临床分离志贺菌的质粒介导磷霉素耐药基因检测及其与β-内酰胺酶基因的相关性研究

发布时间：2018-01-15 22:17

本文关键词：临床分离志贺菌的质粒介导磷霉素耐药基因检测及其与β-内酰胺酶基因的相关性研究　出处：《安徽医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:了解临床分离志贺菌对常用抗菌药物的耐药情况和质粒介导的磷霉素耐药基因的种类、分布及传播方式,为临床合理选择抗生素提供指导。研究临床分离志贺菌质粒介导的磷霉素耐药基因与β-内酰胺酶基因的相关性。材料与方法:菌株来源263株志贺菌为安徽省细菌耐药监测中心2011年9月~2012年10月在临床标本中分离获得的非重复菌株。质控菌株大肠埃希菌ATCC25922,ESBLs阳性菌株肺炎克雷伯菌ATCC700603,转移接合试验受体菌大肠埃希菌J53AZR以及fos A、fos A3和fos C2基因阳性标准株均为安徽省细菌耐药监控中心保存菌株。方法:1.MH琼脂倍比稀释法测定263株志贺菌对磷霉素等12种抗生素的最低抑菌浓度(the minimal inhibitory concentration,MIC),大肠埃希菌ATCC25922为质控菌。2.煮沸法提取磷霉素耐药菌株总基因组DNA为模板,利用fos A、fos A3以及fos C2的特异性引物以聚合酶链反应法(polymerase chain reaction,PCR)扩增质粒介导的磷霉素耐药基因,测序并拼接PCR产物,明确质粒介导的磷霉素耐药基因是否存在突变。3.质粒介导的磷霉素耐药基因阳性菌株与大肠埃希菌J53AZR进行接合实验,M-H琼脂倍比稀释法测定受体菌及接合子对12种常用抗生素的MIC值。4.肠杆菌科基因间的重复序列PCR(enterobacterial repetitive intergenic consensus PCR,ERIC-PCR)法对质粒介导的磷霉素耐药基因阳性菌株进行同源性分析。5.在质粒介导的磷霉素耐药基因阳性志贺菌中进行产ESBLs检测,PCR法明确β-内酰胺酶基因型,接合实验验证质粒介导的磷霉素耐药基因与β-内酰胺酶基因是否同时发生转移。6.煮沸法提取的质粒介导的磷霉素耐药基因及β-内酰胺酶基因均阳性的接合子菌株总基因组DNA为模板,多重PCR法对其携带的质粒进行不兼容性分组。结果:1.263株志贺菌对磷霉素的耐药率为9.5%、中介率为2.7%,氯霉素的耐药率最高、达到87.8%,亚胺培南、哌拉西林/他唑巴坦和头孢哌酮/舒巴坦均有极高的敏感性,敏感率介于97%~100%,三代头孢菌素的敏感率介于50.6%~72.2%,左氧氟沙星和环丙沙星的敏感率分别为60.5%和64.3%。2.25株磷霉素耐药志贺菌中的18株检测到fos A3基因,未检测到fos A及fos C2基因,fos A3基因的Gen Bank注册号为KJ716852。13株fos A3基因阳性志贺菌与大肠埃希菌J53AZR接合成功,接合子均可检测到fos A3基因,接合子与受体菌相比对磷霉素的MIC值有明显提高。ERIC-PCR法证实部分fos A3阳性志贺菌存在同源性。3.18株fos A3阳性志贺菌均产ESBLs,其中1株仅携带OXA基因,3株同时携带OXA、TEM和CTX-M基因,14株同时携带OXA和CTX-M基因;测序分析显示CTX-M型基因分别为CTX-M-15、CTX-M-55、CTX-M-123,CTX-M-123基因的Gen Bank注册号为KJ871006,TEM基因均为TEM-1,OXA基因均为OXA-30;18株志贺菌中的13株接合成功,接合子与受体菌相比对磷霉素和三代头孢的MIC值明显提高,接合子均携带供体菌的相应耐药基因。4.13株接合子均携带Inc F型质粒,其中3株携带HI2型复制子、2株携带Il-Ir型复制子、1株携带N型复制子。结论:1.安徽地区的志贺菌对常用抗菌药物耐药现象严重,但磷霉素仍有较高的敏感性,适合临床运用于治疗细菌性痢疾。2.证实了安徽地区存在质粒介导的志贺菌对磷霉素的耐药现象,且是国内首次在志贺菌中检测到了质粒介导的磷霉素耐药基因。3.携带fos A3的接合子对磷霉素的MIC值与受体菌相比有显著的升高且均高度耐药,表明fos A3可导致志贺菌对磷霉素高度耐药。4.携带fos A3的志贺菌具有较高的接合率,且存在同源性,表明fos A3阳性志贺菌极易传播,导致磷霉素耐药性的扩散。5.可能存在质粒介导的磷霉素耐药基因fos A3与β-内酰胺酶基因位于同一质粒上共同传播,引起多重耐药。
[Abstract]:Objective: to understand the types of clinical isolates of Shigella to fosfomycin antibiotic resistance and plasmid mediated by multidrug resistance gene, distribution and dissemination way, to provide guidance for the reasonable use of antibiotics in clinic. The correlation of fosfomycin in clinical isolates of Shigella plasmid mediated by multidrug resistance gene and beta lactamase genes. Materials and methods: 263 strains of Shigella strains in Anhui province for the bacterial resistance monitoring center in September 2011 October ~2012 in clinical specimens of isolated non repetitive strains. Quality control strains of Escherichia coli ATCC25922, ESBLs positive strains of Klebsiella pneumoniae ATCC700603, conjugation experiment recipient bacterium Escherichia coli J53AZR and Fos A FOS, A3 and Fos positive standard strain C2 gene in Anhui province were the bacterial resistance monitoring center preservation strains. Methods: 1.MH agar dilution method for the determination of 263 Shigella strains of fosfomycin and other 12 kinds of antibiotics The minimum inhibitory concentration (the minimal inhibitory concentration, MIC), Escherichia coli ATCC25922 for fosfomycin extraction control the bacteria.2. boiling method in resistant strains of total genomic DNA as template, using Fos A, Fos A3 and Fos C2 specific primers by polymerase chain reaction (polymerase chain reaction, PCR) - resistant gene amplification fosfomycin plasmid mediated, sequencing and splicing of PCR products, fosfomycin resistance gene in clear plasmid mediated mutations in fosfomycin.3. plasmid mediated by multidrug resistance gene positive strains of Escherichia coli and J53AZR conjugation experiments, M-H agar dilution method for the determination of receptor bacteria and zygote of 12 kinds of antibiotics the MIC value of PCR.4. repeats between genes in Enterobacteriaceae (enterobacterial repetitive intergenic consensus PCR, ERIC-PCR) of fosfomycin plasmid mediated resistance gene in positive bacteria strains were homologous points Analysis of.5. in fosfomycin plasmid mediated resistance gene was positive in Shigella strains producing ESBLs detection, PCR method to determine the beta lactamase genotype, joint fosfomycin experimental verification of plasmid mediated hormone resistance gene and beta lactamase gene plasmid mediated transfer of fosfomycin and.6. boiling extraction the guide element of multidrug resistance gene and beta lactamase genes were positive TRANSCONJUGANT strains total genomic DNA as template, the compatibility grouping of its carrying plasmid by multiplex PCR method. Results: 1.263 strains of Shigella resistance to fosfomycin was 9.5%, the intermediary rate was 2.7%, the resistant rate of chloramphenicol the highest, reaching 87.8%, imipenem and sulbactam have very high sensitivity to piperacillin / tazobactam and Cefoperazone /, sensitive rate is 97%~100%, the three generation cephalosporin sensitivity rate is between 50.6%~72.2%, the sensitive rate of levofloxacin and ciprofloxacin were 60.5% and 64. The 18 strains were detected Fos A3 genes of 3%.2.25 strains fosfomycin resistant Shigella, did not detect the FOS A and Fos C2 Gen, Bank gene, A3 gene is KJ716852.13 FOS registered Fos A3 gene positive strains of Shigella and Escherichia coli J53AZR joint success, zygote FOS could be detected in the A3 gene. Transconjugants and receptor bacteria compared to fosfomycin MIC value increased by.ERIC-PCR FOS confirmed A3 positive Shigella has homology of.3.18 strain Fos A3 positive Shigella were producing ESBLs, which only 1 strains carried OXA gene, 3 strains with both OXA, TEM and CTX-M gene, 14 strains carried at the same time OXA and CTX-M gene; sequencing analysis showed that CTX-M genotypes were CTX-M-15, CTX-M-55, CTX-M-123, Gen, Bank registered CTX-M-123 gene KJ871006, TEM gene was TEM-1, the OXA gene was OXA-30; 18 strains of Shigella in 13 strains of joint success, zygote compared with the receptor bacteria to fosfomycin And the three generation cephalosporin MIC increased obviously, zygote carries the donor of the corresponding resistance genes of.4.13 strains of transconjugants carried the Inc F plasmid, including 3 strains carrying HI2 replicon, 2 strains carrying Il-Ir replicon, 1 strains carrying N type replicon. Conclusion: 1. Shigella strains in Anhui the serious drug resistance to the commonly used antibiotics, but phosphonomycin still has high sensitivity, suitable for clinical application in the treatment of bacterial dysentery.2. confirmed Shigella plasmid mediated resistance to fosfomycin phenomenon in Anhui area, and is the home for the first time in the zygote of Shigella were detected in P by plasmid mediated - resistant gene.3. carrying Fos A3 of fosfomycin MIC value compared with the receptor bacteria increased significantly and were highly resistant, showed that Fos A3 can lead to joint Shigella Shigella to phosphonomycin highly resistant.4. carrying Fos A3 has a higher rate, and in the same The source indicates that Fos A3 positive Shigella is highly susceptible to transmission, leading to the spread of fosfomycin resistance..5. may exist plasmid mediated fosfomycin resistance gene Fos A3 and beta lactamase gene are located on the same plasmid, causing multiple resistance.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

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