【作者】 傅鹰；

【导师】 俞云松；

【作者基本信息】 浙江大学 ， 内科学， 2014， 博士

【摘要】 NDM-1型碳青霉烯酶是2009年发现的一种新型金属p-内酰胺酶,具有对头孢菌素及碳青霉烯类抗生素的高效水解活性。至今,产NDM-1酶菌株已经呈全球范围播散,成为严重威胁人类健康的“超级细菌”。但是目前,我国NDM-1菌株的流行病学数据缺乏,blaNDM-1基因迅速播散的机制仍不明确。本研究第一部分对收集于2009年1月至2010年9月间来自全国18个省市,57家医院的非重复革兰阴性杆菌12024株,其中大肠埃希菌3439株、肺炎克雷伯菌2840株、不动杆菌属细菌2835株以及铜绿假单胞菌2910株,运用PCR技术对blaNDM-1基因进行筛查。结果发现13株菌携带有blaNDM-1基因,均为不动杆菌属细菌。患者流行病学资料回顾性分析结果显示：其中8位患者年龄>60岁、4位<7岁,13位患者中只有2例发生死亡；所有患者半年内均未有出国旅游史。13株菌来自于10个不同的省份及9个不同的科室,7例来源于痰标本、3例血液、2例分泌物及1例尿液标本。筛查结果提示,在我国NDM-1菌株尚未出现流行,不动杆菌属细菌为blaNDM-1基因的重要携带菌,这与国外众多研究结果不同。由于不动杆菌属菌种数目多、菌种间性状相似度高,该属细菌的菌种鉴定一直是个难点。为对13株菌进行准确菌种鉴定,我们除使用生化鉴定外,依次采用了blaOXA-51-like基因、16S-23S rRNA ITS区序列、ARDRA酶切分析及rpoB基因序列分析等四种方法。结果显示13株菌中,A. baumannii4株,A. pittii3株,A. lwoffii、A. johnsonii、A. genospecies10、A. haemolyticus、A. junii及A. genospecies15TU各1株。运用E-test药敏条对菌株进行抗菌药物MIC值测试,结果显示13株不动杆菌菌株对包括碳青霉烯类在内的所有p-内酰胺类抗生素表现出一致的体外耐药表型,对氨基糖苷类(庆大霉素和阿米卡星)、喹诺酮类(环丙沙星)及单环酰胺类(氨曲南)等抗生素的药敏表型有差异,对多粘菌素类(多粘菌素)、甘氨酰环素类(替加环素)及四环素类(米诺环素)均表现为敏感。在此基础上,本研究的第二部分内容通过S1-酶切后脉冲场凝胶电泳(S1-PFGE)联合Southern blot杂交技术对13株不动杆菌属细菌的blaNDM-1基因进行定位,运用质粒接合实验及转化实验验证质粒的可转移性；通过酶切克隆研究blaNDM-1基因的周围结构；进一步运用第二代高通量测序技术完成了ABC8415菌株blaNDM-1质粒的测序,并根据pABC8415质粒序列设计引物进行PCR mapping研究其余12株菌的blaNDM-1质粒序列结构特点,对blaNDM-1质粒结构与耐药基因传递间的关系进行了深入探讨。本部分实验结果提示,所有菌株blaNDM-1基因均定位于质粒上,质粒大小30-55kb。除菌株ABC207外,其余12株菌的blaNDM-1质粒均通过接合成功进入E. coli J53接合受体菌中,药敏结果提示E. coli J53接合子对头孢菌素类抗生素耐药,但对碳青霉烯类仍表现为敏感,推测可能是由于供、受体菌间种属的差异导致NDM-1酶不能在受体菌E. coli J53中充分表达所致。9株非鲍曼不动杆菌blaNDM-1质粒均能通过转化的方式进入电转感受态细菌A. baylyi ADP1,转化子成功获得NDM-1酶介导对头孢菌素类及碳青霉烯类抗生素的耐药性。blaNDM-1基因周围结构分析结果显示该基因位于具有转移功能的大片段转座结构Tn125上,该完整的Tn125转座结构为9个基因的串联排列,顺序为：ISAba125-blaNDM-1-bleo-trpF-dsbc-cutA1-groES-groEL-ISAba125。在13株菌中发现了5种不同大小的转座结构(命名为Ⅰ、Ⅱ、Ⅲ、Ⅳ和Ⅴ型转座结构)。此5种转座结构的主要区别是blaNDM-1基因下游序列发生了不同长度的截短,推测下游拷贝的ISAba125转座酶是参与转座结构产生的主要原因；而上游拷贝的ISAba125与blaNDM-1基因关系密切,构成稳定的ISAba125-blaNDM-1基因簇,是介导blaNDM-1基因在不同菌种间传递的主要转移酶。为明确ISAba125对blaNDM-1基因转移的作用以及blaNDM-1基因在不同种属细菌(包括肠杆菌科细菌及非发酵菌)中的进化关系,我们从NCBI上公布的、来源于不同菌属菌种的、带有ISAba125-blaNDM-1基因簇的参考序列中挑选出了9条序列,与本研究中13条序列进行基因相似性聚类分析,构建系统进化树,分析亲缘关系。纳入的参考序列来源菌种分别为大肠埃希菌2株、弗氏构橼酸杆菌1株、普罗威登菌属2株、鲍曼不动杆菌2株以及铜绿假单胞菌2株。结果发现22条序列分为两簇ClusterⅠ及Ⅱ,9条参考序列与8条本实验序列均归入Cluster I簇,另外5条归入ClusterⅡ簇,两簇间亲缘关系非常近,序列相差最多6bp。也就是说ISAba125-blaNDM-1基因簇在不同种属细菌中具有稳定性,不同种属细菌间ISAba125-blaNDM-1的进化关系是平行的。pABC8415质粒全长39365bp,具有31个CDS,其中已知蛋白20个,未知蛋白11个,未找到质粒复制相关蛋白。质粒结构包括了两个部分：骨架区及可变区。骨架区编码与质粒复制、转移及毒力等功能相关的基本蛋白,GC含量为37.26%；可变区则即blaNDM-1基因所在Tn125转座区,GC含量高达61.42%。PCR mapping结果揭示其余12株细菌blaNDM-1质粒与pABC8415质粒结构非常相似,该类未知型别质粒仅在NDM-1不动杆菌属细菌中特异性存在。根据Tn125转座结构区的GC含量变化推测该区域外源性的可能性大,携带有blaNDM-1基因的Tn125转座子以大片段水平转移的方式进入质粒。通过比对Tn125转座结构序列在不动杆菌属与肠杆菌科细菌间的差异,我们推测blaNDM-1基因是从其他未知高GC物种经由不动杆菌属细菌进入肠杆菌科细菌中的进化顺序。本部分研究可以得出以下几个结论：blaNDM-1质粒及ISAba125转座酶是介导blaNDM-1基因在不动杆菌属不同菌种间水平转移的重要转座元件；而不动杆菌属细菌是blaNDM-1基因进入肠杆菌科细菌的关键中间环节。鉴于国外有关blaNDM-1基因广泛存在于肠杆菌科细菌质粒上的报道,为了解blaNDM-1质粒在肠杆菌科细菌与不动杆菌属细菌之间序列特征上的差异,探索blaNDM-1基因的跨种属水平转移机制,我们对一株来源于弗氏枸橼酸杆菌的blaNDM-1质粒进行了研究。通过基因定位实验发现blaNDM-1基因定位于50-78.2kb大小的可转移性质粒上,质粒型别IncX3型,全质粒GC含量49.03%。该blaNDM-1质粒结构也由骨架区(40kb)及可变区(14kb)两部分组成。质粒的骨架区包括了诸如复制蛋白基因(repB gene)及鞭毛基因(pilX genes)等编码质粒功能蛋白的基因。blaNDM-1基因定位于可变区,该区同时含有另一个β-内酰胺酶耐药基因blaSHV-12,位于blaNDM-1基因下游；可变区有许多与基因转移相关的Is元件或转座子,如IS26,ISAba125、IS26、IS5、tnpA和tnpF；blaNDM-1基因上游的ISAba125转座结构被ⅠS5转座结构插入打断。通过比对该质粒序列与不动杆菌属Ⅰ型Tn125转座结构,我们发现两者均携带有与blaNDM-1基因亲缘关系密切的一簇基因blaNDM-1-bleo-trpF-ds6C-cutA1-groEL-insE-tnpA,片段大小8321bp,该区域的GC含量为58%,命名为NCT (NDM-1composite transposon, NDM-1复合转座子)区；而pCFNDM-CN除NCT区以外的区域与肺炎克雷伯菌pIncX-SHV质粒(序列号JN247852)很相似(覆盖度96%,相似度99%)。因此,我们推测NCT区是外源性的,是由ISAba125转座酶携带从不动杆菌属细菌跨种属水平转移到肺炎克雷伯菌pIncX-SHV质粒中。综上所述,通过本研究发现我国尚未出现产NDM-1酶菌株流行的情况；blaNDM-1基因定位于具有可转移功能的质粒上,且能随由ISAba125转座酶介导跨种属水平转移,这些证据解释了NDM-1菌株迅速引起全球播散的原因,也警示我们blaNDM-1基因所能造成的潜在威胁,必须引起广泛重视。更多还原

【Abstract】 New Delhi metallo-beta-lactamase (NDM-1) is a novel type of metallo-β-lactamase (MBL) reported in2009, which shows highly hydrolytic activity to β-lactams including cephalosporins and carbapenems. NDM-1-producing pathogens have disseminated world widely and developed to "superbugs" threatening human health. However, epidemic data of NDM-1-producing bacteria in our country is absent, even the speading mechanism for blaNDM-1gene spreading is still unclear.In this study, a national wide survey of multidrug resistance in Gram-negative bacteria was undertaken in China from January2009to September2010. A total of12024clinical isolates, including3439E.coli,2840K. pneumonia,2835Acinetobacter spp. and2910P. aeruginosa, were collected from57hospitals representing18provinces and screened by PCR for the presence of blaNDM-1gene.13blaNDM-1positive isolates were identified as Acinetobacter spp.. According to the epidemiological data for the13corresponding patients, eight were over60years old and four were less than7years old. With the exception of two, all of the patients fully recovered. The patient histories were confirmed as lacking any foreign travel. These13isolates were recovered from10provinces in China and were scattered9different wards. The samples obtained for culture included sputum (n=7), blood (n=3), secretion (n=2) and urine (n=1). Our results, different with previous research, suggest that the alarming potential of NDM-1-producing bacteria has yet to be appeared in China. The genus Acinetobacter is the most common bacteria which is prone to carry blaNDM-1currently. The species identification of Acinetobacter genus is difficult to identify owing to the large number of species and high phenotypic similarity between different species. To clarify the species correctly, four methods were utilized in our study beside the phenotypic test using the VITEK2system: blaoXA-51-like gene,16S-23S rRNA intergenic spacer (ITS) sequencing, ARDRA and partial rpoB sequence analysis. By combining the data obtained from the above four methods, we classified the NDM-1-producing strains to eight different Acinetobacter spp., including A. baumannii (n=4), A. pittii (n=3), A. lwoffii (n=1), A.johnsonii (n=1), A. genospecies10(n=1), A. haemolyticus (n=1), A. junii(n=1) and A. genospecies15TU (n=1). All the13isolates were positive for the MBL phenotype and were highly resistant to both carbapenems and broad-spectrum cephalosporins, but showed variable susceptibilities to fluoroquinolones and aminoglycosides by E-test.To unveil the location of the blaNDM-1gene, investigate the surrounding genetic structure, study the plasmid feature and reveal the association between plasmid structure and spread of resistant gene, further works were carried out on these13isolates. S1-PFGE assay and Southern blot hybridization were performed to determine the plasmid location of blaNDM-1. The transferability of blaNDM-1-harbouring plasmid was confirmed by conjugation experiments and electrotransformation. The surrounding genetic structure of the blaNDM-i gene was analysed using a restriction endonucleas-based cloning approach. Genome sequence of the blaNDM-1plasmid from strain ABC8415(pABC8415) was completed by using next-generation sequencing. PCR mapping followed by sequence analysis was done in order to investigate the plasmid structure of the rest12isolates.These13blaNDM-1genes were located on plasmids, with sizes ranging from～30to～55kb. All plasmids except one (strain ABC207) and9non-baumannii Acinetobacter spp. isolates could be transferred to E.coli J53by conjugation and A. baylyi ADP1by electrotransformation. The transconjugants were resistant to all cephalosporins and β-lactam/β-lactamase inhibitor combinations, but susceptible to carbapenems, aztreonam, fluoroquinolones and aminoglycosides, and were negative for the MBL phenotype, a finding that might be because of the differences between the Acinetobacter donor and the Enterobacteriaceae recipient. A. baylyi ADP1 transformants exhibited relatively high resistance to both carbapenems and cephalosporins and presented an MBL-positive phenotype either.The blaNDM-1gene resided in an ISAba125-associated transposon (Tn125) with potential transferability. A series of conserved genes was found in the Tn125structure: ISAba125-blaNDM-1-bleo-trpF-dsbc-cutA1-groES-groEL-ISAba125. Five types of Tn125structure (I, II, III, IV and V) were identified, with differences in downstream of the blaNDM-1gene, which suggested that the ISAba125transposon downstream was very likely responsible for the fragment deletion. While the ISAba125upstream, combined closely with blaNDM-1gene, might be the main transposase mediating horizontal genetic transfer of the blaNDM-1gene among species. To clarify the role of ISAba125-blaNDM-1in the dissemination of the blaNDM-1gene, the same region of other nine reference sequences, which belonged to four different genera (including two Pseudomonas auroginosa isolates, two Providencia spp., two E. coli, one Citrobacter freundii and two A. baumannii), were compared with that of our13sequences. Then Mega5software was applied to draw alignment tree reflecting the evolution of the blaNDMu-1. It suggested all of the involved sequences could be clustered into two groups, cluster I and II, with merely0-6base pair difference within or between the two groups. Nine reference sequences and eight our sequences were grouped to cluster I, while the rest five were grouped to cluster II. The alignment result showed us ISAba125-blaNDM-1is a relatively stable structure in Acinetobacter spp. and other genera such as Enterobacteriaceae spp. and non-fermentive bacteria, which reveals the parallel evolution of the ISAba125-blaNDM-1in different species.pABC8415was39364bp in size, possessed31open reading frames (ORFs) and hold20annotated proteins and11hypothetical proteins. The plasmid consist of a backbone region responsible for plasmid replication and transfer with average G+C content of37.36%, and a variable region harbouring the blaNDM-1gene with G+C content of61.42%. No plasmid replication protein was predicated. All of the reat12 blaNDM-1plasmids showed highly similarity on the plasmid structure with pABC8415. Therefore, we confirm this kind of untyped plasmid was specialized to Acinetobacter spp.. The average G+C content (%) of the region between the two copies of ISAba125, designated as Tn125, was markedly higher than the surrounding sequence which suggested it plays a vital role in the gene mobilization. Our analysis on the genetic environment of blaNDM-1between Acinetobacter spp. and Enterobacteriaceae revealed that Tn125transposon structure might be transferred from a progenitor to Acinetobacter spp. horizontally and that further transfer events mediated by other insertion elements ensued between Acinetobacter spp. and Enterobacteriaceae.We concluded that both blaNDM-1plasmid and Tn125transposon contribute to the gene mobilization within different Acinetobacter species. While Acinetobacter species play as an intermediate link in blaNDM-1gene transmission to Enterobacteriaceae. In order to reveal the epidemiological and evolutional characteristics of blaNDM-1gene in Enterobacteriaecea, as well as to investigate the different feature of blaNDM-1plasmid between Enterobacteriaceae and Acinetobacter spp. in China, further plasmid sequencing and comparative genomic analysis were undertaken on a blaNDM-1-positive Citrobacter freundii isolate.A blaNDM-1-positive C. freundii was isolated from a patient suffering from a urinary tract infection. S1nuclease-based PFGE analysis followed by Southern blot hybridization, a conjugation experiment and electrotransformation confirmed that the blaNDM-i gene was located on a plasmid with size ranging from～50kb to～78.2kb. High-throughput sequencing of the blaNDM-1plasmid (pCFNDM-CN) showed that it was a54kb IncX-type plasmid and contained a backbone region (40kb) and a variable region (14kb). Several conserved gene clusters were annotated in the backbone region, including a repB gene associated with replication and a series of pilX genes related to plasmid mobilization. The blasHV-12gene and the blaNDM-1gene were found within the variable region. In addition, insertion sequences (IS26, ISAba125and IS5) and transposase genes (IS26-tnpA and tnpF) responsible for gene horizontal transfer were also clustered in this region. A series of conserved genes (blaNDM-1-bleo-trpF-dsbC-cutAl-groEL-insE-tnpA) was found in the structure of the NDM-1composite transposon (NCT,8321bp in size). The mean G+C content of the whole plasmid was49.03%, whereas it was significantly higher in the NCT (over58%). The backbone region of pCFNDM-CN presented high similarity to pIncX-SHV (GenBank accession no. JN247852;96%query coverage,99%maximum nucleotide identity) which originated from a K. pneumoniae strain, but showed some differences in the insertion site of the NCT. Our research suggested that the NCT might play an essential role in the mobilization of the blaNDM-1gene from Acinetobacter spp. to Enterobacteriaceae.In summary, we investigated the prevalence of blaNDM-1in China and concluded that NDM-1-producing isolates are rare in our country. However, attention should be given to detecting, monitoring and controlling their dissemination because this resistance gene resides in an ISAba125transposon with potential transferability cross species更多还原