【作者】 许宏涛；

【导师】 李金明；

【作者基本信息】 北京协和医学院 ， 临床检验诊断学， 2011， 博士

【摘要】 研究背景万古霉素耐药肠球菌(VRE)是重要的医院内感染病原菌,由于治疗选择严重受限经常伴有较高的致病率和致死率。近年大量研究显示,VRE菌株主要来自抗生素的压力对肠道菌的筛选,这种抗生素筛选出的定植VRE菌株更易引起医院内感染及播散。甲氧西林耐药金黄色葡萄球菌(MRSA)也是一类重要的医院内感染菌,其具有临床分离率高、致病力强等特点,MRSA一旦接受VRE的vanA基因发展为VRSA,将会给现有的抗感染治疗体系带来巨大困难。研究目的1.我们对从2008年3月至2009年3月卫生部北京医院临床标本中分离得到32株VRE菌株进行了分子特征及遗传背景研究。2.2010年底至2011年初我们对北京医院住院处病人进行了VRE的定植研究,以明确我院VRE菌株的定植情况,及VRE易于出现定植的临床病例特征。3.对我院同一病人同时感染和／或定植MRSA和VRE菌株的病例及菌株进行了研究和分析,以期为医院内感染控制措施的制定提供一定理论依据。研究方法1.对2008年3月至2009年3月临床标本中出现的VRE菌株的研究：1)应用脉冲场凝胶电泳(PFGE)和多位点序列分型技术(MLST)进行遗传背景研究。2)采用重叠PCR技术对转座子Tn1546的结构进行分析,同时检测常见毒性基因esp、hyl,及主要耐药基因van基因和aac(6’)-aph(2")。3)在对菌株进行分子特征及遗传背景研究的同时,收集病人的临床资料并对病房环境进行监测。4)应用转移接合试验对转座子Tn1546的转移接合活性进行研究分析。2.对VRE定植菌株的研究：1)评价自制的含6μg/ml万古霉素血琼脂培养基和法国生物梅里埃公司的chromIDVRE筛选培养基。方法：选取已知VRE菌株30株、VSE菌株30株、甲氧西林耐药金黄色葡萄球菌(MRSA) 20株、其它链球菌20株分别接种两种筛选培养基,评价两种培养基的准确度和特异性；然后应用两种培养基对350份住院处病人便标本进行VRE菌株的筛选,对确证的定植VRE菌株我们应用Etest进行药物敏感试验。2)应用PCR和测序技术对定植VRE菌株的MLST、转座子Tn1546结构、耐药基因及毒性基因等主要分生物学特征进行研究。3)收集病例分析出现VRE定植的病例的主要特征。3.对我院近期临床发现的具有金黄色葡萄球菌及VRE感染和/或定植的病例的研究：1)收集和分析病例资料。2)应用Etest法对金黄色葡萄球菌及VRE进行药物敏感试验。3)采用PCR和测序技术对VRE的主要分子生物学特征进行研究。4)应用接合试验研究Tn1546转座子的转移接合活性。研究结果1.对2008年3月至2009年3月临床标本中出现的VRE菌株的研究：1)32株VRE菌株有21株来自感染病人,11株为定植菌株；从病房分布来看有21株分离自急诊重症监护病房,9株来自老年病房,另2株散在于其它病房。2)所有菌株均为vanA基因阳性,其它基因阴性,但有4株菌表现为VanB表型,即万古霉素耐药,替考拉宁敏感；转座子Tn1546结构分析显示：ISEfa4的插入和vanY或vanZ基因的缺失与基因型与表型不一致相关。3)PFGE分析显示VRE菌株存在一定程度的院内播散；MLST分析提示所有菌株均为CC17克隆复合体,CC17菌株近年已造成了全球的播散。4)对感染病房环境的监测未发现环境污染现象。2.对定植VRE菌株的研究：1)对已知菌株,两种培养基准确度、特异性均可以达到100%。2)对350份临床便标本进行VRE筛查,共筛选出15株VRE菌株,菌株均为屎肠球菌,VRE定植率为4.3%,15株定植VRE菌株全部表现为多重耐药,所有菌株均表现为VanA表型,并拥有vanA基因。3) MLST分型显示所有菌株亦均属于更适合医院内生长的CC17克隆复合体,易形成医院内感染。4)所有检出VRE菌株定植的病人均具有较严重基础疾病,并接受过头孢菌素和糖肽类药物的治疗。3.对同一病人同时感染/定植VRE和MRSA的研究：1)2010年10月-2011年2月我院共发现8例同时感染和/或定植VRE和金黄色葡萄球菌的病例,药敏结果显示：8株金黄色葡萄球菌全部为MRSA,并对多种药物呈现多重耐药,VRE菌株全部为VanA表型并vanA基因。2)VRE菌株毒性基因esp全部阳性,hyl只有1株菌表现阳性；转移接合试验中,有7株VRE菌株含vanA基因的质粒转移接合成功,只有1株菌失败。3)8例病人全部接受过糖肽类药物的治疗,并具有较严重基础疾病。结论1.我们应该重视VRE菌株的监测,同时制定更严格、有效的医院内感染控制措施,以有效降低医院内感染的出现。2.发现了我国罕见的VanB表型-vanA基因型VRE菌株,Tn1546结构的改变影响了vanA基因的表达。3.在国内首次对定植VRE菌株的定植率及定植菌株分子生物学特征进行了研究,结果表明医院内定植VRE菌株比例不能忽视,应该对定植VRE菌株进行严密监测,以有效减少VRE菌株的院内播散。4.对同时感染和/或定植VRE和MRSA的病例的研究国内可查到的文献极少,我们的研究表明这种共感染的现象在我国已不罕见,应该进一步规范抗生素的应用,降低VRE和MRSA比例；并采取适当措施,以阻止或延缓VRSA在我国的出现。更多还原

【Abstract】 BackgroundVancomycin-resistant enterococci (VRE) are established as major nosocomial pathogens worldwide and because treatment options are very limited, VRE are usually with high morbidity and mortality. Many studies showed the pressure of antibiotics, especially the use of vancomycin could select VREs in gastrointestinal tract. And the asymptomatic VRE colonization of the gastrointestinal tract typically precedes infection and VRE spread in the hospital. Methicillin resistant Staphylococcus aureus (MRSA) is another important nosocomial pathogen. And MRSA are usually with high clinical isolation rate and strong pathogenicity. If vanA-mediated vancomycin resistance transfer from VRE to MRSA, the treatment of vancomycin resistant Staphylococcus aureus (VRSA) would be more difficult.Purposes1. We studied the molecular characteristics and genetic background of VRE strains isolated from clinical samples from March 2008 to March 2009.2. We investigated the molecular characteristics of VRE strains colonized in gastrointestinal tract and collected the main clinical features of the patients.3. We also investigated the prevalence of patients co-colonized or infected with MRSA and VRE to improve the infection-control measures in our hospital.Methods1. To study the molecular characteristics and genetic background of VRE strains.1) The genetic background of VRE strains were studied by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).2) Over-lap PCR was used to analyze the structure of Tn1546. And virulence genes and resistant genes were measured by PCR technique.3) The clinical features of the patients were collected and environmental screening was implemented.4) Conjugation experiments were also performed in this study.2. The study of rectal colonization of VRE strains. 1) The two culture mediums for screening for VRE strains were evaluated, one was blood agar containing 6μg/ml vancomycin, the other was chromID VRE agar from bioMerieux. And 30 VRE strains,30 VSE strains,20 MRSA strains and 20 other streptococci strains were inoculate in these two mediums separatly.2) We also used the two mediums to selecte VRE strains from 350 stool samples, and performed antibiotic susceptibility test of VRE isolates.3) The MLST, the structure of Tn1546, resistant genes and virulence genes of VRE isolates were also studied in this part.4) The clinical futures of the patients were collected and studied.3. Investigation of the prevalence of patients co-colonized or infected with MRSA and VRE.1) The clinical features were collected and analyzed.2) The MICs were measured of MRSA and VRE with Etest.3) The molecular feasures of VRE were studied by PCR and sequencing.4) Conjugation experiments were also performed.Results1. Of the 32 VRE isolates:1) There were 21 isolates from infected and 11 from colonized patients. And there were 21 strains found in Emergency Intensive Care Unit (EICU),9 isolates from Geriatric Ward, and two from other units.2) All the isolates harbored the vanA gene, however, four of them exhibited the VanB phenotype. And the analysis of Tn1546 structure showed that the complete deletion of vanY and vanZ and ISEfa4 insertion were related to the variance of phenotype and genotype.3) PFGE analysis indicated there were a nosocomial spread of VRE in our hospital, and MLST analysis revealed all isolates belonged to clonal complex (CC) 17.4) With the environmental screening, no VRE was found.2. Of the VRE colonization.1) Of the two mediums to screen VRE. The sensitivity and specificity of the two mediums were all 100% when we tested the known strains.2) We identified 15 VRE strains from 350 stool samples, they were all Enterococci faecium and the colonization rate was 4.3%. All the 15 VRE strains were multi-resistant, and they were all VanA phenotype with vanA genotype.3) Analysis of MLST showed all VRE isolates were CC 17 strains. 4) The patients were all with chronic underlying illness and under antimicrobial pressure.3. Of the patients who were co-colonized or infected with Staphylococcus aureus and VRE:1) From October 2010 to February 2011 we found 8 patients who were co-colonized or infected with Staphylococcus aureus and VRE. All Staphylococcus aureus were MRSA, and they were multi-resistant to most of the tested antibiotics. The 8 VRE strains were all VanA phenotype with vanA gene.2) The esp gene were detected positive in all VRE strains, and hyl gene were positive in only one VRE strain. The vancomycin resistance of 7 isolates could transfer.3) All patients were with chronic underlying illness and had been used vancomycin therapy.Conclusions1. We should attach more importance to the monitor of VRE strain. And more rigorous and effective infection control policy is needed.2. We found VanB phenotype-vanA genotype VRE strains in this study, which had been unusual in China over the past decade. And the changes of Tn1546 structure interfered the expression of vanA gene.3. It is the first time to study the rectal colonization of VRE strains in China. The results showed that we should monitor VRE colonization routinely to reduce VRE infection in the hospital.4. It is not rare of patients who are co-colonized or infected with MRSA and VRE in our country now. It is urgent to improve infection-control measures to prevent the transmission of the multidrug resistant organism.更多还原