中华预防医学杂志    2018年10期 金黄色葡萄球菌中可移动遗传元件与耐药传播机制研究进展    PDF     文章点击量:44    
中华预防医学杂志2018年10期
中华医学会主办。
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文章信息

王伟 李凤琴
WangWei,LiFengqin
金黄色葡萄球菌中可移动遗传元件与耐药传播机制研究进展
Overview of Staphylococcus aureus mobile genetic elements and horizontal gene transfer of antimicrobial resistance genes
中华预防医学杂志, 2018,52(10)
http://dx.doi.org/10.3760/cma.j.issn.0253-9624.2018.10.020

文章历史

投稿日期: 2017-12-05
上一篇:肠球菌耐药机制与食源性传播研究进展
下一篇:弯曲菌耐药机制研究进展
金黄色葡萄球菌中可移动遗传元件与耐药传播机制研究进展
王伟 李凤琴     
王伟 100021 北京,国家食品安全风险评估中心 卫生部食品安全风险评估重点实验室
李凤琴 100021 北京,国家食品安全风险评估中心 卫生部食品安全风险评估重点实验室
摘要: 金黄色葡萄球菌可移动遗传元件介导的耐药传播机制复杂,给耐药性防控带来重大挑战。本文结合国内外最新文献,综述了金黄色葡萄球菌抗生素耐药传播相关的可移动遗传元件及其传播机制研究进展。金黄色葡萄球菌编码耐药基因的可移动遗传元件种类繁多,不同的可移动遗传元件编码耐药基因不尽相同,其传播机制也具有特异性,人和动物来源的金黄色葡萄球菌可移动遗传元件存在显著差异,金黄色葡萄球菌可移动遗传元件可在人和动物间交互传递,因此又存在一定的趋向性。可通过研究金黄色葡萄球菌耐药基因随可移动遗传元件扩散的传播机制,解析可移动遗传元件转移过程重要的生物学信息,有效防控金黄色葡萄球菌耐药性在动物、人类和环境中的传播。
关键词 :葡萄球菌,金黄色;抗药性;传播机制;可移动遗传元件
Overview of Staphylococcus aureus mobile genetic elements and horizontal gene transfer of antimicrobial resistance genes
WangWei,LiFengqin     
Key Lab of Food Safety Risk Assessment, Ministry of Health, China National Center for Food Safety Risk Assessment, Beijing 100021, China
Corresponding author: Li Fengqin, Email: lifengqin@cfsa.net.cn
Abstract:The mechanism of antimicrobial resistance transmission mediated by mobile genetic elements (MGEs) in Staphylococcus aureus is highly complicated, leading a significant challenge for controlling the spread of the resistant Staphylococcus aureus strains. Based on the latest literature acquired in this work, we have overviewed the transmission mechanism of antimicrobial resistance encoding MGEs. It is notably that there are a number of MGEs, which may encode different antimicrobial resistance determinants and possess specific transmission mechanism. In spite of this specificity of the strains to their host (human or animal), some Staphylococcus aureus strains can be transmitted from animals to humans or vice versa. This ability of cross staphylococci transfer is an additional means to acquire new genetic material encoded by MGE. It was suggested in this review that study on transmission mechanism of MGEs mediated antimicrobial resistance genes could provide important biological information of their spreading and effectively help prevent and control of the resistant strains and/or resistance genes among human, animals and ecologies.
Key words :Staphylococcus aureus;Drug resistance;Horizontal gene transfer;Mobile genetic elements
全文

金黄色葡萄球菌(金葡菌)是引起人类细菌性感染的典型致病菌之一,金葡菌肠毒素引起的食物中毒事件在革兰阳性菌中也高居首位[1,2,3]。随着抗生素在养殖动物和临床的广泛使用,金葡菌耐药率逐年升高,所引起的感染也呈逐年增多的趋势[4,5,6]。同时,耐甲氧西林金葡菌(methicillin-resistant Staphylococcus aureus,MRSA)的出现,尤其在养殖动物、环境和食品中的流行,给其所致感染的治疗带来巨大挑战,使得临床上只能选用万古霉素、达托霉素和利奈唑胺等少数几种替代性药物进行治疗[7]。此外,近年来万古霉素中介金葡菌(vancomycin-intermediate Staphylococcus aureus,VISA)、利奈唑胺耐药及达托霉素非敏感金葡菌在全球范围内的传播,使得MRSA感染的治疗变得更加复杂[8,9,10]。因此,了解和掌握金葡菌尤其是MRSA等多重耐药菌的耐药和传播机制,对预防和控制金葡菌耐药性传播具有重要意义。
        金葡菌耐药性的传播,与其基因组携带的可移动遗传元件(mobile genetic elements, MGEs)密切相关。可移动遗传元件包括质粒(plasmids)、噬菌体(bacteriophages)、转座子(transposons)、插入序列(insertion sequences)、致病岛(staphylococcal pathogenicity islands, SPIs)、金葡菌盒式染色体(staphylococcal cassette chromosome, SCCs)、整合性接合元件(integrative conjugative element,ICE)和整合子(integrons)等,其中除噬菌体外,其他可移动遗传元件都可携带耐药基因(antibiotic-resistance genes, ARGs)[11]。可移动遗传元件使得细菌更容易适应新的环境,尤其是抗生素选择性压力,促进细菌产生抗生素耐药,进而在环境中传播和扩散。本文针对金葡菌可移动遗传元件介导的抗生素耐药基因传播机制综述如下。

一、耐药质粒  

(一)质粒分类  质粒是独立于细菌染色体之外并具有自主复制能力的小分子双链环状DNA,可通过水平基因转移介导耐药或毒力基因在同属内或菌属间转移。金葡菌可携带1个或多个大小为1~60 kb的质粒[12]。根据其质粒大小,可分为3类:(1)大小在1.3~4.6 kb之间的小质粒,一般不携带或仅携带1个耐药基因,如pT181、pS194和pE194等[13,14,15]。(2)大小在15~46 kb之间的低拷贝数质粒(4~6个左右),大多为青霉素类、氨基糖苷类和甲氧苄啶类抗生素耐药质粒,如pSK1和pIP630等[15,16]。(3)大小在30~60 kb之间的接合型多重耐药大质粒,多介导糖苷类抗生素耐药,如pSK41和pG01等[17,18]

(二)质粒接合转移  目前,质粒接合转移是耐药基因在细菌间水平基因转移的主要方式[2]。据报道仅有很少一部分金葡菌质粒(5%)具备接合转移的条件,因此有研究认为金葡菌质粒发生接合转移的频率较低[19]。但研究发现,接合型质粒可促进其他非接合质粒的转移。接合型质粒可通过其编码的DNA释放酶识别并打开转移起始点oriT,并借助偶联蛋白,使质粒DNA通过四型分泌系统(type-IV pretion system,T4SS)进入受体菌细胞[20,21]。接合型质粒还可以识别非接合质粒的oriT位点或oriT外的类似DNA序列,带动非接合质粒发生转移[22,23,24]。最近,O'Brien等[25]的研究发现,接合型质粒pWBG749可以促进含有类似转移起始点oriT序列的可移动型质粒发生转移,值得注意的是,这种携带类似pWBG749质粒oriT序列的质粒在金葡菌质粒中约占50%,包括USA300 p03、pSK41等大质粒,提示这些质粒接合转移在金葡菌中可能经常发生。此外,即便缺少接合转移位点,质粒也可以通过复制释放酶(rep)识别单链复制起始点oriV进行滚环式复制,以实现接合转移[26],这一发现应引起研究人员的重视。

(三)质粒介导的抗生素耐药  目前90%的金葡菌对青霉素耐药,其耐药机制主要有2种:一是质粒介导的β-内酰胺酶,可水解β-内酰胺环抗生素,从而介导细菌对几乎所有类型的β-内酰胺类抗生素耐药[27];二是由mecA基因编码产生的青霉素结合蛋白2a(penicillin-binding protein,PBP2a),其对β-内酰胺类抗生素具有低亲和力,可在转肽酶活性受到抗生素抑制失活后,补偿转肽酶的活性,使细胞壁肽聚糖继续合成,从而使细菌产生耐药[28]。金葡菌的β-内酰胺酶由β-内酰胺结构基因(blaZ)编码,其表达受阻遏蛋白(blaI)和抗阻遏蛋白(blaR)等调控因子严格调控,这些基因多位于一些大质粒(15~45 kb)上,常与其他抗生素耐药基因共同存在;mecA基因则位于染色体可移动遗传元件SCCs上,可介导细菌对包括甲氧西林在内的多种抗生素产生耐药[27]
        万古霉素自20世纪80年代投入使用以来,一直被认为是用于治疗MRSA感染仅有的少数几种抗生素之一[29]。但随着万古霉素在临床上的持续使用,MRSA对万古霉素的敏感性也逐渐下降,1997年,在日本首次分离到VISA[30]。到2002年,在美国首次分离到万古霉素耐药金葡菌(vancomycin-resistant Staphylococcus aureus,VRSA)[31]。近年来,在美国、印度、巴基斯坦、伊朗以及葡萄牙等国家又相继报道检出VRSA[32,33,34],我国尚未有检出VRSA的报道,但已经出现了异质性耐万古霉素金黄色葡萄球菌(heterogeneous vancomycin-resistant Staphylococcus aureus, hetero VISA)[35]。VRSA的出现最初被认为是MRSA菌株在与万古霉素耐药肠球菌(vancomycin-resistant Enterococcus, VRE)合并感染时,从VRE中获得了vanA操纵子所致[36]van操纵子可编码合成低亲和力的粘肽前体D-丙氨酸-D-乳酸(D-Ala-D-Lac),使菌株的粘肽链末端成分发生改变,消除了万古霉素结合的靶位,从而导致VRSA的产生[33]
        利奈唑胺对大多数革兰阳性菌具有很强的抗菌作用,已成为治疗MRSA感染的主要药物之一。然而,利奈唑胺耐药MRSA感染的治疗成为新的难题。目前其耐药机制包括细菌核糖体23S rRNA点突变、核糖体L3或L4蛋白的氨基酸突变以及甲基转移酶基因cfr三个方面。其中cfr基因可使23S rRNA的A2503位核苷酸发生甲基化,导致细菌对氯霉素、氟苯尼考、克林霉素和利奈唑胺等多种抗生素耐药[19,37]。2007年,在哥伦比亚患者分离的一株MRSA菌株(MRSA CM05)中首次检测到cfr基因[38]。目前,除MRSA菌株CM05的cfr基因位于染色体上外,其他有关cfr基因的报道以质粒携带居多[39,40,41,42]。在我国,Cai等[40]在2015年也报道了一株携带cfr基因的利奈唑胺耐药MRSA菌株,全质粒测序结果显示,cfr基因位于1个3.9 kb的质粒上(pLRSA417),相似结构在科氏葡萄球菌和表皮葡萄球菌中也有报道[41,42],说明其具有在种属内或不同属细菌间发生水平转移的能力。

二、噬菌体  噬菌体在细菌致病性进化和环境适应过程中发挥着重要的作用[43]。金葡菌噬菌体可分为溶解性噬菌体、温和噬菌体和慢性感染性噬菌体[44]。一些大小为39.6~45.9 kb的温和噬菌体,可作为其他可移动遗传元件如致病岛发生转移的辅助噬菌体,最常见的就是辅助噬菌体80α介导SaPI1致病岛转移到其他金葡菌或葡萄球菌中[45,46,47]。噬菌体虽不携带耐药基因,但一些温和噬菌体可以将耐药质粒转导整合到宿主细菌的染色体中,如噬菌体φ11和φ11de可介导携带氯霉素和红霉素耐药基因的pS194和pI258质粒发生转导,从而介导耐药基因发生转移[11]

三、转座子和插入序列  金葡菌基因组可携带异质性可移动遗传元件,包括插入序列、转座子和类转座子元件等,这些遗传元件与细菌进化有关[11]。插入序列是最简单的一类转座元件,大小一般在0.6~2 kb,不编码任何耐药基因,但与某些耐药基因的稳定性和重排密切相关[48]。插入序列多参与复合转座子的构成,金葡菌可携带IS256和IS257,如质粒pJ3356,携带两个拷贝的IS257,介导红霉素耐药[49,50]
        金葡菌转座子是相对较小的遗传元件[51],可编码多种耐药基因,如Tn552携带编码青霉素耐药的bla基因,Tn554携带编码红霉素、奇霉素和大环内酯-林可胺-链阳霉素B耐药的基因,通常可被整合到金葡菌盒式染色体、质粒或染色体中[52]。如在金葡菌N315、Mu50和MRSA252的基因组可携带Tn554,而Mu50菌株还含有1个特有的接合转座子Tn5801,其携带的基因tetM可同时介导四环素和二甲胺四环素耐药[51]。甲氧西林耐药基因常位于产青霉素酶质粒pI524的转座子Tn4291上[53]

四、金葡菌致病岛  金葡菌致病岛大小约为12~27 kb,可编码包括整合酶、耐药基因、毒力基因和其他疾病相关的超抗原,对细菌的进化非常重要[8,54]。目前为止,已有20多种致病岛完成测序,其中SPI1长为15.2 kb,两边与17 bp的正向重复序列相连,被认为是原型致病岛[55]。几乎所有的致病岛都编码肠毒素(enterotoxins)和毒性休克综合征毒素(toxic shock syndrome toxin,TSST)[54]。金葡菌致病岛如SaPI1、SaPI3、SaPI5等可编码青霉素结合蛋白,SaPIj50可编码β-内酰胺酶,从而可介导青霉素类和β-内酰胺类抗生素耐药[48]。此外,Φ11、Φ13、80、80α等辅助噬菌体以及85、147等非辅助噬菌体,可参与金葡菌致病岛的切除和转导,从而使宿主细菌基因组获得毒力因子或耐药基因元件[8,51]

五、金葡菌盒式染色体  金葡菌盒式染色体是金葡菌染色体开放读码框orfX内插入的较大的可移动遗传元件[56,57],含有编码抗生素耐药和毒力因子的基因元件,如编码广谱β-内酰胺类抗生素耐药的mecA基因复合物,金葡菌盒式染色体分为携带mecA基因的盒式染色体(SCCmec)和非携带mecA基因的盒式染色体(non-SCCmec)两种[51]
        研究认为SCCmec的获得导致了金葡菌对甲氧西林的耐药,所有MRSA菌株都含有SCCmec,SCCmec类似于致病岛,但它携带的是耐药基因而不是毒力基因,除携带编码β内酰胺酶的mecA基因复合物外,SCCmec还携带介导非β内酰胺类抗生素耐药的可移动遗传元件如转座子和整合质粒等[51]mecA基因的转录表达受上游的mecR1mecI调控,mecI是抑制蛋白,结合在mecA的-10和mecR1-mecI的-35启动子区,抑制mecA的转录。mecR1是相应调控系统的信号转导分子,可直接或间接裂解结合于mecA基因操纵子区的MecI,以解除mecA转录抑制。mecR1mecI可因插入序列的插入而部分或全部缺失,最常见的是mecI缺失,使得mecA转录不再被抑制,从而利于mecA的表达。IS431是金葡菌染色体盒质粒中常见的插入序列,可作为IS431相关质粒和转座子的整合位点,介导对抗生素的耐药。ccr基因复合物是SCCmec的另一个重要组成部分,包含编码2个位点特异重组酶基因ccrAccrB及功能未知的ORF。ccrAccrB基因的存在使SCCmec能够以正确的方向整合到染色体上,以及从染色体上精确的切除[51, 56,57]。目前,SCCmec根据mecA基因复合物和ccr基因复合物的组合,可分为11型(表1),其中Ⅰ~Ⅷ型最常见,值得注意的是,近年来研究发现,SCCmec Ⅳ、Ⅴ和Ⅶ型多见于社区获得性MRSA,而SCCmec Ⅰ、Ⅱ、Ⅲ、Ⅵ和Ⅷ则多见于医院获得性MRSA,Ⅸ-Ⅺ型是近年来新发现的SCCmec型[51]。由于SCCmec存在高度可变的J区,因此未来可能还将会有新的SCCmec型被发现。

六、整合性接合元件  2002年,Pembroke等[58]首次将那些可以从染色体上剪切并整合进宿主染色体,通过接合的方式在细菌之间进行转移的复杂遗传元件进行了分类,称之为整合性接合元件(integrative conjugative element,ICE)。ICE核心基因比较保守,包括intxistra基因,两端一般为反向重复序列attLattR。此外,ICE还含有一些外源基因的高频插入位点和可变区,其携带的基因可以赋予ICE和宿主遗传特异性,同时还能提高宿主的环境适应性[59]。ICE转移机制几乎完全相同,xis基因编码的切离酶可在attLattR位点催化逆向的重组反应,使ICE从宿主的染色体上切除。切除后ICE可自身进行环化,在int基因编码的整合酶的作用下,催化ICE上的特异性位点(attP)与染色体上的靶位点(attB)之间发生同源重组反应,从而特异性地整合进入宿主染色体的基因中去,并形成边界两侧attL和attR位点[59]。目前在金葡菌中已发现2种不同的ICE,即Tn5801和ICE6013,近年来研究发现,Tn5801和ICE6013多存在于动物来源的金葡菌中,如禽类中的ST5和肉猪中的ST398等序列型的金葡菌[60]

七、结语  金葡菌是人类致病菌之一,具有很强的环境适应能力。金葡菌多重耐药现象日益严重,尤其是MRSA甚至VRSA等的出现,给临床金葡菌感染的治疗带来了极大挑战。可移动遗传元件可编码大量的抗生素耐药基因和毒力因子,在细菌的适应性和生存能力中起着重要作用。不同的可移动遗传元件编码抗生素耐药基因不尽相同,其传播机制也具有特异性,人和动物来源的金葡菌可移动遗传元件存在显著差异,金葡菌可移动遗传元件可在人和动物间交互传递,因此又存在一定的趋向性。研究金葡菌耐药基因随可移动遗传元件的转移机制,可提供可移动遗传元件转移过程重要的生物学信息,对预防和控制金葡菌抗生素耐药性的传播至关重要。

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