大肠埃希菌和肺炎克雷伯菌16S rRNA甲基化酶基因的筛选及转移机制研究
发布时间：2018-07-16 20:33
【摘要】：研究背景 大肠埃希菌和肺炎克雷伯菌是临床分离的常见病原菌,主要可以引起呼吸道感染、泌尿道感染、败血症、化脓性感染等,是引起医院感染的重要病原菌,是临床抗感染治疗和医院感染控制的主要对象。随着抗菌药物在临床上的广泛应用,多重耐药的大肠埃希菌和肺炎克雷伯菌引起的感染不断增多,给临床抗感染治疗和医院感染控制造成困难。特别是大肠埃希菌和肺炎克雷伯菌产生超广谱β内酰胺酶(Extended-spectrumβlactamases, ESBLs)后,造成对青霉素类、头孢菌素类以及单环类抗菌药物耐药。除ESBLs外,大肠埃希菌和肺炎克雷伯菌还可产生质粒介导的AmpC酶,导致耐药性更加严重。近年来,在大肠埃希菌和肺炎克雷伯菌中还发现碳青霉烯酶,主要包括KPC酶和金属酶,该类酶可以水解包括碳青霉烯类药物在内的所有β内酰胺类抗菌药物。氨基糖苷类抗生素由于具有耳和肾毒性,在临床上的应用受到一定的限制,但由于氨基糖苷类抗生素对铜绿假单胞菌、肺炎克雷伯菌、大肠埃希菌等常见革兰阴性杆菌的抗生素后效应较长,仍然被用于治疗需氧革兰阴性杆菌所致的严重感染。单独使用氨基糖苷类抗生素治疗时可能疗效不佳,常需联合应用其他对革兰阴性杆菌具有强大抗菌活性的抗菌药物,如第三代头孢菌素及氟喹诺酮类药物等。细菌由于可以产生氨基糖苷类药物钝化酶,如N-乙酰基转移酶、O-磷酸转移酶及O-腺苷转移酶,导致对氨基糖苷类药物产生耐药,这些修饰酶存在于质粒或染色体上。这些修饰酶通常只作用一种或几种结构类似的氨基糖苷类抗生素,不能灭活所有的氨基糖苷类抗生素。 近年来,在革兰阴性杆菌临床分离株中发现一类质粒介导的16S rRNA甲基化酶,该酶能够保护细菌的30S核糖体16S rRNA不被氨基糖苷类抗生素结合,造成对包括阿贝卡星在内的临床上常用的氨基糖苷类抗生素耐药,并且为高水平耐药。自2003年发现首个16S rRNA甲基化酶ArmA以来,到目前为止,在革兰阴性杆菌临床分离株中已发现ArmA、RmtA、RmtB、RmtC、RmtD和NpmA6种16S rRNA甲基化酶,编码这些酶的结构基因通过质粒在不同的菌株之间播散。最近,在牛体内分离的大肠埃希菌中又发现了16S rRNA甲基化酶RmtE,但在临床分离株中没有发现RmtE。据报道,世界许多地方发生由产16S rRNA甲基化酶菌株造成的感染。我国有关产16S rRNA甲基化酶菌株引起感染的流行病学数据不够全面,特别是16S rRNA甲基化酶基因的转移机制研究不够深入本研究拟对分离自温州医学院附属第一医院的大肠埃希菌和肺炎克雷伯菌临床分离株的16S rRNA甲基化酶基因进行筛选,并对16S rRNA甲基化酶基因转移的机制进行研究。 第一部分大肠埃希菌和肺炎克雷伯菌16S rRNA甲基化酶基因的筛选 收集温州医学院附属第一医院2006年1月至2007年9月临床分离的337株肺炎克雷伯菌以及2006年1月至2008年7月临床分离的680株大肠埃希菌,所有菌株均为非重复株。利用全自动微生物分析仪进行菌种鉴定和药敏试验,筛选对庆大霉素(GEN)、妥布霉素(TOB)和阿米卡星(AMK)任一药物耐药的菌株作为被测菌株。共筛选出64株肺炎克雷伯菌至少耐一种上述氨基糖苷类药物,被选为测试菌株。337株肺炎克雷伯菌对GEN、TOB和AMK的耐药率分别为19.0%(64/337)、16.3%(55/337)和8.3%(28/337)；28株菌同时耐GEN、TOB和AMK,所有28株耐AMK的肺炎克雷伯菌同时耐TOB和GEN。365株大肠埃希菌至少耐一种上述氨基糖苷类药物,44株同时对GEN、AMK和TOB耐药,被挑选为被测菌株。680株大肠埃希菌对GEN、TOB和AMK的耐药率分别为52.5%(357/680)、50.9%(346/680)和6.5%(44/680)。所有耐AMK的大肠埃希菌同时耐GEN和TOB。AMK对大肠埃希菌和肺炎克雷伯菌临床分离株的体外抗菌活性强。 经PCR检测和DNA测序证实,21株肺炎克雷伯菌16S rRNA甲基化酶基因阳性,阳性率为6.2%(21/337),其中3株为armA阳性、13株为rmtB阳性及5株为armA和rmlB同时阳性。16S rRNA甲基化酶基因在AMK耐药株、TOB耐药株和GEN耐药株中的检出率分别为75.0%(21/28)、38.2%(21/55)和32.8%(21/64)。37株大肠埃希菌16S rRNA甲基化酶基因阳性,总的阳性率为5.4%(37/680),其中36株为rmtB阳性,l株为armA阳性。所有被测菌株rmtA、rmtC、rmtD和npmA为阴性。所有16S rRNA甲基化酶基因阳性株对GEN、TOB和AMK高度耐药(MIC256mg/L)。19株(90.5%,19/21)16S rRNA甲基化酶基因阳性肺炎克雷伯菌株产ESBLs,经PCR检测和DNA测序证实,21株、15株和19株分别为blaTEM、blasHv和blaCTX-M基因阳性。所有blaTEM为blaTEM-1,blasHv主要为blaSHv-12,19株blaCTX-M阳性株分别为13株blaCTX-M-14阳性和6株blaCTX-M-15阳性。29株(78.4%,29/37)16S rRNA甲基化酶基因阳性大肠埃希菌株产ESBLs,经PCR检测和DNA测序证实,37株全部为blaTEM阳性,经测序全部为blaTEM-1。29株产ESBLs株和2株ESBLs阴性株blaCTX-M基因为阳性,主要为blaCTX-M-14和blaCTX-M-15。所有16SrRNA甲基化酶基因阳性的肺炎克雷伯菌和大肠埃希菌株I类整合酶基因阳性。 16S rRNA甲基化酶基因已经在温州地区分离的大肠埃希菌和肺炎克雷伯菌临床分离株中播散,基因型为rmtB和armA,且以rmtB为主；首次发现rmtB和armA共同存在于同一菌株中；16S rRNA甲基化酶基因阳性株对GEN、TOB和AMK高度耐药,16S rRNA甲基化酶基因通常与ESBL基因和I类整合酶基因共同存在于同一菌株中,造成多重耐药。 第二部分16S rRNA甲基化酶基因转移机制研究 以16S rRNA甲基化酶基因阳性的大肠埃希菌和肺炎克雷伯菌株作为供体菌,以叠氮钠耐药的大肠埃希菌J53为受体菌进行接合试验。13株(61.9%,13/21)肺炎克雷伯菌通过接合试验成功地将携带16S rRNA甲基化酶基因质粒传递给受体菌,其中2株为armA阳性株、7株为rrntB阳性株及4株为armA和rmtB同时阳性株,但5株armA和rmtB同时阳性株的接合子只有rmtB阳性。经反复试验,只有4株(10.8%,4/37)rmtB阳性的大肠埃希菌株通过接合试验成功地将携带16S rRNA甲基化酶基因质粒传递给受体菌。所有接合子对GEN、TOB和AMK高度耐药,且I类整合酶基因全部为阳性,ESBL基因型与供菌相同。利用质粒中量抽取试剂盒提取非接合菌株的质粒,经转化试验把耐药质粒转入大肠埃希菌DH5a获得转化子,所有转化子对GEN、TOB和AMK高度耐药,且I类整合酶基因全部为阳性,所携带的ESBL基因型与供菌相同。 利用DNA分子杂交试验对5株armA和rmtB同时阳性的肺炎克雷伯菌和其接合子的质粒进行DNA杂交,发现rmtB位于一个约75Kb的质粒上,而armA可能位于染色体上。利用分子杂交试验对一株rmtB阳性和另一株armA阳性的大肠埃希菌及其转化子的质粒进行DNA杂交,发现大肠埃希菌中的rmtB和armA位于一个约54Kb的质粒上。利用Walking测序法获得肺炎克雷伯菌rmtB基因周围序列约11Kb大小的片段,发现紧邻rmtB基因的上游存在一个Tn3转座子序列,rmtB基因存在两个插入序列IS26之间,在转座酶tnpA基因的下游存在一个编码与接合功能有关的traI基因。大肠埃希菌中的rmtB的上游与肺炎克雷伯菌中的rmtB基因上游相同,但下游存在一个喹诺酮类药物的外排泵基因qepA。大肠埃希菌和肺炎克雷伯菌中的armA基因的上游存在两个转座酶基因,分别为转座酶基因tnpA和tnpU，，在转座酶基因tnpU的上游存在一个LSCR1插入序列；在armA的下游存在一个转座酶基因tnpD。 存在于肺炎克雷伯菌中的rmtB基因通常与ESBL基因和I类整合子共同位于可转移的质粒上,通过质粒在不同的菌株之间转移。5株armA和rmtB同时阳性株的armA可能位于染色体上,其余菌株的armA位于质粒上。存在于大肠埃希菌中的armA和rmtB基因通常与ESBL基因和I类整合子共同位于非接合性的质粒上。armA和rmtB周围存在插入序列以及转座酶基因,可能通过转座子发生转移。存在于肺炎克雷伯菌中的携带rmtB的质粒可以通过接合发生转移的原因为rmtB下游存在一个与接合相关的traI基因,而存在大肠埃希菌中的rmtB基因下游缺乏traI基因,是质粒不能通过接合发生转移的原因。大肠埃希菌中的rmtB基因下游含有qepA基因,是导致rmtB基因阳性株对环丙沙星和左氧氟沙星同时耐药的原因。 第三部分16S rRNA甲基化酶基因阳性株同源性分析 利用PFGE对21株16S rRNA甲基化酶基因阳性的肺炎克雷伯菌和37株16SrRNA甲基化酶基因阳性的大肠埃希菌进行了同源性分析。21株肺炎克雷伯菌共分成14个PFGE型,主要为A型和I型；5株属于A型,其中4株分离自ICU和1株分离自神经外科的rmtB阳性株；4株属于I型,其中3株分离自胸外科和1株分离自泌尿外科,4株菌为armA和rmtB同时阳性,其余都为每株属于单-的PFGE型并分布在不同的科室。37株16S rRNA甲基化酶基因阳性的大肠埃希菌共分成19个PFGE型,其中H型共12株,A和N型各3株,G、I和P型各2株,其余型各1株。同一PEGE型菌株存在于多个科室,而同一科室存在多个PFGE型。 携带16S rRNA甲基化酶基因的肺炎克雷伯菌和大肠埃希菌都存在克隆株的播散,但大部分菌株分散在遗传上不相关的克隆中；同一个克隆在不同病房中造成感染的播散；同一病房存在多个散在的克隆；rmtB和armA基因阳性株可通过克隆株方式进行播散,也可通过水平方式进行传播。 小结 综上所述,本研究的结论主要有以下几点：1.阿米卡星对大肠埃希菌和肺炎克雷伯菌临床分离株的体外抗菌活性非常强,是筛选16S rRNA甲基化酶基因的理想药物；2.温州地区临床分离的大肠埃希菌和肺炎克雷伯菌的16S rRNA甲基化酶基因阳性率较高,且rmtB基因的阳性率高于armA的阳性率,与国外的报道相反；3.首次发现rmtB和armA基因同时位于同一菌株中；4.在大肠埃希菌中,16S rRNA甲基化酶基因通常与ESBL基因和I类整合子同时位于一个非接合性的质粒上。在肺炎克雷伯菌中,16S rRNA甲基化酶基因通常与ESBL基因和I类整合子同时位于一个可自我转移的接合性质粒上；5.rmtB和armA基因常位于插入序列之间,通过转座子进行转移。存在于大肠埃希菌和肺炎克雷伯菌临床分离中的armA基因周围序列相同,armA的来源及转移机制可能相同,而存在于两种菌中的rmtB基因周围序列不同,两种菌rmtB的来源、转移机制和耐药表型可能不同。6.16S rRNA甲基化酶基因阳性的大肠埃希菌和肺炎克雷伯菌可通过克隆株的方式进行播散,也可通过水平方式进行传播。
[Abstract]:Background of the study
Escherichia coli and Klebsiella pneumoniae are common pathogens of clinical isolation , which can cause respiratory tract infection , urinary tract infection , septicemia , suppurative infection , etc .
In recent years , a kind of plasmid - mediated 16S rRNA methylase has been found in the clinical isolates of Gram - negative bacilli , which can protect the 16S rRNA of the bacterium ' s 30S ribosomal 16S rRNA from aminoglycosides , and it is resistant to high level of resistance . In recent years , the 16S rRNA methylase RmtE has been found in the clinical isolates of Gram - negative bacilli .
Screening of 16S rRNA methylase genes of Escherichia coli and Klebsiella pneumoniae in the first part
The clinical isolates from January 2006 to September 2007 were collected from the First Affiliated Hospital of Wenzhou Medical College from January 2006 to September 2007 . All the strains were non - repeated strains .
All 28 strains of Escherichia coli resistant to AMK were resistant to both GEN , AMK and TOB at the same time , and the resistant rates were 59.5 % ( 357 / 680 ) , 50.9 % ( 346 / 680 ) and 6.5 % ( 44 / 680 ) respectively .
The results of PCR and DNA sequencing showed that the positive rate of 16S rRNA methylase gene of 21 strains of Klebsiella pneumoniae was 6.2 % ( 21 / 337 ) , among which 3 strains were armA positive , 13 strains were rmtB positive and 5 strains were both armA and rmlB .
The 16S rRNA methylase gene was disseminated in the clinical isolates of Escherichia coli and Klebsiella pneumoniae isolated in Wenzhou region . The genotypes were rmtB and armA , and rmtB was dominant .
It was found that rmtB and armA were present in the same strain for the first time .
The 16S rRNA methylase gene positive strain is highly resistant to GEN , TOB and AMK . The 16S rRNA methylase gene is commonly present in the same strain as the ESBL gene and the class I integration enzyme gene , resulting in multiple resistance .
Study on the mechanism of gene transfer of 16S rRNA methylase in the second part
The results showed that only 4 ( 10.8 % , 4 / 37 ) rmtB - positive strains were positive to GEN , TOB and AMK . All transformants were highly resistant to GEN , TOB and AMK . All transformants were highly resistant to GEN , TOB and AMK . All transformants were positive for GEN , TOB and AMK .
Using molecular hybridization , we found that rmtB and armA were located in a plasmid of about 54Kb .
Downstream of armA , there is a transpositional enzyme gene tnpD .
The rmtB gene , which is present in Klebsiella pneumoniae , is usually co - located with ESBL gene and I - type integron in a transferable plasmid . The armA and rmtB genes present in Escherichia coli may be located on the chromosome , and the armA of the other strains is located on the plasmid . The plasmid carrying the rmtB present in Escherichia coli can be transferred by conjugation . The downstream of the rmtB gene in Escherichia coli can be transferred by conjugation . The downstream of the rmtB gene in Escherichia coli contains the qepA gene , which is the cause of the simultaneous resistance of the rmtB gene positive strain to ciprofloxacin and levofloxacin .
Homology analysis of the third partial 16S rRNA methylase gene positive strain
21 strains of Klebsiella pneumoniae and 37 strains of 16SrRNA methylase were analyzed by PFGE . 21 strains of Klebsiella pneumoniae were divided into 14 PFGE types , mainly A and I ;
5 strains belonged to type A , among which 4 were isolated from ICU and 1 isolated from the rmtB positive strain of neurosurgery .
Four strains belong to type I , among which 3 strains isolated from thoracic surgery and 1 isolated from urology , 4 strains were both armA and rmtB , the others were PFGE type belonging to single - PFGE type and distributed in different departments .
The strains of Klebsiella pneumoniae and Escherichia coli carrying 16S rRNA methylase gene all have the spreading of clonal strains , but most of the strains are dispersed in genetically unrelated clones ;
the same clone causes the spread of infection in different wards ;
a plurality of scattered clones exist in the same ward ;
The rmtB and armA gene - positive strains can be disseminated by cloning , and can also be propagated horizontally .
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In conclusion , the conclusions of this study are as follows : 1 . The in vitro antibacterial activity of amikaine to Escherichia coli and Klebsiella pneumoniae clinical isolates is very strong , which is the ideal drug for screening 16S rRNA methylase gene ;
2 . The positive rate of 16S rRNA methylase gene of Escherichia coli and Klebsiella pneumoniae was higher in Wenzhou region , and the positive rate of rmtB gene was higher than that of armA .
3 . The rmtB and armA genes were found in the same strain for the first time .
4 . In Escherichia coli , the 16S rRNA methylase gene is usually located on a non - binding plasmid with the ESBL gene and the I - type integron . In Klebsiella pneumoniae , the 16S rRNA methylase gene is usually located on a self - transferable bond particle at the same time as the ESBL gene and the I - type integron ;
5 . The rmtB and armA genes are often located between the insertion sequences and transferred through the transposon . The source and transfer mechanism of armA are the same around the armA gene in the clinical isolates of Escherichia coli and Klebsiella pneumoniae . The origin , transfer mechanism and drug resistant phenotype of both strains may be different . 6 . The E . coli and Klebsiella pneumoniae positive for 16S rRNA methylase gene may be disseminated by cloning , and can also be propagated horizontally .
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R378
本文编号：2127594
[Abstract]:Background of the study
Escherichia coli and Klebsiella pneumoniae are common pathogens of clinical isolation , which can cause respiratory tract infection , urinary tract infection , septicemia , suppurative infection , etc .
In recent years , a kind of plasmid - mediated 16S rRNA methylase has been found in the clinical isolates of Gram - negative bacilli , which can protect the 16S rRNA of the bacterium ' s 30S ribosomal 16S rRNA from aminoglycosides , and it is resistant to high level of resistance . In recent years , the 16S rRNA methylase RmtE has been found in the clinical isolates of Gram - negative bacilli .
Screening of 16S rRNA methylase genes of Escherichia coli and Klebsiella pneumoniae in the first part
The clinical isolates from January 2006 to September 2007 were collected from the First Affiliated Hospital of Wenzhou Medical College from January 2006 to September 2007 . All the strains were non - repeated strains .
All 28 strains of Escherichia coli resistant to AMK were resistant to both GEN , AMK and TOB at the same time , and the resistant rates were 59.5 % ( 357 / 680 ) , 50.9 % ( 346 / 680 ) and 6.5 % ( 44 / 680 ) respectively .
The results of PCR and DNA sequencing showed that the positive rate of 16S rRNA methylase gene of 21 strains of Klebsiella pneumoniae was 6.2 % ( 21 / 337 ) , among which 3 strains were armA positive , 13 strains were rmtB positive and 5 strains were both armA and rmlB .
The 16S rRNA methylase gene was disseminated in the clinical isolates of Escherichia coli and Klebsiella pneumoniae isolated in Wenzhou region . The genotypes were rmtB and armA , and rmtB was dominant .
It was found that rmtB and armA were present in the same strain for the first time .
The 16S rRNA methylase gene positive strain is highly resistant to GEN , TOB and AMK . The 16S rRNA methylase gene is commonly present in the same strain as the ESBL gene and the class I integration enzyme gene , resulting in multiple resistance .
Study on the mechanism of gene transfer of 16S rRNA methylase in the second part
The results showed that only 4 ( 10.8 % , 4 / 37 ) rmtB - positive strains were positive to GEN , TOB and AMK . All transformants were highly resistant to GEN , TOB and AMK . All transformants were highly resistant to GEN , TOB and AMK . All transformants were positive for GEN , TOB and AMK .
Using molecular hybridization , we found that rmtB and armA were located in a plasmid of about 54Kb .
Downstream of armA , there is a transpositional enzyme gene tnpD .
The rmtB gene , which is present in Klebsiella pneumoniae , is usually co - located with ESBL gene and I - type integron in a transferable plasmid . The armA and rmtB genes present in Escherichia coli may be located on the chromosome , and the armA of the other strains is located on the plasmid . The plasmid carrying the rmtB present in Escherichia coli can be transferred by conjugation . The downstream of the rmtB gene in Escherichia coli can be transferred by conjugation . The downstream of the rmtB gene in Escherichia coli contains the qepA gene , which is the cause of the simultaneous resistance of the rmtB gene positive strain to ciprofloxacin and levofloxacin .
Homology analysis of the third partial 16S rRNA methylase gene positive strain
21 strains of Klebsiella pneumoniae and 37 strains of 16SrRNA methylase were analyzed by PFGE . 21 strains of Klebsiella pneumoniae were divided into 14 PFGE types , mainly A and I ;
5 strains belonged to type A , among which 4 were isolated from ICU and 1 isolated from the rmtB positive strain of neurosurgery .
Four strains belong to type I , among which 3 strains isolated from thoracic surgery and 1 isolated from urology , 4 strains were both armA and rmtB , the others were PFGE type belonging to single - PFGE type and distributed in different departments .
The strains of Klebsiella pneumoniae and Escherichia coli carrying 16S rRNA methylase gene all have the spreading of clonal strains , but most of the strains are dispersed in genetically unrelated clones ;
the same clone causes the spread of infection in different wards ;
a plurality of scattered clones exist in the same ward ;
The rmtB and armA gene - positive strains can be disseminated by cloning , and can also be propagated horizontally .
small knot
In conclusion , the conclusions of this study are as follows : 1 . The in vitro antibacterial activity of amikaine to Escherichia coli and Klebsiella pneumoniae clinical isolates is very strong , which is the ideal drug for screening 16S rRNA methylase gene ;
2 . The positive rate of 16S rRNA methylase gene of Escherichia coli and Klebsiella pneumoniae was higher in Wenzhou region , and the positive rate of rmtB gene was higher than that of armA .
3 . The rmtB and armA genes were found in the same strain for the first time .
4 . In Escherichia coli , the 16S rRNA methylase gene is usually located on a non - binding plasmid with the ESBL gene and the I - type integron . In Klebsiella pneumoniae , the 16S rRNA methylase gene is usually located on a self - transferable bond particle at the same time as the ESBL gene and the I - type integron ;
5 . The rmtB and armA genes are often located between the insertion sequences and transferred through the transposon . The source and transfer mechanism of armA are the same around the armA gene in the clinical isolates of Escherichia coli and Klebsiella pneumoniae . The origin , transfer mechanism and drug resistant phenotype of both strains may be different . 6 . The E . coli and Klebsiella pneumoniae positive for 16S rRNA methylase gene may be disseminated by cloning , and can also be propagated horizontally .
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R378
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1 杨启文;徐英春;陈民钧;胡云建;倪语星;孙景勇;俞云松;孔海深;何林;吴伟元;叶惠芬;杨银梅;朱莲娜;郭世辉;季萍;朱震宏;任健康;张利侠;孙自镛;朱旭慧;童明庆;赵旺胜;梅亚宁;刘勇;张智洁;段琼;李丹;刘蓬蓬;王晶;韩丽霞;王辉;谢秀丽;;2005年中国十五家教学医院院内获得革兰阴性病原菌耐药性分析[J];中华医学杂志;2007年39期
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