本文關(guān)鍵詞：臨床分離志賀菌的質(zhì)粒介導(dǎo)磷霉素耐藥基因檢測及其與β-內(nèi)酰胺酶基因的相關(guān)性研究　出處：《安徽醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 志賀菌 質(zhì)粒 β-內(nèi)酰胺酶 耐藥基因 磷霉素

【摘要】：目的:了解臨床分離志賀菌對常用抗菌藥物的耐藥情況和質(zhì)粒介導(dǎo)的磷霉素耐藥基因的種類、分布及傳播方式,為臨床合理選擇抗生素提供指導(dǎo)。研究臨床分離志賀菌質(zhì)粒介導(dǎo)的磷霉素耐藥基因與β-內(nèi)酰胺酶基因的相關(guān)性。材料與方法:菌株來源263株志賀菌為安徽省細(xì)菌耐藥監(jiān)測中心2011年9月~2012年10月在臨床標(biāo)本中分離獲得的非重復(fù)菌株。質(zhì)控菌株大腸埃希菌ATCC25922,ESBLs陽性菌株肺炎克雷伯菌ATCC700603,轉(zhuǎn)移接合試驗(yàn)受體菌大腸埃希菌J53AZR以及fos A、fos A3和fos C2基因陽性標(biāo)準(zhǔn)株均為安徽省細(xì)菌耐藥監(jiān)控中心保存菌株。方法:1.MH瓊脂倍比稀釋法測定263株志賀菌對磷霉素等12種抗生素的最低抑菌濃度(the minimal inhibitory concentration,MIC),大腸埃希菌ATCC25922為質(zhì)控菌。2.煮沸法提取磷霉素耐藥菌株總基因組DNA為模板,利用fos A、fos A3以及fos C2的特異性引物以聚合酶鏈反應(yīng)法(polymerase chain reaction,PCR)擴(kuò)增質(zhì)粒介導(dǎo)的磷霉素耐藥基因,測序并拼接PCR產(chǎn)物,明確質(zhì)粒介導(dǎo)的磷霉素耐藥基因是否存在突變。3.質(zhì)粒介導(dǎo)的磷霉素耐藥基因陽性菌株與大腸埃希菌J53AZR進(jìn)行接合實(shí)驗(yàn),M-H瓊脂倍比稀釋法測定受體菌及接合子對12種常用抗生素的MIC值。4.腸桿菌科基因間的重復(fù)序列PCR(enterobacterial repetitive intergenic consensus PCR,ERIC-PCR)法對質(zhì)粒介導(dǎo)的磷霉素耐藥基因陽性菌株進(jìn)行同源性分析。5.在質(zhì)粒介導(dǎo)的磷霉素耐藥基因陽性志賀菌中進(jìn)行產(chǎn)ESBLs檢測,PCR法明確β-內(nèi)酰胺酶基因型,接合實(shí)驗(yàn)驗(yàn)證質(zhì)粒介導(dǎo)的磷霉素耐藥基因與β-內(nèi)酰胺酶基因是否同時(shí)發(fā)生轉(zhuǎn)移。6.煮沸法提取的質(zhì)粒介導(dǎo)的磷霉素耐藥基因及β-內(nèi)酰胺酶基因均陽性的接合子菌株總基因組DNA為模板,多重PCR法對其攜帶的質(zhì)粒進(jìn)行不兼容性分組。結(jié)果:1.263株志賀菌對磷霉素的耐藥率為9.5%、中介率為2.7%,氯霉素的耐藥率最高、達(dá)到87.8%,亞胺培南、哌拉西林/他唑巴坦和頭孢哌酮/舒巴坦均有極高的敏感性,敏感率介于97%~100%,三代頭孢菌素的敏感率介于50.6%~72.2%,左氧氟沙星和環(huán)丙沙星的敏感率分別為60.5%和64.3%。2.25株磷霉素耐藥志賀菌中的18株檢測到fos A3基因,未檢測到fos A及fos C2基因,fos A3基因的Gen Bank注冊號為KJ716852。13株fos A3基因陽性志賀菌與大腸埃希菌J53AZR接合成功,接合子均可檢測到fos A3基因,接合子與受體菌相比對磷霉素的MIC值有明顯提高。ERIC-PCR法證實(shí)部分fos A3陽性志賀菌存在同源性。3.18株fos A3陽性志賀菌均產(chǎn)ESBLs,其中1株僅攜帶OXA基因,3株同時(shí)攜帶OXA、TEM和CTX-M基因,14株同時(shí)攜帶OXA和CTX-M基因;測序分析顯示CTX-M型基因分別為CTX-M-15、CTX-M-55、CTX-M-123,CTX-M-123基因的Gen Bank注冊號為KJ871006,TEM基因均為TEM-1,OXA基因均為OXA-30;18株志賀菌中的13株接合成功,接合子與受體菌相比對磷霉素和三代頭孢的MIC值明顯提高,接合子均攜帶供體菌的相應(yīng)耐藥基因。4.13株接合子均攜帶Inc F型質(zhì)粒,其中3株攜帶HI2型復(fù)制子、2株攜帶Il-Ir型復(fù)制子、1株攜帶N型復(fù)制子。結(jié)論:1.安徽地區(qū)的志賀菌對常用抗菌藥物耐藥現(xiàn)象嚴(yán)重,但磷霉素仍有較高的敏感性,適合臨床運(yùn)用于治療細(xì)菌性痢疾。2.證實(shí)了安徽地區(qū)存在質(zhì)粒介導(dǎo)的志賀菌對磷霉素的耐藥現(xiàn)象,且是國內(nèi)首次在志賀菌中檢測到了質(zhì)粒介導(dǎo)的磷霉素耐藥基因。3.攜帶fos A3的接合子對磷霉素的MIC值與受體菌相比有顯著的升高且均高度耐藥,表明fos A3可導(dǎo)致志賀菌對磷霉素高度耐藥。4.攜帶fos A3的志賀菌具有較高的接合率,且存在同源性,表明fos A3陽性志賀菌極易傳播,導(dǎo)致磷霉素耐藥性的擴(kuò)散。5.可能存在質(zhì)粒介導(dǎo)的磷霉素耐藥基因fos A3與β-內(nèi)酰胺酶基因位于同一質(zhì)粒上共同傳播,引起多重耐藥。
[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.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.5

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本文編號：1430307