1800株G-杆菌插入序列共同区和整合子的耐药机制研究
摘要
研究背景与目的
随着抗生素的应用越来越广泛,细菌的耐药性就越来越严重。我国是世界上滥用抗生素最为严重的地区之一,细菌的耐药性问题已经引起人们的高度关注。我国目前临床上分离的细菌对常规抗生素的耐药率逐年上升,而且细菌在异常的抗生素选择压力下,产生多重耐药的情况不断出现,因此研究细菌耐药机制是十分必要和紧迫的。
细菌耐药的生化机制非常复杂,通常是细菌对药物作用靶位、渗透性的改变,产生灭活酶和钝化酶,排外系统的作用以及生物膜的产生等方面。而从分子生物学角度认识细菌的耐药机制,主要集中于基因突变和耐药基因水平传播。基因转移是细菌耐药性迅速扩散的主要原因。携带耐药基因的基因转移元件有：质粒、转座子、整合型噬菌体和整合子,以及最近几年才发现的插入序列共同区(ISCR)。耐药基因可以通过接合、转化、转导和转座等方法在细菌间转移。通过转座的方式,整合子、ISCR和转座子可以导致多个耐药基因在单个质粒中聚集成束,这是多重耐药株产生的重要原因。ISCR由"insertion sequences" (ISs)和"common regions" (CRs)得来。它既具有插入序列(ISs)的特点,也具有共同区(CRs)的特点。插入序列属于高度可移动性转座因子,是对基因组一个或多个靶位点具有插入能力的小分子DNA片段,并常可以移动邻近的基因。共同区域(CRs)是1993年在复杂性Ⅰ类整合子In6 andIn7中作为一个长约2154bp的DNA片段而首先发现的。CRs是一类IS91-like因子家族的延伸成员,它具有与相似位置IS91-like因子的两个主要特点：缺少末端反向重复序列和它们转移邻近耐药基因是通过滚环复制的形式进行的,另外,CR序列还保留着IS91的第三个特点,复制终止子序列terIS。相应地,为了强调它们作为插入序列的同源性和它们被发现的历史,研究假设它们是特定的ISCR因子。用来区分CRs的数字被保留下来以便区分不同的ISCRs,因此,CR1对应ISCR1, CR2对应ISCR2等。
ISCRs包含1个ORF序列,orf513。orf 513基因编码513个氨基酸序列产物,其功能与转座酶相似,可以转移基因盒。在复杂性Ⅰ类整合子中,orf513下游可以有由一种或以上的耐药基因组成的基因盒,紧接着是qac/sul的3'-CS。ISCR常见的可以分为6类,目前研究较多的为1,2,3,4类,研究发现这几种类型的ISCR均与耐药基因的传播相关。其中,最为重要的是ISCR1,它经常出现在复杂性Ⅰ类整合子的基因序列中。ISCR1介导一系列的转座事件发生,转座不同长度的耐药基因盒,如catA2、dfrA、qnr和各种blaCMY基因。ISCR1对耐药基因的传播发挥重要的作用。
另一个重要的基因转移元件是整合子。整合子是细菌的DNA片段,它的独特结构可捕获并整合外源性基因,并使之转变为功能性基因的表达单位。其整合的基因盒大多与耐药基因有关,对细菌基因组进化和耐药性具有重要意义。根据整合酶不同进行分类,整合子主要分为Ⅰ～Ⅳ类。其中,Ⅰ类整合子最常见也是最早被发现的整合子,其基因盒的移动已得到证实。Ⅰ类整合子在革兰阴性杆菌中通过可变区整合一个或多个耐药基因盒介导宿主菌多重耐药,在临床检测中具有重要的意义。这类整合子5’-保守区有编码Ⅰ型整合酶的基因intⅡ、重组位点attⅡ和启动子Pant。3'-保守区由3个开放阅读框组成(qacEΔI, sulI和ORF5),有的整合子没有3’-保守区。Ⅰ类整合子在革兰阴性菌中广泛分布、检出率最高,在许多临床分离的菌株包括肠杆菌科中的埃希菌属、克雷伯菌属和非发酵革兰阴性杆菌中的假单胞菌属和不动杆菌属等都能检出。Ⅰ类整合子可以携带多种耐药基因,如β-内酰胺类、氨基糖昔类、甲氧苄啶、氯霉素、链霉素、喹诺酮和季铵类化合物等耐药基因,对耐药基因在革兰阴性杆菌中的传播十分重要。
ISCR1经常出现在复杂性Ⅰ类整合子的基因序列中。ISCR1上游序列通常是相同的,而在ISCR1与Ⅰ类整合子3’-保守区的连接点序列是不变的。这些都表明ISCR1很符合复杂性Ⅰ类整合子的特征。无论是ISCR1连同耐药基因插入到普通的一类整合子组成的复杂性整合子,还是带有两个拷贝ISCR1因子的复杂性整合子,它们都带有许多耐药性因子,具有广泛传播这些耐药基因的潜能。复杂性Ⅰ类整合子所携带的多重耐药基因是临床研究多重耐药所不可忽视的,这一结构为研究多重耐药的机制提供了更多的途径和方向。
中国是一个抗生素滥用比较严重的国家,细菌的耐药情况也比其他国家或地区严重,细菌所携带的耐药基因种类也相对较多。从科学研究方面看,这是一个丰富的资源。如果对大量耐药菌株进行研究,肯定能发现很多新的耐药相关信息,有利于临床治疗细菌的感染。
ISCR1和Ⅰ类整合子都是细菌携带耐药基因常见的重要基因元件,与耐药基因的水平转移密切相关。国外已经有在多种菌种中的ISCR1的研究报道,但是中国国内还没有相关的报道。而Ⅰ类整合子的研究虽然已经很多,但是在中国还没有研究是同时对大量不同菌种细菌同时进行的。这是本研究的突破点和创新点。
因此,本课题针对常见革兰阴性杆菌进行ISCR1和Ⅰ类整合子耐药基因的筛查,测序,得到新的耐药基因组合,从而了解ISCR1和Ⅰ类整合子的耐药机制,以及初步了解这两者的串联关系。
研究方法
1.菌株的挑选及基因组DNA的提取大肠埃希菌和肺炎克雷伯菌是从2005年到2009年的菌株,分别在各年中挑选不重复的多重耐药菌株,而其他细菌(阴沟肠杆菌、产气肠杆菌、普通变形杆菌、奇异变形杆菌、粘质沙雷菌、居泉沙雷菌、铜绿假单胞菌、鲍曼不动杆菌、嗜麦芽窄食单胞菌、洋葱伯克霍尔德氏菌、恶臭假单胞菌等)是从2008年到2009年两年内在临床中收集的不重复的多重耐药菌株。共收集到1800株细菌,用SDS-蛋白酶K-酚-氯仿方法抽提这些菌株的基因组DNA。
2. ISCR1中orf513基因和可变区耐药基因的检测引用国外已经应用的orf513基因相关引物对所提取的基因组DNA进行扩增,再用ISCR1可变区引物对orf513基因阳性株进行PCR的筛查,然后酶切可变区基因并进行初步分类,挑选各种类型测序,分析同源性和耐药基因组合,GeneBank上申请序列号。
3.Ⅰ类整合子中Ⅰ类整合酶基因和可变区耐药基因的检测引用国内外已经应用的相关Ⅰ类整合酶基因引物进行Ⅰ类整合酶的筛查,得到阳性株后再用国外研究中的引物进行整合子可变区基因的检测,然后酶切可变区基因并进行初步分类,挑选各种类型测序,分析同源性和耐药基因组合,GeneBank上申请序列号。
4.初步了解ISCR1与Ⅰ类整合子之间的串联关系实验设想在既含有Ⅰ类整合子基因又含有ISCR1基因的菌株中,这两者是串联存在的,构成复杂性Ⅰ类整合子。实验中用本研究设计的引物对Ⅰ类整合子靠近3’-保守区的基因和ISCR1的靠近5’-保守区的基因间的序列进行PCR的检测并测序分析。
研究结果
1.检测得到本实验菌株中ISCR1携带的主要耐药基因,并发现了在不同菌种中ISCR1携带的新耐药基因组合总结所有实验菌株,发现ISCR1主要携带喹诺酮(qnrA1、qnrB2和qnrB6)、β-内酰胺类(blaPER、ampC、ampR和blaCTX-M-9)和季铵类化合物(qacEΔ)等耐药基因。同时发现了10个耐药基因组合是ISCR1于相应的菌种在国内外首次报道的,在大肠埃希菌中or/513+sapA-like+qnrB2+qacEdeltal和orf513+short chain dehydrogenase/ reductase +qnrB6+qacEdeltal;阴沟肠杆菌orf513+short chain dehydrogenase/ reductase+qnrB6+qacEdeltal和orf513+blaCTX-M-9+insB+qacEdeltal;产气肠杆菌orf513+ampC+ampR+qacEdeltal;居泉沙雷菌orf513+short chain dehydrogenase /reductase+qnrB6+qacEdeltal;鲍曼不动杆菌orp13+qnrA1+ampR+qacEdeltal和or/513+blaPER-1+GST-like pseudogene+qacEdeltal;在铜绿假单胞菌or/513+qnrA1+ampR+qacEdeltal;嗜麦芽窄食单胞菌orf513+qnrA1+ampR+qacEdeltal。另外还有2个组合是国内首次报道的：在肺炎克雷伯菌中orf513+SapA-like+qnrB2+qacEdeltal和阴沟肠杆菌orf513+qnrA1+ampR+qacEdeltal。
2.检测得到Ⅰ类整合子携带的主要耐药基因,并发现了不同菌种中Ⅰ类整合子携带的新耐药基因组合本实验中检测到Ⅰ类整合子携带的耐药基因主要有氨基糖苷类(addA1、aadA2、aadA5、aadA13、aadB、aac(6')-Ⅱ、aacA4、aacC4、aacC1、ant(3")-Ih-aac(6')-Iid) ;甲氧苄啶类(dfrA1、dfrA12、dfrA16、dfrA17) ;β-内酰胺酶类( blaOXA-10、blaOXA-10a、blaOXA-4、blaOXA-30、blaIMP-9、blaIMP-25、blaPSE-1 )等。同时研究发现16个耐药基因组合在相应的菌种中是国内外是首次报道的：如大肠埃希菌中aacA4+catB8+aadA1 ;肺炎克雷伯菌中dfrA1+aadA 5、dfrA1+orfC、dfrA16+aadA2;阴沟肠杆菌中aacA4+catB8+aadA1、aadB+aadA2、catB8+aadA1、dfrA1+aadA5、dfrA12+orfF+aadA2+ORFⅡ+ORFⅢ、dfrA12+orfF+aadA2 ;产气肠杆菌中aacA4+catB8+aadA1、ant(3")-Ih-aac(6')-Iid +catB8、dfrA12+orfF+aadA2;奇异变性杆菌中dfrA12 +orfF+aadA2 ;嗜麦芽窄食单胞菌中aacA4+blaIMP-25+ blaOXA-30+blacatB3 ;恶臭假单胞菌中blaIMP-9+aacA4+blaOXA-10+aadA2。另外还有6个耐药基因组合是在国内首次报道的：大肠埃希菌中aadB+aadA2;肺炎克雷伯菌中aacC4+cmlA1、aacA4 +blaOXA-4 +aadA2、aacC1/aacCA1 +orfP +orfQ+aadA1、acc(6')-Ⅱ/aac(6')-Ⅱ、arr-3 +ereC +aadA1 +cmlA7。
3. ISCR1和Ⅰ类整合子之间的串联关系的到了初步验证通过对24株既含有ISCR1可变区基因又含有Ⅰ类整合子可变区基因的菌株进行检测,均能扩增出两者间的序列。这提示我们Ⅰ类整合子与ISCR1可以以串联形式存在于细菌基因中,这有利于以后对两者作用机制的研究。
结论
1.不同菌属中ISCR1和Ⅰ类整合子携带的耐药基因种类和数量不等。
2.在不同种属中分别发现了ISCR1和Ⅰ类整合子携带耐药基因的新组合方式。
3.无论是在ISCR1还是Ⅰ类整合子,都出现同种细菌能携带不同的耐药基因组合,不同种细菌能携带相同的耐药基因组合的情况。
4.在本实验中,ISCR1中主要携带喹诺酮(qnr)、β-内酰胺类和季铵类化合物(qacEΔ)等耐药基因。
5.在本实验中,与Ⅰ类整合子相关的耐药基因主要是氨基糖苷类、甲氧苄啶类(dfr)和β-内酰胺类。
6.Ⅰ类整合子与ISCR1可以以串联形式存在于细菌基因中。
Background and objective
The drug resistance has become more and more serious, because of the general use of the antibiotics. Our country is one of the most severe areas in which the antibiotics are misused. The problems of the resistance in the bacterias have taken the attention of the people. At present, the rate of drug resistance in the bacterias which come from the clinic is upgrading year by year. The bacterias product more and more multidrug resistance become of the hingh selection pressure. Therefore, it is very necessary and urgent to research the mechanism of drug resistance in the bacterias.
The mechanism of drug resistance is very complex. Bacterias can product drug resistance because of alteration of target site, change of pervasiveness, inactivation of drug, enhanced drug efflux, biological membrane and so on. To study the mechanism of drug resistance from the site of molecular biology, we focus on gene mutation and gene transfer. Gene transfer is the cause of fast diffusion of the resistance. There are plasmid, transposon, temperate phage, integron and insertion sequences common regions (ISCR), which can get and transfer the resistance genes. Resistance genes can be transferred by conjugation, transformation, transduction and transposition. Many resistance genes in integron, transposon and insertion sequences common regions can integrate in one plasmid by transposition, which is the important reason leading to multidrug resistance.
ISCR comes from "insertion sequences" (ISs) and "common regions" (CRs), which has the feature of insertion sequences and common regions. Insertion sequences are the transposable elements which can move frequently, and the small DNA fragments which can insert one or more fragments to the genome and remove the adjacent genes. Common regions were first discovered and reported in the early 1990s as a DNA sequence of 2,154 bp that was found in two complex class 1 integrons, In6 and In7. CR sequences have two key features of IS91-like elements that are similarly located. CRs lack terminal inverted repeats (IRs) and are thought to transpose by a mechanism termed rolling-circle (RC) transposition. And CR sequences have another feature, terIS, which is a replication terminator. Further, the numbers presently used to distinguish CRs are retained to distinguish different ISCRs. Therefore, CR1 becomes ISCR1, CR2 becomes ISCR2, etc.
ISCRs have an ORF, orf513, encoding a putative product of 513 amino acids. The use of orf513 is similar to the transposase, which can transfer the gene cassettes. In the complex class 1 integrons, downstream of orf513 can have one or more resistance gene cassettes, and qac/sul3'-CS.There are 6 frequent types of ISCR, and 1-4 types are researched in major studies. ISCR elements are notable for their close association with a wide variety of antibiotic resistance genes, and the ISCR1 is the most important one. ISCR1 can mediate transposition, and it can transfer resistance gene cassettes with difference length, such as the genes of catA2、dfrA、qnr and blaCMY and so on. ISCR1 plays an important role on the transmission of the resistance.
Integron is also an important gene transfer element. Integron is genetic fragments that can recognize and mobile gene cassettes by site-specific recombination. Most of the gene cassettes in the integrons relate to resistance, and it is very important to the genome evolution and the resistance. According to the kinds of integrase, there are main four kinds of integrons. Among the total, class 1 integron is the most common and the first discovered, and the movement of the gene cassettes has be confirmed. Class 1 integron can lead to the multidrug resistance in the Gram-negative bacterias by the integration of one or more resistance gene cassettes. Class 1 integron includes 5'-CS,3'-CS and the gene cassettes.In the 5'-CS, there are genes of intll integrase, recombination site attll and promoter. In the 3'-CS, there are three ORFs (qacEΔ1, sull and ORF5). Some integrons have no 3'-CS. Class 1 integron is widespread in the Gram-negative bacterias, such as Escherichia, Klebsierlla, Pseudomonas and Acinetobacter and so on. Class 1 integron can transfer many kinds of resistance gene cassettes, such asβ- lactam, aminoglycosides, amphemycin, phytomycin, Quinolone and quaternary amine, and they are important in the transmission of resistance gene cassettes.
ISCR1 usually exist in the complex class 1 integrons. The upstream sequences of ISCR1 are the same, and the sequences in junction point between ISCR1and class 1 integron are not change, that indicate that ISCR1 consistent with the feature of complex class 1 integrons. The complex class 1 integrons with ISCR and integron, and ones with two ISCRs, are relating to resistance. They have potentiality to transfer the resistance genes. Multidrug resistance is not negligible, and the structure can offer more pathway and direction to the studies of multidrug resistance.
In China, antibiotics are abused acutely. The resistance of bacterias is more serious than other courtries and bacterias can bring more kinds of resistance genes, which is an affluent resource to researchs. If we can take a good use of these bacterias, it makes sure that we can get much message about resistance genes and it is profit for the cure to the bacterias infection.
ISCR1 and class 1 integron are important transposable elements, and they are correlated with resistance genes. In abroad, there are many researches of ISCR1about kinds of bacterias, but in China no one. At the same time, although there are many studies about class 1 integron, in China no study with lots of kinds of bacterias.
Therefore, in our research, we screen the resistance genes in ISCR1and class 1 integron in the Gram-negative bacterias by PCR, and then we can get the gene arrangements, so that we can understand the mechanism and the mutual relationship of ISCR1 and class 1 integron.
Methods
1. The choice of Gram-negative bacterias and extraction of the genome DNA Escherichia coli and Klebsierlla peumoniae strains were from 2005 to 2009, which are multidrug resistance and not reduplicative. Other strains were from 2008 to 2009, such as Enterobcter cloacae, Enterobcter aerogenes, Proteus vulgaris, Proteus mirabilis, Serratia marcescens, Serratia fonticola, Pseudomonas aeruginosa, Acinetobacter baumannii, Pseudomonas maltophilia, Burkholderia cepacia, Pseudomonas putica and so on. There were total 1800 strains, and we used the method of SDS- protease K- alcohol phenyl-trichlormethane to extract the genome DNA.
2. The detection of genes of orf513 and gene cassettes in ISCR1 Used the methods which had been used internal or overseas to detect genes of orf513,then detected the gene cassettes, and then used two incision enzymes to cut the gene cassettes, classified the kinds of gene cassettes, and got some gene sequences to make sequencing, at last analyzed the sequences and submitted to the GeneBank.
3. The detection of genes of integrase and gene cassettes in class 1 integron Used the methods which had been used internal or overseas to detect genes of integrase, then detected the gene cassettes, and then used two incision enzymes to cut the gene cassettes, classified the kinds of gene cassettes, got some gene sequences to make sequencing, at last analyzed the sequences and submitted to the GeneBank.
4. Initial validation of the tandem relationship of ISCR1 and class 1 integron We assumed that ISCR1 and class 1 integron were connection in series.Then we used the PCR primer to detect.
Results
1. Some resistance genes were most frequently associated with ISCR1 and some new resistance gene arrays were discovered in different bacteriums The genes of quinolone(qnr)、β-Lactamase and quaternary amine(qacEA) were most frequently associated with ISCR1 in our study.And there were 10 new resistance gene arrays in ISCR1 in the world, such as orf513+sapA-like+qnrB2+qacEdeltal、orf513+short chain dehydrogenase/reductase+qnrB6+qacEdeltal (Escherichia coli); orf513+short chain dehydrogenase/reductase+qnrB6+qacEdeltal、orf513+blaCTX-M-9+insB+ qacEdeltal(Enterobcter cloacae); orf513+ampC+ampR+qacEdeltal(Enterobcter aerogenes); orf513+short chain dehydrogenase/reductase+qnrB6+qacEdeltal (Serratia fonticola); orf513+qnrA1+ampR+qacEdeltal、orf513+blaPER-1+GST-like pseudogene+qacEdeltal(Acinetobacter baumannii); orf513+qnrA1+ampR+qacEdeltal (Pseudomonas aeruginosa); orf513+qnrA1+ampR+qacEdeltal(Pseudomonas maltophilia).And there were 2 new resistance gene arrays in China in different bacteriums, such as orf513+sapA-like+qnrB2+qacEdeltal (Klebsierlla peumoniae)、orf513+qnrA1+ampR+qacEdeltal(Enterobcter cloacae).
2. Some resistance genes were most frequently associated with class 1 integron and some new resistance gene arrays were discovered in different bacteriums
The genes of aminoglycosides、β-Lactamase and trimethoprim were most frequently associated with class 1 integron in our study.And there were 16 new resistance gene arrays in different bacteriums in the world, such as aacA4+catB8+aadA1 (Escherichia coli); dfrA1+aadA5、dfrA1+orfC、dfrA16+aadA2(Klebsierlla peumoniae); aacA4+catB8+aadA1、aadB+aadA2、catB8+aadA1、dfrA1+aadA5、dfrA12+orfF+aadA2+ORFⅡ+ORFⅢ、dfrA12+orfF+aadA2 (Enterobcter cloacae); aacA4+catB8+aadA1、ant(3")-Ih-aac(6')-Iid+catB8、dfrA12+orfF+aadA2 (Enterobcter aerogenes); dfrA12+orfF+aadA2(Proteus mirabilis); aacA4+blaIMP-25+ blaOXA-30+blacatB3(Pseudomonas maltophilia); blaIMP-9+aacA4+blaOXA-10+aadA2 (Pseudomonas putica).And there were 6 new resistance gene arrays in different bacteriums in China, such as aadB+aadA2(Escherichia coli); aacC4+cmlA1、aacA4 +blaOXA-4+aadA2、aacC1/aacCA1+orfP+orfQ+aadA1、acc(6')-Ⅱ/aac(6')-Ⅱ、arr-3+ereC+aadA1+cmlA 7(Klebsierlla peumoniae).
3. Initial validation of the tandem relationship of ISCR1 and class 1 integron
The results of PCR showed that ISCR1 and class 1 integron could connect in series. It was very important to the studues on the ISCR1and class 1 integron in the fucture.
Conclusion
1. There were different kinds and amount of resistance gene cassettes in different bacteriums.
2. In ISCR1 there were 10 new resistance gene arrays in the world, and 2 new resistance gene arrays in China in different bacteriums; in class 1 integron there were 16 new resistance gene arrays in the world, and 6 new resistance gene arrays in China in different bacteriums.
3. The bacteriums of same kind could bring different resistance gene arrays, and ones of different kinds could bring same arrays, in ISCR1 or class 1 integron.
4. The genes of quinolone(qnr)、β-Lactamase and quaternary amine(qacEΔ) were most frequently associated with ISCR1 in our study.
5. The genes of aminoglycosides、trimethoprim (dfr) andβ-Lactamase were most frequently associated with class 1 integron in our study.
6. Class 1 integron and ISCR1 could connect in series.
随着抗生素的应用越来越广泛,细菌的耐药性就越来越严重。我国是世界上滥用抗生素最为严重的地区之一,细菌的耐药性问题已经引起人们的高度关注。我国目前临床上分离的细菌对常规抗生素的耐药率逐年上升,而且细菌在异常的抗生素选择压力下,产生多重耐药的情况不断出现,因此研究细菌耐药机制是十分必要和紧迫的。
细菌耐药的生化机制非常复杂,通常是细菌对药物作用靶位、渗透性的改变,产生灭活酶和钝化酶,排外系统的作用以及生物膜的产生等方面。而从分子生物学角度认识细菌的耐药机制,主要集中于基因突变和耐药基因水平传播。基因转移是细菌耐药性迅速扩散的主要原因。携带耐药基因的基因转移元件有：质粒、转座子、整合型噬菌体和整合子,以及最近几年才发现的插入序列共同区(ISCR)。耐药基因可以通过接合、转化、转导和转座等方法在细菌间转移。通过转座的方式,整合子、ISCR和转座子可以导致多个耐药基因在单个质粒中聚集成束,这是多重耐药株产生的重要原因。ISCR由"insertion sequences" (ISs)和"common regions" (CRs)得来。它既具有插入序列(ISs)的特点,也具有共同区(CRs)的特点。插入序列属于高度可移动性转座因子,是对基因组一个或多个靶位点具有插入能力的小分子DNA片段,并常可以移动邻近的基因。共同区域(CRs)是1993年在复杂性Ⅰ类整合子In6 andIn7中作为一个长约2154bp的DNA片段而首先发现的。CRs是一类IS91-like因子家族的延伸成员,它具有与相似位置IS91-like因子的两个主要特点：缺少末端反向重复序列和它们转移邻近耐药基因是通过滚环复制的形式进行的,另外,CR序列还保留着IS91的第三个特点,复制终止子序列terIS。相应地,为了强调它们作为插入序列的同源性和它们被发现的历史,研究假设它们是特定的ISCR因子。用来区分CRs的数字被保留下来以便区分不同的ISCRs,因此,CR1对应ISCR1, CR2对应ISCR2等。
ISCRs包含1个ORF序列,orf513。orf 513基因编码513个氨基酸序列产物,其功能与转座酶相似,可以转移基因盒。在复杂性Ⅰ类整合子中,orf513下游可以有由一种或以上的耐药基因组成的基因盒,紧接着是qac/sul的3'-CS。ISCR常见的可以分为6类,目前研究较多的为1,2,3,4类,研究发现这几种类型的ISCR均与耐药基因的传播相关。其中,最为重要的是ISCR1,它经常出现在复杂性Ⅰ类整合子的基因序列中。ISCR1介导一系列的转座事件发生,转座不同长度的耐药基因盒,如catA2、dfrA、qnr和各种blaCMY基因。ISCR1对耐药基因的传播发挥重要的作用。
另一个重要的基因转移元件是整合子。整合子是细菌的DNA片段,它的独特结构可捕获并整合外源性基因,并使之转变为功能性基因的表达单位。其整合的基因盒大多与耐药基因有关,对细菌基因组进化和耐药性具有重要意义。根据整合酶不同进行分类,整合子主要分为Ⅰ～Ⅳ类。其中,Ⅰ类整合子最常见也是最早被发现的整合子,其基因盒的移动已得到证实。Ⅰ类整合子在革兰阴性杆菌中通过可变区整合一个或多个耐药基因盒介导宿主菌多重耐药,在临床检测中具有重要的意义。这类整合子5’-保守区有编码Ⅰ型整合酶的基因intⅡ、重组位点attⅡ和启动子Pant。3'-保守区由3个开放阅读框组成(qacEΔI, sulI和ORF5),有的整合子没有3’-保守区。Ⅰ类整合子在革兰阴性菌中广泛分布、检出率最高,在许多临床分离的菌株包括肠杆菌科中的埃希菌属、克雷伯菌属和非发酵革兰阴性杆菌中的假单胞菌属和不动杆菌属等都能检出。Ⅰ类整合子可以携带多种耐药基因,如β-内酰胺类、氨基糖昔类、甲氧苄啶、氯霉素、链霉素、喹诺酮和季铵类化合物等耐药基因,对耐药基因在革兰阴性杆菌中的传播十分重要。
ISCR1经常出现在复杂性Ⅰ类整合子的基因序列中。ISCR1上游序列通常是相同的,而在ISCR1与Ⅰ类整合子3’-保守区的连接点序列是不变的。这些都表明ISCR1很符合复杂性Ⅰ类整合子的特征。无论是ISCR1连同耐药基因插入到普通的一类整合子组成的复杂性整合子,还是带有两个拷贝ISCR1因子的复杂性整合子,它们都带有许多耐药性因子,具有广泛传播这些耐药基因的潜能。复杂性Ⅰ类整合子所携带的多重耐药基因是临床研究多重耐药所不可忽视的,这一结构为研究多重耐药的机制提供了更多的途径和方向。
中国是一个抗生素滥用比较严重的国家,细菌的耐药情况也比其他国家或地区严重,细菌所携带的耐药基因种类也相对较多。从科学研究方面看,这是一个丰富的资源。如果对大量耐药菌株进行研究,肯定能发现很多新的耐药相关信息,有利于临床治疗细菌的感染。
ISCR1和Ⅰ类整合子都是细菌携带耐药基因常见的重要基因元件,与耐药基因的水平转移密切相关。国外已经有在多种菌种中的ISCR1的研究报道,但是中国国内还没有相关的报道。而Ⅰ类整合子的研究虽然已经很多,但是在中国还没有研究是同时对大量不同菌种细菌同时进行的。这是本研究的突破点和创新点。
因此,本课题针对常见革兰阴性杆菌进行ISCR1和Ⅰ类整合子耐药基因的筛查,测序,得到新的耐药基因组合,从而了解ISCR1和Ⅰ类整合子的耐药机制,以及初步了解这两者的串联关系。
研究方法
1.菌株的挑选及基因组DNA的提取大肠埃希菌和肺炎克雷伯菌是从2005年到2009年的菌株,分别在各年中挑选不重复的多重耐药菌株,而其他细菌(阴沟肠杆菌、产气肠杆菌、普通变形杆菌、奇异变形杆菌、粘质沙雷菌、居泉沙雷菌、铜绿假单胞菌、鲍曼不动杆菌、嗜麦芽窄食单胞菌、洋葱伯克霍尔德氏菌、恶臭假单胞菌等)是从2008年到2009年两年内在临床中收集的不重复的多重耐药菌株。共收集到1800株细菌,用SDS-蛋白酶K-酚-氯仿方法抽提这些菌株的基因组DNA。
2. ISCR1中orf513基因和可变区耐药基因的检测引用国外已经应用的orf513基因相关引物对所提取的基因组DNA进行扩增,再用ISCR1可变区引物对orf513基因阳性株进行PCR的筛查,然后酶切可变区基因并进行初步分类,挑选各种类型测序,分析同源性和耐药基因组合,GeneBank上申请序列号。
3.Ⅰ类整合子中Ⅰ类整合酶基因和可变区耐药基因的检测引用国内外已经应用的相关Ⅰ类整合酶基因引物进行Ⅰ类整合酶的筛查,得到阳性株后再用国外研究中的引物进行整合子可变区基因的检测,然后酶切可变区基因并进行初步分类,挑选各种类型测序,分析同源性和耐药基因组合,GeneBank上申请序列号。
4.初步了解ISCR1与Ⅰ类整合子之间的串联关系实验设想在既含有Ⅰ类整合子基因又含有ISCR1基因的菌株中,这两者是串联存在的,构成复杂性Ⅰ类整合子。实验中用本研究设计的引物对Ⅰ类整合子靠近3’-保守区的基因和ISCR1的靠近5’-保守区的基因间的序列进行PCR的检测并测序分析。
研究结果
1.检测得到本实验菌株中ISCR1携带的主要耐药基因,并发现了在不同菌种中ISCR1携带的新耐药基因组合总结所有实验菌株,发现ISCR1主要携带喹诺酮(qnrA1、qnrB2和qnrB6)、β-内酰胺类(blaPER、ampC、ampR和blaCTX-M-9)和季铵类化合物(qacEΔ)等耐药基因。同时发现了10个耐药基因组合是ISCR1于相应的菌种在国内外首次报道的,在大肠埃希菌中or/513+sapA-like+qnrB2+qacEdeltal和orf513+short chain dehydrogenase/ reductase +qnrB6+qacEdeltal;阴沟肠杆菌orf513+short chain dehydrogenase/ reductase+qnrB6+qacEdeltal和orf513+blaCTX-M-9+insB+qacEdeltal;产气肠杆菌orf513+ampC+ampR+qacEdeltal;居泉沙雷菌orf513+short chain dehydrogenase /reductase+qnrB6+qacEdeltal;鲍曼不动杆菌orp13+qnrA1+ampR+qacEdeltal和or/513+blaPER-1+GST-like pseudogene+qacEdeltal;在铜绿假单胞菌or/513+qnrA1+ampR+qacEdeltal;嗜麦芽窄食单胞菌orf513+qnrA1+ampR+qacEdeltal。另外还有2个组合是国内首次报道的：在肺炎克雷伯菌中orf513+SapA-like+qnrB2+qacEdeltal和阴沟肠杆菌orf513+qnrA1+ampR+qacEdeltal。
2.检测得到Ⅰ类整合子携带的主要耐药基因,并发现了不同菌种中Ⅰ类整合子携带的新耐药基因组合本实验中检测到Ⅰ类整合子携带的耐药基因主要有氨基糖苷类(addA1、aadA2、aadA5、aadA13、aadB、aac(6')-Ⅱ、aacA4、aacC4、aacC1、ant(3")-Ih-aac(6')-Iid) ;甲氧苄啶类(dfrA1、dfrA12、dfrA16、dfrA17) ;β-内酰胺酶类( blaOXA-10、blaOXA-10a、blaOXA-4、blaOXA-30、blaIMP-9、blaIMP-25、blaPSE-1 )等。同时研究发现16个耐药基因组合在相应的菌种中是国内外是首次报道的：如大肠埃希菌中aacA4+catB8+aadA1 ;肺炎克雷伯菌中dfrA1+aadA 5、dfrA1+orfC、dfrA16+aadA2;阴沟肠杆菌中aacA4+catB8+aadA1、aadB+aadA2、catB8+aadA1、dfrA1+aadA5、dfrA12+orfF+aadA2+ORFⅡ+ORFⅢ、dfrA12+orfF+aadA2 ;产气肠杆菌中aacA4+catB8+aadA1、ant(3")-Ih-aac(6')-Iid +catB8、dfrA12+orfF+aadA2;奇异变性杆菌中dfrA12 +orfF+aadA2 ;嗜麦芽窄食单胞菌中aacA4+blaIMP-25+ blaOXA-30+blacatB3 ;恶臭假单胞菌中blaIMP-9+aacA4+blaOXA-10+aadA2。另外还有6个耐药基因组合是在国内首次报道的：大肠埃希菌中aadB+aadA2;肺炎克雷伯菌中aacC4+cmlA1、aacA4 +blaOXA-4 +aadA2、aacC1/aacCA1 +orfP +orfQ+aadA1、acc(6')-Ⅱ/aac(6')-Ⅱ、arr-3 +ereC +aadA1 +cmlA7。
3. ISCR1和Ⅰ类整合子之间的串联关系的到了初步验证通过对24株既含有ISCR1可变区基因又含有Ⅰ类整合子可变区基因的菌株进行检测,均能扩增出两者间的序列。这提示我们Ⅰ类整合子与ISCR1可以以串联形式存在于细菌基因中,这有利于以后对两者作用机制的研究。
结论
1.不同菌属中ISCR1和Ⅰ类整合子携带的耐药基因种类和数量不等。
2.在不同种属中分别发现了ISCR1和Ⅰ类整合子携带耐药基因的新组合方式。
3.无论是在ISCR1还是Ⅰ类整合子,都出现同种细菌能携带不同的耐药基因组合,不同种细菌能携带相同的耐药基因组合的情况。
4.在本实验中,ISCR1中主要携带喹诺酮(qnr)、β-内酰胺类和季铵类化合物(qacEΔ)等耐药基因。
5.在本实验中,与Ⅰ类整合子相关的耐药基因主要是氨基糖苷类、甲氧苄啶类(dfr)和β-内酰胺类。
6.Ⅰ类整合子与ISCR1可以以串联形式存在于细菌基因中。
Background and objective
The drug resistance has become more and more serious, because of the general use of the antibiotics. Our country is one of the most severe areas in which the antibiotics are misused. The problems of the resistance in the bacterias have taken the attention of the people. At present, the rate of drug resistance in the bacterias which come from the clinic is upgrading year by year. The bacterias product more and more multidrug resistance become of the hingh selection pressure. Therefore, it is very necessary and urgent to research the mechanism of drug resistance in the bacterias.
The mechanism of drug resistance is very complex. Bacterias can product drug resistance because of alteration of target site, change of pervasiveness, inactivation of drug, enhanced drug efflux, biological membrane and so on. To study the mechanism of drug resistance from the site of molecular biology, we focus on gene mutation and gene transfer. Gene transfer is the cause of fast diffusion of the resistance. There are plasmid, transposon, temperate phage, integron and insertion sequences common regions (ISCR), which can get and transfer the resistance genes. Resistance genes can be transferred by conjugation, transformation, transduction and transposition. Many resistance genes in integron, transposon and insertion sequences common regions can integrate in one plasmid by transposition, which is the important reason leading to multidrug resistance.
ISCR comes from "insertion sequences" (ISs) and "common regions" (CRs), which has the feature of insertion sequences and common regions. Insertion sequences are the transposable elements which can move frequently, and the small DNA fragments which can insert one or more fragments to the genome and remove the adjacent genes. Common regions were first discovered and reported in the early 1990s as a DNA sequence of 2,154 bp that was found in two complex class 1 integrons, In6 and In7. CR sequences have two key features of IS91-like elements that are similarly located. CRs lack terminal inverted repeats (IRs) and are thought to transpose by a mechanism termed rolling-circle (RC) transposition. And CR sequences have another feature, terIS, which is a replication terminator. Further, the numbers presently used to distinguish CRs are retained to distinguish different ISCRs. Therefore, CR1 becomes ISCR1, CR2 becomes ISCR2, etc.
ISCRs have an ORF, orf513, encoding a putative product of 513 amino acids. The use of orf513 is similar to the transposase, which can transfer the gene cassettes. In the complex class 1 integrons, downstream of orf513 can have one or more resistance gene cassettes, and qac/sul3'-CS.There are 6 frequent types of ISCR, and 1-4 types are researched in major studies. ISCR elements are notable for their close association with a wide variety of antibiotic resistance genes, and the ISCR1 is the most important one. ISCR1 can mediate transposition, and it can transfer resistance gene cassettes with difference length, such as the genes of catA2、dfrA、qnr and blaCMY and so on. ISCR1 plays an important role on the transmission of the resistance.
Integron is also an important gene transfer element. Integron is genetic fragments that can recognize and mobile gene cassettes by site-specific recombination. Most of the gene cassettes in the integrons relate to resistance, and it is very important to the genome evolution and the resistance. According to the kinds of integrase, there are main four kinds of integrons. Among the total, class 1 integron is the most common and the first discovered, and the movement of the gene cassettes has be confirmed. Class 1 integron can lead to the multidrug resistance in the Gram-negative bacterias by the integration of one or more resistance gene cassettes. Class 1 integron includes 5'-CS,3'-CS and the gene cassettes.In the 5'-CS, there are genes of intll integrase, recombination site attll and promoter. In the 3'-CS, there are three ORFs (qacEΔ1, sull and ORF5). Some integrons have no 3'-CS. Class 1 integron is widespread in the Gram-negative bacterias, such as Escherichia, Klebsierlla, Pseudomonas and Acinetobacter and so on. Class 1 integron can transfer many kinds of resistance gene cassettes, such asβ- lactam, aminoglycosides, amphemycin, phytomycin, Quinolone and quaternary amine, and they are important in the transmission of resistance gene cassettes.
ISCR1 usually exist in the complex class 1 integrons. The upstream sequences of ISCR1 are the same, and the sequences in junction point between ISCR1and class 1 integron are not change, that indicate that ISCR1 consistent with the feature of complex class 1 integrons. The complex class 1 integrons with ISCR and integron, and ones with two ISCRs, are relating to resistance. They have potentiality to transfer the resistance genes. Multidrug resistance is not negligible, and the structure can offer more pathway and direction to the studies of multidrug resistance.
In China, antibiotics are abused acutely. The resistance of bacterias is more serious than other courtries and bacterias can bring more kinds of resistance genes, which is an affluent resource to researchs. If we can take a good use of these bacterias, it makes sure that we can get much message about resistance genes and it is profit for the cure to the bacterias infection.
ISCR1 and class 1 integron are important transposable elements, and they are correlated with resistance genes. In abroad, there are many researches of ISCR1about kinds of bacterias, but in China no one. At the same time, although there are many studies about class 1 integron, in China no study with lots of kinds of bacterias.
Therefore, in our research, we screen the resistance genes in ISCR1and class 1 integron in the Gram-negative bacterias by PCR, and then we can get the gene arrangements, so that we can understand the mechanism and the mutual relationship of ISCR1 and class 1 integron.
Methods
1. The choice of Gram-negative bacterias and extraction of the genome DNA Escherichia coli and Klebsierlla peumoniae strains were from 2005 to 2009, which are multidrug resistance and not reduplicative. Other strains were from 2008 to 2009, such as Enterobcter cloacae, Enterobcter aerogenes, Proteus vulgaris, Proteus mirabilis, Serratia marcescens, Serratia fonticola, Pseudomonas aeruginosa, Acinetobacter baumannii, Pseudomonas maltophilia, Burkholderia cepacia, Pseudomonas putica and so on. There were total 1800 strains, and we used the method of SDS- protease K- alcohol phenyl-trichlormethane to extract the genome DNA.
2. The detection of genes of orf513 and gene cassettes in ISCR1 Used the methods which had been used internal or overseas to detect genes of orf513,then detected the gene cassettes, and then used two incision enzymes to cut the gene cassettes, classified the kinds of gene cassettes, and got some gene sequences to make sequencing, at last analyzed the sequences and submitted to the GeneBank.
3. The detection of genes of integrase and gene cassettes in class 1 integron Used the methods which had been used internal or overseas to detect genes of integrase, then detected the gene cassettes, and then used two incision enzymes to cut the gene cassettes, classified the kinds of gene cassettes, got some gene sequences to make sequencing, at last analyzed the sequences and submitted to the GeneBank.
4. Initial validation of the tandem relationship of ISCR1 and class 1 integron We assumed that ISCR1 and class 1 integron were connection in series.Then we used the PCR primer to detect.
Results
1. Some resistance genes were most frequently associated with ISCR1 and some new resistance gene arrays were discovered in different bacteriums The genes of quinolone(qnr)、β-Lactamase and quaternary amine(qacEA) were most frequently associated with ISCR1 in our study.And there were 10 new resistance gene arrays in ISCR1 in the world, such as orf513+sapA-like+qnrB2+qacEdeltal、orf513+short chain dehydrogenase/reductase+qnrB6+qacEdeltal (Escherichia coli); orf513+short chain dehydrogenase/reductase+qnrB6+qacEdeltal、orf513+blaCTX-M-9+insB+ qacEdeltal(Enterobcter cloacae); orf513+ampC+ampR+qacEdeltal(Enterobcter aerogenes); orf513+short chain dehydrogenase/reductase+qnrB6+qacEdeltal (Serratia fonticola); orf513+qnrA1+ampR+qacEdeltal、orf513+blaPER-1+GST-like pseudogene+qacEdeltal(Acinetobacter baumannii); orf513+qnrA1+ampR+qacEdeltal (Pseudomonas aeruginosa); orf513+qnrA1+ampR+qacEdeltal(Pseudomonas maltophilia).And there were 2 new resistance gene arrays in China in different bacteriums, such as orf513+sapA-like+qnrB2+qacEdeltal (Klebsierlla peumoniae)、orf513+qnrA1+ampR+qacEdeltal(Enterobcter cloacae).
2. Some resistance genes were most frequently associated with class 1 integron and some new resistance gene arrays were discovered in different bacteriums
The genes of aminoglycosides、β-Lactamase and trimethoprim were most frequently associated with class 1 integron in our study.And there were 16 new resistance gene arrays in different bacteriums in the world, such as aacA4+catB8+aadA1 (Escherichia coli); dfrA1+aadA5、dfrA1+orfC、dfrA16+aadA2(Klebsierlla peumoniae); aacA4+catB8+aadA1、aadB+aadA2、catB8+aadA1、dfrA1+aadA5、dfrA12+orfF+aadA2+ORFⅡ+ORFⅢ、dfrA12+orfF+aadA2 (Enterobcter cloacae); aacA4+catB8+aadA1、ant(3")-Ih-aac(6')-Iid+catB8、dfrA12+orfF+aadA2 (Enterobcter aerogenes); dfrA12+orfF+aadA2(Proteus mirabilis); aacA4+blaIMP-25+ blaOXA-30+blacatB3(Pseudomonas maltophilia); blaIMP-9+aacA4+blaOXA-10+aadA2 (Pseudomonas putica).And there were 6 new resistance gene arrays in different bacteriums in China, such as aadB+aadA2(Escherichia coli); aacC4+cmlA1、aacA4 +blaOXA-4+aadA2、aacC1/aacCA1+orfP+orfQ+aadA1、acc(6')-Ⅱ/aac(6')-Ⅱ、arr-3+ereC+aadA1+cmlA 7(Klebsierlla peumoniae).
3. Initial validation of the tandem relationship of ISCR1 and class 1 integron
The results of PCR showed that ISCR1 and class 1 integron could connect in series. It was very important to the studues on the ISCR1and class 1 integron in the fucture.
Conclusion
1. There were different kinds and amount of resistance gene cassettes in different bacteriums.
2. In ISCR1 there were 10 new resistance gene arrays in the world, and 2 new resistance gene arrays in China in different bacteriums; in class 1 integron there were 16 new resistance gene arrays in the world, and 6 new resistance gene arrays in China in different bacteriums.
3. The bacteriums of same kind could bring different resistance gene arrays, and ones of different kinds could bring same arrays, in ISCR1 or class 1 integron.
4. The genes of quinolone(qnr)、β-Lactamase and quaternary amine(qacEΔ) were most frequently associated with ISCR1 in our study.
5. The genes of aminoglycosides、trimethoprim (dfr) andβ-Lactamase were most frequently associated with class 1 integron in our study.
6. Class 1 integron and ISCR1 could connect in series.
引文
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[1]Martinez J L. Antibiotics and antibiotic resistance genes in natural environments.[J]. Science,2008,321(5887):365-367.
[2]张珍珍,吴俊伟,杨卫军.细菌耐药性产生的分子生物学机理及控制措施[J].动物医学进展,2008,29(02)：106-109.
[3]党京丹.细菌耐药机制研究新进展[J].临床和实验医学杂志,2009,8(09)134-135.
[4]Guerin E, Cambray G, Sanchez-Alberola N, et al. The SOS response controls integron recombination.[J]. Science,2009,324(5930):1034.
[5]Barlow M. What antimicrobial resistance has taught us about horizontal gene transfer.[J]. Methods Mol Biol,2009,532:397-411.
[6]Stokes H W, Hall R M. A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions:integrons.[J]. Mol Microbiol, 1989,3(12):1669-1683.
[7]Boucher Y, Labbate M, Koenig J E, et al. Integrons:mobilizable platforms that promote genetic diversity in bacteria.[J]. Trends Microbiol,2007,15(7): 301-309.
[8]Hall R M, Stokes H W. Integrons:novel DNA elements which capture genes by site-specific recombination.[J]. Genetica,1993,90(2-3):115-132.
[9][9] Labbate M, Case R J, Stokes H W. The integron/gene cassette system:an active player in bacterial adaptation.[J]. Methods Mol Biol,2009,532:103-125.
[10]Dubois V, Debreyer C, Litvak S, et al. A new in vitro strand transfer assay for monitoring bacterial class 1 integron recombinase Intll activity.[J]. PLoS ONE,2007,2(12):e1315.
[11]Gillings M, Boucher Y, Labbate M, et al. The evolution of class 1 integrons and the rise of antibiotic resistance.[J]. J Bacteriol,2008,190(14):5095-5100.
[12]Ploy M C, Lambert T, Couty J P, et al. Integrons:an antibiotic resistance gene capture and expression system.[J]. Clin Chem Lab Med,2000,38(6):483-487.
[13]Gebreyes W A, Altier C. Molecular characterization of multidrug-resistant Salmonella enterica subsp. enterica serovar Typhimurium isolates from swine.[J]. J Clin Microbiol,2002,40(8):2813-2822.
[14]O'Halloran F, Lucey B, Cryan B, et al. Molecular characterization of class 1 integrons from Irish thermophilic Campylobacter spp.[J]. J Antimicrob Chemother,2004,53(6):952-957.
[15]Mak J K, Kim M J, Pham J, et al. Antibiotic resistance determinants in nosocomial strains of multidrug-resistant Acinetobacter baumannii.[J]. J Antimicrob Chemother,2009,63(1):47-54.
[16]Partridge S R, Tsafnat G, Coiera E, et al. Gene cassettes and cassette arrays in mobile resistance integrons.[J]. FEMS Microbiol Rev,2009,33(4):757-784.
[17]Arakawa Y, Murakami M, Suzuki K, et al. A novel integron-like element carrying the metallo-beta-lactamase gene blaIMP.[J]. Antimicrob Agents Chemother,1995,39(7):1612-1615.
[18]Michael C A, Gillings M R, Holmes A J, et al. Mobile gene cassettes:a fundamental resource for bacterial evolution.[J]. Am Nat,2004,164(1):1-12.
[19]Collis C M, Hall R M. Comparison of the structure-activity relationships of the integron-associated recombination sites attI3 and attI1 reveals common features.[J]. Microbiology,2004,150(Pt 5):1591-1601.
[20]Correia M, Boavida F, Grosso F, et al. Molecular characterization of a new class 3 integron in Klebsiella pneumoniae.[J]. Antimicrob Agents Chemother, 2003,47(9):2838-2843.
[21]Fluit A C, Schmitz F J. Resistance integrons and super-integrons.[J]. Clin Microbiol Infect,2004,10(4):272-288.
[22]Mazel D, Dychinco B, Webb V A, et al. A distinctive class of integron in the Vibrio cholerae genome.[J]. Science,1998,280(5363):605-608.
[23]Rowe-Magnus D A, Guerout A M, Mazel D. Bacterial resistance evolution by recruitment of super-integron gene cassettes.[J]. Mol Microbiol,2002,43(6): 1657-1669.
[24]Mazel D. Integrons:agents of bacterial evolution.[J]. Nat Rev Microbiol,2006, 4(8):608-620.
[25]Labbate M, Case R J, Stokes H W. The integron/gene cassette system:an active player in bacterial adaptation.[J]. Methods Mol Biol,2009, 532:103-125.
[26]Gillings M R, Xuejun D, Hardwick S A, et al. Gene cassettes encoding resistance to quaternary ammonium compounds:a role in the origin of clinical class 1 integrons?[J]. ISME J,2009,3(2):209-215.
[27]Hall R M, Collis C M. Mobile gene cassettes and integrons:capture and spread of genes by site-specific recombination.[J]. Mol Microbiol,1995,15(4): 593-600.
[28]Stokes H W, O'Gorman D B, Recchia G D, et al. Structure and function of 59-base element recombination sites associated with mobile gene cassettes.[J]. Mol Microbiol,1997,26(4):731-745.
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[35]Bennett P M. Plasmid encoded antibiotic resistance:acquisition and transfer of antibiotic resistance genes in bacteria.[J]. Br J Pharmacol,2008,153 Suppl 1:S347-S357.
[36]Mahillon J, Chandler M. Insertion sequences.[J]. Microbiol Mol Biol Rev, 1998,62(3):725-774.
[37]Stokes H W, Tomaras C, Parsons Y, et al. The partial 3'-conserved segment duplications in the integrons In6 from pSa and In7 from pDGO100 have a common origin.[J]. Plasmid,1993,30(1):39-50.
[38]Tavakoli N, Comanducci A, Dodd H M, et al. IS1294, a DNA element that transposes by RC transposition.[J]. Plasmid,2000,44(1):66-84.
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