亚胺培南耐药鲍曼不动杆菌16S rRNA甲基化酶基因检测及流行菌株分析

目的 研究我国多中心亚胺培南耐药的鲍曼不动杆菌的耐药性、16S rRNA甲基化酶的阳性率及分子流行病学特征.方法 收集2004年11月-2005年11月国内6省市19家医院临床分离的342株亚胺培南耐药的鲍曼不动杆菌.采用琼脂稀释法和E test法对18种抗菌药物测定分离株的最低抑菌浓度(MIC)值;脉冲场凝胶电泳(PFGE)分析菌株的同源性;PCR法筛选4种16S rRNA甲基化酶基因armA、rmtA、rmtB、rmtC,克隆测序明确基因型;接合试验、质粒抽提、电转化以及Southern blot确定甲基化酶耐药基因定位.结果 所有菌株均为多重耐药株,其中对阿米卡星、庆大霉素、妥布霉素、异帕米星、奈替米星耐药率分别为92.6%、98.6%、87.4%、90.9%和92.4%.PCR扩增、测序证实221株鲍曼不动杆菌检出甲基化酶armA基因;另3种甲基化酶基因rmtA、rmtB、rmtC均阴性.在上述342株鲍曼不动杆菌临床分离株中,298株可归类为6个流行克隆,44株为散发株.3个主要克隆(A、B、C)在全国广泛播散,分别在国内6家、3家、11家医院内流行.接合试验、质粒抽提、电转化及Southern blot表明armA编码基因存在于染色体上.结论 亚胺培南耐药鲍曼不动杆菌的表型均为多重耐药.介导氨基糖苷类抗生素高度耐药的16S rRNA甲基化酶基因armA在鲍曼不动杆菌中广泛存在,其主要传播方式为克隆播散,这必将引起临床的高度关注.     

鲍曼不动杆菌armA基因的分布与耐药性的研究

目的 调查我院鲍曼不动杆菌中16S rRNA甲基化基因armA的分布以及与鲍曼不动杆菌耐药谱的关系,并初步探讨其在分子流行病学分析中的作用.方法 收集72株鲍曼不动杆菌,采用K-B法对鲍曼不动杆菌进行药物敏感试验,后采用PCR筛选鲍曼不动杆菌的16S rRNA甲基化基因armA,并利用随机扩增多态性DNA法(RAPD)技术进行基因分型.统计各鲍曼不动杆菌菌株对多种氨基糖苷类药物的药敏结果,并分析基因型与耐药性的关系.结果 根据PCR产物片段大小,72株鲍曼不动杆菌共有armA基因阳性菌株20株(27.8%).含有armA基因型鲍曼不动杆菌菌株对庆大霉素、妥布霉素和阿米卡星的耐药率均为90%;随机扩增多态性DNA法显示20株armA基因阳性的鲍曼不动杆菌主要分为7型,A型为优势克隆株.结论 产16S rRNA甲基化基因armA的鲍曼不动杆菌菌株可对多种氨基糖苷类抗生素高水平耐药.同一克隆菌株在病房内和病房间的传播为我院armA基因传播的主要方式.     

60株鲍曼不动杆菌耐药基因携带情况分析

目的研究临床分离的60株鲍曼不动杆菌耐药谱及Ⅰ型整合子和β-内酰胺酶等基因携带情况。方法用微量肉汤稀释法测定16种临床常用抗菌药物的最小抑菌浓度;PCR检测β-内酰胺酶、Ⅰ型整合子和外排泵基因,对阳性基因进行序列分析。结果60株菌中,多重耐药株53株,占88.3%;6株携带OXA-23基因,均对包括碳青霉烯在内的5类以上抗菌药耐药,并具有高耐药特性;38株携带PER-1基因,对头孢菌素类耐药率显著高于PER-1基因阴性菌株(P<0.01);45株检出Ⅰ型整合子结构基因,多重耐药率明显高于Ⅰ型整合子阴性菌株(P<0.01);Ⅰ型整合子和PER-1基因同时阳性25株,与7株两者同为阴性菌株相比,多重耐药率增高(P<0.01),但耐药程度无显著差别。结论Ⅰ型整合子基因及β-内酰胺酶类基因的作用是导致鲍曼不动杆菌多重耐药的重要原因;OXA-23基因阳性菌株多为泛耐药和高耐药株,有必要采取有效措施控制其传播。     

杭州和湖州鲍曼不动杆菌β内酰胺酶和氨基糖苷类修饰酶基因研究

目的了解浙江省杭州和湖州自临床分离的鲍曼不动杆菌中β内酰胺酶(β-lactamases,BLA)基因及氨基糖苷类修饰酶(aminoglycoside-modifying enzymes,AMEs)基因存在状况.方法在2000年7月～2004年2月间从省立同德医院和解放军第98医院各分离20株鲍曼不动杆菌,采用微量稀释法测定其对13种抗菌药物的敏感性,采用聚合酶链反应(PCR)及序列分析的方法分析BLA基因(TEM和SHV)及AMEs基因[aac(3)-Ⅰ、aac(3)- Ⅱ、aac(6′)-Ⅰ和ant(3″)-Ⅰ]类型.结果两地分离株多重耐药严重,但对亚胺培南和美洛培南均无耐药.杭州分离株中TEM、SHV、aac(3)-Ⅰ、aac(3)- Ⅱ、aac(6′)-Ⅰ和ant(3″)-Ⅰ基因阳性率分别为45.0%、0.0%、55.0%、15.0%、35.0%和60.0%,湖州分离株分别为100.0%、30.0%、50.0%、10.0%、55.0%和65.0%.序列分析确认为TEM-1亚型广谱BLA和SHV-12亚型超广谱β内酰胺酶基因.其中98医院HZ40株的TEM-1序列及HZO2株的SHV-12序列均已登录GenBank(GenBank注册号分别为:AY263331和AY259163).结论浙江湖州临床分离的鲍曼不动杆菌中TEM、SHV基因阳性率均高于杭州分离株(Ρ分别＜0.01和＜0.05),但4种AMEs基因阳性率差别不大(Ρ均＞0.05).在鲍曼不动杆菌中发现SHV-12 及TEM-1基因分别为国际及国内首次报道.     

多重耐药鲍曼不动杆菌相关耐药基因检测分析

目的为了解多重耐药鲍曼不动杆菌β-内酰胺酶（BLA）基因、氨基糖苷类修饰酶（AMFs）基因、消毒剂与磺胺类耐药基因（qacE△1-sul1）和1类整合子酶基因（intl1）存在情况。方法对2005年1—6月份临床分离的31株多重耐药菌株（耐哌拉西林、第三代头孢菌素、环丙沙星和阿米卡星），应用聚合酶链反应及序列分析方法分析其BLA、AMEs、qacE△1．sull和intl1基因类型。结果31株多重耐药鲍曼不动杆菌对多黏菌素B敏感，有3株（9．7％）对受试的其他18种抗菌药物均耐药。19株（61．3％）检出了β-内酰胺酶基因，其中TEM、PER、DHA阳性率分别为61．3％、19．4％、3．2％，未检出SHV、OXA-23、OXA-24、GES、IMP和VIM等基因。25株（80．6％）检出氨基糖苷类修饰酶基因，其中aac（3）-I、aac（6’）-I、ant（3”）-I、ant（2”）-I、aac（3）-Ⅱ、aac（6’）-Ⅱ阳性率分别为67．7％、45．2％、29．0％、22．6％、9．7％、3．2％。qacE△A1-sul1、intl1阳性率分别为80．6％、58．1％。分离株常见的基因组合是TEM＋qacE△1-sul1＋intl1和TEM＋PER＋qacE△1-sull＋intl1，分别占25．8％和19．4％。分离株AMEs的常见的基因组合是aac（3）-I＋aac（6’）-I和nnc（3）-I＋aac（6’）-I＋ant（2”）-I，分别占19．4％和12．9％。结论临床分离的多重耐药鲍曼不动杆菌TEM、nnc（3）-I、nnc（6’）-I、ant（3”）-I、ant（2”）-I、qacE△1-sul1和intl1基因携带率高。     

5年间鲍曼不动杆菌产碳青霉烯酶特性与耐药相关性研究

目的 研究2002-2006年每年3-5月临床分离鲍曼不动杆菌中碳青霉烯酶的分布与变迁情况及其与碳青霉烯类抗生素耐药的相关性.方法 琼脂稀释法检测亚胺培南(IPM)和美罗培南(MEN)对174株鲍曼不动杆菌的最低抑菌浓度(MIC);2-巯基丙酸(2-MPA)和乙二胺四乙酸(EDTA)协同试验进行金属β-内酰胺酶表型检测;PCR扩增金属β-内酰胺酶(MBL)VIM、IMP和苯唑西林酶OXA-23、OXA-24、OXA-51、OXA-58相关基因.结果 2002-2006年每年3-5月临床分离的鲍曼不动杆菌对IPM的耐药率分别为0(0/20)、26%(1/38)、0(0/36)、34.9%(15/43)和59.5%(22/37),对MEN的耐药率分别为0(0/20)、5.3%(2/38)、0(0/36)、20.9%(9/43)和51.4%(19/37);15.5%(27/174)的菌株OXA-23阳性,72.4%(126/174)的菌株OXA-5I阳性,其中15.5%(27/174)菌株同时产OXA-51和OXA-23酶,OXA-23的阳性率从2002年的0上升到2006年的48.6%,OXA-51的阳性率从2002年的35.0%上升到2006年的89.2%;所有菌株均未检出OXA-24、OXA-58、IMP及VIM基因.结论 5年间鲍曼不动杆菌中OXA-23、OXA-5I的检出率及其对碳青霉烯类抗生素的耐药性均呈上升趋势;产OXA-23型B-内酰胺酶是本组鲍曼不动杆菌对碳青霉烯类抗生素产生耐药的重要原因,OXA61可能与鲍曼不动杆菌对碳青霉烯类抗生素的低水平耐药有关.     

泛耐药鲍氏不动杆菌氨基糖苷类耐药相关基因研究

目的 探讨泛耐药鲍氏不动杆菌氨基糖苷类抗菌药物耐药机制。方法 收集2014年湖北省黄石市中心医院住院患者中分离的20株泛耐药鲍氏不动杆菌,用mdfA测序确认菌种,再采用聚合酶链反应法分析16种氨基糖苷类药物获得性耐药相关基因。 结果 20株泛耐药鲍氏不动杆菌共检出4种氨基糖苷类获得性耐药基因,氨基糖苷类修饰酶基因aac(3)-Ⅰ阳性率15.00％;ant(3")-Ⅰ阳性率20.00％;aph3'-Ⅰ阳性率100.00％;16SrRNA甲基化酶未检出;外排泵adeB基因阳性率95.00％。 结论　泛耐药鲍氏不动杆菌中携带的耐药基因和耐药表型相对应,对氨基糖苷类药物耐药与产aac(3)-Ⅰ、ant(3")-Ⅰ、aph3'-Ⅰ修饰酶基因和获得外排泵adeB 2种耐药机制有关。     

多重耐药鲍曼不动杆菌的耐药性与AmpC耐药基因的研究

目的了解我院临床分离的60株多重耐药鲍曼不动杆菌（multi-drug resistant Acinetobater baumannii,MDRAB）的耐药性和AmpC耐药基因的存在状况。方法采用VITEK-2全自动细菌鉴定仪检测鲍曼不动杆菌对18种抗生素的药敏结果,对该细菌进行总DNA的提取,用聚合酶链反应（PCR）检测耐药基因,结合药物敏感试验分析菌株的耐药特征。结果检出结构基因ampC型49株,占81.7%;blaADC基因型50株,占83%;ACT基因型2株;41株菌同时携带blaADC和ampC结构基因,占68%;2株同时携带blaADC、ACT和ampC结构基因;未检测到其它AmpC耐药基因（MOX、CIT、FOX、DHA）。携带AmpC耐药基因的菌株耐药率高,特别是对头孢曲松、氨苄西林、头孢替坦、头孢唑啉、呋喃妥因的耐药率已接近或达到100%,而对于丁胺卡那霉素和头孢哌酮/舒巴坦的耐药率最低,分别为5%和20%。结论多重耐药鲍曼不动杆菌的blaADC耐药基因和ampC结构基因同时携带率高,并且是鲍曼不动杆菌多重耐药的主要原因。     

鲍曼不动杆菌多重耐药性与主动外排机制的相关性研究

目的 了解多重耐药鲍曼不动杆菌主动外排系统及双组分调节系统编码基因的携带情况,并观察外排泵抑制剂对多重耐药鲍曼不动杆菌耐药水平的影响程度,以探讨鲍曼不动杆菌多重耐药性与胞膜主动外排系统的关系.方法 PCR方法 扩增外排泵编码基因adeB及双组分调节系统编码基因adeR和adeS.采用琼脂稀释法测定50株多重耐药鲍曼不动杆菌对环丙沙星、阿米卡星、头孢噻肟和亚胺培南的最低抑菌浓度(MIC),并观察在含25μg/ml利血平条件下MIC值的变化程度.结果 50株多重耐药鲍曼不动杆菌adeB、adeR及adeS基因的携带率分别为94%、96%及92%.以环丙沙星、头孢噻肟、阿米卡星和亚胺培南作为底物,分别有49、50、50和46株菌在含25μg/ml利血平的条件下MIC值降低4倍或4倍以上,呈现明显的外排作用.结论 主动外排机制是本地区鲍曼不动杆菌多重耐药的重要原因之一.     

鲍曼不动杆菌对碳青霉烯类抗生素的耐药分析及基因型研究

目的分析20株鲍曼不动杆菌对耐碳青霉烯类抗生素的耐药性及对碳青霉烯酶基因的研究。方法用API鉴定条进行细菌鉴定及K-B法进行药敏试验,用碳青霉烯酶4种基因的特异性引物进行聚合酶链反应（PCR）扩增和基因型的测序分析,并通过网上Genbank进行比对以确定编码酶基因的类型。结果 20株鲍曼不动杆菌对左旋氧氟沙星、丁胺卡那霉素、多粘菌素B的耐药率分别为50%、25%、4%。其它抗生素的耐药率均在90%以上。携带D类碳青霉烯酶OXA-23基因有17株（85%）,携带OXA-51基因有15株（75%）,OXA-24、OXA-58基因引物PCR扩增为阴性,随机各抽取3株OXA-23基因阳性株进行测序后通过在网上Genbank比对发现与OXA-23标准株99%同源,OXA-51基因阳性株与OXA-51标准株98%同源。结论本院耐碳青霉烯类抗生素的鲍曼不动杆菌对多粘菌素B的耐药率最低,其次是丁胺卡那霉素,以携带OXA-23型碳青霉烯酶基因为主,应引起临床高度重视,防止在院内广泛传播。     

Carbapenem-hydrolyzing oxacillinase in high resistant strains of Acinetobacter baumannii isolated from India

Acinetobacter baumannii, a Gram negative bacterium causes nosocomial infections including bacteremia, secondary meningitis and urinary tract infections. Increased resistance of A. baumannii has been global concern. Till recently, carbapenems, latest generation of β-lactams are used for treating infections caused by A. baumannii. Emerging resistance to carbapenem class is an immediate threat to mankind. The objective of present study is to understand the growing carbapenem resistance of A. baumannii. By using iso-electric focusing followed by (in-gel) nitrocefin assay of carbapenem resistant strains of A. baumannii, we could identify three β-lactamases with pIs in the range 5.4-9.5. Expression of the β-lactamase with a pI ≈ 8.5, was found only in very high carbapenem resistant (MIC for imipenem 128 μg/ml) strains. On PCR analysis and sequencing of PCR product, this β-lactamase was confirmed to be OXA-51. Identification of this protein from IEF gel was reconfirmed with the help of Liquid chromatography and Tandem mass spectrometry (LC-MS/MS). Based on the amino acid sequence, OXA-51 found to be a 30 kDa β-lactamase containing conserved functional motifs of class D serine β-lactamase. In the present study, we have established the emergence of OXA-51 in clinical strains of A. baumannii in India which suggests its role in carbapenem resistance.     

Prevalence of OXA-type β-lactamases among Acinetobacter baumannii isolates from Northwest of Iran

Carbapenems have been considered as last line antibiotics for treatment of multidrug-resistant (MDR) Acinetobacter baumannii but carbapenem resistant A. baumannii has been increased during the last decade in many parts of the world. OXA-type β-lactamase enzymes are the most common cause of carbapenem resistance in A. baumannii and presence of ISAba1 in upstream of these genes may increase the expression of these OXA genes. The aim of this study was to determine, for the first time, the antibiotic resistance pattern and prevalence of OXA type β-lactamases among nosocomial A. baumannii isolates from northwest of Iran. A total of 100 A. baumannii isolates were recovered from hospitalized patients in a university hospital in northwest of Iran. Sixty-two percent of isolates were resistant to imipenem. All isolates carried bla(OXA-51)-like gene. Among imipenem resistant isolates, 88.7% carried bla(OXA-23)-like, 1.6% carried bla(OXA-40)-like, and 3.2% had bla(OXA-58)-like resistance genes. Ninety percent of isolates contained ISAba1 element and in 74.2% of imipenem resistant isolates, ISAba1 was located in upstream of bla(OXA-23)-like. The results of this study demonstrated high prevalence of OXA-type carbapenemase among MDR A. bumanii in the Northwest of Iran.     

Characterization of the resistance mechanism to beta-lactams in Acinetobacter baumannii strains isolated in the university hospital Sahloul in Tunisia (2005)

OBJECTIVE: The bacterial multiresistance to beta-lactams and imipenem is an emergent feature in the university hospital Sahloul in Tunisia. This study was conducted to elucidate natural and acquired mechanism of resistance to beta-lactams in strains of Acinetobacter baumannii isolated in different wards of the hospital. MATERIALS AND METHODS: A specimen of 26 clinical strains of Acinetobacter baumannii was studied. beta-lactamases characterization was done by isoelectric focusing on gel of crude enzymatic extract, phenotypic tests for detection of extended spectrum beta-lactamases (ESBL) and metallo-beta-lactamases (MBL) and finally by amplification (PCR) and sequencing of genes encoding naturally occurring AmpC, the insertion sequence ISAbaI and oxacillinase with carbapenemase activity. Study of clonality of strains was performed by analysis of genomic DNA digested by the restriction enzyme ApaI and separated by pulsed field gel electrophoresis (PFGE). RESULTS: The isoelectric focusing on gel revealed two bands of beta-lactamase activity with a pI upper than 8. None ESBL or MBL was detected. PCR for AmpC, ISAbaI and OXA-69 were positive for all studied strains. The sequencing of PCR products show high identity (99-100%) with genes described previously. PFGE analysis has demonstrated clonality of studied strains. CONCLUSION: Resistance to beta-lactams including imipenem is associated to the hyper production of the AmpC enzyme and expression of OXA-69. Those enzymatic mechanisms are associated with the natural low permeability to beta-lactams which characterize Acinetobacter baumannii strains. High clonal relationship of studied strains proved by PFGE analysis has shown the necessity of implementation of strict hygienic rules and rational antibiotic usage.     

Identification of the naturally occurring genes encoding carbapenem-hydrolysing oxacillinases from Acinetobacter haemolyticus, Acinetobacter johnsonii, and Acinetobacter calcoaceticus

Carbapenem resistance is increasingly being reported among Acinetobacter species, and results mostly from the expression of acquired carbapenem-hydrolysing oxacillinases (CHDLs). Several Acinetobacter species intrinsically possess chromosomal CHDL genes: Acinetobacter baumannii (bla(OXA-51) ), Acinetobacter radioresistens (bla(OXA-23) ), and Acinetobacter lwoffii (bla(OXA-134) ). We aimed to identify the progenitors of novel CHDL-encoding genes for identification of potential reservoirs. We performed PCR screening using degenerated internal primers designed from a sequence alignment of the known CHDLs (OXA-23, OXA-40, OXA-51, OXA-58, OXA-134, and OXA-143) applied to a collection of 50 Acinetobacter strains belonging to 23 different species. Two strains of Acinetobacter johnsonii, one strain of Acinetobacter calcoaceticus and two strains of Acinetobacter haemolyticus were found to harbour, respectively, the totally novel bla(OXA-211) -like, bla(OXA-213) -like and bla(OXA-214) -like genes. In addition, the complete genomes of those three species available in GenBank, i.e. one A. johnsonii genome, four A. calcoaceticus genomes, and one A. haemolyticus genome, were analysed and found to be positive for the presence of bla(OXA211) -like, bla(OXA-213) -like and bla(OXA-214) -like genes, respectively. The β-lactamases OXA-211, OXA-213 and OXA-214 are diverse, with amino acid identities ranging from 53% to 76%, as compared with the naturally occurring OXA-51-like CHDL from A. baumannii. These β-lactamases showed a peculiar hydrolysis profile, including mostly penicillins and carbapenems. Regarding bla(OXA-23) in A. radioresistens and bla(OXA-134) in A. lwoffii, these genes were not expressed (or expressed at a non-significant level) in their host. Detection of these β-lactamase genes might be used as a useful tool for accurate identification of these Acinetobacter species.     

Detection of drug resistance-associated genes of multidrug-resistant Acinetobacter baumannii

The beta-lactamase (BLA) genes, the genes for aminoglycosides-modifying enzymes (AMEs), disinfectant-sulfanilamide resistance (qacEDelta1-sul1) genes, class 1 integrase (intl1) gene, and the qnr gene associated with plasmid-mediated quinolone resistance were analyzed using PCR and verified by DNA sequencing for 31 clinical isolates of multidrug-resistant Acinetobacter baumannii (MDRAB). The organism typing was performed by pulsed-field gel electrophoresis (PFGE). The positive rate of ADC, TEM, PER, and DHA of BLA genes were 100%, 61.3%, 19.4%, and 3.2%, respectively; however, the genes of SHV, OXA-23 group, OXA-24 group, GES, VIM, IMP, and qnr gene were negative. The positive rate of the genes of AMEs for aac (3)-I, aac (6')-I, ant (3")-I, ant (2")-I, aac (3)-II, and aac (6')-II were 67.7%, 45.2%, 29.0%, 22.6%, 12.9%, and 3.2%, respectively. The positive rate of qacEDelta1-sul1 and intl1 were 80.6% and 58.1%, respectively. Six different PFGE clones were found, of which two dominated. The findings show that clinical isolates of MDRAB harbor various kinds of resistance genes.     

大肠埃希菌尿液分离株中检出gyrA基因新变异株

目的 调查大肠埃希菌尿液分离株中喹诺酮类耐药相关基因的存在与变化状况.方法 收集宁波市第一医院2008年10月到2009年3月患者尿液标本中分离的大肠埃希菌共28株,采用聚合酶链反应(PCR)及序列分析的方法分析1种染色体介导的喹诺酮类耐药相关基因(gyrA基因)和5种质粒介导的喹诺酮类耐药相关基因[qnrA、qnrB、qnrS、aac(6＇)-Ⅰb、qepA].结果 28株大肠埃希菌检测到1株aac(6＇)-Ⅰb-Cr基因阳性株(经测序比对证实),qnrA、qnrB、qnrS、qepA基因均未检出.gyrA基因83位密码子28株菌都有突变(100.0%),其突变方式为TCG-83→HTG,导致氨基酸从丝氨酸(S)-83→亮氨酸(L);87位密码子22株菌(78.6%)有突变,可分为两种突变方式:21株(75.0%)突变方式为GAC-87→AAC,导致氨基酸从天冬氨酸(D)-87→天冬酰胺(N);5号株gyrA基因(3.6%)为新亚型,其突变方式为GAC-87→TAC,导致氨基酸从天冬氨酸(D)-87→脯氨酸(Y),另6株菌87位密码子无突变.结论 本组大肠埃希菌gyrA基因突变率为100.0%,是喹诺酮类耐药的主要原因.其他耐药相关基因阳性率很低.