天津地区医院感染鲍曼不动杆菌产ESBLs和AmpC酶的检测及耐药性分析

目的 了解天津地区五所三甲医院鲍曼不动杆菌产超广谱β-内酰胺酶(ESBs)和AmpC酶的情况及耐药特点.方法 采用酶提取物三维试验方法分别检测鲍曼不动杆菌产ESBLs和AmpC酶的情况,并以PCR方法检测ESBLs基因.以KB法检测72株鲍曼不动杆菌对12种抗生素的耐药性.结果 72株鲍曼不动杆菌中,共检测到ESBLs阳性株16株.阳性率为22.2%(11/72);AmpC酶阳性株18株.阳性率为25.0%(18/72).11株产TEM型ESBLs,4株产PER型ESBLs,1株产VEB·l型ESBLs.ESBLs阳性株和AmpC酶阳性株均呈多重耐药,产酶株对抗生素的耐药率明显高于非产酶株.结论 产AmpC酶是鲍曼不动杆菌医院感染多重耐药主要原因,应加强对AmpC酶的检测及医院内感染的控制工作.     

产AmpC酶鲍曼不动杆菌的耐药表型及基因型分析

目的通过对鲍曼不动杆菌产AmpCβ-内酰胺酶的耐药表型及基因型分析,研究其耐药特征和流行趋势。方法收集2010年1月至10月两所综合性医院临床分离的多重耐药鲍曼不动杆菌63株,采用琼脂扩散法进行药物敏感试验,采用聚合酶链反应检测AmpC酶。结果 63株鲍曼不动杆菌耐头孢西丁61株(96.8%),而且对临床其他常用抗生素的耐药率也非常高,均在90%以上(除亚胺培南,美罗培南,米诺环素,阿米卡星,头孢哌酮/舒巴坦,替卡西林/棒酸),表现为多重耐药,其中对米诺环素的敏感率最高为56%,头孢哌酮/舒巴坦位列第二为41.3%。所测63株鲍曼不动杆菌中AmpC酶阳性率为67%。结论鲍曼不动杆菌多重耐药有蔓延趋势,出现1株对临床现有抗生素全部耐药的菌株。AmpC酶在鲍曼不动杆菌的耐药机制中起重要作用,其携带率为67%。米诺环素和头孢哌酮/舒巴坦是目前本地区最为有效的抗菌药物。     

38株多重耐药鲍曼不动杆菌超广谱酶基因的研究

目的:了解辽宁医学院附属第一医院38株多重耐药鲍曼不动杆菌超广谱酶基因存在状况。方法:用微量稀释法测定临床分离的38株多重耐药鲍曼不动杆菌对14种抗生素的耐药性,采用多重聚合酶链反应(PCR)、基因测序技术对鲍曼不动杆菌进行超广谱酶基因测定。结果:38株多重耐药鲍曼不动杆菌对14种抗生素高度耐药。检出TEM基因阳性33株(86.84%)、PER-1基因阳性5株(13.16%)、SHV基因阳性1株(2.63%),未检出CTX、GES和VEB基因;其中5株TEM、PER-1基因均阳性,1株TEM、SHV基因均阳性。结论:我院分离的多重耐药鲍曼不动杆菌携带多种超广谱酶基因;应重视合理使用抗生素,减少多重耐药的产生。     

鲍曼不动杆菌质粒上氨基糖苷类耐药基因的研究

目的探讨鲍曼不动杆菌(Acinotobacter baumannii)对氨基糖苷类抗生素的耐药性及其耐药机制。方法对71株MDR A.baumanni用VITEK分析仪进行药敏分析,通过PCR和测序探索质粒上的相关耐药基因(aaaCl,aacC2,aacC3,aacA4和armA)。结果 71株A.baumanni对庆大霉素、妥布霉素和阿米卡星的耐药率分别为100%、73%和20%,其中20%的菌株对3种氨基糖苷类抗生素完全耐药,且aaaCl、aacA4和armA基因阳性率分别为100%、100%和84.5%。结论质粒上广泛携带aacCl、aacA4和armA基因是鲍曼不动杆菌对氨基糖苷类抗生素多重耐药的主要机制之一。     

鲍曼不动杆菌Ⅰ类整合子与多重耐药相关性研究

目的 了解临床分离鲍曼不动杆菌的耐药状况、Ⅰ类整合子的分布情况,探讨Ⅰ类整合子与多重耐药的关系.方法 检测20种临床常用抗菌药物对鲍曼不动杆菌临床分离株的最低抑菌浓度(MIC).PCR扩增Ⅰ类整合酶基因.对部分Ⅰ类整合酶阳性菌株进行耐药基因盒序列分析.结果 鲍曼不动杆菌呈现多重耐药,鲍曼不动杆菌对IMP和MRP耐药率分别为0.9%和1.8%,对CPZ/SB的耐药率为35.7%,对其它抗菌药物的耐药率均大于60%,多重耐药率为76.8%(86/112),但对COL和MIN均敏感.80.4%(90/112)的菌株检测出Ⅰ类整合子.Ⅰ类整合子阳性株对多种药物的耐药率均高于阴性株,且Ⅰ类整合子阳性株多重耐药率(90%)明显高于阴性株(22.7%)(P<0.01).Ⅰ类整合子基因盒序列分析显示,Ⅰ类整合子携带aacA4,catB8和aadA13种耐药基因.结论 Ⅰ类整合子在鲍曼不动杆菌中检出率很高并与其多重耐药性关系密切.     

多重耐药鲍曼不动杆菌临床分布及外排泵基因的检测

目的了解多重耐药鲍曼不动杆菌临床分布并检测其外排泵基因型。方法统计96株多重耐药鲍曼不动杆菌临床分布,用Kirby-Bauer法检测96株多重耐药鲍曼不动杆菌对15种抗菌药物的耐药情况,并用PCR扩增进行外排泵基因的检测。结果 96株多重耐药鲍曼不动杆菌主要分布在重症监护病房(54.2%)和呼吸内科(18.8%);对喹诺酮类、头孢菌素类、氨基糖苷类、四环素类抗菌药物的耐药率均在70%以上;分布在重症监护病房的52株多重耐药鲍曼不动杆菌中检测到ade B、ade R、ade S、ade J、ade E、abe M基因分别有18株(34.62%)、16株(30.77%)、18株(34.62%)、18株(34.62%)、0株、18株(34.62%);分布在呼吸内科的18株多重耐药鲍曼不动杆菌中检测到ade B、ade R、ade S、ade J、ade E、abe M基因分别有9株、8株、8株、8株、0株、8株。结论外排泵基因是分布在重症监护病房和呼吸内科的鲍曼不动杆菌发生多重耐药的重要因素。     

多重耐药鲍曼不动杆菌的β内酰胺酶基因型研究

目的了解临床分离的多重耐药鲍曼不动杆菌β内酰胺酶基因的存在情况。方法采用MicroScan40微生物全自动鉴定系统微量肉汤稀释法测定菌株的耐药性。采用聚合酶链反应(PCR)方法检测β-内酰胺酶基因型。结果 30株鲍曼不动杆菌除对阿米卡星和左氧氟沙星耐药率分别为23.3%和30.0%外,对其他抗菌药物均达80.0%以上。检出β-内酰胺酶基因26株(86.7%),OXA-23样阳性20株(66.7%),OXA-51样阳性18株(60.0%),AMPC酶阳性18株(60.0%),TEM阳性5株(16.6%),SHV阳性4株(13.3%),PER阳性3株(10%),14株(46.6%)同时携带2种以上基因,未检出IMP、VIM、CTX-M-9,DHA、OXA-24、OXA-58。结论我院多重耐药鲍曼不动杆菌携带多种β-内酰胺酶基因,而且同时携带3种以上β-内酰胺酶基因比率高。     

多重耐药鲍曼不动杆菌的主动外排泵基因对β-内酰胺类抗菌药物耐药性的影响

目的 探讨对β-内酰胺类抗菌药物耐药的多重耐药鲍曼不动杆菌主动外排泵基因在耐药机制中表达情况.方法 多重耐药鲍曼不动杆菌96株,分析其对9种β-内酰胺类抗菌药物的耐药性.以加入泵抑制剂羰基氰氯苯腙(CCCP)后最低抑菌浓度(MIC)降低至≤1/4为标准,筛选出34株外排泵表型阳性的鲍曼不动杆菌,用聚合酶联链反应(PCR)扩增法对其行外排泵蛋白基因adeB进行检测和分析.结果 多重耐药鲍曼不动杆菌对头孢噻肟、头孢他啶、头孢曲松、头孢吡肟、头孢哌酮/舒巴坦的耐药率分别为92.3％,86.6％,83.0％,70.8％,68.3％;对氨苄西林、哌拉西林、哌拉西林/他唑巴坦、氨苄西林/舒巴坦的耐药率分别为90.1％,77.5％,72.4％和67.3％.34株外排泵表型阳性的鲍曼不动杆菌检测到adeB基因有33株,检出阳性率97.06％.对33株鲍曼不动杆菌的外排泵基因adeB进行测序,经比对所测序列与Genebank中序列同源性为100.0％.结论 主动外排泵基因是多重耐药鲍曼不动杆菌对β-内酰胺类抗菌药物耐药的一个重要因素.     

耐碳青霉烯类抗生素鲍曼不动杆菌耐药机制的研究

目的：调查和研究南昌地区鲍曼不动杆菌对碳青霉烯类抗生素的耐药机制。方法：收集南昌大学第二附属医院2012年间临床分离的耐亚胺培南鲍曼不动杆菌（CRAB）非重复株。Vitek-32型全自动微生物分析仪进行菌株鉴定,K-B法进行药敏试验,三维试验检测AmpC酶,EDTA协同试验检测金属β-内酰胺酶,采用聚合酶链反应（PCR）扩增耐药基因,并对阳性产物进行双向测序分析,确定其基因型。结果：84株CRAB对临床常用的12种抗菌药物有9种耐药率〉90%,对头孢哌酮-舒巴坦的耐药率最低（44.0%）,所有菌株均为多重耐药株。三维试验在83株菌（98.8%）中检出AmpC酶,协同试验未检出产金属酶菌株。所有菌株均携带blaOXA-23、blaOXA-51及blaADC基因,1株携带blaOXA-58基因,未检测到blaOXA-24及金属酶基因（blaIMP、blaVIM-2、blaNDM-1及blaSIM-1）;外排泵编码基因adeB、调控基因adeS和adeR的检出率分别为98.8%（83/84）、81.0%（68/84）和67.9%（57/84）;所有菌株均检出外膜蛋白carO基因;69株（82.1%）检测出I类整合酶基因（intI1）,全部菌株携带插入序列ISAba1。结论：南昌地区CRAB耐药性及多重耐药性非常严重,鲍曼不动杆菌对碳青霉烯类抗生素耐药的主要机制为产OXA-23型碳青霉烯酶,AmpC酶、外排泵AdeABC、I类整合子和插入序列ISAba1,可能与CRAB的多重耐药性密切相关。     

鲍曼不动杆菌耐药表型与外排泵基因表达水平的研究

目的 探讨鲍曼不动杆菌临床分离株对常用抗菌药物的耐药性及与外排泵adeA基因表达水平之间的关系.方法 琼脂二倍稀释法检测鲍曼不动杆菌临床分离株对17种常用抗菌药物的最低抑菌浓度(MIC);PCR法扩增外排泵编码基因adeA:实时荧光定量RT-PCR(Real Time Fluorescent Quantitative RT-PCR)法检测adeA基因的mRNA表达水平.结果 药敏结果显示86株鲍曼不动杆菌对多粘菌素B全部敏感(100%),其次是美罗培南(73.2%)、亚胺培南(70.9%).耐药率最高的是庆大霉素(84.9%),其次为美罗西林(83.7%)和环丙沙星(79.1%).55株菌为多重耐药株(64%),其中5株(5/86)对多粘菌素B外的所有抗菌药物耐药(5.8%).adeA的检出阳性率为84.9%(73/86),Real Time RT-PCR结果显示多重耐药菌株adeA基因的mRNA相对表达量均高于敏感菌株,其中3株相对表达量为敏感菌株平均表达水平的30倍以上.结论 鲍曼不动杆菌临床分离株耐药情况严重,其主动外排系统adeA基因表达增强在多重耐药性形成中起重要作用.     

Integron gene cassettes in Acinetobacter spp. strains from South China

The epidemiology of emerging antibiotic resistance genes in Asia is inadequately defined and studies within the major pools of transmissible genes such as integron gene cassettes are important. One hundred and twenty-two non-repetitive Acinetobacter spp. isolates were obtained from inpatients of a major hospital in South China. Fifty-three of these isolates contained class 1 integrons, and there is evidence of horizontal gene transfer between unrelated clones. The common pool of gene cassettes was dominated by four cassette arrays: arr3-aacA4 (24 isolates of several unrelated strains); aacC1-orfP-orfQ-aadA1a (11 isolates, probably all the same strain); aacA4-catB8-aadA1 (2 isolates); and dfrVII (1 isolate). We developed a simple restriction fragment length polymorphism (RFLP)-based identification of these and other cassettes reported in China, using readily available enzymes, to facilitate further studies of this type.     

An outbreak of carbapenem-resistant Acinetobacter baumannii producing OXA-23 carbapenemase in western China

Twenty-two non-repetitive carbapenem-resistant Acinetobacter baumannii isolates were obtained from Intensive Care Unit patients. All of the isolates carried bla(OXA-23), bla(OXA-66), a novel cephalosporinase-encoding gene (bla(ADC-25)) and a class 1 integron with an aacC1-orfP-orfQ-aadA1a cassette array and had identical enterobacterial repetitive intergenic consensus (ERIC) profiles. ISAba1 was found upstream of bla(OXA-23), but was not associated with bla(OXA-66) or bla(ADC-25).     

Distribution of the multidrug efflux pump genes, adeABC, adeDE and adeIJK, and class 1 integron genes in multiple-antimicrobial-resistant clinical isolates of Acinetobacter baumannii-Acinetobacter calcoaceticus complex

Of 112 non-repetitive clinical isolates of Acinetobacter baumannii-Acinetobacter calcoaceticus complex, 80% were resistant to a variety of structurally unrelated antimicrobials although all isolates were susceptible to minocycline and polymyxin. Resistance to carbapenems occurred in 8% of the isolates. The presence of adeSR-adeABC, adeDE and adeIJK drug efflux system genes and class 1 integron genes (integrase gene int1) was assessed by polymerase chain reaction (PCR) in relation to the susceptibility of the isolates to 20 antimicrobials. The majority of isolates (75%) with high levels of multidrug resistance were positive for adeSR-adeABC and adeIJK as well as int1 and thus belong to A. baumannii (i.e. genomospecies 2). Positive adeE was only observed in adeSR-adeABC/adeIJK/int1-negative isolates (8%; likely belonging to Acinetobacter genomospecies 3) that were relatively susceptible to several agents, and adeE expression was undetectable. The results reveal a possible association between adeABC/adeIJK and int1 in multidrug-resistant isolates of A. baumannii. In addition, differential distribution of the resistance-nodulation-cell division (RND) genes can likely be used as indicators for differentiating Acinetobacter species.     

Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp

Carbapenem resistance in Acinetobacter baumannii is a growing public health concern and is most often mediated by OXA carbapenemases. We describe a novel multiplex polymerase chain reaction (PCR) assay able to detect and distinguish alleles encoding three subgroups of acquired OXA carbapenemases (OXA-23-like, OXA-24-like and OXA-58-like) that are scattered in Acinetobacter spp., and a fourth subgroup, OXA-51-like, which appears to be intrinsic to Acinetobacter baumannii. Isolates belonging to two prevalent UK A. baumannii 'OXA' clones (OXA-23 clones 1 and 2) had alleles encoding both an intrinsic OXA-51-like and an acquired OXA-23 enzyme, whereas isolates of the 'SE clone' had only an intrinsic bla(OXA-51-like) allele. Genes encoding OXA-58 were detected (with bla(OXA-51-like)) in a cluster of related isolates from a single hospital. This simple assay will assist in monitoring the mechanisms responsible for carbapenem resistance in Acinetobacter spp.     

3株耐碳青霉烯鲍曼不动杆菌临床株耐药机制的研究

目的 研究鲍曼不动杆菌（Acinetobacter baumannii）对碳青霉烯耐药的分子机制。方法 采用琼脂纸片扩散法（KB法）及微量肉汤法筛选出3株对碳青霉烯耐药的鲍曼不动杆菌临床株，PCR扩增blaox-23基因和测序、提取外膜蛋白及进行聚丙烯酰胺凝胶电泳（SDS-PAGE）。结果 3株耐碳青霉烯的鲍曼不动杆菌具有多重耐药性；耐药基因PCR扩增和测序证实3株耐药菌均有blaOX-23 基因；其中1株耐药菌AB173的外膜蛋白电泳条带在28．8ku处出现明显缺失，其余2株耐药菌在该条带处与敏感菌相比较无明显差异。结论 本次试验中对碳青霉烯耐药的鲍曼不动杆菌全部含有blaoxhm基因，其中一株耐药菌还出现了外膜蛋白孔道的缺失可能与其多重耐药性有关。     

多重耐药鲍曼不动杆菌16S rRNA甲基化酶基因研究

目的 调查临床分离多重耐药鲍曼不动杆菌中介导氨基糖苷类高水平耐药的16S rRNA甲基化酶基因armA、rmtA、rmtB的流行情况.方法 收集台州市中心医院、台州市立医院两家多重耐药鲍曼不动杆菌128抹,用K-B法测定18种抗菌药物;采用琼脂稀释法测定4种氨基糖苷类药物MIC值;PCR检测armA、rmtA、rmtB...     

70株鲍曼不动杆菌耐药性及β-内酰胺酶基因型检测

目的调查和研究南京地区70株鲍曼不动杆菌耐药性及β内酰胺酶基因型。方法用KB法测定临床分离的70株鲍曼不动杆菌对5种抗生素的耐药性,采用PCR法检测β-内酰胺酶耐药基因型。结果70株鲍曼不动杆菌对头孢哌酮／舒巴坦耐药率为7．1％,头孢吡肟和头孢他啶的耐药率分别为61．4％和64．3％,头孢唑啉和头孢呋肟的耐药率均在80．0％以上;有40株菌检测到TEM型β-内酰胺酶基因,且均对头孢吡肟等抗生素耐药,2株为GES型,1株是VEB型,未检出CARB、DHA和PER基因。结论南京地区鲍曼不动杆菌以TEM型为主,其对β-内酰胺类的高耐药率与TEM型基因有直接的关系。     

Characterisation and clonal dissemination of OXA-23-producing Acinetobacter baumannii in Tabriz, northwest Iran

The characteristics and molecular epidemiology of carbapenemase genes amongst 68 imipenem-resistant Acinetobacter baumannii isolated from Imam Reza Hospital (Tabriz, Iran) during a 17-month period were studied. All 68 isolates were typed using sequence group-based multiplex polymerase chain reaction (PCR) to compare the clonal relationship of isolates with known international clonal lineages. Repetitive sequence-based PCR was further performed with representative isolates of each clone. PCR and sequencing were performed to detect OXA-type carbapenemases and class 1, 2 and 3 integron genes as well as to confirm the presence of insertion sequence ISAba1 upstream of bla(OXA-23) and bla(OXA-51-like) genes. Sixty-four isolates (94%) belonged to international clone (IC) II, two isolates (3%) belonged to IC I and two isolates (3%) did not belong to known international clones. All isolates carried bla(OXA-51-like), bla(OXA-23) and class 1 integron genes. No other acquired bla(OXA) genes or class 2 or 3 integron genes were detected. Sequence analysis confirmed the presence of bla(OXA-23) as well as the bla(OXA-51-like) variants bla(OXA-66), bla(OXA-69) and bla(OXA-88). ISAba1 was present upstream of the bla(OXA-23) gene in all of the isolates. Clonal spread of OXA-23-producing A. baumannii emphasises the need for appropriate infection control measures to prevent further spread of these multidrug-resistant organisms.     

Worldwide dissemination of acquired carbapenem-hydrolysing class D β-lactamases in Acinetobacter spp. other than Acinetobacter baumannii

The aim of this study was to identify acquired OXA-type carbapenemases in Acinetobacter spp. other than Acinetobacter baumannii. From a total of 453 carbapenem-susceptible and -resistant Acinetobacter isolates collected worldwide, 23 were positive for bla_OXA genes by multiplex PCR. These isolates were identified as Acinetobacter pittii (n = 18), Acinetobacter nosocomialis (n = 2), Acinetobacter junii (n = 1) and Acinetobacter genomic species 14TU/13BJ (n = 2). The bla_OXA genes and associated insertion sequence (IS) elements were sequenced by primer walking. In 11 of these isolates, sequencing of the PCR products revealed that they were false-positive for bla_OXA. The remaining 12 isolates, originating from Europe, Asia, South America, North America and South Africa, harboured OXA-23 (n = 4), OXA-58 (n = 5), OXA-40-like (n = 1) and OXA-143-like (n = 1); one A. pittii isolate harboured both OXA-23 and OXA-58. IS elements were associated with bla_OXA in 10 isolates. OXA multiplex PCR showed a high degree of false-positive results (47.8%), indicating that detection of bla_OXA in non-baumannii Acinetobacter spp. should be confirmed using additional methods.