环丙沙星耐药肠杆菌科细菌临床株qnr和aac(6')-Ⅰ b-cr基因的检测

目的 了解温州地区环丙沙星耐药肠杆菌科细菌临床株的qnr、aac(6')-Ⅰ b-cr基因的分布情况.方法 收集2005年8月-2008年4月间温州医学院附属第一医院环丙沙星耐药的肠杆菌科细菌共461株,其中大肠埃希菌370株,阴沟肠杆菌39株,克雷伯菌属细菌52株.应用PCR方法 检测qnr和aac(6')-Ⅰ b幕因,DNA测序检测qnrA、qnrB、qnrS和aac(6')-Ⅰ b-cr基因;接合传递试验方法 探讨细菌质粒介导的耐药性传递情况.结果 461株环丙沙星耐药的肠杆菌科细菌临床株中检出含qnr基因阳性菌株15株(3.25%),包括qnrA基因阳性株5株(4株阴沟肠杆菌和1株解鸟氨酸克雷伯菌)、qnrB基因阳性株4株(2株肺炎克雷伯菌和2株大肠埃希菌)、qnrS基因阳性株6株(2株肺炎克雷伯菌和4株大肠埃希菌);检出52株细菌(包括42株大肠埃希菌、4株阴沟肠杆菌和6株克雷伯菌属细菌)携带aac(6')-Ⅰ b-cr.15株qnr基因阳性的菌株同时携带aac(6')-Ⅰ b-cr,药敏结果 显示对业胺培南敏感但对多种抗生素耐药.15株qnr基因阳性的菌株中7株质粒接合传递试验成功,临床株对喹诺酮类和氨基糖苷类的耐药性部分传递给了受体株.结论 qnr基因在温州地区环丙沙星耐药的肠杆菌科细菌临床株中较少见,而aac(6')-Ⅰ b-cr基因存在较普遍.     

2017年我院肠杆菌科细菌临床分布及耐药性分析

目的 分析宝山区中西医结合医院2017年所分离肠杆菌科细菌在临床标本中的分布和对常用抗菌药物的耐药性。方法 菌株来源于2017年1月1日~12月31日临床送检各类标本中分离出的肠杆菌科细菌1054株。剔除同一患者相同部位的重复菌株。用西门子的MicroScan WalkAway96全自动微生物鉴定/药敏测试系统进行细菌鉴定和药物敏感试验,K-B纸片扩散法作为药敏补充实验,依照CLSI制定的最新的判断标准判断药敏试验结果。结果 2017年分离出的肠杆菌科细菌1054株,主要来源于痰液标本和中段尿标本,分别占39.85%和31.03%;分离率最高的是肺炎克雷伯杆菌和大肠埃希菌,分别占39.94%和35.67%;其次是奇异变形杆菌和阴沟肠杆菌,分别占6.83%和5.79%;肺炎克雷伯菌亚胺培南和美罗培南的耐药率较高,分别为38.24%和 39.43%,敏感率均为59.86%;奇异变形杆菌亚胺培南的耐药率高于美罗培南。肺炎克雷伯菌ESBLs检出率为45.84%,大肠埃希菌45.74%,奇异变形杆菌为45.83%。结论 我院2017年分离的肠杆菌科细菌中以肺炎克雷伯菌和大肠埃希菌为主,细菌的耐药问题日趋严重,医院应引起高度重视,加强细菌耐药性的监测,指导临床合理用药。     

基质辅助激光解析/电离飞行时间质谱仪检测KPC型碳青霉烯酶的研究

目的 用基质辅助激光解析/电离飞行时间质谱仪(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS)比较产KPC型碳青霉烯酶肠杆菌科细菌在不同药物浓度下水解厄他培南的能力.方法 收集浙江大学医学院附属第二医院临床分离的19株单产KPC肠杆菌科细菌,包括10株奇异变形杆菌,3株产气肠杆菌,2株黏质沙雷菌,2株弗劳地柠檬酸杆菌,1株肺炎克雷伯菌,1株阴沟肠杆菌；杭州市中医院分离的7株产KPC摩根摩根菌,以及上述7株摩根摩根菌和部分奇异变形杆菌(4株)的大肠埃希菌转移接合子.用MALDI-TOF MS检测产KPC肠杆菌科细菌水解厄他培南的能力.结果 当厄他培南浓度为0.1 g/L时,37株单产KPC型碳青霉烯酶的肠杆菌科细菌在1.5h内全部水解厄他培南,质谱图显示厄他培南所呈现的3个峰全部消失,灵敏度高达100％；当厄他培南浓度为0.3 g/L时,1.5h组水解厄他培南的灵敏度为70.3％ (26/37),2.5h组为89.2％ (33/37),3.5h组为94.6％ (35/37)；当厄他培南浓度为0.5 g/L时,1.5h组水解厄他培南的灵敏度为48.6％ (18/37),2.5h组为83.8％ (31/37),3.5h组为94.6％( 35/37).两独立样本非参数检验统计显示:厄他培南0.1 g/L组与0.3 g/L、0.5 g/L组之间的水解时间差异有统计学意义,而0.3 g/L与0.5 g/L组之间的水解时间差异无统计学意义；细菌组内数据统计显示,除奇异变形杆菌组与大肠埃希菌组之间差异有统计学意义外,其他各细菌组之间差异没有统计学意义.结论 MALDI-TOF MS操作简便,可快速检测产KPC型碳青霉烯酶的肠杆菌科细菌,敏感率高,假阳性率低,适合微生物检验中用于产KPC型碳青霉烯酶的检测,推荐使用0.1 g/L的厄他培南作为水解底物来检测产KPC型肠杆菌科细菌.     

313例尿培养病原菌分布及耐药性分析

目的分析313例清洁中段尿培养菌株构成及耐药性分析,为医师合理规范使用抗生素提供依据。方法对医院2012年6月~2013年5月住院泌尿系感染患者尿培养分离出的313株病原菌用法国梅里埃公司VITEK 2 COMPACT全自动微生物鉴定药敏检测系统仪进行鉴定和药敏试验。结果共培养1804份中段尿标本,分离出313例病原微生物,阳性率17.35%。其中革兰阴性杆菌、革兰阳性球菌、真菌、革兰阳性杆菌分别占65.50%、19.81%、7.35%、7.35%。大肠埃希菌仍是泌尿系感染的主要致病菌,其次为肺炎克雷伯杆菌、阴沟肠杆菌、铜绿假单胞菌、奇异变形菌;其中产超广谱β-内酰胺酶(ESBLs)大肠埃希菌占大肠埃希菌41.94%;而在革兰阳性菌中以肠球菌属为主。检出耐亚胺培南的肺炎克雷伯菌1株,耐碳青霉烯类阴沟肠杆菌2株,耐万古霉素屎肠球菌1例。结论泌尿系感染常见病原菌仍为大肠埃希菌,临床应根据尿培养及药敏试验结果有针对性筛选敏感药物进行治疗。     

我院急性阑尾炎手术患者细菌学分析及药敏试验

目的 了解引起急性阑尾炎患者感染的痛原菌分布及对药物的耐药性,有助于指导临床合理选择抗菌药物.方法 回顾分析2013年所有阑尾炎手术患者细菌培养及药敏试验结果.结果 382例标本中有157例检出细菌(其中16例为两种细菌混合感染),阳性率为41.1％,分离到细菌173株,其中革兰阴性菌167株(96 5％),革兰阳性菌6株(3.5％);从分离到的细菌构成前3位是大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌,分别占检出病原菌的67.1％、9 8％、6 9％;大肠埃希菌体外药敏试验结果:对头孢菌素类敏感率＞60％;喹诺酮类敏感率＜50％;头孢替坦、哌拉西林他唑巴坦和阿米卡星敏感率达到99％以上;无对碳青霉烯类耐药株.结论 我院急性阑尾炎患者感染菌中以阴性肠杆菌为主,其中以大肠埃希菌占首位,临床抗菌药物选择可以首选头霉素类和阿米卡星,头孢菌素类亦可以经验选择.     

一株产碳青霉烯酶IMP-26阴沟肠杆菌的分离鉴定

在过去的几年中,产碳青霉烯酶的肠杆菌科细菌不断被发现,这引起了临床工作者的强烈担忧.我院发现一株碳青霉烯类抗菌药物耐药的阴沟肠杆菌,此临床菌株分离自一位9个月的哮喘患儿,对亚胺培南和厄他培南表现为耐药,其最低抑菌浓度分别为4 μg/ml和16μg／ml.对青霉素类,头孢类及头孢与酶的复合制剂都表现为耐药.进一步的实验显示此菌株的Hodge试验阳性,确定该菌株产碳青霉烯酶.通过对亚胺培南与亚胺培南+EDTA的纸片的抑菌圈直径的比较,发现此种碳青霉烯酶能被EDTA抑制.通过PCR扩增,发现此阴沟肠杆菌含有blaInt11、blaIMP和blaTEM基因,通过测序和比对确定blaIMr的基因型为blaIMP-26,balTEM基因的基因型为blaTEM-104.通过等电聚焦电泳,也证实此阴沟肠杆菌产IMP和TEM酶.     

摩根摩根菌中质粒介导KPC-2型碳青霉烯酶的检测

目的 研究碳青霉烯耐药摩根摩根菌的分子流行病学及其耐药机制.方法 2010年10月-2011年2月从杭州市中医院分离到7株碳青霉烯不敏感的摩根摩根菌.脉冲场凝胶电泳(PFGE)分析菌株之间的同源性;琼脂稀释法测定抗生素对细菌的最低抑菌浓度(MIC);接合试验、质粒图谱分析、特异性PCR扩增和序列分析等研究细菌对碳青霉烯耐药的分子机制.结果 分离自急诊监护病房的6株摩根摩根菌PFGE条带完全相同或相差1～3个条带;分离自重症监护病房的1株摩根摩根菌与其他6株PFGE条带差异明显.7株摩根摩根菌的耐药模式基本相同.亚胺培南、美罗培南和厄他培南的MIC值差异较大,分别为8μg/ml(耐药)、1μg/ml(敏感)和0.25～0.50μg/ml(敏感或中介耐药).7株摩根摩根菌对青霉素类、氨曲南和环丙沙星耐药,对头孢菌素类耐药或敏感,对阿米卡星敏感.接合试验使大肠埃希菌EC600对碳青霉烯类抗生素由敏感变为耐药,对其他β-内酰胺类抗生素也均耐药.摩根摩根菌及转移接合子均含有一个约为60kb的质粒.PCR扩增及测序表明摩根摩根菌及转移接合子均产KPC-2型碳青霉烯酶,且携带qnrS1基因.结论 首次在摩根摩根菌中检测到KPC-2型碳青霉烯酶,KPC-2是引起摩根摩根菌对碳青霉烯类不敏感的主要原因.     

大肠埃希菌对碳青霉烯类药物的耐药机制研究

目的 研究我院普外科重症监护病房(ICU)出现的对碳青霉烯类抗菌药物耐药的大肠埃希菌分子流行病学特征和碳青霉烯耐药机制.方法 采用琼脂稀释法检测分离株的抗菌药物敏感性,采用脉冲场琼脂糖凝胶电泳(PFGE)研究碳青霉烯类耐药的大肠埃希菌分子流行病学机制,采用特异性PCR、DNA测序分析、接合试验、质粒提取和质粒转化试验、外膜蛋白分析等技术研究碳青霉烯耐药的分子机制.结果 我院分离的14株碳青霉烯耐药菌分别属于10个流行克隆型,均表现对包括亚胺培南和美罗培南在内多种抗菌药物耐药,碳青霉烯类耐药基因扩增显示均携带KPC-2型碳青霉烯酶基因,质粒提取与转化试验显示KPC-2定位于约56 kb大小的质粒上,SDS-PAGE分析发现耐药株多出现外膜蛋白的改变.结论 我院出现多个流行克隆型的碳青霉烯类耐药的大肠埃希菌,质粒型碳青霉烯酶KPC-2是我院泛耐型大肠埃希菌介导对碳青霉烯类耐药的主要原因,外膜孔蛋白改变参与介导大肠埃希菌对碳青霉烯类高度耐药.     

新生儿感染性肺炎的痰细菌培养检测及免疫功能研究

目的探讨新生儿感染性肺炎痰培养结果,细菌药物耐药情况及其免疫功能的变化。方法负压吸痰,送痰液进行细菌培养及药物敏感试验,同时采集患儿血液检测特种蛋白C3、C4、IgG、IgM、IgA;;,对结果进行分析,选取同期非肺炎新生儿30例作为对照组。结果 79例感染性肺炎患儿痰培养细菌阳性35例,常见大肠埃希菌（31.4%）、金黄色葡萄球菌（22.9%）、肺炎克雷伯菌（20.0%）、阴沟肠杆菌（11.4%）。大肠埃希菌、肺炎克雷伯菌、阴沟肠杆菌对亚胺培南均敏感,金黄色葡萄球菌对万古霉素敏感。各组间C3、C4值两两比较均有统计学意义（P〈0.05）;肺炎细菌感染组与其他感染组IgM值大于对照组（P〈0.05）。结论新生儿肺炎细菌感染中病原菌以革兰氏阴性感染为主,对亚胺培南敏感性最高。革兰氏阳性菌对万古霉素尚未出现耐药性。低补体水平将不能很好发挥补体等生物学功能,从而导致易患肺炎等感染性疾病。不同病原体感染可影响患儿补体合成。IgM能在新生儿体液免疫中发挥重要作用。     

影响消化道来源败血症病原菌分布的相关因素及耐药性分析

目的探讨消化道来源的败血症病原菌分布的相关因素及耐药性。方法采用回顾性研究方法,收集91例病例,进行单因素和多因素logistic分析。结果消化道来源的败血症以大肠埃希菌感染为主(45%),以肝硬化作为原发首要疾病(33%);单因素研究发现,原发疾病中肝硬化、胆囊炎是感染大肠埃希菌、肺炎克雷伯杆菌及阴沟肠杆菌的主要危险因素(P0.05);多因素研究发现,肝硬化、高血压、冠心病、术前抗生素使用、PPI使用及操作史是感染大肠埃希菌的危险因素。耐药性大肠埃希菌、肺炎克雷伯杆菌及阴沟肠杆菌对美洛培南、替考拉宁、亚胺培南和哌拉西林/他唑巴坦普遍敏感,其它均产生耐药性。结论 2009~2012年我院消化道来源的败血症患者病原菌分布和耐药性都发生了明显变迁,不同原发疾病的患者病原菌具有相应的特点。     

Occurrence and characterization of carbapenemase-producing Enterobacteriaceae: report from the SENTRY Antimicrobial Surveillance Program (2000-2004)

Emergence and dissemination of Enterobacteriaceae isolates harboring carbapenemases in various geographic regions represents a significant threat to the management of nosocomial infections. Enterobacteriaceae isolates from the SENTRY Antimicrobial Surveillance Program (2000-2004) demonstrating decreased susceptibility to imipenem and meropenem (minimum inhibitory concentration [MIC], > or =2 mg/L) were evaluated for the production of metallo-beta-lactamases and serine carbapenemases using disk approximation and polymerase chain reaction (PCR) tests. Carbapenemase-producing strains were epidemiologically typed by automated riboprinting and pulsed-field gel electrophoresis (PFGE) to establish clonality. Among 37,557 Enterobacteriaceae (5 genus groups) evaluated, 119 (0.32%) had increased carbapenem MIC values, and a carbapenemase was identified in 51 (42.9%) of these strains. KPC-2 and KPC-3 were the most frequently occurring carbapenemases (24 isolates, 20.2%) in the United States and were detected in Klebsiella spp, Citrobacter spp., Enterobacter spp., and Serratia marcescens strains isolated in New York, Arkansas, and Virginia. SME-2-producing S. marcescens were isolated in the New York City area, Texas, and Ohio, while NMC-A was found in one E. cloacae strain from New York. In contrast, metallo-beta-lactamases were prevalent in Europe. IMP-1-producing E. cloacae (11 isolates) were detected in Turkey, while VIM-1-producing strains were found in Italy (Enterobacter spp.) and Greece (Klebsiella pneumoniae). Clonal dissemination of carbapenemase-producing strains was observed in several medical centers on both continents. The occurrence of carbapenemases in various Enterobacteriaceae remains rare but appears to be spreading geographically (not in Latin America), mainly with metallo-beta-lactamases being found in Mediterranean Europe and KPC enzymes in the New York City area.     

Evaluation of methods to identify the Klebsiella pneumoniae carbapenemase in Enterobacteriaceae

The Klebsiella pneumoniae carbapenem (KPC) beta-lactamase occurs in Enterobacteriaceae and can confer resistance to all beta-lactam agents including carbapenems. The enzyme may confer low-level carbapenem resistance, and the failure of susceptibility methods to identify this resistance has been reported. Automated and nonautomated methods for carbapenem susceptibility were evaluated for identification of KPC-mediated resistance. Ertapenem was a more sensitive indicator of KPC resistance than meropenem and imipenem independently of the method used. Carbapenemase production could be confirmed with the modified Hodge test.     

Carbapenemases among Klebsiella pneumoniae: sensitivity, E-test and Hodge test

Carbapenems are the major weapons in the treatment of nosocomial infections. Since 1996, there have been an emergence and dissemination of carbapenem resistance in the world, especially among Enterobacteriaceae. Resistance is due to one or several mechanisms including production of carbapenemases. A prospective study was carried out from April to September 2011 at the Laboratory of Microbiology of Military Teaching Hospital in Rabat. The aim of this study was to search by phenotypic methods carbapenemases production in Klebsiella pneumoniae at Laboratory of Microbiology of the HMIMV of Rabat by disc diffusion for susceptibility to imipenem (IMP) and ertapenem (ETP), E-test and Hodge test; 211 strains of K. pneumoniae were analyzed. The samples were most frequently obtained from the urinary tract (63.8%); pus (16.5%) and blood cultures (11.8%);?27% of the strains produced extended spectrum of beta-lactamase (ESBL); 3 strains were resistant to ertapenem by disc diffusion. Investigations of these 3 strains in E-test shown that they were resistant to ertapenem but susceptible to imipenem. Tested by Hodge method, 3 strains gave repeatedly positive results. Thus, rate of K. pneumoniae producing KPC type in our study is 1.42%. Our study shows the evidence of K. pneumoniae type carbapenemase (KPC) in Military Teaching Hospital of Rabat. However, the gene encoding the type of resistance must be determined by molecular methods.     

Metallo-β-lactamase-producing Enterobacteriaceae isolates at a Taiwanese hospital: lack of distinctive phenotypes for screening

We investigated the prevalence of metallo-β-lactamases (MBLs) among 1,827 Enterobacteriaceae isolates collected in 2006 and evaluated the VITEK 2 microbiology system, modified Hodge test, and 2 combined disk tests as the screening tools for MBLs by using these isolates and 77 previously characterized IMP-8 producers. The IMP-8 MBL was identified in 18 isolates of 2006, and the IMP-8-positive isolates represented 0.2%, 1.1%, and 5.0% of all Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae isolates, respectively. Only one-third of all MBL producers could be recognized by either VITEK 2 or the Hodge test. MBL production could be identified in 38 (40%) of the 95 IMP-8-producing isolates by the combined disk test using meropenem disks supplemented by phenylboronic acid and EDTA, and only 2 (2.1%) isolates gave positive results in the combined disk test using meropenem disks supplemented with dipicolinic acid. Of all IMP-8 producers, 37.9%, 50.5%, and 32.6% were nonsusceptible to tigecycline, fluoroquinolones, and both, respectively. In conclusion, this study demonstrated the lack of distinct phenotypes that could be easily identified among the IMP-8-producing Enterobacteriaceae isolates at a Taiwanese hospital. Continuous surveillance and monitoring are needed because the widespread of tigecycline- and fluoroquinolone-coresistant MBL producers may become a serious therapeutic problem.     

Accuracy of carbapenem nonsusceptibility for identification of KPC-possessing Enterobacteriaceae by use of the revised CLSI breakpoints

Using the updated 2010 CLSI carbapenem breakpoints for the Enterobacteriaceae, nonsusceptibility to ertapenem and imipenem predicted the presence of bla(KPC) poorly, especially among Escherichia coli and Enterobacter species. In regions where KPC-producing bacteria are endemic, testing for nonsusceptibility to meropenem may provide improved accuracy in identifying these isolates.     

Emergence of New Delhi metallo-beta-lactamase (NDM-1) and Klebsiella pneumoniae carbapenemase (KPC-2) in South Africa

This report documents emergence of New Delhi metallo-beta-lactamase (NDM-1) and Klebsiella pneumoniae carbapenemase (KPC-2) in K. pneumoniae and Enterobacter cloacae in South Africa. NDM-1 producers have not been described in South Africa, and this is the first instance that KPC producers have been identified in Africa. The two patients infected with these carbapenemase-producing bacteria demised.     

Reproducibility of the MS-2 system for identification of members of the family Enterobacteriaceae: a collaborative study with blindly assigned reference stains.

The reproducibility of identification and biochemical reactions for five different reference organisms of Enterobacteriaceae; Proteus vulgaris, Klebsiella pneumoniae, Escherichia coli, Serratia marcescens, and Enterobacter cloacae, were evaluated using the updated MS-2 system software (Abbott Laboratories, Diagnostic Division, Irving, Tex.) in a collaborative study involving 11 laboratories. When a total of 220 randomly coded test organisms were blindly examined, the MS-2 system correctly identified 92.7 and 86.8% for over 80 and 90% probability identification, respectively. Four organisms, P. vulgaris, K. pneumoniae, E. coli, and S. marcescens, were correctly identified in all laboratories with high probability, but 9 of 44 tests of Enterobacter cloacae resulted in misidentifications or low-likelihood (less than 80%) identifications. Accuracy was directly related to level of experience and familiarity with the MS-2 system in the individual laboratories. Biochemical reactions varied among the identification trials, especially in the identification of S. marcescens and Enterobacter cloacae. Among a total of 44 subcultures for each organism, 10 different biochemical patterns for P. vulgaris, 6 for K. pneumoniae, 9 for E. coli, 15 for S. marcescens, and 14 for Enterobacter cloacae were obtained. The results indicate that the MS-2 system performs with high accuracy and reproducibility in identifying Enterobacteriaceae, except for Enterobacter cloacae.     

对碳青霉烯类抗生素敏感性下降的肠杆菌科细菌耐药机制的研究及流行病学调查

目的 对临床分离的碳青霉烯类抗生素耐药的肠杆菌科细菌进行耐药机制研究及流行病学调查.方法 收集2010年1月至2010年8月,对碳青霉烯类抗生素敏感性下降的肠杆菌科细菌18株,全自动微生物鉴定仪检测细菌对常见抗生素最低抑菌浓度(MIC),纸片法检测细菌产超广谱β-内酰胺酶、头孢菌素酶、碳青霉烯酶的情况,并用PCR扩增、DNA测序确定所产碳青霉烯酶基因型.脉冲场凝胶电泳对耐药菌进行同源性分析.结果 8株耐药菌全部检出ESBLs酶、AmpC酶,17株检出KPC-2酶,3株EDTA纸片法阳性提示产其他金属碳青霉烯酶,其中两株合并产KPC-2.脉冲场凝胶电泳结果显示15株肺炎克雷伯菌分为6种带型.结论 KPC-2碳青霉烯酶是造成肠杆菌科细菌对碳青霉烯类抗生素敏感性下降的主要原因,并在我院局部短暂流行,携带KPC-2基因的临床菌株同时携带多种耐药基因.     

Dissemination of extended-spectrum beta-lactamase-producing Enterobacteriaceae in intensive care units of a medical center in Taiwan

The prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in a tertiary hospital in Taiwan was assessed over a 16-month period. A total of 125 nonrepetitive ESBL-producing isolates of Enterobacter cloacae, Escherichia coli, and Klebsiella pneumoniae were available for investigation using molecular methods. Four predominant intensive care units (ICUs) were identified, and SHV-12 (59%), CTX-M- 3 (36%), and CTX-M-14 (14%) were the three most frequent ESBLs. SHV-12 was predominant among E. cloacae in the burn unit and K. pneumoniae in the other three chest medicine-related ICUs. CTX-M-3 was predominant among E. coli and K. pneumoniae in three other ICUs. The dissemination of ESBL-producing Enterobacteriaceae in four ICUs of a medical center in Taiwan is a consequence of the clonal dissemination of a few epidemic strains along with the horizontal transmission of resistance genes-carrying plasmids among bacterial organisms.