整合子与细菌耐药性的相关研究进展

整合子是广泛存在于细菌中的一种可移动基因元件,它可以捕获外来基因盒并使其在细菌体内得到表达,在细菌耐药性的传播过程中扮演着重要角色。过去研究认为,细菌耐药是在质粒及转座子~([1])等基因水平上广泛传播,近几年大量研究表明,细菌可通过位点特异性重组的方式将耐药基因盒捕获并整合到自身的染色体或者质粒DNA上,即细菌体内存在一种天然的基因克隆表达系统——整合子。细菌耐药的高频次出现已成为临床医疗工作中的瓶颈,整合子不仅在细菌耐药中起关键作用,而且在细菌适应性及基因进化中具有普遍又重要的意义。     

整合子与细菌耐药性

整合子是携带编码抗生素耐药基因盒的DNA片段,在细菌获得和传播耐药基因方面起着重要的作用。本文对整合子和基因盒种类、结构以及在耐药基因获得和传播中的作用机制进行了综述。     

整合子与细菌耐药性

整合子是由1个编码整合酶的intI基因、2个基因重组位点、启动子和耐药基因盒组成,根据整合酶的DNA碱基序列的不同分为4类,它能通过位点的基因重组机制使耐药基因移动,传递细菌耐药性,并与多重耐药性相关。     

细菌染色体第1类整合子捕获耐药性基因盒反应模型的构建

【背景】整合子在细菌耐药性的获得及传播中占据重要地位,对于整合反应检测方法的改良及反应机制的研究,可以加深我们对细菌耐药性产生和播散的理解,为遏制耐药菌株的产生和播散提供新的途径。【目的】在细菌染色体上构建第1类整合子反应模型,用于评价整合酶介导的基因盒位点特异性重组。【方法】 PCR分别扩增含氯霉素耐药基因cat的CM片段、含基因盒aadA5的LacA5片段、含整合子重组位点attI1及强可变区启动子的PcS片段和插入位点两侧的同源臂,重叠延伸聚合酶链反应连接上述5个片段制备整合子模型插入片段,通过同源重组将构建好的整合子模型片段插入大肠埃希菌JM109染色体中。转入高表达第1类整合酶的质粒pHSint,在链霉素平板上筛选发生整合的菌株,并经聚合酶链反应和测序验证。【结果】构建的整合子模型片段经测序与预期一致,整合子模型片段成功插入大肠埃希菌JM109染色体中。转入高表达整合酶的质粒pHSint后,在链霉素平板上成功筛选出基因盒aadA5发生整合的菌株,经聚合酶链反应扩增并测序与预期一致。【结论】在大肠埃希菌染色体上成功构建第1类整合酶介导基因盒位点特异性重组反应模型,为进一步揭示整合子捕获耐药性基因盒的反应机制奠定基础。     

革兰阴性杆菌基因盒-整合子系统与耐药性的研究进展

革兰阴性杆菌耐药机制十分复杂,既有天然耐药又有获得性耐药。其遗传物质基础在于细菌染色体、质粒、转座子及近年来发现的基因盒．整合子系统,后者是一种可以移动的基因元件系统,被认为是革兰阴性细菌多重耐药性迅速发展的主要原因。     

革兰阴性菌中整合子携带超广谱β-内酰胺酶的研究进展

整合子是一种主要存在于革兰阴性菌中的基因捕获和表达的遗传单位,能够选择性捕获或去除各种特异性耐药基因盒,从而加速了细菌耐药性的扩散.近年来,整合子介导的细菌耐药机制引起了研究人员的极大关注,成为研究细菌耐药传播机制的一大热点.目前已明确的位于整合子上的抗性基因已超过70多种[1],其中包括相继发现的一些超广谱β-内酰胺酶(ESBL)被整合在整合子上,这无疑加速了ESBL的传播速度.本文就整合子的分类、结构,革兰阴性菌中整合子携带ESBL的流行病学特征、检测和协同耐药基因的表达等作一综述.     

质粒介导的喹诺酮耐药基因qnr的分类、耐药机制及其在国内的流行状况北大核心CSCD

喹诺酮类抗菌药物从早期主要用于治疗尿道感染发展到后来治疗肠道感染和呼吸道感染,目前已在临床、畜牧业和水产业中广泛使用,细菌对其耐药性也逐渐呈蔓延趋势,耐药机制日趋复杂。喹诺酮类耐药机制主要分为染色体介导的耐药和质粒介导的耐药,后者对细菌耐药性的广泛传播起着重要作用。1998年首次报道了质粒介导的喹诺酮类耐药机制,即质粒上qnr基因介导的细菌对氟喹诺酮耐药机制,qnr基因可在不同细菌中迅速水平传播,引发的感染不易控制,使得院内感染大范围的流行。此外,qnr基因通常与β-内酰胺类耐药基因相关或存在于复杂整合子中与其它多重耐药基因共同整合,缩小了临床医生治疗相关细菌感染时选药或联合用药的空间,给我们带来了严峻的挑战。本文就qnr基因的发现历史、耐药机理及在国内的流行状况做了详细概述。     

产超广谱β-内酰胺酶细菌中I类整合子的研究进展

产超广谱β-内酰胺酶(Extended-spectrum beta-lactamase, ESBLs)细菌的多重耐药性是临床用药的一大难题, 近年研究发现其耐药性的产生与整合子密切相关, 其中临床最常见、研究最深入的是I类整合子。整合子是一种可移动基因元件, 在整合酶的作用下捕捉外源基因盒并使之表达, 是具有基因整合和切除功能的天然克隆和表达系统。研究表明I类整合子可连续捕捉和整合多种耐药基因, 以质粒或转座子为载体在细菌之间传播耐药性, 使ESBLs细菌多重耐药趋势十分严峻。本文就I类整合子的结构特征、I类整合子对耐药基因盒的整合作用及其与ESBLs细菌耐药性的关系等方面进行综述。     

细菌整合子与移动性基因盒的研究进展

整合子是由整合酶基因、基因盒和基因盒附着位点三者组成的遗传元件.在整合酶介导下,整合子通过位点特异的重组系统获取并交换外源DNA(基因盒),即将基因盒整合到整合子上或将之从整合子上剪切下来,但是整合子本身不能够移动.本文综述了国外近年来关于整合子与基因盒的结构特征及其分类的研究近况,对了解整合子及与之密切相关的移动性基因盒在细菌的多重耐药和毒力研究中,尤其是在适应选择性压力下细菌基因组进化中的作用具有重要的意义.     

整合子基因盒系统及β-内酰胺酶介导的细菌耐药

整合子是一个能捕获并整合细胞外游离基因盒,并可使之转化为功能性基因的新型DNA元件。这种可移动的基因元件通过水平基因转移的方式极大地加速了抗性基因在同种及不同种属之间的传播,造成细菌的耐药以至多重耐药问题日益严重,耐药机制日趋复杂。尤其对临床上使用较多的头孢菌素类、青霉素类等β-内酰胺类抗生素的耐药,已给人类健康造成巨大威胁,急需阐明其复杂的耐药机制。     

喹啉降解反应器菌群内整合子检测和菌株耐药性分析

运用PCR技术及克隆文库方法,对一个实验室规模的喹啉降解反应器生物膜系统中的整合子进行了分析。结果表明,在该反硝化喹啉降解反应器的生物膜群落中,整合子携带着丰富多样的基因盒。主要为编码与抗生素耐药性相关的基因盒,如氨基糖苷类耐药基因(aadA基因等),也带有与工业废水环境发现的整合子中可能与芳香族化合物降解有关的基因(如FldF基因)。还有一些功能未知的基因。鉴于耐药性相关基因的广泛存在,对该反应器中分离的优势菌株进行了耐药性分析。结果表明,44.1%的菌株存在耐药性,29.4%的菌株有多重耐药性。它们对4种抗生素的耐药率分别为:氨苄青霉素29.4%、卡那霉素23.5%、氯霉素20.6%、链霉素23.5%。不存在抗生素选择压力环境的微生物群落中分离的群落优势菌株普遍具有抗生素耐药性,而且群落基因组的整合子中携带多种抗生素抗性基因的基因盒。这一现象还未曾见报道,其成因值得进一步研究。     

Bacterial superintegrons, a source of new genes with adaptive functions

Data on the structural organization of the platform of integrons, gene cassettes, and integrons with integrated cassettes of genes encoding drug resistance are briefly summarized. Data obtained during recent years about superintegrons or chromosomal integrons, characteristics of their organization, the presence of genes with known adaptive and unidentified functions in them, as well as data on the differences between superintegrons and previously described multiple resistance integrons, are considered in more detail. Studies that provide evidence for translocations of gene cassettes from stationary chromosomal integrons into integrons associated with mobile elements resulting in gene flows in natural bacterial populations, which favors their survival and adaptation to changing environment, are also reviewed.     

Bacterial superintegrons, a source of new genes with adaptive functions

Data on the structural organization of the platform of integrons, gene cassettes, and integrons with integrated cassettes of genes encoding drug resistance are briefly summarized. Data obtained during recent years about superintegrons or chromosomal integrons, characteristics of their organization, the presence of genes with known adaptive and unidentified functions in them, as well as data on the differences between superintegrons and previously described multiple resistance integrons, are considered in more detail. Studies that provide evidence for translocations of gene cassettes from stationary chromosomal integrons into integrons associated with mobile elements resulting in gene flows in natural bacterial populations, which favors their survival and adaptation to changing environment, are also reviewed.     

Class 1 integrons potentially predating the association with tn402-like transposition genes are present in a sediment microbial community

Integrons are genetic elements that contribute to lateral gene transfer in bacteria as a consequence of possessing a site-specific recombination system. This system facilitates the spread of genes when they are part of mobile cassettes. Most integrons are contained within chromosomes and are confined to specific bacterial lineages. However, this is not the case for class 1 integrons, which were the first to be identified and are one of the single biggest contributors to multidrug-resistant nosocomial infections, carrying resistance to many antibiotics in diverse pathogens on a global scale. The rapid spread of class 1 integrons in the last 60 years is partly a result of their association with a specific suite of transposition functions, which has facilitated their recruitment by plasmids and other transposons. The widespread use of antibiotics has acted as a positive selection pressure for bacteria, especially pathogens, which harbor class 1 integrons and their associated antibiotic resistance genes. Here, we have isolated bacteria from soil and sediment in the absence of antibiotic selection. Class 1 integrons were recovered from four different bacterial species not known to be human pathogens or commensals. All four integrons lacked the transposition genes previously considered to be a characteristic of this class. At least two of these integrons were located on a chromosome, and none of them possessed antibiotic resistance genes. We conclude that novel class 1 integrons are present in a sediment environment in various bacteria of the beta-proteobacterial class. These data suggest that the dispersal of this class may have begun before the "antibiotic era."     

Gene cassettes and cassette arrays in mobile resistance integrons

Gene cassettes are small mobile elements, consisting of little more than a single gene and recombination site, which are captured by larger elements called integrons. Several cassettes may be inserted into the same integron forming a tandem array. The discovery of integrons in the chromosome of many species has led to the identification of thousands of gene cassettes, mostly of unknown function, while integrons associated with transposons and plasmids carry mainly antibiotic resistance genes and constitute an important means of spreading resistance. An updated compilation of gene cassettes found in sequences of such 'mobile resistance integrons' in GenBank was facilitated by a specially developed automated annotation system. At least 130 different (<98% identical) cassettes that carry known or predicted antibiotic resistance genes were identified, along with many cassettes of unknown function. We list exemplar GenBank accession numbers for each and address some nomenclature issues. Various modifications to cassettes, some of which may be useful in tracking cassette epidemiology, are also described. Despite potential biases in the GenBank dataset, preliminary analysis of cassette distribution suggests interesting differences between cassettes and may provide useful information to direct more systematic studies.     

Towards more virulent and antibiotic-resistant Salmonella?

Salmonella are well-known pathogens. Virulence determinants can be present on the chromosome, usually encoded on pathogenicity islands, or on plasmids and bacteriophages. Antibiotic resistance determinants usually are encoded on plasmids, but can also be present on the multidrug resistance region of Salmonella Genomic Island 1 (SGI1). Virulence plasmids show a remarkable diversity in the combination of virulence factors they encode, which appears to adapt them to specific hosts and the ability to cause gastroenteritidis or systemic disease. The appearance of plasmids with two replicons may help to extend the host range of these plasmids and thereby increase the virulence of previously non- or low pathogenic serovars. Antibiotic resistance among Salmonella is also increasing. This increase is not only in the percentage isolates resistant to a particular antibiotic, but also the development of resistance against newer antibiotics. The increased occurrence of integrons is particularly worrying. Integrons can harbour a varying set of antibiotic resistance encoding gene cassettes. Gene cassettes can be exchanged between integrons. Although the gene cassettes currently present in Salmonella integrons encode for older antibiotics (however, some still frequently used) gene cassettes encoding resistance against the newest antibiotics has been documented in Enterobacteriaceae. Furthermore, beta-lactamases with activity against broad-spectrum cephalosporins, which are often used in empiric therapy, have been found associated with integrons. So, empiric treatment of Salmonella infections becomes increasingly more difficult. The most worrisome finding is that virulence and resistance plasmids form cointegrates. These newly formed plasmids can be selected by antibiotic pressure and thereby for virulence factors. Taken together these trends may lead to more virulent and antibiotic-resistant Salmonella.     

Integrons: natural tools for bacterial genome evolution

Integrons were first identified as the primary mechanism for antibiotic resistance gene capture and dissemination among Gram-negative bacteria. More recently, their role in genome evolution has been extended with the discovery of larger integron structures, the super-integrons, as genuine components of the genomes of many species throughout the gamma-proteobacterial radiation. The functional platforms of these integrons appear to be sedentary, whereas their gene cassette contents are highly variable. Nevertheless, the gene cassettes for which an activity has been experimentally demonstrated encode proteins related to simple adaptive functions and their recruitment is seen as providing the bacterial host with a selective advantage. The widespread occurrence of the integron system among Gram-negative bacteria is discussed, with special focus on the super-integrons. Some of the adaptive functions encoded by these genes are also reviewed, and implications of integron-mediated genome evolution in the emergence of novel bacterial species are highlighted.     

A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons

A family of novel mobile DNA elements is described, examples of which are found at several independent locations and encode a variety of antibiotic resistance genes. The complete elements consist of two conserved segments separated by a segment of variable length and sequence which includes inserted antibiotic resistance genes. The conserved segment located 3' to the inserted resistance genes was sequenced from Tn21 and R46, and the sequences are identical over a region of 2026 bases, which includes the sulphonamide resistance gene sull, and two further open reading frames of unknown function. The complete sequences of both the 3' and 5' conserved regions of the DNA element have been determined. A 59-base sequence element, found at the junctions of inserted DNA sequences and the conserved 3' segment, is also present at this location in the R46 sequence. A copy of one half of this 59-base element is found at the end of the sull gene, suggesting that sull, though part of the conserved region, was also originally inserted into an ancestral element by site-specific integration. Inverted or direct terminal repeats or short target site duplications, both of which are characteristics of class I and class II transposons, are not found at the outer boundaries of the elements described here. Furthermore, the conserved regions do not encode any proteins related to known transposition proteins, except the DNA integrase encoded by the 5' conserved region which is implicated in the gene insertion process. Mobilization of this element has not been observed experimentally; mobility is implied from the identification of the element in at least four independent locations, in Tn21, R46 (IncN), R388 (IncW) and Tn1696. The definitive features of these novel elements are (i) that they include site-specific integration functions (the integrase and the insertion site); (ii) that they are able to acquire various gene units and act as an expression cassette by supplying the promoter for the inserted genes. As a consequence of acquiring different inserted genes, the element exists in a variety of forms which differ in the number and nature of the inserted genes. This family of elements appears formally distinct from other known mobile DNA elements and we propose the name DNA integration elements, or integrons.     

Molecular characterization of class 3 integrons from Delftia spp

Two environmental strains, Delftia acidovorans C17 and Delftia tsuruhatensis A90, were found to carry class 3 integrons, which have seldom been reported and then only from pathogens in which they are associated with antibiotic resistance genes. The Delftia integrons comprised a highly conserved class 3 integrase gene, upstream and oppositely oriented from a set of three or four gene cassettes that encoded unidentified functions. The A90 integron had one more gene cassette than the C17 integron, but the two were otherwise the same; furthermore, they were located within regions of sequence identity in both strains and linked to chromosomal genes. A screen of other Delftia and related strains did not reveal the presence of additional class 3 integrons. The observations suggest that these integrons were horizontally transferred to Delftia as part of a larger region and reside as chromosomal elements that probably predate transposon dissemination, as has been proposed for certain class 1 integrons.     

Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes.

The three classes of enzymes which inactivate aminoglycosides and lead to bacterial resistance are reviewed. DNA hybridization studies have shown that different genes can encode aminoglycoside-modifying enzymes with identical resistance profiles. Comparisons of the amino acid sequences of 49 aminoglycoside-modifying enzymes have revealed new insights into the evolution and relatedness of these proteins. A preliminary assessment of the amino acids which may be important in binding aminoglycosides was obtained from these data and from the results of mutational analysis of several of the genes encoding aminoglycoside-modifying enzymes. Recent studies have demonstrated that aminoglycoside resistance can emerge as a result of alterations in the regulation of normally quiescent cellular genes or as a result of acquiring genes which may have originated from aminoglycoside-producing organisms or from other resistant organisms. Dissemination of these genes is aided by a variety of genetic elements including integrons, transposons, and broad-host-range plasmids. As knowledge of the molecular structure of these enzymes increases, progress can be made in our understanding of how resistance to new aminoglycosides emerges.