临床多重耐药铜绿假单胞菌中VEB-1基因盒结构及其对整合子捕获频率影响的研究

发布时间：2018-01-16 15:17

本文关键词：临床多重耐药铜绿假单胞菌中VEB-1基因盒结构及其对整合子捕获频率影响的研究　出处：《复旦大学》2010年硕士论文　论文类型：学位论文

【摘要】： 近年来由于抗生素在人类、动物、植物中的滥用,病原体对抗生素耐药日趋严重,传染性疾病的治疗逐年变得更加有挑战性,特别是由条件致病菌铜绿假单胞菌所引起的感染,它能够迅速产生多重耐药性,使多个抗生素失去了临床效用。虽然移动元件所引起耐药的机制一直都是我们关心的一个问题,但最困难的挑战是我们如何面对治疗感染过程中铜绿假单胞菌迅速引起的耐药。对细菌抗生素耐药机制的研究,导致了包括转座子和共轭质粒在内的许多自然移动元件的发现,而通过对这些元件序列进行比较分析,最终发现了整合子(integron)的存在。整合子起初在病原菌的移动性元件中被发现和鉴定,整合子现在被认为是一种古老的结构并且能够存在于不同菌种中,在如今已经被测序鉴定的菌种中,有大约9%含有整合子。总的来说,基因盒有着非常高的多样性,暗示着整合子-基因盒系统在提高大多数细菌对环境适应性上都起到很大的作用。整合子定位于革兰阴性杆菌的染色体、转座子或具有广泛宿主的质粒上,整合子是近年来新发现的一种可水平传递耐药基因的DNA元件。整合子在细菌中通过位点特异性重组来捕获基因盒,并且通过启动子来确保这些耐药基因的表达。同时整合子可谓与质粒,或者自身周围转座子的一个组成部分而发生转移,使耐药基因发生播散。本研究通过对临床多重耐药铜绿假单胞菌整合子中的VEB-1基因盒的筛查与结构分析,并利用一种简便的实时定量PCR的方法,来研究VEB-1基因盒的结构特点以及其中的某些结构对整合子捕获效率的影响,从而分析解释一些临床耐药性基因盒的播散现象。第一部分：临床菌株中含有VEB-1型基因盒的整合子筛选 VEB-1基因盒存在于整合子上,并且编码超广谱内酰胺酶VEB-1 (Vietnamese extended-spectrum b-lactamase)。VEB-1基因盒最初在越南临床分离的一株大肠埃希菌株中被发现,随后也是在越南的2株临床分离的铜绿假单胞菌中被检测和鉴定。在泰国、中国等东南亚地区的铜绿假单胞菌中也检测到该型ESBL。本研究选取了2004年6～11月间,华山医院微生物实验室经梅里埃仪鉴定后收集并鉴定的37株全耐药铜绿假单胞菌株,设计引物扩增其中含有VEB-1基因盒的菌株,再利用高分辨率敏感曲线HRM来分型,辨别VEB型基因盒中的VEB-1与VEB-3基因盒。设计引物扩增临床分离VEB-1基因盒的5cs端,了解其基因环境,然后进行测序分析。最后,我们在37株全耐药铜绿假单胞菌中,发现有35株存在VEB型基因,说明VEB型基因盒在多重耐药细菌中分布很广泛。经过hrm分型,并测序确定型别后,得到9株VEB-1型基因盒。经过PCR扩增以及测序并Blast比对之后,了解到9株细菌中有8株5cs都含有IS1999插入序列。再经过ERIC PCR鉴定后,只有3株为同一型别,其他几株都互为不同型别。提示IS1999广泛分布于多重耐药铜绿假单胞菌VEB-1基因盒上游。第二部分：研究VEB-1上游IS1999结构对整合子捕获效率的影响根据第一部分的研究结果,所有VEB-1基因盒都位于整合上,并且为第一位整合基因盒,另外有一段长lkb左右的插入序列广泛存在于多重耐药铜绿假单胞菌中VEB-1基因盒的上游,引起了我们对此结构与整合子整合效率的兴趣。有前人的研究显示,IS1999本身编码402氨基酸蛋白的转座酶,其中与IS10有71%的氨基酸同源性,所以也属于IS4插入序列家族。IS1999片段总共有1,328bp,有21-bp的不完全重复片段位于两侧,并且可以在转座之后生成一个9bp的重复目标片段,介导转座子以及转座片段的水平移动。本研究用长引物法构建lacZα基因盒,并连接到载体pACYC184质粒中,命名为pAC-lac。再从临床分离铜绿假单胞菌684号菌株中扩增出含有IS1999插入序列的attI位点VEB-1基因盒片段,从961号菌株中扩增出上游是普通attI位点的VEB-1基因盒片段。把这两个片段构建进入pAC-lac,利用相对定量PCR的方法,比较出两者不同的基因盒捕获频率。结果发现,含有IS1999片段的整合子,其捕获基因盒插入到VEB-1基因盒之前的能力与普通的整合子结构相比大大下降,由此可推测,IS1999结构可阻断有效整合的途径,阻止整合子捕获其他基因盒,从而保持VEB-1在整合子的优势位置。第三部分：比较不同attC位点结构对VEB-1基因盒整合效率的影响 Ⅰ类整合子包括编码整合酶的基因(IntI)和毗邻的一个重组位点(attI)。基因盒并不是整合子必要的元件,不过一旦被整合,则他们也成为了整合子的一部分,依赖在整合子上的启动子(Part)来表达,启动子也是整合子5’保守区间的一部分。另外,整合子中的其他序列(比如3’端)可以是保守的,但并不是所有整合子中都具有同源性。基因盒包含了一个编码序列。在3’末端存在一段碱基,被称作59-碱基序列,或者attC位点。本研究比较VEB-1与VIM-2这两种临床上常见的播散程度不相同的基因盒,并且构建将VEB-1基因盒中的attC位点换成VIM-2的attC位点后的基因盒,然后利用一种简单的定量PCR方法,设计合理引物,来扩增发生整合的基因盒,从而测定不同基因盒之间整合频率是否有差别；并且可以分析在基因盒中哪一部分对整合效率有所影响。并且考虑到本研究之前的实验,菌株皆处于营养丰富的理想环境中,这与临床上菌株的生存情况不符,所以我们检测对照菌株进行的MIC,然后取低于MIC的抗生素浓度,来进行诱导,观察他们的整合频率还是否存在区别,可基本上去除抗生素的选择效应。结果表明,在没有抗生素的情况下,VEB-1的整合频率小于VIM-2基因盒以及置换attC位点的59VIM-VEB基因盒,说明整合效率与attC存在一定关系,而与编码的序列关系不明显。在加入Amp1μg/ml,其他变量都控制的条件下,这三者之间的整合效率的差别又消失了。虽然这些基因盒的attC元件以及结构基因序列都不尽相同,但可能加入抗生素之后,细菌自身调节触发其他自救系统发挥作用,从而影响到了整合的过程,这一部分原因还需要一些后续的研究来分析。
[Abstract]:In recent years , due to the abuse of antibiotics in humans , animals and plants , pathogens are becoming more and more challenging for antibiotic resistance , and the treatment of infectious diseases has become more challenging year by year , especially by the condition pathogenic bacteria Pseudomonas aeruginosa , which can rapidly generate multiple drug resistance , which can cause multiple antibiotics to lose clinical utility . Although the mechanism of resistance caused by moving elements has always been a problem of our concern , the most difficult challenge is how we face the rapid drug resistance caused by Pseudomonas aeruginosa in the course of treatment infection . The research on the mechanism of resistance to antibiotics has led to the discovery of many natural moving elements , including transposon and conjugate plasmids , and the existence of integron has been found by comparing these elements . The integrons are now considered to be an ancient structure and can be present in different strains . Through screening and structure analysis of VEB - 1 gene cassette in clinical multiple drug resistant Pseudomonas aeruginosa integron , a simple and convenient method of real - time quantitative PCR was used to study the structural characteristics of VEB - 1 gene cassette and the influence of some structures on the efficiency of integrated sub - capture , thus the dissemination of some clinical drug - resistant gene cassettes was analyzed . Part I : Screening of Integrals Containing VEB - 1 Gene Box in Clinical Strain The VEB - 1 gene cassette is present on the integron and encodes the extended - spectrum b - 1 gene . VEB - 1 gene cassette was originally found in a strain of E . coli isolated clinically in Viet Nam and was subsequently detected and identified in 2 clinically isolated strains of Pseudomonas aeruginosa in Vietnam . This type of ESBL was also detected in Pseudomonas aeruginosa in Southeast Asia , such as Thailand , China . In this study , 37 full - resistant Pseudomonas aeruginosa strains were collected and identified in the microbiological laboratory of Huashan Hospital from June to November 2004 . The primers were designed to amplify the strains of VEB - 1 and VEB - 3 . The second part : study the effect of the IS1999 structure upstream of VEB - 1 on the efficiency of integrated sub - capture According to the results of the first part , all VEB - 1 gene cassettes are located on the integration , and the first integration gene cassette is the first integration gene box . In addition , a length of about lkb insertion sequence exists in the upstream of VEB - 1 gene cassette in multiple drug - resistant Pseudomonas aeruginosa , which leads to our interest in integrating the structure with the integrated sub - integration efficiency . The IS1999 fragment has a total of 1,328 bp , a 21 - bp incomplete repeat fragment is located on both sides , and a 9bp repeat target fragment can be generated after the transposition , thereby mediating the horizontal movement of the transposon and the transposition fragment . The recombinant plasmid pACYC184 was cloned into vector pACYC184 . The vector pACYC184 was inserted into pACYC184 plasmid and named pAC - lac . The third part : Compare the effect of different attC site structure on the integration efficiency of VEB - 1 gene cassette The class I integrons include a gene ( IntI ) encoding an integration enzyme and a contiguous one of the recombination sites ( attI ) . The gene cassette is not an integral element , but once integrated , they also become part of the integron , depending on the promoter ( Part ) on the integron . In addition , other sequences in the integron ( such as the 3 ' end ) may be conserved but not all of the integrons . The gene cassette contains a coding sequence . A base sequence is present at the 3 ' end , referred to as a 59 - base sequence , or attC site . In this study , we compare VEB - 1 gene cassette with vim - 2 gene cassette , and construct a gene cassette after the attC site in VEB - 1 gene cassette is changed to attC locus of vim - 2 , and then use a simple quantitative PCR method to amplify the integrated gene cassette , so as to determine whether the integration frequency is different between different gene cassettes . The results showed that the integration efficiency of VEB - 1 was less than that of vib - 2 gene cassette and substitution attC locus in the absence of antibiotics . The difference of integration efficiency between these three genes was not obvious . Although the attC elements and structural gene sequences of these gene cassettes were different , it was possible to adjust the function of the other self - rescue system after the addition of Amp1 渭g / ml and other variables , thus affecting the integration process , which also needed some subsequent studies to analyze .

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

【相似文献】