【作者】 徐建国；

【导师】 俞云松；

【作者基本信息】 浙江大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 第一部分临床分离肠球菌耐药性分析研究目的了解临床分离肠球菌耐药特征,给临床诊断和治疗肠球菌感染提供依据,为进一步开展肠球菌耐药机理和分型研究奠定基础。研究方法1菌株来源397例肠球菌全部分离自杭州市中医院2005年2月至2008年3月期间的住院患者,其中粪肠球菌339株,屎肠球菌58株。2药敏实验所有菌株均经生物梅里埃公司API鉴定板条鉴定。应用法国生物梅里埃公司VITEK 2全自动细菌分析仪配套试剂进行药物敏感试验。以粪肠球菌ATCC29212为质控菌。研究结果药敏试验表明,肠球菌已成为医院感染重要的耐药菌,397株肠球菌属临床分离株中,粪肠球菌和屎肠球菌对11种抗菌药物具有不同的耐药谱。除对万古霉素外,两种肠球菌耐药情况较为严重,屎肠球菌对氯霉素、氨苄西林、四环素的敏感率为89.66％、46.55％、39.66%,而粪肠球菌为73.82％、46.31％、39.35%。对万古霉素耐药率较低,分别为7.72％、7.94%。从MIC的分布情况来看,90%粪肠球菌和91%屎肠球菌对万古霉素的MIC≤4μg/ml.结论屎肠球菌和粪肠球菌耐药情况较为严重,屎肠球菌对氯霉素、氨苄西林、四环素的敏感率较高,而粪肠球菌敏感率较低。屎肠球菌和粪肠球菌对万古霉素耐药率均较低。第二部分临床分离肠球菌毒力基因及多位点序列分型研究研究目的比较杭州地区万古霉素敏感屎肠球菌(vancomycin susceptible Enterococcus faecium, VSEF)和万古霉素耐药屎肠球菌(vancomycin resistant Enterococcus faecium, VREF)分离菌株的遗传背景、耐药机理和分子分型。研究方法1.菌株来源收集2008年5月至2009年8月期间临床分离万古霉素耐药屎肠球菌(VREF)19株,来自杭州地区三家医院；收集2008年10月至2009年10月期间浙江大学医学院附属第一医院临床分离万古霉素敏感屎肠球菌(VSEF)21株,作为对照菌株。以粪肠球菌ATCC29212为质控菌。2.药敏实验按照NCCLS推荐的K-B法或E-test法检测21株VSEF和19株VREF对利奈唑胺(LZD)、万古霉素(VAN)、替考拉宁(TEC)、奎奴普丁/达福普汀(QD)、利福平(RD)、替加环素(TGC)、磷霉素(FOS)、氨苄西林(AM)的敏感性；3.毒力基因检测采用PCR方法检测所收集到的21株VSEF和19株VREF的常见毒力基因asal、gel、cyt、hyln、esp,了解并比较各基因出现的频率及分布特征；4.多序列位点分型采用多位点序列分型(MLST)技术测定21株VSEF和19株VREF菌株7种管家基因atp、ddl、gdh、purk、gyd、psts、adk的基因序列,将其结果按MLST方法进行分类,与相应数据库进行比较,获得杭州地区菌株的序列型(ST),并将VSEF与VREF的序列型(ST)进行比较。研究结果1．19株临床分离的VREF多重耐药情况严重,对糖肽类抗生素万古霉素MIC均≥256μg/ml,替考拉宁MIC均≥24μg/ml,所有菌株对阿米卡星、氨苄西林耐药,对庆大霉素高水平耐药,但所有VREF菌株对利奈唑胺和替加环素敏感。21株临床分离的VSEF菌株耐药情况较轻,所有21株VSEF菌株对替考拉宁、利奈唑胺和替加环素敏感,1株VSEF菌株对奎奴普丁/达福普汀耐药,3株VSEF菌株对磷霉素耐药。2.毒力基因检测结果显示,21株万古霉素敏感屎肠球菌(VSEF)中有15株检出毒力基因,阳性率为76.20%,不同毒力基因阳性率由高至低依次为esp (76.20%)、hyln(4.76%),其余3种毒力基因皆未检出。19株万古霉素耐药屎肠球菌(VREF)中有13株检出毒力基因,阳性率为68.42%,不同毒力基因阳性率由高至低依次为esp (68.42%)、hyln(10.52%), asal、gel、cyt毒力基因各有一株阳性,阳性率皆为5.30%.从以上数据可以看出,此次检测的万古霉素耐药及敏感屎肠球菌在毒力基因表达方面无明显差异。近年来,肠球菌毒力基因或潜在的毒力基因在肠球菌中的分布研究较多,不同来源、不同种肠球菌毒力基因检出率存在较大差异,可能是地区、人群、标本等构成不同而造成的。3.19株VREF分离株总共分为6个ST型,其中12株等位基因谱相同,为ST78型；ST203型、ST341型各2株,其它3株菌分属3个不同的ST型；21株VSEF分离株总共可以分为14个ST型,其中5株菌序列型相同,为ST78型,其余16株菌分属于13个不同的ST型。比较以上两类屎肠球菌ST型,我们发现有两个ST型,即ST78和ST203型在两类屎肠球菌中均有出现。VREF和VSEF中ST78型分别出现12株与5株,各占63.2%与23.8%。从中我们可以看出,在VREF中ST78序列型占有明显的优势,是优势型别。对于VREF菌株,采用eBURST软件分析后,发现ST78、ST341、ST18等15株菌为同一组分型,属于克隆复合体CC17。而在VSEF中,共出现14个型别,5株为ST78型,占23.8%。本次实验共40株屎肠球菌,共检出ST78型19株,占47.5%，其余的序列型仅散在分布,散在分布在17个序列型中。因此可以看出,ST78型菌株分布最为广泛,来源于不同的临床标本类型,尤其是杭州地区VREF的优势克隆,是否是我国VREF的优势克隆,需扩大样本量进一步研究。而VSEF分离株存在广泛的遗传多样性,多数菌株亲缘关系较远。结论VREF与VSEF在携带毒力基因方面无明显差异,阳性率最高的是esp。ST78是杭州地区VREF的优势克隆,而VSEF分离株存在广泛的遗传多样性。更多还原

【Abstract】 Part one drug resistance analysis on clinical enteroccociObjectiveThis thesis aimed to determine the antibiotic resistance patterns of clinical isolates of enterococci, which can provide the basis for further diagnosis and treatment of enterococcal infections and resistance mechanism and sequence typing study on Enterococcus in Hangzhou.MothodsA total of 397 E.faecalis and E.faecalis isolates were collected from clinical samples, all from the Hospital of Hangzhou Traditional Chinese Medicine from February 2005 to March 2008 including 339 E.faecalis and 58 E.faecalis. All isolates were identified by bio-Merieux API re-identification and drug resistance were tested by the the French company bioMerieux VITEK 2 automatic analyzer. The quality control strain is Enterococcus faecalis ATCC29212. ResultsEntero.coccus has become the important antibiotic resistant bacteria which results in nosocomial infection. A total of 397 E.faecalis and E.faecium isolates were collected, including stains origin from clinical samples. A different pattern of resistance to 11 antibiotics was found between E.faecalis and E.faecium isolates. Except vancomycin E.faecalis and E.faecium isolates showed high percentages of drug resistance.The prevalence of E.faecium susceptiblity to chloramphenicol, ampicillin and tetracyline were 89.66%,46.55%,39.66%, respectively,whereas 73.82%,46.31% and 39.35% of the E.faecalis isolates were susceptible to chloramphenicol, ampicillin and tetracyline. E.faecalis and E.faecium isolates showed low percentages of drug resistance to vancomycin,7.72% and 7.94%, respectively. However, the minumum inhibitory concentration(MIC) was less than 4μg/ml to 90% E.faecalis and 91% E.faecium.ConclusionsE.faecalis and E.faecium showed high percentages of drug resistance. E. faecium to chloramphenicol, ampicillin, tetracycline had a higher sensitivity, but lower sensitivity of E.faecalis. E.faecalis and E.faecium had a low resistance rate to vancomycin. Part two virulence genes and multilocus sequence typing analysis on alinical EnterococciObjectiveVirulence genes profiles and multilocus squence testing(MLST) was performed for VREF and VSEF isolates to obtain insights into the genetic relationships in Hangzhou, which can provide the basis for the genetic background, resistance mechanism and sequence typing study on VREF and VSEF.MothodsA total of 19 VREF were collected from three hospitals in Hangzhou from May 2008 to October 2009.21 strains of VSEF were collected from the First Affiliated Hospital, Zhejiang University from October 2008 to October 2009, as a control strain.All samples were identified by conventional metheds. The antibiotic resistance of isolates against linezolid(LZD),vancomycin(VAN),teicoplanin(TEC),quinupristin/dalfopristin(QD),rifa-mpicin(RD),tigecycline(TGC),fosfomycin(FOS), ampicillin(AM), were determined by Kirby-Bauer or E-test method, and interpreted as recommended by Clinical and Laboratory Standards Institute 2006(CLSI2006). Presence of virulence determinants encoding aggregation substance asal, gelatinase gel, cytolysin activator cyⅠ, hyalronidase hyln, and enterococcus surface protein esp was tested by PCR.These 40 E. faecium strains were analysed by a standard set of E.faecium multilocus sequence typing primers as described(http://efaecium.mlst.net). The seven house-keeping genes evaluated are atp, ddl, gdh, purk, gyd, psts and adk. Sequence types of isolates are denfined by the allelic profile at these seven loci,with unique combination of alleles assigned a distinct sequence type number. Isolates with the similar allelic profile,and therefore the similar sequence type, are regarded as members of a single clone by eBURST software.ResultsA total of 19 VREF isolates collected from three hospitals showed higher percentages of multidrug resistance than VSEF strains.The MIC of the total 19 VREF isolates to vancomycin and teicoplanin was more than 256μg/ml and 24μg/ml, respectively. The total 19 VREF isolates was resistant to amikacin, ampicillin and gentamicin and susceptible to linezolid and tigecycline. A total of 21 VSEF isolates collected from four hospitals showed lower percentages of drug resistance than VREF strains. All VSEF isolates was susceptible to teicoplanin, linezolid and tigecycline.1 VSEF strain was resistant to quinupristin / dalfopristin and 3 VSEF strains was resistant to fosfomycin.In this study, the data showed that there are 15 strains were detected virulence genes from the total 21 VSEF isolates,and the positive rate is 76.20%. The esp gene was the most frequently detected gene(76.20%) followed by hyln(4.76%).The other three genes were not detected. There are 13 strains were detected virulence genes from the total 19 VREF isolates, and the positive rate is 68.42%, The esp gene was the most frequently detected gene(68.42%) followed by hyln(10.52%).The other three genes asal, gel, cyt tested only one was positive stain,then the positve rate is 5.30%.The above results have showed that there was no significant difference between the VREF and VSEF isolates of the expression of the virulence genes. In recent years, virulence genes or potential virulence genes in Enterococcus virulence gene distribution in many different sources, different species of Enterococcus virulence genes there is a big difference in detection rates, which may be areas, populations, samples, etc.that constitutes a different result.19 strains VREF can be divided into six STs, of which 12 were the same allelic profile as ST78-type; ST203 type, ST341-type of the two, the other 3 strains belong to three STs.21 strains VSEF can be divided into 14 STs, of which 5 series model of the same strains, the ST78-type, the other 16 strains belonged to 13 STs. Comparing the above two types of Enterococcus sequence type, we found that two ST-type, that type of ST78 and ST203 in the two types of Enterococcus were encountered. VRE Fand VSEF ST78 respectively,12 and 5 times, each 63.2% and 23.8%. We can see that the ST78 of the VREF sequence typing has obvious advantages,which is the dominant type. For VREF strains, using eBURST software analysis, we found that ST78, ST341, ST18 and other 15 strains are the same component type, belonging to clone complex CC17. In VSEF, a total of 14 types occur, up to ST78 appears 5 times, accounting for 23.8%. In this experiment,a total of 40 enterococci,19 strains were found ST78-type, accounting for 47.5%, the rest of the equences-type appears only less than 4 times, scattered in the 17 sequences typing. It can be seen, ST78 strains are widely distributed, from a different type of specimen, the First Affiliated Hospital of Zhejiang University in hospitalized patients in a large proportion of an advantage cloning, whether it is in China, dominant clonal need to expand the sample size further to study. In addition, from 40 strains of Enterococcus detected 18 ST types indicating that isolates of Enterococcus extensive genetic diversity exists, most are distantly related strains.ConclusionsVREF and VSEF power in portable carriers of the virulence genes were no significant differences. The highest positive rate is esp gene. ST78 is the advantage of VREF clone in Hangzhou. E.faecium has a wide range of genetic diversity.更多还原