湖南省铜绿假单胞菌四种外排泵在多重耐药菌株中表达的分布及作用

目的 探讨湖南省多重耐药(multi-drug resistance,MDR)铜绿假单胞菌(Pseudomonasaeruginosa,Pa)中4种外排泵表达的分布和作用,以及相关调控基因的突变情况.方法 收集2008年湖南省临床首次分离非重复多重耐药Pa株40株,以外排泵抑制剂苯丙氨酸-精氨酸-β萘酰胺及4种药敏纸片进行外排泵表型初步筛选,PCR扩增膜融合蛋白基因及相关外排泵调控基因并测序;以18组非多重耐药菌株为对照研究分析Pa外排泵基因表达在多重耐药中的作用.结果 表型筛选MDR组菌株MexAB-OprM、MexCD-OprJ、MexEF-oprN、MexXY-OprM阳性率分别为45.0%(18/40)、30.0%(12/40)、42.5%(17/40)、12.5%(5/40);RT-PCR显示mexA、mexC、mexE和mexX表达阳性率分别为100%(58/58)、22.5%(9/40),45.0%(18/40)和22.5%(9/40).Real-time PCR半定量分析菌株超表达mexA和mexX阳性率分别为55.0%(22/40)和22.5%(9/40).非多重耐药组中,mexA与mexX real-time PCR超表达阳性率均为0(0/18).mexC和mexE RT-PCR表达阳性率分别为11.1%(2/18)、38.9%(7/18).两组相比,mexA与mexX超表达差异有统计学意义(P<0.001,P=0.045).随机挑选超表达mexA菌株Pa20出现mexR的164GTG→GAG、126Val→Gh替代;超表达mexX菌株Pa34出现mexZ的164GCG→GAG、55Ala→Glu.结论 湖南省多重耐药Pa株大多存在主动外排的耐药机制,其耐药与外排基因Mex-AB-OprM和MexXY-OprM超表达有关.主动外排泵MexAB-OprM和MexXY-OprM超表达大多存在其调控基因突变.     

铜绿假单胞菌临床分离株主动外排泵的检测

目的了解铜绿假单胞菌临床分离株的耐药特点及其外排泵存在的状况。方法采用K-B法检测铜绿假单胞菌临床分离株对9种常用抗生素（庆大霉素、氨曲南、头孢他啶、亚胺培南、阿米卡星、哌拉西林/他唑巴坦、环丙沙星、左氧氟沙星、多粘菌素B）的耐药情况;通过外排泵抑制剂氰氯苯腙（CCCP）对四种药物（羧苄青霉素、红霉素、亚胺培南、庆大霉素）的琼脂稀释法抑制试验分别检测铜绿假单胞菌的四种外排泵（MexAB-OprM、MexCD-OprJ、MexEF-OprN、MexXY-OprM）的表型;应用PCR检测MexAB-OprM外排泵调节基因mexR的存在情况。结果铜绿假单胞菌对9种常用抗生素的耐药率依次为：庆大霉素36.67%（22/60）,氨曲南33.33%（20/60）,头孢他啶31.67%（19/60）,亚胺培南28.33%（17/60）,阿米卡星26.67%（16/60）,哌拉西林/他唑巴坦23.33%（14/60）,环丙沙星21.67%（13/60）,左氧氟沙星11.67%（7/60）,多粘菌素B为0。外排泵表型阳性株依次为：MexAB-OprM35株,阳性率58.33%（35/60）;MexXY-OprM13株,阳性率21.67%（13/60）;MexCD-OprJ11株,阳性率18.33%（11/60）;MexEF-OprN10株,阳性率16.67%（10/60）。35株MexAB-OprM外排泵表型阳性株中有28株扩增出mexR基因片段,检出率为80.0%（28/35）。结论铜绿假单胞菌临床分离株对庆大霉素耐药率最高,对多粘菌素B耐药率最低;MexAB-OprM外排泵的存在是铜绿假单胞菌临床分离株的重要耐药机制。     

铜绿假单胞菌耐药性在生物被膜形成过程中的变化

目的 研究铜绿假单胞菌遗传性耐药(gyrA基因突变和MexAB-OprM主动外排)在生物被膜形成过程中对药物抗性的作用。方法 通过PCR技术鉴定gyrA基因突变株和MexB-OprM主动外排株，利用三亲杂交的方法将绿色荧光蛋白(GFP)基因转到铜绿假单胞菌药物敏感株中，测定gyrA基因突变株、MexAB-OprM主动外排株以及带有绿色荧光蛋白的铜绿假单胞菌在特氟隆上产生生物被膜耐药的规律。结果 筛选出gyrA基因突变株和MexAB-OprM主动外排株。通过三亲杂交成功构建了带有pGFPuv质粒的铜绿假单胞菌。将突变株和敏感株培养在最低杀菌浓度(MBC)的环丙沙星溶液中形成生物被膜使菌株的存活率均在50％以上。结论 比较遗传性耐药和生物被膜耐药的作用表明gyrA基因突变和主动外排在生物被膜耐药形成前期发挥主要作用，当生物被膜完全形成之后，铜绿假单胞菌的耐药性有可能是由生物被膜介导的。     

鲍曼不动杆菌临床株多重药物外排泵AdeABC的研究

目的 研究鲍曼不动杆菌(Acinetobacter baumannii)临床株外排泵AdeABC的表达与耐药的关系及表达调控.方法 微量肉汤稀释法检测鲍曼不动杆菌临床株对抗菌药物的敏感性及泵抑制剂作用,RT-PCR检测泵基因adeB的mRNA表达水平,PCR扩增泵调控基因adeRS并测序分析.结果 30株多重耐药的鲍曼不动杆菌临床株及5株敏感株均存在泵结构基因片段adeB和调控基因adeRS,随机选取15株多重耐药株中均检测到adeB的mRNA表达,而在5株敏感株中无表达.测定2株多重耐药株调控基因adeRS序列均出现基因突变,发生氨基酸替代及缺失.结论 鲍曼不动杆菌临床株外排泵AdeABC的表达可能与耐药性有关,在多重耐药株中存在着调控基因adeRS的基因序列变化.     

siRNA干扰铜绿假单胞菌MexA-MexB-OprM外排泵mexB基因表达的研究

目的 探讨RNA干扰技术对铜绿假单胞菌MexA-MexB-OprM外排泵mexB基因表达及有关功能的影响.方法 设计并合成4条小分子干扰RNA(small interfering RNA,siRNA)(siRNA1、siRNA2、siRNA3和siRNA4)序列,构建干扰RNA( short hair pin RNA,shRNA)表达载体,将带有siRNA质粒电转入PAO1和临床耐药株PAO3.应用E-test方法检测PAO1和PAO3抗生素MIC的变化.用半定量RT-PCR和定量PCR方法检测mexB的表达变化.结果 经酶切鉴定siRNA表达载体构建成功,并明显提高PAO和临床耐药株PAO3对抗生素的敏感性.RT-PCR结果显示干扰后的PAO1和临床耐药株PAO3 mexB基因表达明显下降(P＜0.05).结论 siRNA成功干扰MexA-MexB-OprM外排泵mexB基因的表达,提高铜绿假单胞菌对抗生素的敏感性,为临床上治疗铜绿假单胞菌感染,降低其耐药性提供了新的思路.     

外排泵抑制剂对泛耐药铜绿假单胞菌作用的体内研究

铜绿假单胞菌(Psedumonas aeruginosa,PA)是主要的医院内感染致病菌,对抗假单胞菌作用的所有药物全部耐药的即为泛耐药PA[1](pandrug-re-sistance,PDR).主动外排系统的过度表达是导致PA耐药的重要机制.Henrichfreise等[2 ]报道多耐药的22株PA中,有82％的菌株高表达MexXYOprM.衣美英等[3 ]发现主动外排系统在多耐药PA中表达超过60％.     

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.     

应用siRNA干扰技术沉默铜绿假单胞菌外排泵基因mexB的蛋白表达

目的 观察siRNA干扰质粒转入铜绿假单胞菌后,MexB蛋白表达量的变化.方法 针对mexB基因设计合成特异性siRNA分子,与pGPU6/GFP/Neo载体连接,构建pGPU6/GFP/NeosiRNA重组质粒.构建重组表达质粒pET22b+/mexB,并转化大肠杆菌BL21( DE3) plysS,诱导表达MexB蛋白,蛋白纯化后免疫家兔制备多克隆抗体.siRNA质粒分别电转化铜绿假单胞菌野生株、临床耐药株及mexB基因高表达株,运用Western blot观察转化8、12、24h后MexB蛋白表达量的变化.结果 成功构建pGPU6/GFP/Neo-siRNA重组质粒.成功表达铜绿假单胞菌外排泵蛋白MexB,并制备多克隆兔抗.siRNA质粒对铜绿假单胞菌野生株、临床耐药株及mexB基因高表达株的mexB基因均具有良好的沉默效果,而且沉默效果存在时间差异性.结论 siRNA质粒转化3株铜绿假单胞菌后,在8h及12 h时均可观察到MexB蛋白表达量明显减少,而在24 h时MexB蛋白表达量无改变.     

志贺菌主动外排泵调控基因marOR突变与泵表达水平的关系

目的 了解临床分离志贺菌调控基因marOR的突变情况,并探讨其与主动外排泵表达水平的关系.方法 对100株临床分离志贺菌和本实验室筛选的敏感菌及标准株的marOR基因进行聚合酶链反应(PCR),对扩增产物进行限制性内切酶酶切与单链构象多态性(SSCP)分析,对marOR基因突变株进行测序,利用RT-PCR测定其泵基因acrA、acrB和tolC的表达水平,并对其进行四环素、氯霉素、氨苄青霉素、庆大霉素、诺氟沙星、复方新诺明等6种药物的药敏试验.结果 100株临床分离志贺菌、敏感株及标准株均扩增出marOR基因,未发现该基因缺失株;SSCP分析发现marOR基因的突变率为23%;随机选择11株marOR基因突变株和1株敏感株及标准株进行测序,其中有9株存在4个碱基的缺失和3个位点的碱基突变,RT-PeR分析表明11株marOR基因突变株的acrAB-tolC主动外排泵表达水平高于敏感株和标准株,且该11株均为多蕈耐药株.结论 在该实验中志贺菌marOR基因突变率较高,其突变株的acrAB-tolC主动外排泵基因高表达,可能增加耐多药性.     

汕头地区耐亚胺培南铜绿假单胞菌耐药分析

目的：了解汕头地区对亚胺培南耐药的铜绿假单胞菌（PA）的耐药情况及耐药机制。方法：收集临床分离耐亚胺培南的PA共141株，双纸片协同实验检测金属酶表型，PCR法检测外膜孔蛋白OprD2和金属β内酰胺酶（IMP、VIM、SPM）基因。结果：耐亚胺培南的铜绿假单胞菌均为多重耐药茵，对头孢哌酮／舒巴坦的耐药率较低，未发现产金属酶菌株，仅22株菌株扩增出OprD2基因。结论：头孢哌酮／舒巴坦可作为本地区临床治疗耐亚胺培南铜绿假单胞茵所致感染的首选经验用药，OprDa表达减少或不表达可能是临床分离铜绿假单胞茵对亚胺培南耐药的主要机制。     

Evaluation of fluoroquinolone resistance mechanisms in Pseudomonas aeruginosa multidrug resistance clinical isolates

Efflux transporters have a considerable role in the multidrug resistance (MDR) of Pseudomonas aeruginosa, an important nosocomial pathogen. In this study, 45 P. aeruginosa clinical strains, with an MDR phenotype, have been isolated in a hospital of Northern Italy and characterized to identify the mechanisms responsible for their fluoroquinolone (FQ) resistance. These isolates were analyzed for clonal similarity, mutations in genes encoding the FQ targets, overexpression of specific Resistance Nodulation-cell Division efflux pumps, and search for mutations in their regulatory genes. The achieved results suggested that the mutations in genes encoding ciprofloxacin targets represented the main mechanism of FQ resistance of these strains; 97.8% of these isolates showed mutations in gyrA, 28.9% in gyrB, 88.9% in parC, and 6.7% in parE. Another mechanism of resistance was overexpression of the efflux pumps in some representative strains. In particular, overexpression of MexXY-OprM drug transporter was found in five isolates, whereas overexpression of MexCD-OprJ was detected in two isolates; surprisingly, in one of these last two isolates, also overexpression of MexAB-OprM pump was identified.     

Low-level resistance to fluoroquinolones conferred by efflux overproduction in Pseudomonas aeruginosa: clinical significance and routine detection

The aim of this study was (i) to assess the impact of stable overproduction of efflux systems MexAB-OprM and MexXY-OprM on the bacteriostatic activities of fluoroquinolones in clinical Pseudomonas aeruginosa strains and (ii) to find a convenient test for screening isolates with a low level resistance to fluoroquinolones. The minimal inhibitory concentrations (MICs) of ciprofloxacin and levofloxacin were determined for clinical isolates of P. aeruginosa overexpressing MexAB-OprM or MexXY-OprM. Efflux pumps derepression was associated with a modest two- to fourfold increase in resistance to the tested fluoroquinolones. Clinical significance of low level resistance conferred by the efflux mechanism was evaluated with a Monte Carlo simulation with various fluoroquinolone regimens. With this model, low levels of resistance to ciprofloxacin (MIC > or =0.25 mg/L) or levofloxacin (MIC > or =1 mg/L) such as those due to overproduced MexAB-OprM or MexXY-OprM were predicted to result in poor clinical outcomes. Altogether these data strongly suggest that when derepressed MexAB-OprM or MexXY-OprM provides P. aeruginosa with a resistance that may be sufficient to impair the efficacy of single therapy with highly potent fluoroquinolones such as ciprofloxacin and levofloxacin. Routine detection of clinical strains that displayed low-level resistance to fluoroquinolones with a Mueller Hinton agar containing 0.20 mg/L of ciprofloxacin will help clinician in his therapeutical choice.     

Rapid identification of mutations in a multidrug efflux pump in Pseudomonas aeruginosa

The gene mexR regulates negatively the expression of the MexA-MexB-OprM efflux pump in Pseudomonas aeruginosa, and mutations in mexR cause a multiple antibiotic resistance phenotype. Five hundred and forty resistant clones of P. aeruginosa PAO503 were isolated after selection for resistance to chloramphenicol or tetracycline. All isolates showed similar phenotypes and were resistant to tetracycline, chloramphenicol and norfloxacin. Nineteen randomly selected isolates were analyzed. Since mutational analysis by direct sequencing of all regions of interest in several strains is time-consuming and expensive, a screening method, Non-Isotopic RNase Cleavage Assay (NIRCA), was applied to identify mutant genes so that they could be targeted for DNA sequencing. NIRCA is a simple but rapid method for mutational analysis and can be performed in 3-4 h. Results of NIRCA analysis were compared with DNA sequencing. Both NIRCA and DNA sequencing analysis showed mexR gene mutations in 11 of 19 isolates but no alterations in 8 strains. An immunoblot assay showed overexpression of OprN, a component of another multidrug efflux pump, MexE-MexF-OprN, in those eight isolates. Nucleotide sequencing of quinolone resistance-determining regions of DNA gyrase (gyrA) or topoisomerase IV (parC) showed no alterations in any of the 19 mutants. The data indicate that two efflux pump systems, MexA-MexB-OprM and MexE-MexF-OprN, were involved in multidrug resistance including quinolones and that NIRCA is a sensitive method for screening mutations.     

Mechanisms of resistance in clinical isolates of Pseudomonas aeruginosa less susceptible to cefepime than to ceftazidime

The MIC of cefepime determined with the MicroScan WalkAway system was ≥2 times higher than that of ceftazidime for 105 clinical isolates of Pseudomonas aeruginosa. This phenotype was confirmed by reference microdilution in 68 (64.8%) isolates, corresponding to 48 different rep-PCR patterns. The PSE-1 blactamase was identified in only 13.2% isolates, while oxacillinases were not identified in any of the 68 isolates. The level of expression of mexB, mexD and mexY was determined by real-time RT-PCR in eight clinical isolates representative of the different clones and patterns of susceptibility to cefepime and ceftazidime and in strain PAO1. All clinical strains overexpressed the mexY gene (18.3- to 152.7-fold in comparison with PAO1), although there was not a linear relationship between MIC of cefepime and level of mexY expression. Five of these strains contained mutations in the regulatory gene mexZ. mexD and mexB were also overexpressed in three and two isolates, respectively. Different mutations were observed in the regulatory genes nalD, mexR, nfxB and nalC. In conclusion, we have documented in our institution a polyclonal spread of P. aeruginosa with higher MICs of cefepime than of ceftazidime, related to overexpression of Mex-XY-OprM, coincident in some isolates with the production of PSE-1, Mex-CD-OprJ or MexAB-OprM.     

Expression of the MexXY efflux pump in amikacin-resistant isolates of Pseudomonas aeruginosa

The MexZ-MexX-MexY multidrug efflux system in Pseudomonas aeruginosa was studied to determine its contribution to aminoglycoside resistance. Amikacin-resistant (AR) mutants were generated from P. aeruginosa strain PAO1, and clinical isolates of P. aeruginosa were collected from cystic fibrosis patients. The regulatory gene mexZ and the intergenic region (mexOZ) between mexZ and mexX were investigated for mutation by PCR and DNA sequence analysis. The results showed that 14 of 15 AR clinical isolates and one of ten laboratory mutants had at least one mutation in mexZ and/or mexOZ. To study the effect of mexZ and mexOZ mutations, the production of MexY mRNA was investigated quantitatively by real-time PCR. Seven of ten AR mutants (MIC 4-8 mg/L) produced 8-21-fold more MexY mRNA than PAO1. These isolates were sensitive to fluoroquinolones, carbapenems and ceftazidime. One AR mutant (MIC 64 mg/L) that produced > 200-fold more MexY mRNA than PAO1 was also resistant to fluoroquinolones, carbapenems and ceftazidime. Thirteen of 15 AR clinical isolates produced 3.4-727-fold more MexY mRNA. No evidence was found for the aminoglycoside-modifying enzymes 6'-N-acetyltransferase type Ib, 4'-O-nucleotidyltransferase type IIb or aminoglycoside 3'-phosphotransferase IIps in these strains. Nine AR mutants overproduced MexY without mutations in mexZ or mexOZ, suggesting that MexXY efflux is also regulated by gene(s) other than mexZ.