本文選題：大腸桿菌 + β-內(nèi)酰胺酶類　； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究目的了解臨床分離的產(chǎn)超廣譜-β內(nèi)酰胺酶大腸埃希菌(ESBLs-producing Escherichia coli,ESBL-EC)的散布特性及其耐藥情況,在合理應(yīng)用抗菌藥物方面供臨床參考;通過對(duì)ESBL-EC菌株β-內(nèi)酰胺類耐藥基因和可移動(dòng)遺傳元件進(jìn)行檢測(cè),了解ESBL-EC菌株基因分型情況和可移動(dòng)遺傳標(biāo)記存在狀況;通過轉(zhuǎn)化來驗(yàn)證ESBL可否通過質(zhì)粒來轉(zhuǎn)移播散,并研究轉(zhuǎn)化子特征,探討ESBL-EC菌株主要的耐藥傳播機(jī)制,以此作為臨床防控ESBL的播散的參考依據(jù)。研究方法1收集2014年1月-2014年12月從臨床各個(gè)科室分離的大腸埃希菌(Escherichia coli,E.coli)共409株,應(yīng)用VITEK-Compact 2微生物分析儀(法國bio Merieux)所配套的細(xì)菌鑒定卡和藥敏卡對(duì)細(xì)菌進(jìn)行鑒定及藥敏。ESBLs菌的檢測(cè)采取雙紙片法,由美國臨床實(shí)驗(yàn)室標(biāo)準(zhǔn)化委員會(huì)(NCCLs)推薦使用。最終判讀根據(jù)美國臨床實(shí)驗(yàn)室標(biāo)準(zhǔn)化研究所(CLSI 2014年版)的標(biāo)準(zhǔn)進(jìn)行。2采用聚合酶鏈?zhǔn)椒磻?yīng)(Polymerase Chain Reaction,PCR)對(duì)臨床分離的ESBL-EC菌株的19種β-內(nèi)酰胺酶基因,3種插入序列元件(ISEcp、ISCR1、IS26),6種轉(zhuǎn)座子遺傳標(biāo)記(mer A、tnp M、tnp R、tnp U、tnp A7、tnp A21),3種整合子遺傳標(biāo)記(int I 1、int I 2、int I 3)進(jìn)行檢測(cè),對(duì)陽性基因PCR產(chǎn)物進(jìn)行測(cè)序,并對(duì)測(cè)序結(jié)果進(jìn)行序列比對(duì)驗(yàn)證。3以ESBLs基因陽性的E.coli為質(zhì)粒供體菌,感受態(tài)大腸埃希菌DH5α為質(zhì)粒受體菌,進(jìn)行質(zhì)粒轉(zhuǎn)化試驗(yàn),VITEK-Compact 2系統(tǒng)對(duì)轉(zhuǎn)化子進(jìn)行細(xì)菌鑒定與藥敏試驗(yàn)。研究結(jié)果1臨床分布情況:409株臨床分離的E.coli中檢出ESBL-EC菌株98株,23.96%(98/409)。ESBL-EC菌株主要來源于50-69歲年齡段患者(36.73%),其次是≥70歲年齡段的患者(28.57%);主要來源科室為泌尿外科(22.45%),其次是婦科(10.20%)、骨科(9.18%)和普通外科(9.18%);標(biāo)本主要分離自尿液(47.96%),其次是陰道分泌物(11.22%)和血液(10.20%)。2藥敏情況:ESBL-EC菌株對(duì)臨床經(jīng)常應(yīng)用的抗生素的耐藥情況如下:對(duì)氨芐西林、哌拉西林、頭孢噻肟、頭孢曲松、頭孢唑啉和頭孢他啶的耐藥率均高于90%,對(duì)氨曲南、復(fù)方新諾明、環(huán)丙沙星、妥布霉素、慶大霉素和左氧氟沙星的耐藥率均高于60%,對(duì)頭孢吡肟、阿莫西林/克拉維酸和呋喃妥因的耐藥率為分別為51.02%、46.93%和34.69%,對(duì)頭孢西丁和哌拉西林/他唑巴坦耐藥率相對(duì)較低,分別為16.33%和11.22%,以上幾種抗生素的耐藥率與非產(chǎn)ESBLs菌株耐藥率的差異均具有統(tǒng)計(jì)學(xué)意義(P0.05)。ESBL-EC菌株對(duì)阿米卡星、替加環(huán)素、亞胺培南均較敏感。3 ESBL-EC基因型檢出情況:98株菌中β-內(nèi)酰胺酶基因bla TEM、bla CTX-M-9、bla CTX-M-1、bla CTX-M-2和bla SHV陽性率分別為61.22%、53.06%、32.65%、4.08%和3.06%,7株未檢出基因型(7.14%)。基因分布以TEM+CTX-M-9基因陽性組(共31株)最多見,占31.63%。4 ESBL基因PCR產(chǎn)物測(cè)序顯示:61株bla TEM陽性菌株均為bla TEM-1亞型。52株bla CTX-M-9群陽性菌株中,主要的基因型為bla CTX-M-14,占94.2%(49/52),其他3株基因型分別為bla CTX-M-19、bla CTX-M-21、bla CTX-M-38。32株bla CTX-M-1群陽性菌株中,主要的基因型為bla CTX-M-55,占59.4%(19/32);其次為bla CTX-M-15和bla CTX-M-64型,分別占31.3%(10/32)和9.4%(3/32)。5可移動(dòng)遺傳元件攜帶情況:98株ESBL-EC中,47.96%(47/98)的分離菌株為Ⅰ類整合子陽性,2.04%(2/98)的分離菌株為Ⅱ類整合子陽性,此中有1株同時(shí)含Ⅰ類和Ⅱ類整合子,未檢出Ⅲ類整合子。整合子陽性菌株僅對(duì)四環(huán)素和復(fù)方新諾明的耐藥率高于整合子陰性的菌株(P0.05)。插入序列元件和轉(zhuǎn)座子遺傳標(biāo)記檢出ISEcp1、IS26、ISCR1、Tnp A21和Tnp M基因,以ISEcp1檢出率最高,達(dá)97.96%。6轉(zhuǎn)化試驗(yàn)結(jié)果顯示:42株ESBL-EC菌中,有39株菌轉(zhuǎn)化后能在在篩選平板上生長,其中有36株成功把ESBL基因轉(zhuǎn)到轉(zhuǎn)化子中,一些野生株攜帶的ESBL基因并不能完全轉(zhuǎn)到轉(zhuǎn)化子中,bla CTX-M轉(zhuǎn)移成功者較多見,bla TEM多數(shù)未能成功轉(zhuǎn)移7所有ESBL-EC野生株和相應(yīng)的轉(zhuǎn)化子對(duì)頭孢噻肟、頭孢曲松和氨芐西林完全耐藥,可見耐藥性在兩者之間進(jìn)行了傳播。研究結(jié)論1 ESBL-EC菌株耐藥情況嚴(yán)重,具有多耐藥性,對(duì)青霉素類、頭孢菌素類、氨曲南、氨基糖苷類(除阿米卡星外)、喹諾酮類等抗菌素耐藥率均較高,對(duì)碳青霉烯類抗生素、替加環(huán)素及哌拉西林/他唑巴坦極敏感。2本院ESBLs基因型以CTX-M型(81.63%)是最常見,TEM型次之(61.22%),大部分臨床分離菌株檢出2種或2種以上的基因型(60.20%),以TEM+CTX-M-9陽性組(31.63%)最常見,可見多重耐藥在臨床分離菌廣泛存在。同時(shí)攜帶TEM和CTX-M-9基因是本院ESBL-EC菌株產(chǎn)生的主要原因。本院流行的bla CTX-M基因亞型主要為bla CTX-M-14,其次是bla CTX-M-55和bla CTX-M-15;bla TEM基因亞型主要是bla TEM-1。3Ⅰ類整合子在ESBL-EC菌株中分布廣泛,本院ESBLs的播散可能與ISEcp1基因的存在密切相關(guān)。4位于質(zhì)粒上的ESBL基因可通過轉(zhuǎn)化的方式進(jìn)行傳播,以bla CTX-M尤為明顯;ESBL-EC菌株對(duì)頭孢噻肟、頭孢曲松和氨芐西林的耐藥性與bla CTX-M的存在密切關(guān)聯(lián)。
[Abstract]:The purpose of this study is to understand the dispersal characteristics of ESBLs-producing Escherichia coli (ESBL-EC) and its drug resistance in clinical isolation, and to provide clinical reference for the rational use of antibiotics, and the detection of the beta lactam resistance gene and mobile genetic components of the ESBL-EC strain to understand ESBL-EC The genotyping of strains and the existence of mobile genetic markers; through transformation to verify whether ESBL can transfer broadcasting through plasmids, and study the characteristics of the transformants, and explore the main mechanism of drug resistance of ESBL-EC strains, as a reference for the dissemination of clinical prevention and control of ESBL. Method 1 collected from December January 2014, from clinical to clinical 409 strains of Escherichia coli (Escherichia coli, E.coli) isolated from various departments were used for identification of bacteria and the detection of drug sensitive.ESBLs bacteria by VITEK-Compact 2 microorganism analyzer (France bio Merieux), which were recommended by the United States national clinical laboratory standardization committee (NCCLs). Finally, 19 beta lactamase genes, 3 insertion sequences (ISEcp, ISCR1, IS26), and 6 transposon genetic markers (ISEcp, ISCR1, IS26), and 6 kinds of transposon genetic markers (ISEcp, ISCR1, IS26), were carried out according to the standards of the American clinical laboratory standardization institute (CLSI 2014 Edition) and.2 used polymerase chain reaction (Polymerase Chain Reaction, PCR). U, TNP A7, TNP A21), the 3 integron genetic markers (int I 1, int I 2, int I 3) were detected, and the positive gene PCR products were sequenced. Bacterial identification and drug sensitivity test were performed on the transformants. Results 1 Clinical Distribution: 98 strains of ESBL-EC strains were detected in 409 clinical isolates of E.coli. 23.96% (98/409).ESBL-EC strains were mainly from 50-69 age group (36.73%), followed by older age group (28.57%); the main source section was Department of Urology (22.45%). The second was gynecologic (10.20%), Department of orthopedics (9.18%) and general surgery (9.18%); specimens were mainly separated from urine (47.96%), followed by vaginal secretions (11.22%) and blood (10.20%).2 drug sensitivity: the drug resistance of the ESBL-EC strain to clinical frequently used antibiotics was as follows: ampicillin, piperacillin, cefotaxime, ceftriaxone, cefazolin and The drug resistance rate of ceftazidime was higher than 90%. The resistance rates for amamethamine, compound noxoxin, ciprofloxacin, tobramycin, gentamicin and levofloxacin were all higher than 60%. The resistance rates for cefepime, amoxicillin / clavulanic acid and furantoin were 51.02%, 46.93% and 34.69%, respectively, and resistance to cefoxitin and piperacillin / tazobactam. The rates were relatively low, 16.33% and 11.22% respectively. The differences of resistance rates between the above antibiotics and non producing ESBLs strains were all statistically significant (P0.05).ESBL-EC strains were more sensitive to Amikacin, tegocyclin and imipenem than the sensitive.3 ESBL-EC genotypes: 98 strains of beta lactamase gene bla TEM, BLA CTX-M-9, BLA C. The positive rates of TX-M-1, BLA CTX-M-2 and Bla SHV were 61.22%, 53.06%, 32.65%, 4.08% and 3.06%, 7 strains were not genotypes (7.14%). The distribution of the gene distribution in the TEM+CTX-M-9 gene positive group (31 strains) was the most, and the 31.63%.4 ESBL gene PCR product sequencing showed that 61 bla TEM positive strains were all of the BLA isolates. The main genotypes were bla CTX-M-14, accounting for 94.2% (49/52), and the other 3 genotypes were bla CTX-M-19, BLA CTX-M-21, BLA CTX-M-38.32 strain bla CTX-M-1 group, and the main genotype was bla, accounting for 59.4%. Among the 98 strains of ESBL-EC, 47.96% (47/98) isolates were positive for class I integron, and 2.04% (2/98) isolates were type II integron positive. 1 of them included class I and class II integrons, and no class integrons were detected. The resistance rate of integron positive strains only to four cyclin and compound novomine was higher than that of integron negative bacteria. Strain (P0.05). ISEcp1, IS26, ISCR1, Tnp A21 and Tnp M genes were detected by inserting sequence elements and transposon genetic markers. The detection rate of ISEcp1 was the highest. The result of 97.96%.6 transformation test showed that among 42 ESBL-EC bacteria, 39 strains could grow on the screen, and 36 of them succeeded in transferring ESBL genes to the transformants and some wild plants. The ESBL gene could not be completely transferred to the transformant. The successful transfer of BLA CTX-M was more common, and most of the BLA TEM failed to successfully transfer 7 of all ESBL-EC wild strains and corresponding transformants to cefotaxime, ceftriaxone and ampicillin, and the resistance was transmitted between the two. The study concluded that the 1 ESBL-EC strains were resistant to drug resistance. The rate of resistance to penicillins, cephalosporins, aminoglycosides, aminoglycosides (except Amikacin), quinolones and other antibiotics were high. The ESBLs genotypes of carbapenems, tetracycline and piperacillin / tazobactam were the most common and TEM type (81.63%) was the most common and TEM type (61) was the most common (81.63%). .22%), most clinically isolated strains detected 2 or more than 2 genotypes (60.20%), the most common in TEM+CTX-M-9 positive group (31.63%), and multidrug resistance widely existed in clinical isolates. Meanwhile, TEM and CTX-M-9 genes were the main causes of ESBL-EC strain in our hospital. The main bla CTX-M subtype in our hospital was BLA CTX-M-14. The second is bla CTX-M-55 and Bla CTX-M-15, and the BLA TEM gene subtype is mainly the BLA TEM-1.3 I integron in the ESBL-EC strain. The dissemination of ESBLs is probably closely related to the existence of ISEcp1 gene. The drug resistance of cefotaxime, ceftriaxone and ampicillin is closely related to the presence of BLA CTX-M.
【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】：2017
【分類號(hào)】：R446.5

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