产ESBLs大肠埃希菌耐药基因水平转移元件的研究

发布时间：2018-06-01 17:29

本文选题：大肠杆菌 + β-内酰胺酶类　；参考：《安徽医科大学》2017年硕士论文

【摘要】：研究目的了解临床分离的产超广谱-β内酰胺酶大肠埃希菌(ESBLs-producing Escherichia coli,ESBL-EC)的散布特性及其耐药情况,在合理应用抗菌药物方面供临床参考;通过对ESBL-EC菌株β-内酰胺类耐药基因和可移动遗传元件进行检测,了解ESBL-EC菌株基因分型情况和可移动遗传标记存在状况;通过转化来验证ESBL可否通过质粒来转移播散,并研究转化子特征,探讨ESBL-EC菌株主要的耐药传播机制,以此作为临床防控ESBL的播散的参考依据。研究方法1收集2014年1月-2014年12月从临床各个科室分离的大肠埃希菌(Escherichia coli,E.coli)共409株,应用VITEK-Compact 2微生物分析仪(法国bio Merieux)所配套的细菌鉴定卡和药敏卡对细菌进行鉴定及药敏。ESBLs菌的检测采取双纸片法,由美国临床实验室标准化委员会(NCCLs)推荐使用。最终判读根据美国临床实验室标准化研究所(CLSI 2014年版)的标准进行。2采用聚合酶链式反应(Polymerase Chain Reaction,PCR)对临床分离的ESBL-EC菌株的19种β-内酰胺酶基因,3种插入序列元件(ISEcp、ISCR1、IS26),6种转座子遗传标记(mer A、tnp M、tnp R、tnp U、tnp A7、tnp A21),3种整合子遗传标记(int I 1、int I 2、int I 3)进行检测,对阳性基因PCR产物进行测序,并对测序结果进行序列比对验证。3以ESBLs基因阳性的E.coli为质粒供体菌,感受态大肠埃希菌DH5α为质粒受体菌,进行质粒转化试验,VITEK-Compact 2系统对转化子进行细菌鉴定与药敏试验。研究结果1临床分布情况:409株临床分离的E.coli中检出ESBL-EC菌株98株,23.96%(98/409)。ESBL-EC菌株主要来源于50-69岁年龄段患者(36.73%),其次是≥70岁年龄段的患者(28.57%);主要来源科室为泌尿外科(22.45%),其次是妇科(10.20%)、骨科(9.18%)和普通外科(9.18%);标本主要分离自尿液(47.96%),其次是阴道分泌物(11.22%)和血液(10.20%)。2药敏情况:ESBL-EC菌株对临床经常应用的抗生素的耐药情况如下:对氨苄西林、哌拉西林、头孢噻肟、头孢曲松、头孢唑啉和头孢他啶的耐药率均高于90%,对氨曲南、复方新诺明、环丙沙星、妥布霉素、庆大霉素和左氧氟沙星的耐药率均高于60%,对头孢吡肟、阿莫西林/克拉维酸和呋喃妥因的耐药率为分别为51.02%、46.93%和34.69%,对头孢西丁和哌拉西林/他唑巴坦耐药率相对较低,分别为16.33%和11.22%,以上几种抗生素的耐药率与非产ESBLs菌株耐药率的差异均具有统计学意义(P0.05)。ESBL-EC菌株对阿米卡星、替加环素、亚胺培南均较敏感。3 ESBL-EC基因型检出情况:98株菌中β-内酰胺酶基因bla TEM、bla CTX-M-9、bla CTX-M-1、bla CTX-M-2和bla SHV阳性率分别为61.22%、53.06%、32.65%、4.08%和3.06%,7株未检出基因型(7.14%)。基因分布以TEM+CTX-M-9基因阳性组(共31株)最多见,占31.63%。4 ESBL基因PCR产物测序显示:61株bla TEM阳性菌株均为bla TEM-1亚型。52株bla CTX-M-9群阳性菌株中,主要的基因型为bla CTX-M-14,占94.2%(49/52),其他3株基因型分别为bla CTX-M-19、bla CTX-M-21、bla CTX-M-38。32株bla CTX-M-1群阳性菌株中,主要的基因型为bla CTX-M-55,占59.4%(19/32);其次为bla CTX-M-15和bla CTX-M-64型,分别占31.3%(10/32)和9.4%(3/32)。5可移动遗传元件携带情况:98株ESBL-EC中,47.96%(47/98)的分离菌株为Ⅰ类整合子阳性,2.04%(2/98)的分离菌株为Ⅱ类整合子阳性,此中有1株同时含Ⅰ类和Ⅱ类整合子,未检出Ⅲ类整合子。整合子阳性菌株仅对四环素和复方新诺明的耐药率高于整合子阴性的菌株(P0.05)。插入序列元件和转座子遗传标记检出ISEcp1、IS26、ISCR1、Tnp A21和Tnp M基因,以ISEcp1检出率最高,达97.96%。6转化试验结果显示:42株ESBL-EC菌中,有39株菌转化后能在在筛选平板上生长,其中有36株成功把ESBL基因转到转化子中,一些野生株携带的ESBL基因并不能完全转到转化子中,bla CTX-M转移成功者较多见,bla TEM多数未能成功转移7所有ESBL-EC野生株和相应的转化子对头孢噻肟、头孢曲松和氨苄西林完全耐药,可见耐药性在两者之间进行了传播。研究结论1 ESBL-EC菌株耐药情况严重,具有多耐药性,对青霉素类、头孢菌素类、氨曲南、氨基糖苷类(除阿米卡星外)、喹诺酮类等抗菌素耐药率均较高,对碳青霉烯类抗生素、替加环素及哌拉西林/他唑巴坦极敏感。2本院ESBLs基因型以CTX-M型(81.63%)是最常见,TEM型次之(61.22%),大部分临床分离菌株检出2种或2种以上的基因型(60.20%),以TEM+CTX-M-9阳性组(31.63%)最常见,可见多重耐药在临床分离菌广泛存在。同时携带TEM和CTX-M-9基因是本院ESBL-EC菌株产生的主要原因。本院流行的bla CTX-M基因亚型主要为bla CTX-M-14,其次是bla CTX-M-55和bla CTX-M-15;bla TEM基因亚型主要是bla TEM-1。3Ⅰ类整合子在ESBL-EC菌株中分布广泛,本院ESBLs的播散可能与ISEcp1基因的存在密切相关。4位于质粒上的ESBL基因可通过转化的方式进行传播,以bla CTX-M尤为明显;ESBL-EC菌株对头孢噻肟、头孢曲松和氨苄西林的耐药性与bla CTX-M的存在密切关联。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5

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