【作者】 周宏伟；

【导师】 陈丽荣； 陈功祥；

【作者基本信息】 浙江大学 ， 肿瘤学， 2009， 博士

【摘要】 目的研究临床分离的产酸克雷伯菌和阴沟肠杆菌对碳青霉烯类抗生素耐药和/或敏感性降低的分子机制。方法临床分离到一株对碳青霉烯类抗生素耐药的产酸克雷伯菌ZC101和一株对碳青霉烯类抗生素不敏感的阴沟肠杆菌ZY106。采用琼脂稀释法进行抗生素最低抑菌浓度（MIC）的测定。以大肠杆菌EC600或C600作为受体菌进行接合实验。等电聚焦电泳（IEF）检测产酸克雷伯菌ZC101、阴沟肠杆菌ZY106和它们的大肠杆菌接合子产β-内酰胺酶情况。PCR和DNA测序确定耐药基因的基因型。质粒消除实验证实外膜蛋白缺失是否可以引起对碳青霉烯类抗生素感性下降。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析产酸克雷伯菌ZC101外膜蛋白表达情况,并对OmpK35和OmpK36基因进行PCR扩增和DNA序列分析。Urea-SDS-PAGE分析阴沟肠杆菌ZY106的外膜蛋白表达情况。结果亚胺培南、美罗培南和厄他培南对产酸克雷伯菌ZC101的MIC分别为16μg/ml、16μg/ml和128μg/ml;对阴沟肠杆菌ZY106的MIC分别为2μg/ml、4μg/ml和16μg/ml。接合实验证实产酸克雷伯菌ZC101可将耐药质粒传递给受体菌大肠杆菌EC600,使其对碳青霉烯类抗生素的敏感性明显降低（MIC值至少上升4倍）。产酸克雷伯菌ZC101的bla_IMP-4编码质粒被消除后菌株仍然对碳青酶烯类抗生物素有较高的耐药性。阴沟肠杆菌ZY106可将耐药质粒传递给大肠杆菌C600,使碳青霉烯类抗生素的MIC由接合前的≤0.0625μg/ml变为接合后的0.25μg/ml～0.5μg/ml。IEF、PCR扩增和序列分析证实ZC101产IMP-4型金属β-内酰胺酶和CTX-M-14型超广谱β-内酰胺酶,而转移接合子只产IMP-4。bla_IMP-4基因位于大小约3000 bp的Ⅰ类整合子上,该整合子位于大小约55 kb的质粒上。同时证实ZY106中存在IMP-1型金属β-内酰胺酶和CTX-M-3型超广谱β-内酰胺酶,转移接合子只产IMP-1。ZY106还同时携带有质粒介导喹诺酮耐药基因qnrS1,将编码该基因的质粒通过接合试验转移到受体菌大肠杆菌EC600中可使后者对环丙沙星中介耐药（MIC:2μg/ml）,。质粒图谱分析发现bla_IMP-1位于大小约50 kb的质粒上,qnrS1基因位于大小约60 kb的质粒上。产酸克雷伯菌ZC101外膜蛋白的SDS-PAGE和ompK35和ompK36基因序列分析发现ZC101由于ompK36基因中存在插入序列IS5而导致OmpK36膜孔蛋白缺失。对阴沟肠杆菌ZY106外膜蛋白进行Urea-SDS-PAGE分析发现有38 kDa蛋白的缺失。结论1、质粒介导的IMP-4型金属β-内酰胺酶合并OmpK36膜孔蛋白缺失是引起产酸克雷伯菌ZC101对碳青霉烯类抗生素耐药的主要原因。2、质粒介导的IMP-1型金属β-内酰胺酶合并外膜蛋白缺失是引起阴沟肠杆菌ZY106对碳青霉烯类抗生素敏感性降低的主要原因,该菌株同时携带有质粒介导的喹诺酮耐药基因qnrS1。目的了解尿路感染细菌分布情况,分析尿路感染大肠杆菌的耐药性和超广谱β-内酰胺酶（ESBLs）检出率,研究其对头孢菌素和喹诺酮类抗生素的耐药机制;了解尿道致病基因在其中的分布情况;通过HeLa细胞模型筛选尿道致病性大肠杆菌强毒株,分析其对HeLa细胞的致病性。方法回顾性调查浙江省59家医院2007年细菌分离状况,以及不同地区大肠杆菌ESBLs检出率和耐药性;同时回顾性调查我院2000年～2008年9年间,我院临床标本和尿液标本细菌分离情况、耐药性和ESBLs检出率,比较尿液分离大肠杆菌与不同标本来源的大肠杆菌的耐药性;采用特异性PCR检测耐药菌株的耐药基因,DNA测序后序列与GenBank中已知序列对比确定基因型和基因突变;采用PFGE分析菌株同源性,并通过PCR对尿路感染分离的大肠杆菌进行分子分型;采用PCR检测尿路感染患者分离的大肠杆菌的致病基因,分析致病基因的分布;通过黏附实验和胎盼兰染色对尿路感染患者分离的大肠杆菌进行强致病菌株的初步表型筛选,采用流式细胞术检测强致病菌株诱导HeLa细胞早期凋亡的能力,在电子显微镜观察细胞凋亡形态学改变。结果2007年浙江省59家医院调查结果显示,大肠杆菌的分离率处与首位;而且浙江省各地区大肠杆菌ESBLs检出率均在50%以上;对头孢菌素的耐药率在45%以上,对喹诺酮类抗生素耐药率最高,达60%以上;我院9年问回顾性调查显示,尿液分离的细菌中大肠杆菌连续九年都处于首位;大肠杆菌的ESBLs的检出率由2000年的7.2%上升到2008年的53.7%;头孢菌素的耐药率也由20%左右上升到50%以上,而环丙沙星和庆大霉素一直处于较高的耐药水平,耐药率分别为70%和60%左右。尿液分离的大肠杆菌的耐药性与其它标本来源的大肠杆菌比较显示,喹诺酮类抗生素和氨基糖苷类抗生素的耐药率基本相同,而尿液分离的大肠杆菌对头孢菌素的耐药率明显低于其它标本分离的大肠杆菌。通过病例查询,我们选定了28例住院患者,其尿常规检查白细胞均在2+以上,并且有尿路感染的症状。28株尿路感染患者分离的大肠埃希菌中,对头孢噻肟耐药率为64.29%,对环丙沙星的耐药率为71.43%。耐药基因PCR检测和DNA测序分析显示,同时携带CTX-M-15和CTX-M-14基因的有2株,只携带CTX-M-14的有16株。DNA测序分析喹诺酮耐药菌株gyrA和parC发现,在QRDRs存在突变。而且检测2株携带qnr基因,分别为qnrA1和qnrS1。PFGE结果显示,尿液分离的大肠杆菌同源性比较低,只有两株为同一克隆株,PCR分子分型显示以D型为主,占60.71%,其次为B2型,占35.71%。致病基因PCR检测阳性率最高的为Ⅰ型菌毛基因,为96.43%,只检测到6株usp基因阳性。通过致病表型筛选实验筛选到2株强致病性大肠埃希菌（6N和27N）,携带多种致病基因。其中6N菌株携带usp基因,而27N不携带usp基因,其它致病基因与6N相同。PCR分子分型显示,6N属于B2型,而27N属于D型。它们可以在3h破坏大量HeLa细胞,使HeLa死亡;流式细胞仪检测凋亡结果显示,6N菌株在1.5h可以诱导20.75%的HeLa细胞发生早期凋亡,而27N只有1.55%的HeLa细胞发生早期凋亡。电镜结果从形态学证实6N菌株可以快速引起HeLa细胞发生早期凋亡。结论1、大肠杆菌在细菌性感染中占有主要地位,尤其是在尿路细菌感染中。2、大肠杆菌产ESBLs是引起头孢菌素耐药的主要原因,ESBLs的主要基因型是CTX-M-14型。3、介导喹诺酮耐药的主要是gyrA和parC基因碱基突变引起,并且伴有qnr质粒介导的耐药。4、携带usp基因的强毒力株可以引起HeLa细胞快速早期凋亡。更多还原

【Abstract】 ObjectiveTo investigate the mechanisms of carbapenems resistance and/or reduced susceptibility to carbapenems in clinical isolates of Klebsiella oxytoca and Enterobacter cloacae.MethodsA carbapenem-resistant K.oxytoca ZC101 strain and a carbapenem-non-susceptible E. cloacae ZY106 strain isolated in our hospital were investigated.Minimum inhibitory concentration（MIC） values of various antibiotics were determined by agar dilution method.Conjugation experiment was carried out in mixed broth cultures,and Escherichia coli EC600 or C600 strain was used as recipient.The crudeβ-lactamase extracts of ZC101,ZY106 and their E.coli transconjugants were subjected to analytical isoelectric focusing（IEF）.PCRs and DNA sequence analysis were preformed to confirm the genotype of drug resistance genes.Plasmid currying experiment was performed to confirm the attribution of reduced susceptibility to carbapenems.Outer membrane proteins（OMPs） of K.oxytoca ZC101 were isolated and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） and the ompK35 and ompK36 genes were amplified by using PCR and were sequenced.OMPs of E.cloacae ZY106 were isolated and examined by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis（Urea-SDS-PAGE）.ResultsMICs of imipenem,meropenem and ertapenem for ZC101 were 16μg/ml,16μg/ml, and 128μg/ml,respectively,and those for ZY106 were 2μg/ml,4μg/ml,and 16μg/ml, respectively.Conjugation study with E.coli EC600 resulted in the transfer of significant reduced carbapenem susceptibility from ZC101（MICs increased at least four-fold）.The IMP-4-plasmid eliminated strain of ZC101 still has high resistance against the carbapenems.Conjugation experiments with E coli C600 resulted in the transfer of drug resistance gene from ZY106,and MICs of carbapenems were increased from 0.0625μg/ml to a range of 0.25 to 0.5μg/ml.IEF,PCRs and DNA sequence analysis confirmed that ZC101 produced IMP-4 metallo-β-lactamase and CTX-M-14 extended-spectrumβ-lactamase,while E.coli transconjugant produced a single IMP-4.Amplification of integron revealed that blaIMP-4 gene located within a class I integron that was carried on a plasmid with a size of approximately 55 kb.Meanwhile,the results of IEF,PCRs and DNA sequence analysis showed that ZY106 produced IMP-1 metallo-β-lactamase and CTX-M-3 extended-spectrumβ-lactamase,but E.coli transconjugant produced a single IMP-1.A plasmid-mediated quinolone resistance determinant qnrS1 was detected in ZY106. Transfer of the qnrS1-encoding-plasmid into E.coli EC600 by conjugation resulted in intermediate resistance to ciprofloxacin（MIC,2μg/ml） in E.coli transconjugant. Plasmid analysis indicated that the bla_IMP-1 was located in a plasmid with a size of approximately 50 kb while the size of qnrS1-encoding-plasmid was approximately 60 kb. SDS-PAGE analysis of outer membrane proteins of K.oxytoca ZC101 indicated its lack of OmpK36.Sequence analysis of ompK36 gene of K.oxytoca ZC101 showed disruption by an insertion sequence IS5.Urea-SDS-PAGE analysis of OMPs showed that ZY106 lacked an OMP of approximately 38 kDa.Conclusions1.Carbapenem resistance in K.oxytoca ZC101 is mainly due to production of IMP-4 metallo-β-lactamase combined with the loss of the OmpK36.2.Reduced susceptibility to carbapenems in E.cloacae ZY106 is mainly due to production of IMP-1 metallo-β-lactamase combined with the loss of an OMP.A plasmid-mediated quinolone resistance determinant qnrS1 is detected in the same isolate. Objectives To investigate the bacteria flora composiotn of the urinary tract（UT） in the UT infected patients;To analyze the drug resistance profile and extended spectrumβ-lactamases（ESBLs） detection rate of the Escherichia coli that caused the urinary tract infection,and to study its drug resistance for cephalosporin and quinolones;To understand the distribution of pathogenic genes in uropathogenic E.coli（UPEC）;To screen the high virulent UPEC strains through HeLa cell model and to analyze the pathogenicity of UPEC to HeLa cell.Methods We retrospectively studied the data of bacteria isolating,ESBLs detection rate from different regions and drug resistant profile from 59 hospitals of Zhejiang Province in 2007.Meanwhile,we investigated the statistical data of bacteria isolated from urine specimen and other clinical specimen,drug resistance and ESBLs detection rate in the past nine years ranging from 2000 to 2008 in our hospital.We have compared the drug resistance of E coli strains isolated from urine specimens with those from other specimens.Drug resistant genes of the resistant strains were detected by specific PCR and the products were sequenced by PCR.Genotypes and mutations of those genes were confirmed by blasting with the known genes in GenBank.Genetic relationship of isolated strains was examined by pulsed field gel electrophoresis（PFGE）.Molecular typing of E coli strains isolated from urinary tract infected patients was determined by a PCR method.Virulence genes of those E coli strains were determined by using PCR method to analyze the distribution of pathogenic genes in those E coli strains.The E coli strains with high pathogenicity isolated from urinary tract infected patients were initially screened by using adhesion experiments and Trypan-blue staining.HeLa cell early apoptosis induced by the high pathogenic strains was examined by flow cytometry and the morphology changes were confirmed by using transmission electron microscopy.Results The results from 59 hospitals of Zhejiang province in 2007 showed that E coli is the most common cause of infection.ESBLs detection rate of E coli from different regions of Zhejiang province is above 50%.The resistance rate against cephalosporins is above 45%,while the resistance rate of quinolones is the highest,above 60%. Retrospective study of data from our hospital in the past nine years indicated that E coli is the most frequently isolated strain from urine specimen for the past nine years.The ESBLs positive E coli strain detection increased from 7.2%in 2000 to 53.7%in 2008. The resistant rate of cephalosporins also increased from 20%to 50%.However, resistance to ciprofloxacin and gentamicin was maintained at high levels since nine years ago,around 70%and 60%respectively.The drug resistant rate against cephalosporins in E coli isolated from urinaray tract specimens is significantly lower than that in the strains isolated from other sources,while the resistant rate against quinolones and aminoglycoside is similar in the strains from different specimen sources.Through case inquiring,we selected 28 cases of hospitalized patients,who showed a leukocyte level at above ++ by routine urianalysis,and had symptoms of urinary tract infection.Among the 28 E coli strains isolated from the urinary tract infected patients, 64.29%of the isolates were resistant to cefotaxime and 62.49%was resistant to ciprofloxacin.Results of detection of drug resistance by PCR and DNA sequencing indicated that two isolates harbored both CTX-M-15 and CTX-M-14 genes,and 16 isolates only had CTX-M-14 gene.DNA sequence analysis of gyrA and parC in the quinolones resistant strains found that there were several mutations in the QRDRs.The qnr gene in two isolates was detected as qnrA1 and qnrS1,respectively.PFGE analysis showed that the E coli strains isolated from urine specimen had high genetic heterogeneity,with only two strains belonging to the same clone according to PFGE profile.PCR molecular typing indicated that those isolates mainly belong to type D （60.71%）,following type B2（35.71%）.TypeⅠpilus gene was the most common virulence gene with a positive rate at about 96.43%,while only six strains were positive for usp gene.We obtained two high virulent isolates through the pathogenic screening experiment（strain 6N and strain 27N）. Both strains had various virulence genes.Strain 27N and 6N contained very similar virulence gene profile except that strain 27N did not contain usp gene.PCR molecular typing showed that strain 6N and 27N belonged to group B2 and group D,respectively. Both strains can destroy HeLa cell within 3 hours causing cell death.Results of apoptosis detected by flow cytometer revealed that strain 6N induced 20.75%of HeLa cells to an early stage apoptosis within 1.5 hours.On the other hand,strain 27N induced only 1.55%of HeLa cells to the early stage apoptosis.Morphology changes observed by transmission electron microscopy confirmed that the strain 6N can induce HeLa cells early apoptosisConclusions1.E.coli has an important role in bacterial infection,especially in urinary tract bacterial infection.2.Resistance of E coli to cephalosporin is mainly caused by the ESBLs production.The primary genotype of ESBLs in E coli is CTX-M-14 type.3.Resistance to quinolones is mainly caused by the mutations in gyrA and parC gene, along with qnr plasmid mediated drug resistance.4.High virulent UPEC strain carrying usp gene can induce HeLa cell rapid early apoptosis.更多还原