菌曲线。结果表明乙酰甲喹对大肠杆菌呈现浓度依赖性的杀菌作用(MBC=1-2MIC),氟苯尼考对大肠杆菌呈现浓度依赖性的抑菌作用(MBC=4-16MIC)两种药物对大肠杆菌都有一定的体外PAE,尽管它们的突变选择窗(Mutant Selection Window, MSW)较宽。70年代到2013年之间,国内动物源大肠杆菌对乙酰甲喹和氟苯尼考的敏感性呈下降趋势,低敏感菌株的比例逐年上升。鸡源大肠杆菌相对猪源大肠杆菌对乙酰甲喹敏感,而两种动物来源的大肠杆菌对氟苯尼考的敏感性情况比较接近。通过分别绘制两种药物对1123株大肠杆菌MIC分布的直方图,运用统计学方法确定了乙酰甲喹对大肠杆菌的野生型折点COWT为32μg/mL,氟苯尼考对大肠杆菌的野生型折点COWT为16μg/mL。通过检测这些菌株中的喹乙醇耐药基因oqxAB和相关氟苯尼考耐药基因(fexA、fexB、floR和cfr),发现oqxAB和floR基因分别是介导大肠杆菌对乙酰甲喹和氟苯尼考低敏感表型的主要基因,分别占MIC≥64μg/mL大肠杆菌的94.4%和MIC≥32μg/mL大肠杆菌的93.7%,并且基因的检出率随着年代的推移呈上升趋势。首次在一株猪源大肠杆菌中检测到了cfr基因,检出率为0.09%,cfr基因定位于质粒上,可介导大肠杆菌对氟苯尼考的低敏感表型。耐药基因的检测结果与我们制定的CCWT满足耐药表型和基因型的一致。通过基因测序,发现同一动物来源不同大肠杆菌菌株或不同动物来源的大肠杆菌中oqxAB和floR基因的DNA同源性都很高,分别达到了99.2%和99.6%以上,说明大肠杆菌中这两种基因很少发生变异。通过研究oqxAB基因和cfr基因在大肠杆菌中的基因环境发现,这些基因的两侧都有方向相同的IS26,形成了一个复合转座子结构。这种复合转座子结构不能稳定存在,可以通过两侧的IS26发生同源重组,从而将oqxAB和cfr基因以环状中间体的形式从质粒或染色体上游离下来,加大了耐药基因传播的风险。通过构建粒细胞减少小鼠大腿肌肉感染模型研究了乙酰甲喹注射剂对小鼠大肠杆菌感染的PK-PD同步关系,结果发现与临床效力最为相关的PK-PD参数为AUC/MIC,并且当AUC/MIC分别为0.85h和4.3h时,可以达到抑菌或杀菌效应。通过猪肠瘘和血清半体内模型研究氟苯尼考粉剂对猪大肠杆菌感染的PK-PD同步关系,结果发现猪肠道环境复杂,所构建的猪肠瘘模型并不能考察氟苯尼考对大肠杆菌的半体外杀菌效力,故只检测了氟苯尼考在回肠中的药物浓度变化,结果表明氟苯尼考在猪回肠中存在时间较短,3h后基本检测不到原药。使用Hill模型模拟猪血清半体内氟苯尼考对大肠杆菌的PK-PD关系,当AUC24h/MIC分别为15.79h和49.49h时,能够达到抑菌和杀菌效应。考虑到野生型大肠杆菌氟苯尼考MIC的分布情况(MIC≤16μg/mL),目前一天给药两次的方案可能对部分野生型菌株引起的系统感染起不到治疗效果(杀菌效应)。综上所述,国内动物源大肠杆菌对乙酰甲喹和氟苯尼考的敏感性随着年代推移呈现下降趋势,介导两种药物低敏感表型的主要基因分别为oqxAB和floR,使用统计学方法分别建立了乙酰甲喹和氟苯尼考对大肠杆菌的COWT。研究得到了乙酰甲喹治疗小鼠大肠杆菌感染的PK-PD靶值为AUC/MIC≥4.3h,初步探讨了氟苯尼考治疗大肠杆菌引起的猪肠道感染和系统感染的PK-PD关系并给出用药建议。该研究结果为最终敏感性折点的制定提供了理论依据和数据支持,有利于下一步耐药性监测工作的进行,从而最终为耐药性的风险评估提供科学依据。更多还原

【Abstract】 The interpretive criteria or breakpoint for antimicrobial susceptibility testing was the key technical criteria to conduct resistance surveillance and to analyze the resistance rate and resistance level. The authoritative organizations to set the breakpoint included the CLSI in the US and EUCAST in the Europe. For the lack of interpretive criteria, the breakpoint abroad was employed in China. However, there was no interpretive criteria for the new antimicrobials or formulations firstly devoloped in China such as mequindox and florfenicol powder. These two antimicrobials were extensively used in China and has played an important role in the control of infections caused by Escherichia coli. However, the lack of breakpoint hindered the implement of resistance surveillance for mequindox and florfenicol. Thus, this study aimed to investigate the susceptibility phenotype and resistance genotype as well as to set the susceptible breakpoint for these two drugs against E. coli.In vitro efficacy of these two drugs were studied including minimum inhibitory concentration (MIC^minimum bactericidal concentration (MBC)、mutant prevention concentration (MPC) and post-antimicrobial effect (PAE) It showed that mequindox presented a concentration-dependent bactericidal activity against enteropathogenic E. coli (MBC=1-2MIC), while florfenicol showed concentration-dependent bacteriostatic activity against enteropathogenic E. coli (MBC=4-16MIC). Both drugs have an in vitro PAE although their mutation selective window (MSW) was wide. A total of1123E. coli isolates were collected from domestic animals in China from the1970s to2013, and mequindox and florfenicol susceptibility was tested by broth microdilution. The percentages of E. coli isolates with increased mequindox MICs (≥64μg/ml) and florfenicol MICs (≥32μg/ml) showed a rising trend each year throughout the study period. The E. coli from chicken was more sensitive to mequindox than that from pigs while strains of both origins have the similar susceptibility pattern towards florfenicol. By plotting the histogram of MIC distribution for mequindox and florfenicol and also using the statisticial analysis, the wide-type cut-coff (COWT) was set for these two drugs against E. coli.PCR was performed to detect the olaquindox resistance gene oqxAB and all the florfenicol resistance genes in these strains. It revealed that the oqxAB was the dominant resistance gene (94.4%) mediating increased mequindox MICs (MIC≥64μg/ml), while floR gene was the most prevalent gene mediating increased florfenicol MICs (MIC≥32μg/ml) and percentages of these genes showed a rising trend in E. coli during the70s to2013. The cfr gene was detected for the first time in the plasmid of one porcine E. coli isolate and the cfr-positive strain showed florfenicol MICs of64μg/ml. The genotype tested was in agreement with the COWT.By sequencing, we found that both oqxAB and floR genes showed high identities (99.2%and99.6%, respectively) in strains of different animal origins. The genetic environment of oqxAB and cfr was investigated and both genes were found to be bracketed by the IS26and the element was not stable and was readily to be looped out via IS26mediated recombination in the form of a circular intermediate. The PK-PD relationship for mequindox injection against E. coli was studied in neutropenic murine thigh infection model. The most relevant PK-PD parameter is the AUC/MIC and the PK-PD targets were0.85h for bacteriostatic and4.3h for bactericidal effect, respectively. The PK-PD relationship for florfenicol powder against E. coli was investigated in porcine fistula and the serum ex vivo model. However, the bactericidal test was impracticable ex vivo for the complicated environment in intestinal tract. The concentration-time process of florfenicol in the intestinal tract was investigated and it revealed that the florfenicol was present in the ileum for a short time and could not be detected after3h. The PK-PD relationship in porcine serum ex vivo was simulated using Hill model and it showed that the bacteriostatic and bactericidal effect was achieved when the AUC24h/MIC attained15.79h and49.49h, respectively. Considering the MIC distributions for wild-type E. coli strains (MIC≤16μg/mL), the regime of administration twice one day may not be practical to cure the system infection caused by some of the wild-type strains.In conclusion, the percentage of E. coli isolates with decreased mequindox and florfenicol susceptibility showed a rising trend during the70s and2013. The oqxAB and floR gene was the dominant resistance gene mediating low-level susceptibility of mequindox and florfenicol, respectively. The COwTwas set for mequindox and florfenicol against E. coli, respectively. The PK-PD target for mequindox against E. coli infection in mice is AUC/MIC≥4.3h. PK-PD relationship in porcine intestinal tract as well as serum ex vivo was explored and administration regime was recommended. The findings in this study provided theory basis and data support for the final breakpoint and may promote the implement of resistance surveillance as well as the resistance risk assessment of these two drugs against E. coli.更多还原