细菌遗传元件水平转移与抗生素抗性研究进展*

细菌可移动遗传元件包括噬菌体、质粒、转座子、插入序列、整合子、基因组岛(genomic island and genomic islet)等,其中接合性质粒、转座子、整合子及基因组岛等是与抗生素抗性有关的元件,可以在同种甚至于不同种菌株间水平转移,加速了临床上耐药及多重耐药菌株的产生。综述了细菌与抗生素抗性有关的可移动遗传元件的种类、特征及转移机制的研究进展。     

多耐药肺炎克雷伯菌获得性耐药基因检测及指标聚类分析

目的调查多耐药肺炎克雷伯菌中65种获得性耐药基因和7种可移动遗传元件遗传标记基因的存在状况,以及获得性耐药基因和可移动遗传元件遗传标记基因的相关性。方法收集绍兴地区六家医院分离的肺炎克雷伯菌共20株,采用PCR的方法分析65种β-内酰胺类、氨基糖苷类、喹诺酮类获得性耐药基因和7种转座子、插入序列、接合性质粒遗传标记基因,并用指标聚类分析(SPSS法)分析β-内酰胺类、氨基糖苷类和喹诺酮类获得性耐药基因与整合子、转座子、插入序列、接合性质粒遗传标记基因的相关性。结果 20株肺炎克雷伯菌共检测到14种获得性耐药基因(包括6种β-酰胺类获得性耐药基因、6种氨基糖苷类获得性耐药基因、2种喹诺酮类获得性耐药基因)和6种可移动遗传元件遗传标记基因(包括1种整合子遗传标记基因、3种转座子和插入序列基因遗传标记基因、2种接合性质粒遗传标记基因),其余52种基因均未检测到。SPSS法将上述阳性检出基因分成两大簇群。结论绍兴地区六家医院的多耐药肺炎克雷伯菌菌株对抗菌药物的耐药表型与获得性耐药基因相关,且可移动遗传元件的水平转移使细菌的耐药性在同种细菌菌株之间甚至不同种细菌菌株之间得以快速传播。获得性耐药基因与可移动遗传元件遗传标记基因的指标聚类分析显示:OXA-1、aac(6’)-Ⅰb、qnrB、IMP、aadA5、VEB、KPC、qnrS等基因与接合性质粒遗传标记traA相关,提示这些基因在F接合性质粒上;DHA、aph(3′)-Ⅰ等基因与转座子遗传标记tnpU、tnp513相关,提示它们位于转座子上;TEM-1、aac(3)-Ⅱ、qacE△1与接合性质粒遗传标记trbC相关,提示TEM-1、aac(3)-Ⅱ等基因和Ⅰ类整合子可能位于宽范围接合性质粒上;ant(3″)-Ⅰ、rmtB等基因与ISEcp1较为相关,提示这些基因位于插入序列上。     

细菌中基因组岛的转移与调控机制研究进展

基因水平转移可导致细菌不同种属间个体DNA的交换,从而使细菌对环境的适应性增强,是细菌进化的重要途径之一。基因组岛是基因水平转移的重要载体,可移动的基因组岛能够整合到宿主的染色体上,并在特定的条件下切除,进而通过转化、接合或转导等方式转移到新的宿主中。基因组岛具有多种生物学功能,如抗生素抗性、致病性、异源物质降解、重金属抗性等。基因组岛的转移造成可变基因在不同种属细菌间的广泛传播,例如毒力和耐药基因的传播导致了多重耐药细菌的产生,威胁人类健康。基因组岛由整合酶介导转移,同时在转移的过程受到多种不同转录因子的调控。本文对细菌中基因组岛的结构特点、转移和调控机制以及预测等方面进行了综述,并最终阐明基因组岛的转移及其调控机制是遏制基因组岛传播的重要策略。     

细菌中介导多重耐药的Tn7转座子研究进展

转座子是介导细菌耐药性传播的重要可移动遗传元件。Tn7转座子与细菌耐药密切相关,其携带转座模块和Ⅱ类整合子系统。Tn7编码转座相关蛋白TnsABCDE进行“剪切-粘贴”机制转座,转座核心TnsABC也可与三链DNA或Cas-RNA复合物结合实现转座。近年来新发现了多种介导多重耐药的Tn7转座子,其在介导细菌抗生素、消毒剂和重金属抗性基因的获得、传播扩散等方面发挥了重要作用。本文综述了细菌中Tn7转座子的遗传结构、转座机制、流行以及新发现的介导多重耐药的Tn7转座子,以期为细菌中Tn7转座子的深入研究提供参考。     

一株污水源多重耐药大肠杆菌的耐药性检测及全基因组测序分析

【背景】水体环境分布广、流动性强,是耐药菌和耐药基因传播的主要媒介。【目的】了解北方污水厂大肠杆菌携带的耐药基因及可移动遗传元件情况。【方法】从北方污水厂筛选出一株多重耐药大肠杆菌,通过药敏试验进行耐药性检验,采用96孔板法测定菌株的最小抑菌浓度,利用酶标仪探究亚抑菌浓度抗生素对菌株生长的影响,并对菌株进行全基因组测序,对其携带的耐药基因及可移动遗传元件进行预测。【结果】大肠杆菌WEC对四环素、环丙沙星、诺氟沙星和红霉素具有耐药性,亚抑菌浓度的四环素、环丙沙星和诺氟沙星能够延缓或抑制菌株的生长。WEC菌株的基因组中包含一条大小为4 782 114 bp的环状染色体和2个大小分别为60 306 bp (pWEC-1)和92 065 bp (pWEC-2)的环状质粒。菌株共携带129个耐药基因,其中128个位于染色体上,在染色体上预测到原噬菌体、基因岛及插入序列的存在,部分可移动遗传元件携带有耐药基因。质粒pWEC-1中无耐药基因,pWEC-2含有1个耐药基因,在质粒基因组中预测到原噬菌体和插入序列。【结论】污水源大肠杆菌WEC是一株多重耐药菌株,其基因组中携带耐药基因和多种可移动遗传元件...     

沙门氏菌抗生素抗性机理研究进展

沙门氏菌的多重耐药性问题已经成为世界范围内的公共卫生和经济问题.目前沙门氏菌抗生素抗性机理的研究主要集中以下方面:(1)基因突变与抗生素抗性;(2)外排泵与抗生素抗性:(3)耐药基因编码的钝化酶和灭活酶引起的抗生素抗性;(4)可移动的细菌遗传耐药基因元件及其转移与抗生素抗性.本文基于以上几个方面综述了与沙门氏菌抗生素抗性机理研究相关的研究动态和研究进展.     

泛耐药鲍氏不动杆菌看家基因和水平转移基因样本聚类分析

目的了解20株泛耐药鲍氏不动杆菌的菌株亲缘性。方法完成该组泛耐药鲍氏不动杆菌2种与耐药相关的看家基因和54种水平转移获得与β-内酰胺类、氨基糖苷类、喹诺酮类耐药相关基因以及13种接合性质粒、转座子、插入序列、整合子等可移动遗传元件遗传标记检测,并对检测结果作样本聚类分析。结果20株泛耐药鲍氏不动杆菌已经发生演化,并存在2个克隆传播：1-4—6号菌株和2—5—7—8—9—10—11—12—13—14—15-16—17—18-19号菌株。结论与耐药相关的看家基因和水平转移获得的耐药基因均为显性遗传,本研究耐药菌所观察的表型与之相对应,为追溯耐药菌传播途径提供了方便。     

携带armA基因的铜绿假单胞菌耐药性及基因周边环境

目的了解多重耐药(MDR)铜绿假单胞菌armA基因与可移动遗传元件的携带情况及其相关性;分析armA基因的周边环境,探讨armA基因转移的可能机制。方法收集MDR铜绿假单胞菌98株,琼脂稀释法测定MIC,PCR方法检测16S rRNA甲基化酶基因armA、I型整合子、可移动元件IS26及重要耐药基因侧翼基因环境,测序并拼接PCR产物明确耐药基因座位排列,并对armA基因进行周边序列分析。结果 98株MDR铜绿假单胞菌检出5株armA基因PCR扩增阳性,携带armA基因的菌株对庆大霉素和阿米卡星全耐药;检出20株携带I型整合子,17株携带可移动元件IS26;armA基因扩增阳性的菌株均携带I型整合子和IS26;序列测序显示armA定位于Tn1548相关区域,位于插入序列ISCR1的下游,该序列含多种移动元件。结论大连市氨基糖苷类高水平耐药基因armA广泛分布在MDR铜绿假单胞菌中,均对庆大霉素和阿米卡星高度耐药;该基因定位在转座子Tn1548的质粒上,提示16S rRNA甲基化酶基因armA的广泛播散可能是可移动元件ISCR1-armA-IS26结构参与其中。     

细菌中整合性接合元件的研究进展

整合性接合元件是近年来在细菌中发现的一种可移动的基因元件,它位于染色体上,可通过接合转移的方式介导细菌间基因的水平转移。这种基因的水平转移有助于细菌适应特定的环境条件,但许多整合性接合元件包含耐药基因,这些遗传元件的水平转移极大地加速了耐药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;同时整合性接合元件与基因岛有着密切的联系,因此对其特征及转移机制进行研究很有必要。     

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。     

Transposon Tn21, flagship of the floating genome.

The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.     

Transposon Tn21, Flagship of the Floating Genome

The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.     

Antibiotic resistance gene spread due to manure application on agricultural fields

The usage of antibiotics in animal husbandry has promoted the development and abundance of antibiotic resistance in farm environments. Manure has become a reservoir of resistant bacteria and antibiotic compounds, and its application to agricultural soils is assumed to significantly increase antibiotic resistance genes and selection of resistant bacterial populations in soil. The genome location of resistance genes is likely to shift towards mobile genetic elements such as broad-host-range plasmids, integrons, and transposable elements. Horizontal transfer of these elements to bacteria adapted to soil or other habitats supports their environmental transmission independent of the original host. The human exposure to soil-borne resistance has yet to be determined, but is likely to be severely underestimated.     

The 79,370-bp conjugative plasmid pB4 consists of an IncP-1beta backbone loaded with a chromate resistance transposon,the strA-strB streptomycin resistance gene pair,the oxacillinase gene bla(NPS-1), and a tripartite antibiotic efflux system of the resistance-nodulation-division family

Plasmid pB4 is a conjugative antibiotic resistance plasmid, originally isolated from a microbial community growing in activated sludge, by means of an exogenous isolation method with Pseudomonas sp. B13 as recipient. We have determined the complete nucleotide sequence of pB4. The plasmid is 79,370 bp long and contains at least 81 complete coding regions. A suite of coding regions predicted to be involved in plasmid replication, plasmid maintenance, and conjugative transfer revealed significant similarity to the IncP-1beta backbone of R751. Four resistance gene regions comprising mobile genetic elements are inserted in the IncP-1beta backbone of pB4. The modular 'gene load' of pB4 includes (1) the novel transposon Tn 5719 containing genes characteristic of chromate resistance determinants, (2) the transposon Tn 5393c carrying the widespread streptomycin resistance gene pair strA-strB, (3) the beta-lactam antibiotic resistance gene bla(NPS-1) flanked by highly conserved sequences characteristic of integrons, and (4) a tripartite antibiotic resistance determinant comprising an efflux protein of the resistance-nodulation-division (RND) family, a periplasmic membrane fusion protein (MFP), and an outer membrane factor (OMF). The components of the RND-MFP-OMF efflux system showed the highest similarity to the products of the mexCD-oprJ determinant from the Pseudomonas aeruginosa chromosome. Functional analysis of the cloned resistance region from pB4 in Pseudomonas sp. B13 indicated that the RND-MFP-OMF efflux system conferred high-level resistance to erythromycin and roxithromycin resistance on the host strain. This is the first example of an RND-MFP-OMF-type antibiotic resistance determinant to be found in a plasmid genome. The global genetic organization of pB4 implies that its gene load might be disseminated between bacteria in different habitats by the combined action of the conjugation apparatus and the mobility of its component elements.     

水环境中耐药菌污染与危害研究进展

细菌在抗菌药选择性压力下产生耐药性并可传代,通过质粒和整合子等可移动基因元件将耐药基因在相同或不同种属中广泛传播,导致细菌多重耐药,并可通过多种途径进入水体,水环境日益成为庞大的耐药基因库,为致病菌及条件致病菌提供获得大量耐药基因的机会,若多重耐药菌再次侵入人体,可能引发严重的公共卫生问题。     

Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment

The impact of human activity on the selection for antibiotic resistance in the environment is largely unknown, although considerable amounts of antibiotics are introduced through domestic wastewater and farm animal waste. Selection for resistance may occur by exposure to antibiotic residues or by co-selection for mobile genetic elements (MGEs) which carry genes of varying activity. Class 1 integrons are genetic elements that carry antibiotic and quaternary ammonium compound (QAC) resistance genes that confer resistance to detergents and biocides. This study aimed to investigate the prevalence and diversity of class 1 integron and integron-associated QAC resistance genes in bacteria associated with industrial waste, sewage sludge and pig slurry. We show that prevalence of class 1 integrons is higher in bacteria exposed to detergents and/or antibiotic residues, specifically in sewage sludge and pig slurry compared with agricultural soils to which these waste products are amended. We also show that QAC resistance genes are more prevalent in the presence of detergents. Studies of class 1 integron prevalence in sewage sludge amended soil showed measurable differences compared with controls. Insertion sequence elements were discovered in integrons from QAC contaminated sediment, acting as powerful promoters likely to upregulate cassette gene expression. On the basis of this data, >1 × 10¹⁹ bacteria carrying class 1 integrons enter the United Kingdom environment by disposal of sewage sludge each year.     

Multidrug-resistant Salmonella enterica serovar paratyphi A harbors IncHI1 plasmids similar to those found in serovar typhi

Salmonella enterica serovars Typhi and Paratyphi A cause systemic infections in humans which are referred to as enteric fever. Multidrug-resistant (MDR) serovar Typhi isolates emerged in the 1980s, and in recent years MDR serovar Paratyphi A infections have become established as a significant problem across Asia. MDR in serovar Typhi is almost invariably associated with IncHI1 plasmids, but the genetic basis of MDR in serovar Paratyphi A has remained predominantly undefined. The DNA sequence of an IncHI1 plasmid, pAKU_1, encoding MDR in a serovar Paratyphi A strain has been determined. Significantly, this plasmid shares a common IncHI1-associated DNA backbone with the serovar Typhi plasmid pHCM1 and an S. enterica serovar Typhimurium plasmid pR27. Plasmids pAKU_1 and pHCM1 share 14 antibiotic resistance genes encoded within similar mobile elements, which appear to form a 24-kb composite transposon that has transferred as a single unit into different positions into their IncHI1 backbones. Thus, these plasmids have acquired similar antibiotic resistance genes independently via the horizontal transfer of mobile DNA elements. Furthermore, two IncHI1 plasmids from a Vietnamese isolate of serovar Typhi were found to contain features of the backbone sequence of pAKU_1 rather than pHCM1, with the composite transposon inserted in the same location as in the pAKU_1 sequence. Our data show that these serovar Typhi and Paratyphi A IncHI1 plasmids share highly conserved core DNA and have acquired similar mobile elements encoding antibiotic resistance genes in past decades.     

Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria.

Many multiresistance plasmids and transposons of gram-negative bacteria carry related DNA elements that appear to have evolved from a common ancestor by site-specific integration of discrete cassettes containing antibiotic resistance genes or sequences of unknown function. The site of integration is flanked by conserved segments coding for an integraselike protein and for sulfonamide resistance, respectively. These segments, together with the antibiotic resistance genes between them, have been termed integrons (H. W. Stokes and R. M. Hall, Mol. Microbiol. 3:1669-1683, 1989). We report here the characterization of an integron, In0, from Pseudomonas aeruginosa plasmid pVS1, which has an unoccupied integration site and hence may be an ancestor of more complex integrons. Codon usage of the integrase (int) and sulfonamide resistance (sul1) genes carried by this integron suggests a common origin. This contrasts with the codon usage of other antibiotic resistance genes that were presumably integrated later as cassettes during the evolution and spread of these DNA elements. We propose evolutionary schemes for (i) the genesis of the integrons by the site-specific integration of antibiotic resistance genes and (ii) the evolution of the integrons of multiresistance plasmids and transposons, in relation to the evolution of transposons related to Tn21.