山东省济南市区废水环境中整合子细菌筛选及耐药性机制研究
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摘要
近年来由于抗生素的广泛应用治疗各类感染,使细菌耐药问题越来越严重,从而导致了细菌多重耐药性(Multidrug resistance, MDR)的现象。不断的滥用抗生素使得人体乃至自然环境为细菌进化为多重耐药性细菌提供了选择压力并使细菌耐药性得以广泛的传播。整合子是一种可移动元件,在整合酶作用下,识别重组位点,通过位点特异性重组剪切、捕获并整合细胞外游离的基因盒,并借助整合子的强启动子使功能性基因得以表达的遗传系统；整合子的基因盒大部分是具编码多种水解酶或修饰酶、外排泵等与细菌耐药性相关基因。
临床上具有不同耐药基因的致病菌会以各种方式流入环境中,环境细菌通过基因的水平转移,可以从流入环境中的临床致病菌中获得耐药性基因。在耐药基因转移过程中,往往会发生突变或重组,进而产生新的耐药基因或新的多重耐药性基因的组合。反过来,这些新的耐药因子有可能再从环境细菌转入临床致病菌,经过空气、食物或水来感染人体,在临床上产生耐药性,对人类健康具有极大的危害。在抗菌药物选择压力环境下,整合子随转座子、接合性质粒一同发生水平转移具有使耐药性在细菌之间传播的功能,是细菌竞争生存、适应环境的机制之
也是耐药性传播的重要途径。
我们这项研究是通过调查济南地区医院周边生活废水中整合子细菌的发生率以及其含有的耐药基因盒结构,目的是发掘、鉴定一些新型的整合子结构。从基因水平上分析细菌的耐药及其转移的机制,说明抗菌药物滥用的危害性从而指导抗菌药物的正确使用,防止抗菌药物的滥用；根据细菌耐药的机制,有目的的开发一些新型抗菌药物。
本文主要的工作内容及结果如下：
1.通过滤法从济南市区废水环境中筛选耐药性细菌,对所有耐药性菌株的1型、2型、3型整合酶通过PCR分别进行检测
从2008年至2009年各大医院废水处理厂周边的废水样品；济南齐鲁制药厂下游小清河地下水、工程改造地下抽去的排水及药厂猪圈废水；废水处理厂处理前废水及部分天然少污染的济南周边泉水共15个不同时间的样品进行分析。筛选出391株不同耐药性肠杆菌菌株,根据CLSI标准,通过抗菌药物敏感性实验分析菌株对常用抗菌药物耐药性,发现菌株对氨苄西林、甲氧苄啶、硫代异唑的耐药性大于85%。通过1、2、3型整合酶引物intI1F/R,intI2F/R和intI3F/R进行PCR检测,其中1型整合子比率为59.3%,2型整合子的比率为9.2%,未筛选到3型整合酶阳性的菌株。391株菌株药物敏感性结果与整合子筛选结果分析发现,含有整合子的耐药细菌对氨苄西林、甲氧苄啶和硫代异唑三种抗菌药物的耐药率高达90%以上,非整合子细菌氨苄西林的耐药性也高达87.2%,除了磷霉素外,耐药性含整合子细菌比不具有该结构的菌株耐药性的比率高10%-20%。
2.对1型整合子可变区采用PCR扩增,通过PCR产物大小、PCR产物RFLP图谱分析及测序结果机型分析
根据1型整合子可变区根据其保守的5'CS和3'CS设计引物hep58,hep59进行扩增。根据PCR产物大小从0.2kb至6.3kb,PCR产物EcoRⅡ酶切图谱分析,发现34种不同酶切图谱,测序结果发现35种不同的基因盒阵列,具有42种不同的基因盒。35种基因盒阵列中九种基因盒阵列属于新的排列组合,16SrRNA分析,九株菌包含6个属：气单胞菌属、克雷伯菌属、大肠杆菌属、从毛假单胞菌属、普罗维登斯菌属和奇异变形杆菌属。9种新型基因盒阵列如下所示：orfI；arr3--dfrA27；aadA△16—acc(6')-Ⅱ；aac(6')-Ib—bIaoXA-1—catB3；aacA4—orfun—aacA4—catB3 dhfrV—aac.(6—Ⅱ—nitr1—nit2—catB3—blaOXA-1；aac(6')-Ib-cr—arr3—dfrA27—aadA16—IS26；aadB—cat—blaOXA-10—aadAl—dfrAl—aacA4；aadB—cat—blaOXA-10—aadAl—orfll.232株具有1型整合子的基因盒阵列中,dfrA17—aadA5排列方式是最多的一种,占43.5%,其次是dfrA12—orfF—aadA2阵列,占19.3%。
3.对共筛选出36株2型整合子进行分析,其中含有5种不同的基因盒排列组合,接合转移及转化方法分析2型整合子的水平转移性
用于扩增2型整合子可变区hep74和hep51引物成功扩增到5种不同的PCR产物4.3kb,3.0kb,2.5kb和2.2kb及时隐时现的2.5kb片段大小。PCR产物连接至pMD19-T测序结果显示分别为linF—dfrAl—aadAl—orf441；dfrA1—catB2—sat2—aadA1; estXvr—sat—aadA1; dfrA1—sat2—aadA1及杂合型的dfrA1—sat2—aadA1—qacH基因盒阵列。36株2型整合子细菌根据REP图谱进行分类,发现其可以分为15类,16S rRNA测序结果显示19株属于奇异变形杆菌属,大肠杆菌属、普罗威登斯菌属各7株,费氏柠檬酸杆菌、志贺氏菌属、不动杆菌属各1株。接合转移、转化实验及脉冲场电泳及Southern杂交实验结果表明除E.coli C4杂合型2型整合子外,其余新型2型基因盒阵列均位于染色体上。采用Tn7类转座子特有序列tnsD, tnsE进行检测,除2株杂合型2型整合子不具有此类保守序列外,其余34株2型整合子均属于Tn7转座子家族。在杂合型2型整合子下游鉴定存在IS26, tnp440和sul3基因。
4. Aeromonas puctata中新型喹诺酮耐药性基因qnrVC4的克隆及活性分析
从Aeromonas puctata基因盒3.3kb aacA4—orfun—aacA4—aatB3比对结果发现中间lkb序列功能未知,具有218氨基酸的开放阅读框,GenBank比对结果显示其与QnrB6, QnrA1, QnrS1, QnrC, QnrVC1和QnrVC3具有45%-81%的相似性。根据核苷酸、氨基酸序列相似性及其attC重组位点进行了详细的分析,根据qnr基因命名的法则,将此基因命名为qnrVC4。接合转移和转化实验均未获得该菌株相应的转移子。采用碱裂解法提取质粒,脉冲场电泳,23S rRNA探针杂交对染色体定位,然后用qnrVC4探针进行杂交分析,结果表明该基因位于
大质粒上,并且该质粒不可以发生水平转移。利用pBC KS(+)的Lac启动子及整合子本身的启动子PcWTGN-10进行表达。将657bp的qnrVC4及含有启动子、aacA4、qnrVC4基因的1.8kb的PCR产物克隆至质粒pBC KS(+)中,在E.coli Top 10中成功构建重组子pBCKS-qnrVC4和pBCKS-Pt-qnrVC4。重组子pBCKS-qnrVC4对环丙沙星、加替沙星、萘啶酮酸最小抑菌浓度(MICs)结果分别为0.032,0.047和4 gg/mL；重组子pBCKS-Pt-qnrVC4结果分别为0.008,0.006和2μg/mL。分析此菌株染色体上GyrA, GyrB和ParC的喹诺酮耐药性决定区域(QRDR)发现GyrA Ser-83-Ile突变是该菌株喹诺酮耐药性的主要原因。
5.分析391株耐药细菌中质粒介导的喹诺酮耐药性基因qnrA, qnrB, qnrS的分布
虽然QnrA, QnrB和QnrS蛋白在喹诺酮耐药性的贡献非常小,但是qnrA,qnrB和qnrS通常位于质粒上并成为喹诺酮耐药性水平转移的主要方式,并且此类基因的水平转移为获得高喹诺酮耐药性提供可能。从391株筛选的不同耐药菌株中31株具有qnr类基因,其中2株具有qnrA基因；9株具有qnrS基因阳性菌株；17株qnrB基因阳性菌株,3株同时含有qnrB, qnrS基因。通过两对引物进行PCR,并且通过PCR-RFLP的方法对qnrB基因变体进行了详细的分析,此分析法的结果与测序结果相一致,可以用来大量快速鉴定qnrB基因变体并发现新型qnrB变体。与喹诺酮耐药性相关的aac(6')-Ib-cr基因与qnr基因同时存在可以提高喹诺酮耐药性,在31株菌中检测到12株菌具有该基因。与喹诺酮相关的外排泵基因qepA仅在1株qnrB菌株中和4株qnrS菌株中检测到。接合转移和转化实验结果发现18株菌株的qnr基因可以发生水平转移。
6.调查391株耐药性菌株中ISCR1介导的复杂结构的1型整合子及具有该元件菌株的耐药性机制分析
本实验调查了391株菌株含有ISCR1元件的概率,发现其中有41株具有ISCR1元件,包括所有含有qnrA, qnrB的菌株。采用ISCR1元件的保守序列设计引物513BF和3'CS的qac△EB引物及SEFA-PCR分析ISCR1元件下游所携带的耐药性基因,从中发现了11种不同的结构,包括ISCR1—qnrA—ampR—qac△E—sull, ISCR1—qnrB2—qac△E—sull, ISCR1—qnrB6—qac△E—sull, ISCR1—dfrA10—qac△E—sull, ISCR1—tnpU—armA, ISCR1—dfrL—qac△E—sull, ISCR1—dfrM—qac△E—sull, ISCR1—dfrN—qac△E—sull, ISCR1—dfrA19—intI1—aatB3—aadB—qac△E—sull, ISCR1—blaPER-1—gst—ABC transporter—orf252—qac△E, ISCR1—IS630—blaPER-1—gst—ABC transporter—arf52—qac△E等结构。模拟3种新型的DHFR家族(DfrL, DfrM, DfrN)位于ISCR1元件下游的遗传结构,对甲氧苄啶最小抑菌浓度进行分析,耐药性均大于1024μg/mL。
Today, because antibiotic agents are wildely used to treat infection in our daily life caused the bacteria resistance problem and the multidrug resistance (MDR) phenomenon. The abuse of antibiotic agents was not only the way of bacteria multidrug resistance dissemination but also the important factor for bacteria evolution of multidrug resistance, which provided a selection pressure making human body and the natural environment to be a reservoir for the bacteria to evolve into multi-drug resistant strains. Integron is a mobile element which is referred the recombinase and a recombination site. The recombinase recognizes the specific recombination site, then excises or captures the gene cassettes which can be expressed by a promoter of integron and encoding a variety of hydrolytic enzymes or modifying enzymes, efflux pumps and bacterial resistance related genes.
Clinical pathogenic bacteria with different resistance genes released into the environment by many ways and the resistance mechanism can be transferred by daughter colony and the other bacteria can obtain the resistance genes through the horizontal gene transfer. Mutations or recombinations were occurred frequently in the gene transfer process to form the new resistant genes, and novel arrays of multiple drug resistance genes were formed by recombination involving integrases. Consequently, these new resistance factors may be transferred to the clinical pathogenic bacteria from the environment and through air, food or water to infect the human body to form new resistance factors in clinical and threat to human health. Integron can be transferred horizentally by transposon and conjugation plasmids, especially under the aquatic environment with antibiotic selection pressures, which is one of the mechanisms for competition and adaption in bacteria and the most important way for resistance scattering.
This study, we investigate the integrons dissemination and the related resistance gene cassette array structures in the wastewater nearby hospital to explore and identify the novel integron genetic structures. We analysis the bacteria resistance mechanisms and the related gene transfer mechanisms by genetic level to illustrate the risk of antibiotic abuse and guide the proper use of antibiotics to prevent the overuse antibiotics. As well, we could develope a number of new antibacterial drugs according to the mechanisms of bacterial resistance. The main methods and results are as follows:
1. Bacteria were selected from contaminated water in Jinan by mebrane filter method, all the resistant isolates were also investigated by polymerase chain reactions of class 1,2 and class 3 integrases
Fifteen different water samples were collected from 2008 to 2009, including the waste water nearby Thousand Buddha hospital, Qilu hospital, the XiaoQing River, a pigpen wastewater in Qilu pharmaceutical factory, water sample pollutated by petroleum, Waste Water Treatment Plant, and the river nearby the Shandong Traditional Chinese Medicine Hospital, the spring of Heihu, and the Xi Lake at different times.391 different resistant Enterobacteriaceae were selected and the antibiotics susceptibility test was used to distinguish the bacteria resistance profiles by CLSI criterion. More than 85% isolates were resistant to ampicillin, trimethoprim and sulfisoxazole. According to the conserved sequences of class 1, class 2 and class 3 integrase, primers intI1F/R, intI2F/R and intI3F/R were designed to investigate the prevalence of integron and 59.3% belonged to class 1 integron,9.2% identified to class 2 integron but no class 3 integron were detected. Comparing the susceptibility between integron bacteria and non-integron bacteria, the ampicillin, trimethoprim and sulfisoxazole resistances are more than 90% in integron bacteria and 87.2% in non-integron bacteria. In all, except the fosmycin, all the other antibiotics in containing integron structure are 10%-20% more than the resistance in non-integron bacteria.
2. We analyzed the structure of class 1 integron by PCR amplification and the RFLP type by EcoRⅡ
According to class 1 integron 5'CS and 3'CS, the variable region were obtained by primers hep58 and hep59. The sizes of PCR production were from 0.2kb to 6.3kb, and the related profiles of EcoRⅡrestriction RFLP map, we found 34 different restriction maps, sequencing results showed that 35 different gene cassette arrays with 42 kinds of gene cassettes. Nine of 35 different gene cassette arrays were belonged to the novel rearragement.16S rRNA sequencing results showed the nine isolates were belonged to Aeromonas sp., Klebsiela sp., Escherichia sp., Comanionas sp., Providencia sp. and Proteus sp. The nine novel gene cassette arrays are showed as following:orfI; arr3—dfrA27；aadA△16—acc(6')-Ⅱ; aac(6')-Ib-cr—blaoxA-1—catB3；aacA4—orfun—aacA4—catB3; dhfrV—aac(6)-Ⅱ—nitl—nit2—catB3—blaOXA-1; aac(6')-Ib-cr—arr3—dfrA27—aadA16—IS26; aadB—cat—blaoxA-10—aadA1—dfrA1—aacA4; aadB—cat—blaoxA-10—aadA1—orfll. The gene cassette array about dfrAl7—aadA5 was the most prevelance in our study, contained 43.5%, and followed arrays was 19.3% of dfrA12—orfF—aadA2.
3. Thirty six isolates were positive to the class 2 integrons and the structures of class 2 integrons were analysised to five different gene cassette arrays. The transformation and cojugation expriments were conducted to analysist the horizontal gene transfer
PCR products of class 2 variable regions were carried out by the conservative primers hep74 and hep51 and show five different sizes,4.3kb,3.0kb,2.5kb and 2.2kb, as well as a 2.5kb amplicon either negative to the primers or appeared a weaker band. Sequencing results showed the arrays are linF—dfrA1—aadA1—orf441; dfrA1—catB2—sat2—aadA1; estXvr—sat—aadA1; dfrA1—sat2—aadA1 and the hybrid array dfrA1—sat2—aadA1—qacH. Rep-PCR results showed that the 36 bacteria containing 15 different profiles,16S rRNA identification results showed that 19 isolates are Proteus spp., Escherichia spp.,and Providencia spp. are both 7 isolates and Citrobacter freundii, Shieglla sp., and Acinetobacter sp. are only one isolate, respectively. Cojugation and transformation experiment, PFGE and southern hybrid results were showed that except the hybrid class 2 integron, E.coli C4, the other novel class 2 integrons were chromosomally located. The specific primers about tnsD and tnsE were used to identify the Tn7 family. As a result, all the class 2 integrons except the hybrid class 2 integrons were the members of typical Tn7 family. The elements about IS26, tnp440 and sul3 were located downstream of the hybrid class 2 integrons.
4. The new variant of quinolone resistance gene qnrVC4 was identified in Aeromonas puctata isolates and anlaysis of the mechanisms of quinolone resistance
The 3.3 kb aacA4—orfun—aacA4—catB3 array was identified in class 1 gene cassette array and orfun was a lkb gene cassette encoded an open reading frame of 218aa which was 45%-81% similarities with QnrB6, QnrAl, QnrS1, QnrC, QnrVCl, and QnrVC3 by GenBank Blast suit analysis. According to the similarity of nucleotides, amino acids and the attC recombination site, the gene was named qnrVC4, and its protein was designated QnrVC4 based on the qnr nomenclature. Conjugative transfer and transformation expriments were both unsuccessful. The location of qnr VC4 was carried out by southern bolting with probes of qnr VC4 gene and 23 S rRNA, respectively. Both hybridizations were carried out using plasmids isolated by alkaline lysis, followed by agarose gel electrophoresis and total genetic material digested with S1 nuclease and I-CeuI, followed by pulsed-field gel electrophoresis (PFGE). The results indicated that qnrVC4 was located on a large plasmid. The qnrVC4 gene and qnrVC4 with the promoter of integron and aacA4 gene were both subcloned in plasmid pBC KS (+), both of which expressed by Lac promoter or promoter of integron (PcWTGN-10)-The minimum inhibitory concentrations (MICs) of ciprofloxicin, gatifloxacin, and nalidixic acid were 0.032, 0.047 and 4μg/mL with the recombinant pBCKS-qnrVC4 and 0.008,0.006 and 2μg/mL in the recombinant pBCKS-Pt-qnrVC4, but the MIC of the original isolate Aeromonas punctata was 0.38,0.19 and 96μg/mL, respectively. The analysis of the GyrA, GyrB, and ParC about the quinolone resistance determining regions (QRDR) showed the GyrA mutation at 83 Ser to Ile was the main facter of quinolone resistance.
5. Quinolone resistances genes qnrA, qnrB, qnrS were detected in the 391 isolates
Because of qnrA, qnrB and qnrS gene were reported on plasmids, which provided the easier way of low quinolone resistance transfer and the possibility to form the high quinolone resistance. Eventhough they make little contribute to the quinolone resistance in quinolone resistance isolates but many researchers are more trendency to research the dissemination of these genes and their variants. In 391 isolates,2 isolates were positive qnr A,17 isolates contained qnrB gene,9 qnrS isolates and 3 isolates were positive to both qnrB and qnrS. The PCR with two specific primers and the PCR-RFLP method to analysis the qnrB variants were successful to identify the qnrB variant and which were identical to the sequencing results. The aac(6')-Ib-cr gene identified with qnr gene can promote the quinolone resistance, and 12 isolates were both contained aac(6')-Ib-cr and one kind of qnr gene. The efflux pump gene qepA related quinolone resistance was also detected in one qnrB isolate and four qnrS isolates.18 isolates of qnr gene can be transferred by horizontal gene transfer.
6. ISCR1 element of the complex class 1 integron was detected in 391 isolates and the related resistance mechanisms were analysised
The ISCR1 element was detected in the 391 resistance isolates,41 isolates were showed with ISCR1, including all the qnrA and qnrB isolates. Primers of conserved regions (513BF, qac△EB and SEFA-PCR primers) were designed to analysis the downstream genetic structures of ISCR1 element.11 different kinds of genetic structures were illustrated, including ISC1—qnrA—ampR—qac△E—sull, ISCR1—qnrB2—qac△E—sull, ISCRl—qnrB6—qac△E—sull, ISCR1—dfrA10—qac△E—sull, ISCR1—tnpU—armA, ISCR1—dfrL—qac△E—sull, ISCR1—dfrM—qac△E—sull, ISCR1—dfrN—qac△E—sull, ISCR1—dfrA19—int11—catB3—aadB—qac△E—sull, ISCR1—blaPER-1—gst—ABC transporter—orf252—qac△E, ISCR1—IS630—blaPER-1—gst——ABC transporter—orf252—qac△E. Three dihydrofolate reductase (DfrL, DfrM and DfrN) were analysised the MICs of trimethoprom by the mimic genetic structure with ISCR1, and showed the high resistance more than 1024μg/mL。
临床上具有不同耐药基因的致病菌会以各种方式流入环境中,环境细菌通过基因的水平转移,可以从流入环境中的临床致病菌中获得耐药性基因。在耐药基因转移过程中,往往会发生突变或重组,进而产生新的耐药基因或新的多重耐药性基因的组合。反过来,这些新的耐药因子有可能再从环境细菌转入临床致病菌,经过空气、食物或水来感染人体,在临床上产生耐药性,对人类健康具有极大的危害。在抗菌药物选择压力环境下,整合子随转座子、接合性质粒一同发生水平转移具有使耐药性在细菌之间传播的功能,是细菌竞争生存、适应环境的机制之
也是耐药性传播的重要途径。
我们这项研究是通过调查济南地区医院周边生活废水中整合子细菌的发生率以及其含有的耐药基因盒结构,目的是发掘、鉴定一些新型的整合子结构。从基因水平上分析细菌的耐药及其转移的机制,说明抗菌药物滥用的危害性从而指导抗菌药物的正确使用,防止抗菌药物的滥用；根据细菌耐药的机制,有目的的开发一些新型抗菌药物。
本文主要的工作内容及结果如下：
1.通过滤法从济南市区废水环境中筛选耐药性细菌,对所有耐药性菌株的1型、2型、3型整合酶通过PCR分别进行检测
从2008年至2009年各大医院废水处理厂周边的废水样品；济南齐鲁制药厂下游小清河地下水、工程改造地下抽去的排水及药厂猪圈废水；废水处理厂处理前废水及部分天然少污染的济南周边泉水共15个不同时间的样品进行分析。筛选出391株不同耐药性肠杆菌菌株,根据CLSI标准,通过抗菌药物敏感性实验分析菌株对常用抗菌药物耐药性,发现菌株对氨苄西林、甲氧苄啶、硫代异唑的耐药性大于85%。通过1、2、3型整合酶引物intI1F/R,intI2F/R和intI3F/R进行PCR检测,其中1型整合子比率为59.3%,2型整合子的比率为9.2%,未筛选到3型整合酶阳性的菌株。391株菌株药物敏感性结果与整合子筛选结果分析发现,含有整合子的耐药细菌对氨苄西林、甲氧苄啶和硫代异唑三种抗菌药物的耐药率高达90%以上,非整合子细菌氨苄西林的耐药性也高达87.2%,除了磷霉素外,耐药性含整合子细菌比不具有该结构的菌株耐药性的比率高10%-20%。
2.对1型整合子可变区采用PCR扩增,通过PCR产物大小、PCR产物RFLP图谱分析及测序结果机型分析
根据1型整合子可变区根据其保守的5'CS和3'CS设计引物hep58,hep59进行扩增。根据PCR产物大小从0.2kb至6.3kb,PCR产物EcoRⅡ酶切图谱分析,发现34种不同酶切图谱,测序结果发现35种不同的基因盒阵列,具有42种不同的基因盒。35种基因盒阵列中九种基因盒阵列属于新的排列组合,16SrRNA分析,九株菌包含6个属：气单胞菌属、克雷伯菌属、大肠杆菌属、从毛假单胞菌属、普罗维登斯菌属和奇异变形杆菌属。9种新型基因盒阵列如下所示：orfI；arr3--dfrA27；aadA△16—acc(6')-Ⅱ；aac(6')-Ib—bIaoXA-1—catB3；aacA4—orfun—aacA4—catB3 dhfrV—aac.(6—Ⅱ—nitr1—nit2—catB3—blaOXA-1；aac(6')-Ib-cr—arr3—dfrA27—aadA16—IS26；aadB—cat—blaOXA-10—aadAl—dfrAl—aacA4；aadB—cat—blaOXA-10—aadAl—orfll.232株具有1型整合子的基因盒阵列中,dfrA17—aadA5排列方式是最多的一种,占43.5%,其次是dfrA12—orfF—aadA2阵列,占19.3%。
3.对共筛选出36株2型整合子进行分析,其中含有5种不同的基因盒排列组合,接合转移及转化方法分析2型整合子的水平转移性
用于扩增2型整合子可变区hep74和hep51引物成功扩增到5种不同的PCR产物4.3kb,3.0kb,2.5kb和2.2kb及时隐时现的2.5kb片段大小。PCR产物连接至pMD19-T测序结果显示分别为linF—dfrAl—aadAl—orf441；dfrA1—catB2—sat2—aadA1; estXvr—sat—aadA1; dfrA1—sat2—aadA1及杂合型的dfrA1—sat2—aadA1—qacH基因盒阵列。36株2型整合子细菌根据REP图谱进行分类,发现其可以分为15类,16S rRNA测序结果显示19株属于奇异变形杆菌属,大肠杆菌属、普罗威登斯菌属各7株,费氏柠檬酸杆菌、志贺氏菌属、不动杆菌属各1株。接合转移、转化实验及脉冲场电泳及Southern杂交实验结果表明除E.coli C4杂合型2型整合子外,其余新型2型基因盒阵列均位于染色体上。采用Tn7类转座子特有序列tnsD, tnsE进行检测,除2株杂合型2型整合子不具有此类保守序列外,其余34株2型整合子均属于Tn7转座子家族。在杂合型2型整合子下游鉴定存在IS26, tnp440和sul3基因。
4. Aeromonas puctata中新型喹诺酮耐药性基因qnrVC4的克隆及活性分析
从Aeromonas puctata基因盒3.3kb aacA4—orfun—aacA4—aatB3比对结果发现中间lkb序列功能未知,具有218氨基酸的开放阅读框,GenBank比对结果显示其与QnrB6, QnrA1, QnrS1, QnrC, QnrVC1和QnrVC3具有45%-81%的相似性。根据核苷酸、氨基酸序列相似性及其attC重组位点进行了详细的分析,根据qnr基因命名的法则,将此基因命名为qnrVC4。接合转移和转化实验均未获得该菌株相应的转移子。采用碱裂解法提取质粒,脉冲场电泳,23S rRNA探针杂交对染色体定位,然后用qnrVC4探针进行杂交分析,结果表明该基因位于
大质粒上,并且该质粒不可以发生水平转移。利用pBC KS(+)的Lac启动子及整合子本身的启动子PcWTGN-10进行表达。将657bp的qnrVC4及含有启动子、aacA4、qnrVC4基因的1.8kb的PCR产物克隆至质粒pBC KS(+)中,在E.coli Top 10中成功构建重组子pBCKS-qnrVC4和pBCKS-Pt-qnrVC4。重组子pBCKS-qnrVC4对环丙沙星、加替沙星、萘啶酮酸最小抑菌浓度(MICs)结果分别为0.032,0.047和4 gg/mL；重组子pBCKS-Pt-qnrVC4结果分别为0.008,0.006和2μg/mL。分析此菌株染色体上GyrA, GyrB和ParC的喹诺酮耐药性决定区域(QRDR)发现GyrA Ser-83-Ile突变是该菌株喹诺酮耐药性的主要原因。
5.分析391株耐药细菌中质粒介导的喹诺酮耐药性基因qnrA, qnrB, qnrS的分布
虽然QnrA, QnrB和QnrS蛋白在喹诺酮耐药性的贡献非常小,但是qnrA,qnrB和qnrS通常位于质粒上并成为喹诺酮耐药性水平转移的主要方式,并且此类基因的水平转移为获得高喹诺酮耐药性提供可能。从391株筛选的不同耐药菌株中31株具有qnr类基因,其中2株具有qnrA基因；9株具有qnrS基因阳性菌株；17株qnrB基因阳性菌株,3株同时含有qnrB, qnrS基因。通过两对引物进行PCR,并且通过PCR-RFLP的方法对qnrB基因变体进行了详细的分析,此分析法的结果与测序结果相一致,可以用来大量快速鉴定qnrB基因变体并发现新型qnrB变体。与喹诺酮耐药性相关的aac(6')-Ib-cr基因与qnr基因同时存在可以提高喹诺酮耐药性,在31株菌中检测到12株菌具有该基因。与喹诺酮相关的外排泵基因qepA仅在1株qnrB菌株中和4株qnrS菌株中检测到。接合转移和转化实验结果发现18株菌株的qnr基因可以发生水平转移。
6.调查391株耐药性菌株中ISCR1介导的复杂结构的1型整合子及具有该元件菌株的耐药性机制分析
本实验调查了391株菌株含有ISCR1元件的概率,发现其中有41株具有ISCR1元件,包括所有含有qnrA, qnrB的菌株。采用ISCR1元件的保守序列设计引物513BF和3'CS的qac△EB引物及SEFA-PCR分析ISCR1元件下游所携带的耐药性基因,从中发现了11种不同的结构,包括ISCR1—qnrA—ampR—qac△E—sull, ISCR1—qnrB2—qac△E—sull, ISCR1—qnrB6—qac△E—sull, ISCR1—dfrA10—qac△E—sull, ISCR1—tnpU—armA, ISCR1—dfrL—qac△E—sull, ISCR1—dfrM—qac△E—sull, ISCR1—dfrN—qac△E—sull, ISCR1—dfrA19—intI1—aatB3—aadB—qac△E—sull, ISCR1—blaPER-1—gst—ABC transporter—orf252—qac△E, ISCR1—IS630—blaPER-1—gst—ABC transporter—arf52—qac△E等结构。模拟3种新型的DHFR家族(DfrL, DfrM, DfrN)位于ISCR1元件下游的遗传结构,对甲氧苄啶最小抑菌浓度进行分析,耐药性均大于1024μg/mL。
Today, because antibiotic agents are wildely used to treat infection in our daily life caused the bacteria resistance problem and the multidrug resistance (MDR) phenomenon. The abuse of antibiotic agents was not only the way of bacteria multidrug resistance dissemination but also the important factor for bacteria evolution of multidrug resistance, which provided a selection pressure making human body and the natural environment to be a reservoir for the bacteria to evolve into multi-drug resistant strains. Integron is a mobile element which is referred the recombinase and a recombination site. The recombinase recognizes the specific recombination site, then excises or captures the gene cassettes which can be expressed by a promoter of integron and encoding a variety of hydrolytic enzymes or modifying enzymes, efflux pumps and bacterial resistance related genes.
Clinical pathogenic bacteria with different resistance genes released into the environment by many ways and the resistance mechanism can be transferred by daughter colony and the other bacteria can obtain the resistance genes through the horizontal gene transfer. Mutations or recombinations were occurred frequently in the gene transfer process to form the new resistant genes, and novel arrays of multiple drug resistance genes were formed by recombination involving integrases. Consequently, these new resistance factors may be transferred to the clinical pathogenic bacteria from the environment and through air, food or water to infect the human body to form new resistance factors in clinical and threat to human health. Integron can be transferred horizentally by transposon and conjugation plasmids, especially under the aquatic environment with antibiotic selection pressures, which is one of the mechanisms for competition and adaption in bacteria and the most important way for resistance scattering.
This study, we investigate the integrons dissemination and the related resistance gene cassette array structures in the wastewater nearby hospital to explore and identify the novel integron genetic structures. We analysis the bacteria resistance mechanisms and the related gene transfer mechanisms by genetic level to illustrate the risk of antibiotic abuse and guide the proper use of antibiotics to prevent the overuse antibiotics. As well, we could develope a number of new antibacterial drugs according to the mechanisms of bacterial resistance. The main methods and results are as follows:
1. Bacteria were selected from contaminated water in Jinan by mebrane filter method, all the resistant isolates were also investigated by polymerase chain reactions of class 1,2 and class 3 integrases
Fifteen different water samples were collected from 2008 to 2009, including the waste water nearby Thousand Buddha hospital, Qilu hospital, the XiaoQing River, a pigpen wastewater in Qilu pharmaceutical factory, water sample pollutated by petroleum, Waste Water Treatment Plant, and the river nearby the Shandong Traditional Chinese Medicine Hospital, the spring of Heihu, and the Xi Lake at different times.391 different resistant Enterobacteriaceae were selected and the antibiotics susceptibility test was used to distinguish the bacteria resistance profiles by CLSI criterion. More than 85% isolates were resistant to ampicillin, trimethoprim and sulfisoxazole. According to the conserved sequences of class 1, class 2 and class 3 integrase, primers intI1F/R, intI2F/R and intI3F/R were designed to investigate the prevalence of integron and 59.3% belonged to class 1 integron,9.2% identified to class 2 integron but no class 3 integron were detected. Comparing the susceptibility between integron bacteria and non-integron bacteria, the ampicillin, trimethoprim and sulfisoxazole resistances are more than 90% in integron bacteria and 87.2% in non-integron bacteria. In all, except the fosmycin, all the other antibiotics in containing integron structure are 10%-20% more than the resistance in non-integron bacteria.
2. We analyzed the structure of class 1 integron by PCR amplification and the RFLP type by EcoRⅡ
According to class 1 integron 5'CS and 3'CS, the variable region were obtained by primers hep58 and hep59. The sizes of PCR production were from 0.2kb to 6.3kb, and the related profiles of EcoRⅡrestriction RFLP map, we found 34 different restriction maps, sequencing results showed that 35 different gene cassette arrays with 42 kinds of gene cassettes. Nine of 35 different gene cassette arrays were belonged to the novel rearragement.16S rRNA sequencing results showed the nine isolates were belonged to Aeromonas sp., Klebsiela sp., Escherichia sp., Comanionas sp., Providencia sp. and Proteus sp. The nine novel gene cassette arrays are showed as following:orfI; arr3—dfrA27；aadA△16—acc(6')-Ⅱ; aac(6')-Ib-cr—blaoxA-1—catB3；aacA4—orfun—aacA4—catB3; dhfrV—aac(6)-Ⅱ—nitl—nit2—catB3—blaOXA-1; aac(6')-Ib-cr—arr3—dfrA27—aadA16—IS26; aadB—cat—blaoxA-10—aadA1—dfrA1—aacA4; aadB—cat—blaoxA-10—aadA1—orfll. The gene cassette array about dfrAl7—aadA5 was the most prevelance in our study, contained 43.5%, and followed arrays was 19.3% of dfrA12—orfF—aadA2.
3. Thirty six isolates were positive to the class 2 integrons and the structures of class 2 integrons were analysised to five different gene cassette arrays. The transformation and cojugation expriments were conducted to analysist the horizontal gene transfer
PCR products of class 2 variable regions were carried out by the conservative primers hep74 and hep51 and show five different sizes,4.3kb,3.0kb,2.5kb and 2.2kb, as well as a 2.5kb amplicon either negative to the primers or appeared a weaker band. Sequencing results showed the arrays are linF—dfrA1—aadA1—orf441; dfrA1—catB2—sat2—aadA1; estXvr—sat—aadA1; dfrA1—sat2—aadA1 and the hybrid array dfrA1—sat2—aadA1—qacH. Rep-PCR results showed that the 36 bacteria containing 15 different profiles,16S rRNA identification results showed that 19 isolates are Proteus spp., Escherichia spp.,and Providencia spp. are both 7 isolates and Citrobacter freundii, Shieglla sp., and Acinetobacter sp. are only one isolate, respectively. Cojugation and transformation experiment, PFGE and southern hybrid results were showed that except the hybrid class 2 integron, E.coli C4, the other novel class 2 integrons were chromosomally located. The specific primers about tnsD and tnsE were used to identify the Tn7 family. As a result, all the class 2 integrons except the hybrid class 2 integrons were the members of typical Tn7 family. The elements about IS26, tnp440 and sul3 were located downstream of the hybrid class 2 integrons.
4. The new variant of quinolone resistance gene qnrVC4 was identified in Aeromonas puctata isolates and anlaysis of the mechanisms of quinolone resistance
The 3.3 kb aacA4—orfun—aacA4—catB3 array was identified in class 1 gene cassette array and orfun was a lkb gene cassette encoded an open reading frame of 218aa which was 45%-81% similarities with QnrB6, QnrAl, QnrS1, QnrC, QnrVCl, and QnrVC3 by GenBank Blast suit analysis. According to the similarity of nucleotides, amino acids and the attC recombination site, the gene was named qnrVC4, and its protein was designated QnrVC4 based on the qnr nomenclature. Conjugative transfer and transformation expriments were both unsuccessful. The location of qnr VC4 was carried out by southern bolting with probes of qnr VC4 gene and 23 S rRNA, respectively. Both hybridizations were carried out using plasmids isolated by alkaline lysis, followed by agarose gel electrophoresis and total genetic material digested with S1 nuclease and I-CeuI, followed by pulsed-field gel electrophoresis (PFGE). The results indicated that qnrVC4 was located on a large plasmid. The qnrVC4 gene and qnrVC4 with the promoter of integron and aacA4 gene were both subcloned in plasmid pBC KS (+), both of which expressed by Lac promoter or promoter of integron (PcWTGN-10)-The minimum inhibitory concentrations (MICs) of ciprofloxicin, gatifloxacin, and nalidixic acid were 0.032, 0.047 and 4μg/mL with the recombinant pBCKS-qnrVC4 and 0.008,0.006 and 2μg/mL in the recombinant pBCKS-Pt-qnrVC4, but the MIC of the original isolate Aeromonas punctata was 0.38,0.19 and 96μg/mL, respectively. The analysis of the GyrA, GyrB, and ParC about the quinolone resistance determining regions (QRDR) showed the GyrA mutation at 83 Ser to Ile was the main facter of quinolone resistance.
5. Quinolone resistances genes qnrA, qnrB, qnrS were detected in the 391 isolates
Because of qnrA, qnrB and qnrS gene were reported on plasmids, which provided the easier way of low quinolone resistance transfer and the possibility to form the high quinolone resistance. Eventhough they make little contribute to the quinolone resistance in quinolone resistance isolates but many researchers are more trendency to research the dissemination of these genes and their variants. In 391 isolates,2 isolates were positive qnr A,17 isolates contained qnrB gene,9 qnrS isolates and 3 isolates were positive to both qnrB and qnrS. The PCR with two specific primers and the PCR-RFLP method to analysis the qnrB variants were successful to identify the qnrB variant and which were identical to the sequencing results. The aac(6')-Ib-cr gene identified with qnr gene can promote the quinolone resistance, and 12 isolates were both contained aac(6')-Ib-cr and one kind of qnr gene. The efflux pump gene qepA related quinolone resistance was also detected in one qnrB isolate and four qnrS isolates.18 isolates of qnr gene can be transferred by horizontal gene transfer.
6. ISCR1 element of the complex class 1 integron was detected in 391 isolates and the related resistance mechanisms were analysised
The ISCR1 element was detected in the 391 resistance isolates,41 isolates were showed with ISCR1, including all the qnrA and qnrB isolates. Primers of conserved regions (513BF, qac△EB and SEFA-PCR primers) were designed to analysis the downstream genetic structures of ISCR1 element.11 different kinds of genetic structures were illustrated, including ISC1—qnrA—ampR—qac△E—sull, ISCR1—qnrB2—qac△E—sull, ISCRl—qnrB6—qac△E—sull, ISCR1—dfrA10—qac△E—sull, ISCR1—tnpU—armA, ISCR1—dfrL—qac△E—sull, ISCR1—dfrM—qac△E—sull, ISCR1—dfrN—qac△E—sull, ISCR1—dfrA19—int11—catB3—aadB—qac△E—sull, ISCR1—blaPER-1—gst—ABC transporter—orf252—qac△E, ISCR1—IS630—blaPER-1—gst——ABC transporter—orf252—qac△E. Three dihydrofolate reductase (DfrL, DfrM and DfrN) were analysised the MICs of trimethoprom by the mimic genetic structure with ISCR1, and showed the high resistance more than 1024μg/mL。
引文
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