發(fā)布時(shí)間：2018-03-13 22:19

  本文選題：黏質(zhì)沙雷菌　切入點(diǎn)：16S　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

【摘要】：目的了解201株安徽地區(qū)臨床分離黏質(zhì)沙雷菌藥敏情況和質(zhì)粒介導(dǎo)的16S r RNA甲基化酶檢出率與基因型,為臨床合理選擇抗菌藥物提供依據(jù)。研究臨床分離黏質(zhì)沙雷菌質(zhì)粒介導(dǎo)的16S r RNA甲基化酶耐藥基因與β-內(nèi)酰胺酶基因的共轉(zhuǎn)移情況。材料與方法菌株來源201株黏質(zhì)沙雷臨床分離菌株,來源于安徽省細(xì)菌耐藥監(jiān)測(cè)中心監(jiān)測(cè)網(wǎng)所屬34所醫(yī)院(由皖南、皖中、皖北不同級(jí)別的醫(yī)院組成)2005年~2012年每年9月份住院和門診患者的各類臨床標(biāo)本。藥敏實(shí)驗(yàn)質(zhì)控菌株大腸埃希菌ATCC25922,轉(zhuǎn)移接合試驗(yàn)受體菌大腸埃希菌J53 AZR,二者均為安徽省細(xì)菌耐藥監(jiān)控中心保存。方法1.采用MH瓊脂倍比稀釋法測(cè)定臨床分離黏質(zhì)沙雷菌株對(duì)抗菌藥物的敏感性,質(zhì)控菌采用E.coli ATCC 25922。2.煮沸法提取細(xì)菌的總DNA,堿裂解法提取質(zhì)粒DNA;用聚合酶鏈反應(yīng)(PCR)方法擴(kuò)增質(zhì)粒介導(dǎo)16S r RNA甲基化酶基因;PCR產(chǎn)物純化測(cè)序,測(cè)序結(jié)果在Gen Bank中經(jīng)Blast程序比對(duì)分析,以明確16S r RNA甲基化酶基因的基因型別以及是否突變。3.用16S r RNA甲基化酶基因陽性菌株與大腸埃希菌J53AzR行轉(zhuǎn)移接合試驗(yàn);對(duì)接合子進(jìn)行生化鑒定并經(jīng)PCR檢測(cè)耐藥基因,PCR擴(kuò)增產(chǎn)物DNA測(cè)序確認(rèn);采用Muller-Hinton瓊脂對(duì)比稀釋法測(cè)定大腸埃希菌J53AzR與接合子對(duì)抗菌藥物的最低抑菌濃度(MIC)。4.以16S r RNA甲基化酶基因陽性臨床分離菌株的總DNA為模板,采用PCR方法擴(kuò)增β-內(nèi)酰胺酶基因;PCR產(chǎn)物純化測(cè)序,測(cè)序結(jié)果在Gen Bank中經(jīng)Blast程序比對(duì),比較分析16S r RNA甲基化酶基因型別與β-內(nèi)酰胺酶基因型別關(guān)系。5.以16S r RNA甲基化酶基因陽性接合子菌株質(zhì)粒DNA為模板,PCR擴(kuò)增β-內(nèi)酰胺酶基因;PCR產(chǎn)物純化測(cè)序,測(cè)序結(jié)果在Gen Bank中經(jīng)Blast程序比對(duì),明確基因型。探究16S r RNA甲基化酶基因與β-內(nèi)酰胺酶基因是否共轉(zhuǎn)移。結(jié)果1.201株黏質(zhì)沙雷菌中,對(duì)17種抗菌藥物的敏感率及耐藥率可以看出,黏質(zhì)沙雷菌對(duì)亞胺培南和美羅培南最敏感,敏感率高達(dá)98.51%。相比于其他三代頭孢類抗菌藥物,酶抑制劑復(fù)合制劑如哌拉西林-他唑巴坦和頭孢哌酮-舒巴坦以及四代頭孢菌素頭孢吡肟的敏感性較好,分別可達(dá)76.62%、65.63%及74.13%。喹諾酮類抗菌藥以加替沙星抗菌藥物敏感性最高,為77.11%,其次為左氧氟沙星敏感性達(dá)75.12%。本研究中檢測(cè)出135株對(duì)慶大霉素耐藥、58株對(duì)阿米卡星耐藥,二者耐藥率分別為:67.16%及28.8%,58株對(duì)阿米卡星耐藥的菌株同時(shí)對(duì)慶大霉素耐藥。58株阿米卡星耐藥菌株中45株來自痰,5株來自尿液,3株來自血液,3株來自分泌物,1株來自胸水,1株來自大便。2.58株同時(shí)對(duì)阿米卡星和慶大霉素耐藥的黏質(zhì)沙雷菌經(jīng)PCR檢測(cè)和DNA測(cè)序,7株為16S r RNA甲基化酶基因陽性,其中5株為arm A陽性,2株為rmt B陽性,所有菌株rmt A、rmt C、rmt D和npm A基因檢測(cè)均為陰性。16S r RNA甲基化酶基因陽性菌株數(shù)占總臨床分離黏質(zhì)沙雷菌株數(shù)的陽性率為3.5%(7/201),在耐阿米卡星菌株中的陽性率為12.1%(7/58)。3.7株16S r RNA甲基化酶基因陽性的菌株中有6株轉(zhuǎn)移接合成功,接合子經(jīng)生化鑒定為大腸埃希菌。經(jīng)PCR擴(kuò)增及DNA測(cè)序分析證實(shí),5株接合子攜帶arm A基因;1株接合子攜帶rmt B基因。藥敏結(jié)果表明,這6株接合子與受體菌相比,對(duì)阿米卡星等五種氨基糖苷類抗生素耐藥性明顯提高。接合子對(duì)第四代頭孢菌素以及碳青霉烯類均敏感。4.7株16S r RNA甲基化酶基因陽性的菌株經(jīng)PCR檢測(cè)β-內(nèi)酰胺酶基因顯示5株臨床分離株檢測(cè)出β-內(nèi)酰胺酶基因,最常見的基因型別為CTX-M。且PCR產(chǎn)物經(jīng)DNA測(cè)序表明4株CTX-M基因陽性,均為CTX-M-14型;2株TEM基因陽性,均為TEM-1型;2株OXA基因陽性,均為OXA-1型;1株SHV基因陽性,為SHV-5型。其中有3株菌株檢測(cè)出二種以上β-內(nèi)酰胺酶基因。5.對(duì)6株攜帶有16S r RNA甲基化酶基因的轉(zhuǎn)移接合成功接合子,以質(zhì)粒DNA為模板,經(jīng)PCR檢測(cè)出4株β-內(nèi)酰胺酶基因,其PCR產(chǎn)物經(jīng)DNA測(cè)序證實(shí):3株CTX-M基因陽性,均為CTX-M-14型;2株TEM基因陽性,均為TEM-1型;1株SHV基因陽性,為SHV-5型。結(jié)論1.藥敏結(jié)果顯示安徽地區(qū)臨床分離黏質(zhì)沙雷菌對(duì)多種抗菌藥物呈現(xiàn)不同程度耐藥,耐氨基糖苷類抗菌藥物的菌株呈現(xiàn)多重耐藥現(xiàn)象。2.研究報(bào)道了安徽地區(qū)存在攜帶有質(zhì)粒編碼的16S r RNA甲基化酶耐藥基因的黏質(zhì)沙雷菌,也是國內(nèi)第一次在黏質(zhì)沙雷菌中檢出16S r RNA甲基化酶基因。3.轉(zhuǎn)移接合實(shí)驗(yàn)成功的接合子對(duì)氨基糖苷類抗菌藥物的MIC較受體菌相比,對(duì)阿米卡星等五種氨基糖苷類抗生素耐藥性明顯提高。4.我國黏質(zhì)沙雷菌中已經(jīng)出現(xiàn)了質(zhì)粒介導(dǎo)的16S r RNA甲基化酶,且多與β-內(nèi)酰胺酶基因同時(shí)存在。5.質(zhì)粒介導(dǎo)的16S r RNA甲基化酶耐藥基因與β-內(nèi)酰胺酶常同時(shí)存在于同一株黏質(zhì)沙雷菌中,并可同時(shí)通過可接合性質(zhì)粒發(fā)生共轉(zhuǎn)移,造成耐藥基因在不同菌屬間擴(kuò)散。
[Abstract]:Objective to understand the Anhui region of 201 strains of clinical isolates of Serratia marcescens susceptibility and plasmid mediated 16S R RNA methylase detection rate and genotype, provide the basis for clinical rational use of antibiotics. The transfer of clinical isolates of Serratia marcescens plasmid mediated 16S R RNA methylase gene and beta lactamase genes. Materials and methods 201 strains of Serratia strains from clinical isolates from Anhui Province, bacterial resistance monitoring network monitoring center belongs to 34 hospitals (from Anhui, Anhui, Anhui different levels of hospitals) 2005 ~2012 in September of each year of inpatient and outpatient all kinds of clinical specimens. Drug sensitivity the quality control strains of Escherichia coli ATCC25922, conjugation experiment recipient bacterium Escherichia coli J53 AZR, the two are Anhui province bacterial resistance monitoring center preservation. Methods 1. MH agar dilution method was used measuring The sensitivity of clinical isolated strains to antimicrobial agents Sarre clay, total DNA extracted by E.coli ATCC bacteria and control the bacteria 25922.2. boiling method, alkaline lysis method of extracting plasmid DNA; polymerase chain reaction (PCR) amplification of plasmid mediated 16S R RNA methylase gene; PCR sequencing in Gen pure product, Bank by Blast the program comparison and analysis of sequencing results, with 16S R RNA genotype specific methylase gene and whether mutations in the.3. joint test with 16S R RNA methylase gene positive strains of Escherichia coli and J53AzR metastasis; biochemical identification of exconjugants and detected by PCR resistance gene, PCR DNA PCR product sequencing and determination; zygotic minimal inhibitory concentrations of antibiotics of Escherichia coli J53AzR by Muller-Hinton agar dilution method compared with total DNA (MIC).4. 16S R RNA methylase gene positive clinical isolates as template And the amplification of beta lactamase genes using PCR method; purification of PCR products sequencing, sequencing results in Gen Bank by Blast program comparison, analysis and comparison of 16S R RNA methylase genotypes and beta lactamase genotypes in 16S r relationship between.5. RNA methylase gene positive strains plasmid DNA as the zygote the template, PCR amplification of beta lactamase genes; purification of PCR products sequencing, sequencing results in Gen Bank by Blast program comparison, clear genotype. R explore 16S RNA methylase gene and beta lactamase genes were transferred. Results 1.201 strains of Serratia marcescens, sensitive and resistant rates of 17 antimicrobial rate can be seen, Serratia marcescens is most sensitive to imipenem and meropenem, the sensitive rate of 98.51%. compared to the other three generation cephalosporins, enzyme inhibitor such as piperacillin tazobactam and cefoperazone - sulbactam and Good sensitivity to the four generation cephalosporin cefepime, respectively 76.62%, 65.63% and 74.13%. of quinolones with gatifloxacin antibiotic sensitivity was 77.11%, the highest, followed by levofloxacin sensitivity of 75.12%. in this study detected 135 strains of gentamicin resistant, 58 strains of Amikacin resistance, the two resistance rates were: 67.16% and 28.8%, 58 strains of Amikacin resistant strains resistant to gentamicin and Amikacin.58 strains resistant strains 45 strains from 5 strains from sputum, urine, blood from 3 strains, 3 strains from 1 strains from secretions, pleural effusion, and 1 strains from stool.2.58 strains of Serratia marcescens in Amikacin and gentamicin resistance by PCR detection and DNA sequencing, 7 strains were 16S R RNA methylase gene positive, 5 of them were arm A positive, 2 were RMT B positive, all A RMT C strain RMT, RMT, D and NPM detection of A gene were The positive rate of.16S negative R RNA methylase gene positive strains in total clinical isolates of Serratia marcescens was 3.5% (7/201), the positive rate of resistant strains in Amikacin 12.1% (7/58) strain 16S R RNA methylase gene.3.7 positive strains in 6 strains by conjugation, zygote biochemical identification of Escherichia coli. After PCR amplification and DNA sequencing analysis confirmed that 5 strains of transconjugants carrying arm A gene; 1 strains of transconjugants carrying RMT B gene. Drug sensitivity results showed that these 6 strains of zygote and compared to recipient strains of amikacin five kinds of aminoglycoside antibiotic resistance was significantly improved. Zygote were sensitive to fourth generation cephalosporins and carbapenems.4.7 strain 16S R RNA methylase gene positive strains were detected by PCR beta lactamase gene showed that 5 strains of clinical isolates detected beta lactamase gene, genotype is the most common and CTX-M. The PCR products by DNA sequencing showed that the CTX-M gene of 4 strains were positive for type CTX-M-14; TEM gene was positive in 2 strains, were type TEM-1; OXA gene was positive in 2 strains, were type OXA-1; SHV gene was positive in 1 strains, SHV-5 type. There are 3 strains detected more than two kinds of beta lactamase.5. 16S R gene transfer RNA methyltransferase genes successfully engage transconjugants against 6 strains carried by plasmid DNA as template, the PCR detection of 4 strains of beta lactamase genes, the PCR product was confirmed by DNA sequencing: 3 strains of CTX-M gene were CTX-M-14 positive, 2 strains of TEM gene positive type; that is TEM-1 type; SHV gene was positive in 1 strains, SHV-5 1.. Conclusion according to the results of drug susceptibility in Anhui clinical isolates of Serratia marcescens to antibiotics showed different degrees of resistance, resistance to aminoglycoside antibiotics showed multidrug resistance strains of.2. have reported carrying the plasmid encoding 16S R R in Anhui area Serratia marcescens NA methylase gene, is also the first time in Serratia marcescens in detection of 16S r methylase gene RNA.3. conjugation experiment successfully zygote to aminoglycoside antibiotics MIC compared with the host strain of amikacin five kinds of aminoglycoside antibiotic resistance has been significantly improved China's.4. of Serratia marcescens plasmid mediated 16S R RNA methylase, and with beta lactamase genes exist at the same time 16S R RNA methylase gene and plasmid mediated.5. beta lactamases often coexist in the same strains of Serratia marcescens in sticky matter, and at the same time the conjugative plasmid transfer occurred, resulting in diffusion resistance genes in different bacterial species.

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

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本文編號(hào)：1608360