细菌演化的天然工具——超级整合子

已有证据表明整合子是细菌发生水平基因转移和产生耐药机制的主要因素。目前知道的整合子可分为两类：多重抗性整合子和超级整合子。多重抗性整合子基因盒可编码一种或多种耐抗生素和消毒剂基因。存在于转座子、质粒和细菌染色体；超级整合子有的可以同时携带数百个基因盘，可以编码很多不同的功能基因，它们只在细菌的染色体上存在，目前只在特定菌株中发现超级整合子。研究表明，整合子上的基因盒可能最初都来之于超级整合子。本文就超级整合子的结构、分布、起源及它对基因进化产生的影响等几个方面的研究进展进行讨论。     

整合子系统与细菌多重耐药研究进展

细菌的多重耐药已成为临床治疗的难题，整合子是携带编码抗生素耐药基因盒的DNA片断，在细菌获得和传播耐药基因方面起着重要作用。本文就整合子及其基因盒的分类、分布、结构、来源、整合子对基因盒的捕获表达、整合子与细菌的多重耐药性关系及多重耐药菌感染的治疗等作一阐述。     

第一类整合子整合酶基因intI1的定位分析

目的　整合子 ( integrons)介导的细菌耐药特性已成为研究细菌耐药机制的热点 ,在研究了来自正常人携带沙门氏菌中整合子的分布和特性的基础上 ,进一步探讨整合子的基因定位。方法　从已鉴定的整合子阳性菌株出发 ,分别提取其质粒和染色体 DNA,进行质粒的接合转移试验。对染色体 DNA进行限制性酶切 ,以第一类整合酶基因 int I1( DIG标记 )为探针 ,进行 Southern杂交。结果　 4株整合酶阳性菌株不存在含有第一类整合子的接合性质粒 ,确定 4株整合子阳性菌株的整合酶基因 int I1基因位于染色体上。结论　本文发现的整合子阳性菌株对耐药基因的捕获是通过染色体 DNA介导的。     

整合子与细菌耐药性关系的研究进展

细菌耐药性是临床用药的一大难题,对其耐药机制探讨也就成为医学界研究热点,近年研究发现细菌耐药性产生与一种克隆表达载体——整合子密切相关。整合子是一种可移动基因元件,在整合酶的作用下捕获外源基因盒并使之表达,同时整合子又可整合到质粒上,或自身作为转座子的一个组成部分而参与转移,使耐药基因在不同种属间传播,目前整合子已成为革兰阴性杆菌产生耐药性的重要机制。本文就整合子与细菌多重耐药性关系作一综述。     

整合子与铜绿假单胞菌多重耐药性研究进展

铜绿假单胞菌（PA）多重耐药性是临床抗感染治疗的难点，以往的研究多注重PA的天然耐药机制，如外膜渗透性降低及泵出机制。近年来，在PA中发现了多种β-内酰胺酶，如广谱酶中的PSE-1。SHV-1．超广谱酶中的VEB-1。对碳青霉烯类抗生紊（如亚胺培南）耐受的金属β-内酰胺酶。但上述研究只局限于细菌的单一耐药机制。不能解释为什么没有接触过抗生素的病原菌也会出现对抗生紊的多重耐药。基因盒一整合子系统是细菌基因组中可移动的基因克隆和表达单位，能携带位点特异性重组系统组分，形成多种基因的组合、排列，对细菌及质粒基因组的进化具有重要意义。并且通常涉及到抗生素的耐药基因。与细菌产生多重耐药和耐药基因的水平传播密切相关。目前发现，细菌整合子携带的耐药基因有70余种，整合子作为一个移动遗传元件，通过质粒、转座子在细菌同种或不同种属间进行基因水平转移。使细菌的耐药性在病原菌中广泛传播。整合子结构是国际上研究细菌多重耐药机制、监测细菌耐药性播散趋势的热点。     

细菌II型、III型整合子在耐药性传播中的作用#br#

整合子是可以定位于染色体、质粒或转座子上的可移动遗传元件，它可以通过位点特异性基因重组来捕获、整合或剪切基因盒，使耐药基因在细菌间进行水平转移，从而增强细菌的生存适应性。目前发现的整合子主要分为I、II和III型，针对I型整合子的研究已较广泛和深入，而对II型和III型整合子的研究并不多见，可能是因为它们在菌株中的存在并不常见。目前发现含有III型整合子的菌种不足10种。尽管如此，II型和III型整合子在耐药性传播中起的作用却不可忽视。本文通过参考近几年来国内外相关文献，对报道的II型和III型整合子的结构以及它们在细菌耐药性传播中的作用进行综述。     

多重耐药临床菌株中整合子结构的检测与分析

目的研究广州暨南大学附属第一医院2004年部分临床菌株样本的整合子及其基因盒的分布特性。方法多重PCR检测与细菌耐药关系密切的1、2、3类整合酶基因,进一步对阳性样本可变区的基因盒序列鉴定分析。结果随机抽取109株临床菌株,整合酶阳性检出率为97.2%(106/109),其中1类整合酶阳性菌100株(91.7%),2类整合酶阳性菌1株(0.92%),此外有5株(4.6%)同时检出1、2类整合酶,没有检测到3类整合酶;基因盒鉴定结果显示,插入基因盒以dfrA(甲氧苄氨嘧啶耐药相关)和aadA(氨基糖苷类耐药相关)基因家族为主,也在少数菌株中发现了aacA4、cmlA1、catB3以及sat1基因盒。其中又以dfrA12、orfF和aadA2组合最为常见,耐药基因盒PCR扩增片段为1913bp(64.6%);此外,还发现了同时存在两种整合子结构的菌株。结论整合子普遍存在于临床菌株中,可通过基因水平转移在不同菌属间传播,提示各医药单位必须加强耐药监测及合理选择抗菌药物,以减少多重耐药细菌的发生和发展。     

鲍曼不动杆菌对亚胺培南耐药分子机制的研究

目的了解鲍曼不动杆菌对亚胺培南产生耐药的分子机制。方法收集对亚胺培南耐药的鲍曼不动杆菌(imipenem resistant Acinetobacter baumannii，IRAB)无重复株共9株，采用琼脂稀释法进行药敏检测，协同抑制试验、质粒接合试验、Southern杂交、等电聚焦电泳、PCR扩增blavIM、blaIMP、baooxA-23、blaoA-24相关基因及整合子编码序列及其分子克隆和测序，以阐述其分子耐药机制。结果本组IRAB具有多重耐药性；耐药基因分子克隆、测序并结合等电聚焦分析证实均产OXA-23型碳青霉烯酶，质粒接合试验、Southern杂交显示其编码基因定位在染色体上。9株细菌均检测出Ⅰ型整合子基因结构(大小约1．2～4kb)；3株菌检测出Ⅱ型整合子基因结构(1．8～2kb)，均携带了多种耐药基因。结论产OXA-23型p内酰胺酶是本组鲍曼不动杆菌对碳青霉烯类抗生素产生耐药性的重要原因；IRAB整合子基因携带的多种耐药基因与其多重耐药性相关。     

临床分离革兰阴性菌整合子研究

目的 了解临床分离革兰阴性菌整合子的产生情况及整合子在细菌耐药中的作用.方法 E实验测定细菌对11种抗生奈及抗生素复合制剂的最低抑菌浓度,PCR扩增三种整合酶基因及Ⅰ型整合子的可变区,并对可变区的扩增产物进行克隆、测序.结果 16株革兰阴性菌中,6株含Ⅰ型整合子,所含基因盒主要是妒dfr/dhf及aadA基因盒.结论 整合子在不同革兰阴性菌中广泛存在,与细菌的多重耐药尤其是对氨基糖苷类抗生素的耐药密切相关.     

产ESBLs并高产AmpC酶肺炎克雷伯氏杆菌中整合子的基因研究

目的 :观察 4株从临床分离产超广谱内 β内酰胺酶 (ESBLs)及AmpC酶肺炎克雷伯氏杆菌的多重耐药情况 ,分析其中整合子的存在 ,对整合子的特性进行研究 ,探讨整合子基因盒表达对肺炎克雷伯杆菌耐药表型的影响。方法 :采用微量稀释法测定复方新诺明等 15种抗菌药物对细菌的最低抑菌浓度 (MIC)。PCR技术检测整合子基因 ,DNA测序研究整合子插入耐药基因盒情况。结果 :这 4株菌对多种抗菌素耐药。其中 1株存在整合子 (扩增片段 2 0 0 0bp左右 ) ,携有dhfrXII和aadA2基因。结论 :4株菌中仅 1株存在整合子结构 ,尽管产ESBLs和AmpC酶基因未位于整合子的基因盒上 ,但整合子参与多重耐药的产生。     

Antibiotic resistance, integrons and Salmonella genomic island 1 among non-typhoidal Salmonella serovars in The Netherlands

The objective of this study was to investigate the antimicrobial resistance patterns, integron characteristics and gene cassettes as well as the presence of Salmonella genomic island 1 (SGI1) in non-typhoidal Salmonella (NTS) isolates from human and animal origin. Epidemiologically unrelated Dutch NTS strains (n=237) originating from food-producing animals and human cases of salmonellosis were tested for their susceptibility to 15 antimicrobial agents. Resistance to 14 of these antimicrobials, including the third-generation cephalosporins, was detected. Resistance to sulphonamides, ampicillin, tetracycline, streptomycin, trimethoprim and nalidixic acid was common (>/=10% of the strains were resistant). Resistance against three or more antimicrobials was observed in 57 isolates. The same 237 strains were studied for the prevalence of class 1 integrons, their gene cassettes and the presence of SGI1. Thirty-six isolates (15.2%) carried class 1 integrons. These integrons had ten distinct profiles based on the size of the integron and restriction fragment length polymorphism analysis. Integrons were detected for the first time in serovars Indiana and Senftenberg. Multidrug resistance was strongly associated with the presence of class 1 integrons in which the aadA2, aadA1, bla(PSE-1), dfrA1, dfrA5, dfrA14 or sat genes were present, as determined by nucleotide sequence determination. The presence of gene cassettes or combinations of gene cassettes not previously found in integrons in Salmonella was observed. SGI1 or its variants (SGI-B, -C and -F) were present in 16 isolates belonging to either serovar Typhimurium, Derby or Albany. Regardless of whether the isolate was of human or animal origin, the same resistance phenotype, integron profile and SGI1 structure could be observed.     

Antibiotic resistance in gram-negative bacteria: the role of gene cassettes and integrons.

Resistance of gram-negative organisms to antibiotics such as beta-lactams, aminoglycosides, trimethoprim and chloramphenicol is caused by many different acquired genes, and a substantial proportion of these are part of small mobile elements known as gene cassettes. A gene cassette consists of the gene and a downstream sequence, known as a 59-base element (59-be), that acts as a specific recombination site. Gene cassettes can move into or out of a specific receptor site (attl site) in a companion element called an integron, and integration or excision of the cassettes is catalysed by a site-specific recombinase (Intl) that is encoded by the integron. At present count there are 40 different cassette-associated resistance genes and three distinct classes of integron, each encoding a distinct Intl integrase. The same cassettes are found in all three classes of integron, indicating that cassettes can move freely between different integrons. Integrons belonging to class I often contain a further antibiotic resistance gene, sull, conferring resistance to sulphonamides. The sull gene is found in a conserved region (3'-CS) that is not present in all members of this class. Class I integrons of the sull type are most prevalent in clinical isolates and have been found in many different organisms. Even though most of them are defective transposon derivatives, having lost at least one of the transposition genes, they are none the less translocatable and consequently found in many different locations. The transposon Tn7 is the best known representative of class 2 integrons, and Tn7 and relatives are also found in many different species.     

Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria

Bacteria have existed on Earth for three billion years or so and have become adept at protecting themselves against toxic chemicals. Antibiotics have been in clinical use for a little more than 6 decades. That antibiotic resistance is now a major clinical problem all over the world attests to the success and speed of bacterial adaptation. Mechanisms of antibiotic resistance in bacteria are varied and include target protection, target substitution, antibiotic detoxification and block of intracellular antibiotic accumulation. Acquisition of genes needed to elaborate the various mechanisms is greatly aided by a variety of promiscuous gene transfer systems, such as bacterial conjugative plasmids, transposable elements and integron systems, that move genes from one DNA system to another and from one bacterial cell to another, not necessarily one related to the gene donor. Bacterial plasmids serve as the scaffold on which are assembled arrays of antibiotic resistance genes, by transposition (transposable elements and ISCR mediated transposition) and site-specific recombination mechanisms (integron gene cassettes).The evidence suggests that antibiotic resistance genes in human bacterial pathogens originate from a multitude of bacterial sources, indicating that the genomes of all bacteria can be considered as a single global gene pool into which most, if not all, bacteria can dip for genes necessary for survival. In terms of antibiotic resistance, plasmids serve a central role, as the vehicles for resistance gene capture and their subsequent dissemination. These various aspects of bacterial resistance to antibiotics will be explored in this presentation.     

大肠埃希菌中Ⅰ类整合子耐药基因的分析

目的 对临床分离大肠埃希菌株携带的Ⅰ类整合子及相关耐药基因进行筛选和分析.探讨Ⅰ类整合子在大肠埃希菌耐药中的作用.方法 对43株大肠埃希菌临床分离株做药敏试验;采用PCR扩增、DNA测序、DNA序列比对的方法 对其携带的Ⅰ类整合子相关耐约基因进行分析.结果 43株大肠埃希菌分离株对10种抗菌药物的耐药率依次为亚胺培南4.7%、阿米卡星18.6%、头孢他啶、27.9%、头孢吡肟37.2%、头孢呋辛55.8%、复方磺胺甲嗯唑58.1%、妥布霉素74.4%、庆大霉素79.1%、头孢噻肟81.4%和哌拉两林83.7%.在43株大肠埃希菌分离株中有25株含有Ⅰ类整合子,其中18株携带整合子相关耐药基因,如介导对磺胺类和氨基糖苷类约物的耐药基因等;某些菌株携带的整合子相关耐药基因相同.结论 Ⅰ类整合子在大肠埃希菌中广泛存在,整合子相关耐约基因在该菌耐药性的形成和播散中发挥作用.     

Detection of integrons and antibiotic-resistance genes in Salmonella enterica serovar Typhimurium isolates with resistance to ampicillin and variable susceptibility to amoxicillin-clavulanate

We characterized 29 antimicrobial-resistant Salmonella enterica serovar Typhimurium strains, including four belonging to the monophasic variant 4,5,12:i:-, mostly isolated from infants. They were selected from 3230 strains isolated in the years 1990-2001 on the basis of resistance to ampicillin and variable susceptibility to the amoxicillin-clavulanate combination. Twenty-three strains were resistant to more than four antibiotics. All the strains carried the bla(TEM) gene and most were able to transfer this gene by conjugation. Sequencing of the gene from one of the amoxicillin-clavulanate-resistant strains allowed identification of the encoded beta-lactamase as TEM-1; all of these strains carried a second gene encoding beta-lactamase production, either pse-1 or oxa1. However, the association of bla(TEM) plus pse-1 genes did not always confer resistance to amoxicillin-clavulanate. The pse-1 gene, found in 17 strains, was located in the Salmonella Genomic Island-1 (SGI1), which carries two integrons and encodes multiple drug-resistance. None of the oxa1-bearing strains had the SGI1, yet this gene was found as part of an integron that also carried the aadA1 gene and was not plasmid-associated. Thirteen of the strains harbouring SGI1 belonged to the definitive phage type (DT) 104, and most of those remaining to DT104b and U302; particularly, strains carrying the oxa1-aadA1 integron belonged to the last two phage types. Pulsed field electrophoresis confirmed the clonal organization of DT104 strains, whereas U302 strains fell into different groups, depending on their resistance determinants.     

The functions and structure of ABC transporters: implications for the design of new inhibitors of Pgp and MRP1 to control multidrug resistance (MDR)

Multidrug resistance (MDR) is a kind of acquired resistance of microorganisms and cancer cells to chemotherapic drugs that are characterized by different chemical structure and different mechanism of action. Classic MDR is the consequence of the over-expression of a variety of proteins that extrude the chemotherapic from the cell, lowering its concentration below the effective one. The ABC (ATP Binding Cassette) is a ubiquitous and important family of such transporter proteins. Members of this super family are present in mammals as well as in prokaryotic organisms and use ATP as the energy source to activate the extrusion process. P-glycoprotein (Pgp) and Multidrug Resistance Proteins (MRP1 and sister proteins) are the most important and widely studied members of ABC super family. Our knowledge about the structures and functions of transporter proteins has definitely improved in recent years, following the resolution of the structure of bacterial pumps which opened the way to the building of homology models for the more complex Pgp and MRP. It can be anticipated that these results will have a strong impact on the design of more potent and safer MDR reverters. A huge number of small molecules, many of natural origin, are able to reverse multidrug resistance by inhibiting the functions of Pgp, MRP1 and sister proteins and their action has been considered a possible way to reverse MDR. However, while a few compounds have reached clinical trials, none of them has, so far, been cleared for therapeutic use. Two main reasons are at the base of this difficulty: i) MDR is a complex phenomenon that may arise from several different biochemical mechanisms, with the consequence that inhibition of transporter proteins may be insufficient to reverse it; ii) the physiological role of Pgp and sister proteins requires more potent modulators with proper selectivity and pharmacokinetic in order to avoid unwanted side effects. This paper first reviews the most recent discoveries on the structures and functions of the ABC super family, in particular Pgp and MRP. Then, the medicinal chemistry of MDR reverters, in light of these findings, is discussed and the molecules that are presently in development are reviewed.