基层医疗机构大肠埃希菌及肺炎克雷伯菌氟喹诺酮耐药分子流行及遗传特征研究

发布时间：2019-06-26 13:10

【摘要】：氟喹诺酮是一类人工合成药物,抗菌谱广,是临床使用最广泛的一类抗菌药物,其耐药也尤为突出。自20世纪90年代以来,我国呈现出大肠埃希菌高频耐药(64.5%-71.8%)现象,有关耐药调查以及机制研究大多集中在中心城市三级医院,县级医院及社区感染细菌耐药研究极少,开展县级医疗机构社区感染患者细菌对喹诺酮类的耐药研究,有助于指导这类医疗机构感染治疗和抗菌药物合理使用。本研究第一部分收集来自全国七个地区30家县级医院社区感染非重复大肠埃希菌590株,肺炎克雷伯菌569株。细菌体外药敏试验检测其对17种抗菌药物的敏感性；PCR方法检测质粒介导的喹诺酮耐药(PMQR)基因：qnrA, qnrB, qnrC, qnrD, qnrS, aac(6')-Ib-cr, qepA和oqxAB及喹诺酮耐药决定区(QRDR)DNA旋转酶gyrA, gyrB亚基和拓扑异构酶IV parC, parE亚基突变,采用DNASTAR软件分析测序结果,突变比较参照E. coli k-12和Klebsiella pneumoniae MGH78578;对非QRDR介导耐药菌株进行外排泵抑制试验。试验结果显示590株大肠埃希菌对环丙沙星的耐药率为51.2%,PMQR基因检出率为37.3%,qnr类的流行率为3.7%,aac(6')Ib-cr19.7%, qepA14.1%, oqxAB3.9%。24株同时携带2种以上PMQR基因。环丙沙星耐药组100%存在QRDR突变,93.7%是3个以上位点突变；敏感组66.7%存在QRDR突变。569株肺炎克雷伯菌对环丙沙星耐药率为8.3%,PMQR检出率为70.0%,qnr类流行率为36.6%,aac(6')Ib-cr9.1%, qepA43.8%。环丙沙星耐药组73.9%存在QRDR突变,19.6%是3个以上位点突变。敏感组4%存在QRDR突变。12株(26.1%)肺炎克雷伯菌为非QRDR介导耐药,环丙沙星最低抑菌浓度(MIC)可被外排泵抑制剂N-甲基吡咯烷酮(NMP)降为敏感水平。分子流行病学研究发现,县级医院社区感染大肠埃希菌和肺炎克雷伯菌氟喹诺酮耐药率相差悬殊(51.2%versus8.3%),原因除肺炎克雷伯菌氟喹诺酮耐药率相比大肠埃希菌长期处于较低水平外,还与肺炎克雷伯菌24.5%(大肠埃希菌9.9%)来自0-14岁儿童有关,该组环丙沙星的耐药率仅为4.2%。大肠埃希菌在耐药组(100%)甚至敏感组(66.7%)都有很高的QRDR突变率,对QRDR突变的积累明显高于肺炎克雷伯菌(耐药组73.9%,敏感组4%)。本研究第二部分对302株环丙沙星耐药大肠埃希菌和46株环丙沙星耐药肺炎克雷伯菌遗传特征及多PMQR菌株耐药质粒传播特征进行研究。大肠埃希菌MLST分型选用UCC数据库(http://mlst.ucc.ie/mlst/dbs/Ecoli),肺炎克雷伯菌MLST分型选用pasteur数据库(http://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html)。测序结果在数据库中在线比对,获得等位基因型和序列型(ST)。氟喹诺酮耐药菌株遗传进化分析采用eBURST V3和商业化Bionumerics软件。多PMQR耐药质粒复制子分型采用基于PCR的分型方法PBRT+ColE+IncR,可对肠杆菌科20种常见复制子进行分型。并对这些多PMQR耐药质粒进行接合传播试验。试验结果显示302株环丙沙星耐药大肠埃希菌共有60种ST型。常见流行型为ST131(14.6%), ST1193(7.9%), ST405(6.6%), ST69(6.3%), ST648(6.3%)。华东、中南、华北、东北、西北、华南和西南地区分别有15、26、28、11、17、22和13种。46株环丙沙星耐药肺炎克雷伯菌共有23种ST型。克隆复合体CC11(ST11, ST340, ST258)占15.2%,其次为ST1(13.0%), ST15(10.9%), ST147(10.9%)和ST875(8.7%)。华东、中南、华北、东北、西北、华南和西南地区分别有3、2、5、4、8、5和7种。大肠埃希菌多PMQR耐药质粒复制子83.3%为IncF,大多与其他复制子共存,45.8%可接合转移耐药基因。肺炎克雷伯菌多PMQR耐药质粒复制子70%为ColE-like,75%可接合转移耐药基因。本研究通过第二部分耐药氟喹诺酮遗传特征分析和多PMQR耐药质粒复制子分型和传播特征研究发现氟喹诺酮耐药大肠埃希菌序列型多样,在各地区呈散在分布,没有暴发流行克隆型。肺炎克雷伯菌虽然仅46株对环丙沙星耐药,但序列型也很丰富,达23种。也呈散在分布,无克隆传播趋势。说明大肠埃希菌氟喹诺酮耐药菌株呈多点起源,散在分布。本研究通过对中国七个地区30家县级医院社区感染大肠埃希菌和肺炎克雷伯菌氟喹诺酮耐药分子流行及遗传特征研究发现：大肠埃希菌对氟喹诺酮高频耐药,大肠埃希菌和肺炎克雷伯菌对QRDR突变积累存在差异可能是两者耐药率悬殊的一个重要影响因素。氟喹诺酮耐药菌株呈多点起源,散在分布。高氟喹诺酮耐药水平是长期耐药基因突变积累的结果,非优势克隆传播引起。
[Abstract]:Flufenone is a kind of synthetic medicine, and its antibacterial spectrum is wide, it is the most widely used anti-bacterial drug in clinical use, and its drug resistance is especially prominent. Since the 1990 's, the high-frequency drug resistance (64.5%-71.8%) of E. coli is present in our country. The study on the drug-resistance of the bacteria of the community-infected patients in the county-level medical institution to the noonone class can help to guide the treatment of such medical institutions and the rational use of the anti-bacterial drugs. The first part of the study collected 590 strains of non-repeated E. coli from 30 county-level hospital communities in seven regions of the country, and K. pneumoniae 569 The susceptibility of the strain to the 17 antibacterial drugs was tested by the in vitro drug sensitivity test of the strain. The PCR method was used to detect the plasmid-mediated gene of the drug resistance (PQR): qnrA, qnrB, qnrC, qnrD, qnrS, aac (6 ')-Ib-cr, qeA and oqxAB, and the drug-resistance-determining region (QRDR), gyrA, gyrB subunit and topoisomerase IV par. C, pare subunit mutation, DNA STAR software was used to analyze the sequencing result, and the mutation was compared with E. coli k-12 and Klebsiella pneumonae MGH78578; and the non-QRDR-mediated drug-resistant strain was subjected to efflux pump inhibition test. The test results showed that the resistance rate of the 590 strains of E. coli to ciprofloxacin was 51.2%, the detection rate of PQR gene was 37.3%, the prevalence rate of qnr was 3.7%, aac (6 ') Ib-cr19.7%, qpA1.4%, oqxAB3.9%.24 strains carried 2 or more PMB simultaneously. The resistance of ciprofloxacin to ciprofloxacin was 8.3%, the detection rate of PQR was 70.0%, the prevalence of qnr was 36.6%, aac (6 ') Ib-cr9.1%, qpA43. The QRDR mutation was found in 73.9% of the ciprofloxacin-resistant group and 19.6% in the ciprofloxacin-resistant group. Point mutations.4% of the sensitive groups had a QRDR mutation.12 (26.1%) of Klebsiella pneumoniae were non-QRDR-mediated resistance, and the minimum inhibitory concentration (MIC) of ciprofloxacin was reduced to sensitivity by the efflux pump inhibitor N-methyl-tridecanone (NMP). The study of molecular epidemiology found that the drug-resistant rates of E. coli and Klebsiella pneumoniae in the community of the county-level hospital were significantly different (51.2% and 83.3%), because of the long-term relationship between E. coli and E. coli than that of Klebsiella pneumoniae. In addition to the lower level, it was also associated with 24.5% of klebsiella pneumoniae (9.9% of E. coli) from 0 to 14 years of age, and the rate of resistance to ciprofloxacin was only The mutation rate of QRDR was higher in the drug-resistant group (100%) and even in the sensitive group (66.7%), and the accumulation of QRDR mutation was significantly higher than that of Klebsiella pneumoniae (73.9% in the drug-resistant group). The genetic characteristics of two strains of ciprofloxacin-resistant E. coli and 46 ciprofloxacin-resistant Klebsiella pneumoniae and the transmission of drug-resistant plasmids of multiple PQR strains in 302 ciprofloxacin-resistant E. coli and 46 ciprofloxacin-resistant strains of ciprofloxacin in the second part of the study The characteristics of the seeding were studied. The UCC database (http://mlst.ucc.ie/mlst/dbs/Ecoli) was used for MLST typing of E. coli. The model of MLST of Klebsiella pneumoniae was selected as the pateur database (

http://bigsdb.web.pastor. fr/klebsiella/ klebsilla.html) ). The sequencing results are online specific in the database to obtain the allelic type and Sequence-type (ST). The analysis of the genetic evolution of the strain-resistant strain of the fluoronone-resistant strain uses eBURST V3 and commercial Bionum. The multi-PQR-resistant plasmid replicon type is based on the PCR-based typing method (PBRT + ColE + IncerR), which can be used for 20 species of Enterobacteriaceae. The replicon was genotyped and these multiple PQR-resistant plasmids were fed The results of the test show that 302 ciprofloxacin-resistant E. coli A total of 60 ST-types were found in the bacteria. The common prevalence was ST131 (14.6%), ST1193 (7.9%), ST405 (6.6%), ST69 (6.3%), ST 648 (6.3%). There are 15,26,28,11,17,22 and 13 species in East China, South China, North China, Northeast, Northwest, South China and Southwest.46 strains of ciprofloxacin-resistant Klebsiella pneumoniae There were 23 types of ST-type. The clone complex CC11 (ST11, ST340, ST258) was 15.2%, followed by ST1 (13.0%), ST15 (10.9%), ST147 (10.9%), and ST. 875 (8.7%). East China, Central South, North China, Northeast, Northwest, South China and Southwest China are 3,2,5, respectively. 4,8,5 and 7. The MPMQR-resistant plasmid replicon of E. coli 83.3% was IncF, most of which co-existed with other replicon, 45.8%. The transfer-resistant gene can be engaged. The multi-PQR-resistant plasmid replicon of Klebsiella pneumoniae is ColE-like,75% In this study, the genetic characteristics of the second part of the drug-resistant fluobanone and the type and the characteristics of the multi-PQR-resistant plasmid replicon were studied. The results of the study found that the sequence type of the drug-resistant E. coli was diverse, and it was scattered in each area. in that case of Klebsiella pneumoniae, only 46 of the strains were resistant to ciprofloxacin, but the order Nematic is also very rich, up to 23 species. No clonal propagation trend in the distribution and no clonal propagation trend. The prevalence and genetic characteristics of the drug-resistant E. coli and Klebsiella pneumoniae in the community of 30 county-level hospitals in seven regions of China have been found in this study. The difference between the high-frequency resistance of E. coli to the high-frequency, E. coli and K. pneumoniae in the QRDR mutation may be both. One of the most important factors of the disparity in drug rate. The drug-resistant level of high-flurolenin is a long-term drug-resistant gene mutation accumulation.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R446.5

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