鲍曼不动杆菌临床株的外排泵研究

目的 探讨鲍曼不动杆菌临床株外排泵AdeABC、AdeIJK、AdeFGH、AbeM、AbeS、CraA、MdtL的表达与耐药的关系.方法 收集多重耐药鲍曼不动杆菌临床株32株和敏感株10株,PCR扩增泵基因;选取主要克隆型的21株多重耐药株和10株敏感株,实时荧光定量RT-PCR方法检测泵基因adeB、adeJ、adeG、abeM、abeS、craA、mdtL的mRNA相对表达水平,PCR扩增泵调控基因adeRS、adeL并测序分析.结果 32株多重耐药鲍曼不动杆菌临床株中携带泵结构基因片段adeB100％、adeJ 100％、adeG 100％、abeM 96.88％、abeS 100％、craA 100％、mdtL 93.75％,10株敏感株均存在7种泵结构基因片段;主要克隆型的21株多重耐药株和10株敏感株adeB、abeM、mdtL的mRNA相对表达水平的差异有统计学意义( P＜0.001,P=0.001,P=0.013),选取多重耐药株AbR3和AbR11检测外排泵AdeABC调控基因adeRS序列出现氨基酸替代及缺失,而外排泵AdeFGH调控基因adeL序列无基因突变.结论 鲍曼不动杆菌临床株外排泵AdeABC、AbeM、MdtL的表达可能与耐药性有关.     

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.     

不同抗菌药物组合对多重耐药鲍曼不动杆菌的作用研究

目的 评价18组抗菌药物组合对临床分离的多重耐药鲍曼不动杆菌( MDRAB)的体外抗菌效应.方法 收集2009年10月-2010年5月首都医科大学附属北京友谊医院临床检验中心细菌室分离的非重复鲍曼不动杆菌,肉汤微量稀释法测定抗菌药物单药MIC,棋盘肉汤微量稀释法测定联合用药的部分抑菌浓度( FIC)值.根据FIC值判别药物组合的作用类型.对实验菌株,同一药物组合表现矛盾作用的,用PCR扩增其外排泵基因.结果 体外联合作用中利福平+多黏菌素B、亚胺培南+庆大霉素、头孢吡肟+左氧氟沙星协同作用比例大,分别达到68.1％、45.5％、40.9％,米诺环素+利福平、氨苄西林/舒巴坦+妥布霉素、头孢他啶+环丙沙星具有较高的相加作用,分别为81.8％、68.2％、68.2％.有些组合对实验菌株出现协同和拮抗的矛盾作用.选择协同作用的No.19和拮抗作用的No.21、No.26进行耐药基因扩增,基因型表现不同,其中No.19扩增adeS( -),No.21和No.26扩增adeS(+).结论 18种药物组合里利福平+多黏菌素B存在较高的协同作用,在严重MDRAB感染情况下可以考虑应用该组合.亚胺培南+庆大霉素、头孢吡肟+左氧氟沙星也具有较高的体外协同作用,但是在部分菌株的试验中存在拮抗作用,可能是由于菌株存在adeS基因,某些抗菌药物的应用会激活adeS,使外排泵表达或者过量表达,反而使进入细菌细胞内的药物被泵出,表现为拮抗作用.     

多重聚合酶链反应技术快速鉴定鲍曼不动杆菌

目的建立快速鉴定鲍曼不动杆菌菌株的方法。方法本研究建立多重PCR实验技术快速鉴定170株醋酸钙-鲍曼不动杆菌复合体以及对照组的其他菌属14株。结果138株菌的PCR产物扩增出2条条带,为鲍曼不动杆菌,另外32株只扩增出1条条带,为醋酸钙-鲍曼不动杆菌复合体的其他基因型,对照组的菌株没有扩增出条带。结论多重PCR技术的建立为快速鉴定鲍曼不动杆菌提供了一个快速而简便的方式。     

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。     

鲍曼不动杆菌临床分布及耐药性分析

目的 了解鲍曼不动杆菌的感染情况及其耐药性,为医院控制感染及临床合理应用抗生素提供科学依据。方法 回顾性分析2013年1月~2014年12月我院临床分离的154株鲍曼不动杆菌的药敏试验数据和各科分布情况。结果 在分离出的154株鲍曼不动杆菌中,重症监护病房（PICU）最多,为48株（31.17%）,其中以痰液分离最多,为116株（75.32%）,多重耐药菌分离率为7.14%,耐碳青霉烯类菌分离率为2.66%。结论 鲍曼不动杆菌极易对抗菌药产生耐药,应加强医院耐药菌株的监测,指导临床合理使用抗生素,以便采取有效的防范措施,最大限度减少鲍曼不动杆菌感染。     

鲍曼不动杆菌的分布与耐药性分析

目的了解鲍曼不动杆菌在医院内的分布状况与耐药谱,指导临床有效预防并合理用药。方法对中国中医科学院望京医院2009年至2011年三年间临床送检标本中,分离的254株鲍曼不动杆菌,采用纸片扩散法进行药敏试验,并使用WHONET 5.5对药敏试验数据进行分析统计。结果 254株鲍曼不动杆菌主要来源于痰标本,占90.59%。对哌拉西林和头孢曲松耐药率很高,分别为79.38%和89.33%,对头孢他啶、头孢吡肟、阿米卡星、妥布霉素、以及两种加β内酰胺酶抑制剂的复合抗生素的耐药率也都在51%～57%,多粘菌素B和头孢哌酮/舒巴坦对其具有较强的抗菌活性,耐药率分别为0.63%和17.88%。结论鲍曼不动杆菌是目前医院感染重要的条件致病菌,并且多重耐药率高,应引起广泛关注并严格按照临床用药方案进行抗生素使用,防止耐药性的进一步产生和院内传播。     

371株鲍曼不动杆菌的临床耐药状况

本文对临床分离出的鲍曼不动杆菌的耐药性进行回顾性分析,为临床经验性治疗首选抗生素提供依据。对我院2006年10月至2009年10月分离出的371株鲍曼不动杆菌,用VITEK32细菌鉴定及药敏分析系统进行细菌鉴定和药敏试验,结合K-B法作补充对照。在分离出的371株鲍曼不动杆菌中,以老年煤工尘肺患者及重症监护病人集中的科室比率较高,在呼吸道标本中的构成比高于其他种类的标本。该菌对亚胺培南、哌拉西林/他唑巴坦,氨苄西林/舒巴坦的耐药率相对较低,各为5.0%、19.5%、19.5%。对复方新诺明、妥布霉素、左氧氟沙星、庆大霉素、环丙沙星、头孢吡肟、头孢他啶的耐药率分别为32.7%、43.2%、44.6%、44.9%、47.3%、53.8%、56.5%。对头孢曲松、氨曲南、氨苄西林、呋喃妥因、头孢唑啉有较高的耐药性,耐药率均在90%以上。鲍曼不动杆菌对多种抗生素耐药现象严重,根据药敏试验合理应用抗生素十分重要。     

鲍曼不动杆菌临床分离株表型、基因型和耐药基因的对比研究

目的 对比研究鲍曼不动杆菌临床分离株基因型和编码耐药基因的差异,并分析其与临床多重耐药性的关系.方法 随机收集中南大学湘雅二医院2008年9月至2009年9月分离的77株鲍曼不动杆菌,采用WHO推荐的K-B法对鲍曼不动杆菌进行临床常见15种抗生素药物敏感试验,并对药敏谱进行分析.用随机扩增多态性DNA法(RAPD)技术进行基因分型.并利用PCR对β-内酰胺酶基因TEM-1、IMP、OXA-23、OXA-24、AmpC和氨基糖苷类修饰酶基因aac(3)-Ⅰ、aac(6')-Ⅰ、ant(3")-Ⅰ和16S rRNA甲基化基因armA、rmtA、rmtB进行扩增及序列分析.对比分析鲍曼不动杆菌耐药基因的携带情况,以及与基因型和耐药性的关系.结果 77株鲍曼不动杆菌中敏感菌株有31株,对5种或5种以上抗生素耐药的多重耐药菌株46株,内含全耐药菌株10株.RAPD技术将其分为17型,为A-G型,多重耐药株中E型为优势克隆株(17株),在重症监护病房(ICU)中流行最广,占47.1%(8/17).敏感株中各型散在分布.PCR扩增结果显示,多重耐药株和敏感株携带TEM-1、IMP、OXA-23、OXA-24、AmpC、aac(3)-Ⅰ、aac(6')-Ⅰ、ant(3")-Ⅰ和armA耐药基因的比率分别为95.7%、39.1%、84.8%、54.3%、87.0%、89.1%、84.8%、45.7%、63.0%和58.1%、9.7%、32.3%、48.4%、48.4%、29.0%、45.2%、12.9%、9.7%,未发现rmtA和rmtB基因阳性菌株.经x2检验,除OXA-24外,其余各耐药基因携带率比较差异有统计学意义(P＜0.05).药敏分析提示携带以上耐药基因的鲍曼不动杆菌菌株的耐药率明显高于未携带该基因的菌株,其中对阿米卡星和庆大霉素耐药的菌株,其氨基糖苷类酶基因均阳性(34.8%),含所测的所有β-内酰胺酶基因的菌株均为全耐药株.结论 与临床分离的敏感鲍曼不动杆菌相比,多重耐药株耐药谱广,耐药率高,其携带β-内酰胺酶基因和氨基糖苷类酶基因种类多,分离率高,且同一克隆的多重耐药株可在病室内和病室间传播.     

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。     

Efflux pump inhibitory activity of biologically synthesized silver nanoparticles against multidrug-resistant Acinetobacter baumannii clinical isolates

This study was carried out to investigate the possible efflux pump inhibitory activity of biologically synthesized silver nanoparticles (AgNPs) against multidrug-resistant (MDR) Acinetobacter baumannii isolates. In this study, the physicochemical characteristics of synthesized AgNPs were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectrophotometer (FTIR) methods. Subsequently, MDR A. baumannii isolates were recovered from clinical samples and the phenotypic cartwheel efflux assay and polymerase chain reaction (PCR) were used to elucidate the possible presence of efflux pump in MDR strains. After treatment of MDR strains with sub-minimum inhibitory concentration (MIC) concentration of AgNPs, the expression level of efflux pump genes was evaluated using a quantitative real-time PCR technique. The synthesized AgNPs had a spherical nanostructure, with mean size 38.89 nm, according to SEM and TEM data. XRD and FTIR results confirmed the synthesis of AgNPs. The results of PCR revealed that among 50 strains, 12 A. baumannii strains had efflux pump genes and the expression level of AdeA, AdeC, AdeS, AdeR, AdeI, AdeJ, and AdeK efflux pump genes was downregulated significantly after the treatment with AgNPs. In addition, the inhibitory effect of AgNPs on efflux pumps can be detected when the MIC of ethidium bromide (EtBr) with AgNPs is lower than that of EtBr alone. According to the results, the biologically synthesized AgNPs exhibit efflux pumps inhibitory activity, which may be one of the possible mechanisms of their antibacterial activity against MDR A. baumannii strains.     

Gene expression analysis of the SdeAB multidrug efflux pump in antibiotic-resistant clinical isolates of Serratia marcescens

Purpose: Many isolates of Serratia marcescens, a well-known opportunistic pathogen, can be multidrug resistant. Fluoroquinolones are among the most important groups of antibiotics used for treatment of these organisms. However, fluoroquinolone resistance among S. marcescens isolates is fast increasing. Drug extrusion through efflux pumps like SdeAB/ HasF is one of the major mechanisms of resistance to fluoroquinolones. This study was carried out to analyze, through gene expression analysis of sdeB, the relative contribution of this mechanism toward fluoroquinolone resistance in clinical isolates of Serratia. Materials and Methods: Total RNA from 45 clinical isolates of S. marcescens was isolated. Quantitative real-time RT PCR was performed on the extracted RNA to study the gene expression of sdeB and was normalized to the sdeB expression in the standard strain of S. marcescens. Results: Of the 45 isolates analyzed, sdeB expression was found to be elevated in 20 isolates (44%). Of these 20 isolates, eight (40%) were fully resistant to at least one of the fluoroquinolones studied. Conversely, of the 20 isolates that over-expressed sdeB, 12 (60%) were fully sensitive to all fluoroquinolones tested. Conclusions: Drug efflux pumps are an important means of fluoroquinolone resistance among clinically important species ofSerratia. The expression of these pumps can be up-regulated in the presence of antibiotics and have the potential for changing the phenotype from sensitive to resistant, thus contributing to therapeutic failures.     

Integron-mediated antibiotic multiresistance in Acinetobacter baumannii clinical isolates from Spain

Objective  To determine whether non-epidemiologically related, antibiotic-resistant isolates of Acinetobacter baumannii from different geographical origins posses common type 1 integrons.
Methods  The epidemiologic relationships between seven A. baumannii strains recovered from different Spanish hospitals were established by pulsed-field gel electrophoresis, the presence of integrons being determined by PCR and DNA sequencing.
Results  Integron analysis showed the presence of four different integrons, containing six different known genes ( aacC1, aacA4 , aadA1 , aadB , oxa21 and oxa37 ) plus an ORF. It was found that the same integron was present in different unrelated strains and that related strains could have different integrons.
Conclusion  These results show the potential risk of integron dissemination among different strains of A. baumannii .     

Multiple carbapenem hydrolyzing genes in clinical isolates of Acinetobacter baumannii

Purpose: Carbapenem resistance in Acinetobacter baumannii has become highly rampant, which has been ascribed to the presence of multiple carbapenemases. The objective of the present study was to prospectively investigate the presence of multiple carbapenemase encoding genes in clinical isolates of A. baumannii. Materials and Methods: A total of 30 imipenem resistant, consecutive non-repeat clinical isolates A. baumannii from a Tertiary Care Centre of Delhi were subjected to antimicrobial susceptibility testing (AST), screening for carbapenemase production by modified Hodge test (MHT) and determination of minimum inhibitory concentration for imipenem by E-Test®. These were subjected to Real time PCR for bla_{IMP-1 and 2}, bla_{VIM-1 and 2}, bla_{OXA23, 24, 51 and 58} using SYBR green-I. These were grouped together on the basis of their genotype as each isolate harboured multiple carbapenemases and correlated with their AST profile. Detection of the novel carbapenemase bla_NDM-1 was performed by real time PCR using TaqMan probes on 14 isolates. Results: Colistin appeared to be the most effective drug in vitro, followed by tetracycline and beta lactam/beta lactamase inhibitor combinations. All, but one isolate were positive for the MHT. All 30 isolates were positive for bla_OXA-51 like gene as well as bla_IMP-1 and bla_VIM-1 genes. bla_{OXA 24 and 58} were not detected in any of the isolates. bla_IMP-2, bla_VIM-2, bla_OXA-23 were present in 15, 6 and 14 isolates respectively. Grouping based on the genotypic profile did not correlate with susceptibility pattern. Nine among the 14 isolates also harboured the novel bla_NDM-1 gene. Conclusions: This is the first study from North India, which comprehensively detected the presence of multiple carbapenemases as well the bla_NDM-1 gene. The presence of the novel gene bla_NDM-1indicated ability of A. baumannii to acquire new carbapenemase genes despite the existence of multiple carbapenemase genes. The present study confirmed the presence of multiple genetic mechanisms for carbapenemases production among the clinical isolates of A. baumannii in north India.     

Hidden VIM-1 metallo-beta-lactamase phenotypes among Acinetobacter baumannii clinical isolates

A total of 87 Acinetobacter baumannii nonrepetitive consecutive clinical isolates were tested for the presence of metallo-β-lactamases (MBLs). Results of phenotypic assays (MBL Etest, imipenem/imipenem-EDTA combined-disk test, and imipenem/EDTA double-disk synergy test) were negative in all cases, but molecular testing revealed the presence of two bla_VIM-1-carrying isolates. One isolate had bla_VIM-1 preceded by a weak P1 promoter, and both had inactivated P2 promoters and reduced bla_VIM-1 expression, partially justifying the results revealing hidden MBL phenotypes.     

Effect of iron on expression of efflux pump (adeABC) and quorum sensing (luxI,luxR) genes in clinical isolates of Acinetobacter baumannii

Resistance‐nodulation‐division efflux system (RND) adeABC contributes to intrinsic resistance to various drug classes in Acinetobacter baumannii. Similarly, quorum sensing (QS) plays an important role in the biofilm formation and pathogenicity of this bacterium. The aims of this study were to evaluate the influence of iron limitation on the expression of efflux pump (adeABC) genes and QS (luxI, luxR) system by relative quantitative real‐time polymerase chain reaction (qRT‐PCR). In addition, DNA sequence and phylogenetic relatedness of biofilm‐associated protein (Bap) gene was also investigated. Sixty‐five multidrug‐resistant isolates of A. baumannii were recovered from ICU patients of three hospitals in Kerman, Iran. The isolates were highly resistant to at least 11 antibiotics (MIC ≥64 μg/mL); however, 87% and 89% were susceptible to colistin and tigecycline, respectively (MIC 0.05 μg/mL) (p ≤ 0.05). We detected the presence of RND efflux pump, QS, and bap genes with the frequencies of 92% (adeA), 61.5% (adeB), 84.6% (adeC), 80% (luxI), 61% (luxR), and 66% (bap), respectively. qRT‐PCR analysis showed that in some isolates, expression of both adeABC and luxI/R was increased more than fourfold in the presence of low iron (20 μm ), suggesting the additional regulatory role of iron on both efflux pump and QS system. Alignment and phylogenetic analysis on the strong biofilm forming isolates confirmed that the fragments amplified were indeed part of bap gene and deduced sequence was similar to A. baumannii K9B410.     

Epidemiological characterization and distribution of carbapenem-resistant Acinetobacter baumannii clinical isolates in Italy

This study was aimed at tracing the molecular characteristics of carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates in Italy with both pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Two hundred and two CRAB isolates were collected during 2004–2009, in two different surveillance periods, from 22 Italian hospitals that were representative for both distribution and infection. PFGE was performed, and the MLST scheme used was based on the gene sequence as published on the MLST Pasteur website http://www.pasteur.fr/mlst. Representatives of the major European clones I (RUH 875) and II (RUH 134) were used as controls. The two groups of isolates were characterized for their carbapenem resistance genes: 154 of 202 carried bla_OXA-58 alone, 21 of 202 also carried bla_OXA-23, and 27 of 202 carried bla_OXA-23 alone. No isolates were positive for bla_OXA-24. Genotype analysis of all isolates identified four distinct patterns by PFGE, which correlated with four distinct sequence types (STs) by MLST. The distribution of these four clusters in Italy confirmed the propensity of A. baumannii for nosocomial cross-transmission in a vast geographical area. We observed that clones A and B had similarities with European clone II and I respectively. By MLST, clone A was ST2, like European clone II, and clone B was ST1, like European clone I. PFGE and MLST showed the same discriminatory power and reproducibility. In addition, the two methods were concordant in defining CRAB Italian clones and in correlating them with the two pan-European clones.