【作者】 沈继录；

【导师】 朱德妹； 王明贵； 徐晓刚；

【作者基本信息】 复旦大学 ， 内科学， 2008， 博士

【摘要】 第一部分铜绿假单胞菌药物敏感性试验及同源性分析铜绿假单胞菌（PA）是医院感染的主要病原菌之一,引起临床多种感染。本课题对复旦大学附属华山医院2005年1月至2005年12月一年间临床分离的141株耐碳青霉烯类铜绿假单胞菌进行了研究。琼脂稀释法测定141株耐碳青霉烯类铜绿假单胞菌菌株对14种抗菌药物的敏感性,结果显示其中有141株耐碳青霉烯类铜绿假单胞菌对亚胺培南和美罗培南耐药（MIC≥16μg/ml）。药敏试验结果显示141株耐碳青霉烯类铜绿假单胞菌具三种耐药模式。其中亚胺培南和美罗培南均耐药的菌株占最多为94株（66.7%）。141株铜绿假单胞菌对各种抗菌药物的耐药率依次为:多粘菌素B＜头孢吡肟＜哌拉西林—他唑巴坦＜阿米卡星＜头孢哌酮—舒巴坦＜头孢他啶＜美罗培南＜哌拉西林＜氨曲南＜左氧氟沙星＜环丙沙星＜头孢哌酮＜庆大霉素＜亚胺培南。141株铜绿假单胞菌对各种抗菌药物的体外抗菌活性比较结果显示,耐亚胺培南的菌株对哌拉西林—他唑巴坦和阿米卡星仍有46.1%和45.4%的菌株敏感,未发现多粘菌素B耐药株。提示在严重铜绿假单胞菌感染患者的治疗中,β内酰胺类加氨基糖苷类仍是一个很好的联合用药组合,多粘菌素B也可以作为一个备选药物。ERIC—PCR分析141株耐碳青霉烯类菌株的同源性,研究结果显示141株碳青霉烯类耐药铜绿假单胞菌具有A、B、C、E、D、F、G、H、I、J、K共11个型别,其中以A、B、C 3个型别为主,分别有64,31和29株,提示本院主要存在该3种克隆。A型在各科室均有存在,但主要分布于脑外科,高干病房和中心ICU3个科室;B型在6个科室内有分布,主要存在于脑外科,高干病房,神经内科;C型在9个科室内有分布主要分布于脑外科。在脑外科,高干病房,神经内科、中心ICU和内科均存在A、B、C 3个主要克隆。141株耐碳青霉烯类铜绿假单胞菌,均为多重耐药株（MDR）,其中19株为泛耐药株,占13.5%（19/141）。19株PDR菌株主要分布在脑外科、ICU、肾内科和手外科。ERIC—PCR分型结果分别显示具有5个型别,以A和B型为主,分别有6株和7株。该5个型别分布于多个科室,未呈现出集中于某一科室的特点。因此,加强对这些MDR菌株尤其是PDR菌株的监测,对防止耐药铜绿假单胞菌在各病区的传播,降低医院感染的发生率具有十分重要的意义。第二部分铜绿假单胞菌金属β内酰胺酶研究近年来由于亚胺培南等碳青霉烯类抗生素在临床上的广泛应用,细菌产生了一类能广泛水解β—内酰胺类抗生素的金属β—内酰胺酶,产该类酶的细菌对β—内酰胺类抗生素产生耐药性,给临床抗感染治疗造成极大困难。本研究用亚胺培南和乙二胺四乙酸（ED7A）纸片法协同试验和E-试验对2005年间141株碳青霉烯类耐药铜绿假单胞菌进行碳青霉烯酶筛选;并对产酶株进行碳青酶烯酶基因的PCR扩增和DNA序列分析;采用脉冲场凝胶电泳（PFGE）分析产酶株同源性;Southern Blot进行耐药基因的定位;转移接合试验和PCR扩增产酶株整合子,以研究耐药基因的可转移性。结果显示141株碳青霉烯耐药铜绿假单胞菌中,仅4株PA菌株的EDTA协同试验和E-试验为金属酶检测阳性,只占141株耐药铜绿假单胞菌的2.8%;另有137株耐碳青霉烯类铜绿假单胞菌中均未检测到任何碳青霉烯酶（包括丝氨酸碳青酶烯酶）,因此推测产碳青酶烯酶不是导致铜绿假单胞菌对碳青霉烯类抗菌药物耐药的主要机制。设计编码金属酶（IMP型、VIM型）基因的引物经PCR扩增获得的扩增产物经DNA序列分析确认为4株铜绿假单胞菌均产生VIM—2金属酶。4株细菌药敏试验结果显示对亚胺培南和美罗培南均耐药,提示金属酶能够同时水解该两种抗菌药物,但均对氨曲南和多粘菌属B敏感。接合试验结果显示4株产VIM—2金属酶的铜绿假单胞菌均可通过接合转移方式传递其产VIM—2金属酶的特性,提示该基因位于质粒上,并可以通过质粒转移其耐药性。此外本研究结果显示4株产金属酶菌株均带有Ⅰ类整合子,提示Ⅰ类整合子可能在细菌耐药及多重耐药中扮演了重要角色。第三部分耐碳青霉烯类铜绿假单胞菌外膜蛋白机制研究碳青霉烯类抗生素是治疗铜绿假单胞菌感染的重要抗生素,但随着该抗菌药物在临床上的广泛应用,铜绿假单胞菌对碳青霉烯类的耐药性在快速上升。碳青霉烯类是一类分子量较小亲水性的β-内酰胺类抗生素,可以通过细菌外膜上具有通透性功能的孔蛋白OprC、OprD₂、OprE扩散。本研究对141株碳青霉烯类耐药（其中140株亚胺培南耐药）的铜绿假单胞菌的外膜孔蛋白采用PCR方法扩增外膜蛋白Opr D₂编码基因和SDS—PAGE分析opr D₂蛋白的缺失,结果显示136株耐药株Opr D₂编码基因发生显著变异,变异位点呈现多样性。其中34株Opr D₂编码基因存在大片段缺失,6株显示Opr D₂编码基因中有插入片段,96株有小片段缺失或不同位置的多点突变。另4株产金属酶菌株及1株亚胺培南敏感（美罗培南耐药）株Opr D₂编码基因未发现异常。提示这些菌株中Opr D₂编码基因的缺失或突变是引起铜绿假单胞菌对亚胺培南耐药的主要机制。采用SDS—PAGE分析15株亚胺培南耐药株Opr D₂蛋白缺失情况,结果显示这些菌株中Opr D₂蛋白均发生了缺失或减少。对该15株Opr D₂编码基因测序分析发现有3株发生了大片段缺失,其余12株均有小片段缺失或不同位置的多点突变,提示Opr D₂基因缺失突变是导致Opr D₂蛋白丢失的分子基础。第四部分耐碳青霉烯类铜绿假单胞菌多药外排泵机制研究为了解多药外排泵与耐碳青霉烯类抗菌药之间的关系,我们对141株碳青霉烯类耐药（其中95株美罗培南耐药）的铜绿假单胞菌的多药外排泵进行了研究。采用琼脂稀释法分别测定了外排泵抑制剂MC207110（20ug/ml）与美罗培南协同时141株多重耐药铜绿假单胞菌的美罗培南MIC值。结果显示外排泵抑制剂MC207110可以使94株美罗培南耐药株的美罗培南MIC值较单药时降低4倍或以上,占69.1%;随机选取外排泵抑制试验中美罗培南MIC值降低4倍或以上菌株20株,运用real-timePCR方法检测4种外排泵mRNA的表达,结果显示20株美罗培南耐药铜绿假单胞菌中有4种外排泵基因mRNA表达水平有增高。其中以MexEF-OprN为最多,共有10株。其次,为MexCD—oprJ,有5株。MexAB-OprM有4株,MexXY-OprM最少,只有3株。其中有2株细菌MexA和MexC基因mRNA表达水平同时增高。提示在这些临床耐药菌中,对美罗培南耐药有可能是这些外排泵的过度表达所致。PA外排泵的过度表达常常由其上游的调控基因突变所致。20株美罗培南耐药铜绿假单胞菌的4种外排泵调控基因测序结果显示MexAB-OprM过度表达的菌株中有3株系mexR突变,1株系nalC突变所致,未发现nalD突变。5株MexCD-OprJ过度表达的菌株均发生了nfxB突变,10株MexEF-OprN高表达株均有mexT突变,3株MexXY-OprM高表达株均有mexZ基因突变,提示这些外排泵系统的过度表达主要是由于其相应的调节基因突变所致。第五部分泛耐药铜绿假单胞菌耐药机制研究近年来铜绿假单胞菌对碳青霉烯类抗生素的耐药率上升显著,甚至出现了对所有抗菌药耐药的泛耐药铜绿假单胞菌（PDR-PA）。为了解这些菌株泛耐药的机制,本研究通过对19株泛耐药铜绿假单胞菌产ESBLs的检测,结果显示17株为产VEB-3型ESBL,其中1株同时产OXA—10型ESBL。19株泛耐药铜绿假单胞菌的质粒AmpC酶检测均阴性,碳青霉烯酶检测亦均阴性。19株细菌的OprD₂编码基因测序分析结果显示Opr D₂编码基因均发生小片段缺失。泵抑制剂（MC207110）与美罗培南协同联合检测19株泛耐药菌铜绿假单胞菌外排泵试验结果显示有16株美罗培南的MIC较单药时的MIC值降低了4倍以上,提示这些菌株具有外排泵机制。19株泛耐药铜绿假单胞菌均发生gyrA突变,其中14株同时发生了parC突变,未发现gyrB和parE突变,Qnr基因的检测结果均为阴性。采用16种氨基糖苷修饰酶基因引物,经PCR扩增,结果显示19株泛耐药铜绿假单胞菌均含有氨基糖苷修饰酶,其中含有ant（3″）Ⅰ、aac（3）Ⅱ、ant（4′）Ⅱ、ant（2″）和ant（6）Ⅰ5种饨化酶的菌株分别有19株、18株、1株、1株和1株。15株泛耐药铜绿假单胞菌的Ⅰ类整合子检测阳性;提示泛耐药铜绿假单胞菌的耐药性是由多重耐药机制综合所致。更多还原

【Abstract】 1.Drug sensitivity testing and homology analysis of Pseudomonas aeruginosa.Pseudomonas aeruginosa（PA） is one of nosocomial infection germs,and causes the clinical variety of infections.This study focused on 141 carbapenem-resistant Pseudomonas aeruginosa from January 2005 to December 2005 in Huashan Hospital,Fudan University.141 carbapenem-resistant Pseudomonas aeruginosa sensitivity testing showed that three models.Imipenem and meropenem which are resistant strains accouted for a maximum of 94（66.7%）.The results of resistance rates of 141 Pseudomonas aeruginosa to antibiotics were:Polymyxin B＜cefepime＜piperacillin-tazobactam＜amikacin＜cefoperazone-sulbactam＜ceftazidime＜meropenem＜piperacillin＜aztreonam＜levofloxacin＜ciprofloxacin＜cefoperazone＜gentamicin＜imipenem.141 PA to various antibiotics in vitro activity of the comparison results showed that sensitivity of the imipenem-resistant strains to piperacillin-tazobactam and amikacin are still 46.1%and 45.4%,and not found polymycin B-resistant strains,suggesting that serious PA infection in patients with the treatment ofβ-lactams and aminoglycosides anti- Pseudomonas is a good medication combination.Polymyxin B can also be used as an alternative drug.Homology of 141 carbapenem-resistant strains were analysis by ERIC-PCR.the results showed that 141 carbapenem-resistant PA have 11 type which were A、B、C、E、D、F、G、H、I、J、K、and A、B、C type were mainly threetypes,respectively,64,31 and 29,suggesting that the hospital mainly to spread three kinds of cloning.Type A has existed in various departmens,but mainly distributed in brain surgery,the senior cadres ward and departments of the Center ICU;type B in the six departments,mainly in brain surgery,the senior cadres ward and neurology;type C in the nine departments,mainly distributed in brain surgery.In brain surgery,senior cadres ward,neurology,ICU and medicine there are A、B、C three major cloning.All of 141 carbapenem resistant PA are MDR,accounting for 100%;PDR for 19, accounting for 13.5%.19 PDR strains are mainly distributed in brain surgery,ICU,renal medicine and hand surgery.ERIC-PCR results showed five types,the main type A and B, respectively,six and seven.The five type are distributed in a number of departments,not showing a focus on the characteristics of a particular department.Therefore,the strengthening of these strains especially PDR and MDR strains monitoring,control drug-resistant PA in the spread of the ward and reduce the incidence of nosocomial infection is the urgent affairs.2.Study on Carbapenem-Resistant Pseudomonas aeruginosa Producing Metallo-β-Lactamase.In recent years,carbapenem antibiotics,such as imipenem,were the wider used in clinical practice,as a result,metallo-β-lactamases were produced,which can extensive hydrolyseβ-lactam antibiotics,bacterial producted MBLs were resistant to all ofβ-lactama antibiotics,and caused great difficulties to the clinical anti-infective chemotherapy.Since 1991,genotype and quantity of metallo-β-lactamases in PA were increased continuously. In this study,141 Carbapenem-Resistant Pseudomonas aeruginosa,metallo-β-lactamases were detected by EDTA synergy and Etest test.Genotype of bacterial producted MBLs were amplified by PCR and sequenced analysis,homology analysis using PFGE, resistance gene positioning by Southern Blotting and transconjection testing,integron of MBLs strains were amplified by PCR and sequenced analysis.The study results of EDTA synergy and Etest test showed that 4 strains is positive results in 141 carbapenem resistant PA.accounting for 2.8%.Another 137 resistance carbapenem PA was not detected in any of carbapenem,therefore,to product MBLs are not the main cause of resistance PA to carbapenem antibacterial in our hospital.IMP,VIM- type MBLs was amplified by PCR,and DNA sequencing was identified as PA produced VIM-2 type enzyme.Drug sensitivity test results show that imipenem and meropenem were resistant,suggested that metal enzyme can hydrolysis the two antimicrobial agents at the same time,but both sensitivity to Polymyxin B and aztreonam.4 strains of PA,are producing metal VIM-2 enzyme,transconjection testing showed that the donor bacteria can be transferred resistance,suggesting that the gene lied in the plasmid,and can transfer their resistance by plasmid.In addition,the study indicated that 4 MBLs strains are carried a classⅠintegron,and plays an important role in multi-drug resistant bacterial.3.study on outer membrane protein mechanism of Carbapenem-Resistant Pseudomonas aeruginosa.Carbapenem antibiotic is an important antibiotics to treatment of PA infection,with the antimicrobial agents in a broad clinical application,resistance of PA to carbapenem are the rapidly rising.Carbapenem is a kind of small molecular weight hydrophilic ofβ-lactam antibiotics,and can be passed on the outer membrane of the bacteria which are OprC,OprD₂,OprE.In this study,of 141 carbapenem resistant（140 meropenem resistancee） PA,outer membrane OprD₂ encoding gene was amplified and studied by PCR,useing SDS-PAGE analysis OprD₂ deletion,Results showed that the gene encoding of OprD₂ happened significant gene mutation in 141 imipenem resistant strains,variable sites show diversity.OprD₂ gene encoding of 34 strains has a large fragment missing,OprD₂ encoding gene of 6 strains has insert fragment,OprD₂ the gene encoding sequencing of 96 resistant strains were found small fragments missing or different location of multi-point mutation. the gene encoding of 4 MBLs strains and 1 imipenem sensitive（meropenem resistant） strains is normal,suggesting that OprD₂ gene mutation is the main mechanism for PA to imipenem resistance.The results revealed that OprD₂ of 15 imipenem resistant strains were reduced by SDS-PAGE.the sequencing of the encoding gene of OprD₂ were found three fragments in a big loss,and the remaining 12 are small fragments missing or different location of multi-point mutation,suggesting that OprD₂ gene mutation is the molecular basis of OprD₂ loss.4.Study on multi-drug effiux pump mechanism of Carbapenem-resistant Pseudomonas aeruginosaFor understanding the relationship between efflux pump system and carbapenem resistance,multidrug efflux pump of 141 carbapenem resistance PA（of which 95 imipenem resistance） have been studied.MIC of 141 multi-drug resistant PA to meropenem is determined by Agar dilution method with and without efflux pump inhibitor MC207110（20μg/ml）.The results indicated that effiux pump inhibitor MC207110 can make MIC of 94 meropenem resistant strains to meropenem lower four times or more than single drug,accounting for 69.1%.Randomly 20 meropenem resistant strains were selected,which is meropenem MIC decreased four times or more by effiux pump inhibitor test.using real-time PCR,four kinds of effiux pump mRNA expression levels are increased in 20 meropenem resistant strains.MexEF-OprN have ten,MexCD-oprJ have five,MexAB-OprM have four,MexXY-OprM at least,only three.Among them,two MexA and MexC gene mRNA expression levels increased at the same time.Results indicated resistance of clinical resistant bacteria to Meropenem may caused by the over-expression of these efflux pump.efflux pump over-expression often contribute to the upper control gene mutation.,of 20 meropenem resistant PA,the four types of effiux pump control gene sequencing results showed,three are MexR mutation,one is nalC mutation,not found nalD mutation in over-expression of MexAB-OprM.5 MexCD-OprJ over-expression strains have occurred nfxB mutation,10 MexEF-OprN high expression strains have mexT mutation,3 MexXY-OprM high expression strains have mexZ gene mutation,suggesting that these efflux pump system over expression was mainly due to the corresponding regulation gene mutation.5.Study on resistant mechanism of Pandrug-resistant Pseudomonas aeruginosaResistance rate of Pseudomonas aeruginosa to the carbapenem increased significantly in recent years,even appeared the pandrug resistant PA（PDR-PA）to all of the anti-bacteria drug resistance.To understand the pandrug resistant mechanism,we screened 19 PDR-PA ESBLs,and results showed that 17 VEB-3 positive,1 OXA-10 positive at the same time. Plasmid AmpC enzyme were negative in 19 pandrug resistant strains,carbapenem enzyme tests were negative.OprD₂ encoding gene sequencing analysis revealed that mutation were in all of strains.Pump inhibitor（MC207110） and meropenem collaborative detected 19 pandrug resistant strains effiux pump showed that 16 meropenem MIC values more than four-fold lower,suggesting that these strains have effiux pump mechanism.19 have gyrA mutation occurred,14 of them have parC mutation simultaneously,and found no gyrB and parE mutation,Qnr gene tests were negative.16 kinds of aminoside modifying enzyme gene test results show that 19 strains pandrug PA have ant（3"）Ⅰ、aac（3）Ⅱ、ant（4’）Ⅱ、ant（2"） and ant（6）Ⅰfive kinds were positive,respectively,19,18,1,1 and 1.Ⅰtype integron of 15 PDR-PA were positive.These results suggested that the resistance of pan-resistant Pseudomonas aeruginosa was caused by comprehensive the multi-drug resistance mechanism.更多还原