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.     

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.     

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。     

铜绿假单胞菌主动外排系统研究进展

铜绿假单胞菌(Pseudomonas aeruginosa，PA)是一种重要的院内感染条件致病菌，常常感染癌症病人、严重烧伤和AIDS患者等免疫力低下的病人，且由于它具有先天的和后天获得的多药耐药性(multidrug resistance．MDR)．临床治疗十分困难，一旦感染，死亡率很高．已经成为临床医生面临的治疗难题之一。近年来的研究发现，PA内存在着外排多种物质的的主动外排系统，在MDR中起着极其重要的作用。下面就对铜绿假单胞菌的主动外排系统研究进展进行综述。     

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.     

多重耐药铜绿假单胞菌MexAB-OprM、MexXY-OprM主动外排系统的耐药作用

目的 探讨MexAB-OprM、MexXY-OprM主动外排系统（外排泵）在多重耐药铜绿假单胞菌耐药机制中的作用.方法 选取25株外科监护室分离的多重耐药铜绿假单胞菌,用实时定量RTPCR测定结构基因mexA、mexX的mRNA表达水平来判断MexAB-OprM、MexXY-OprM外排泵表达情况;用PCR分别扩增其调控基因mexR、mexZ,并对其产物测序,用Blast软件在GenBank与已知序列比较,研究其过度表达的机理.结果 25株多重耐药铜绿假单胞菌中,14株高表达MexAB-OprM系统（56%）,3株高表达MexXY-OprM系统（12%）,这3株菌同时高表达2种外排泵.在8株mexA高表达的菌株中,5株菌发生mexR基因突变,出现氨基酸替代,1株mexR提前出现终止密码子,2株菌未发现mexR基因突变.2株mexX高表达的菌株均发生基因mexZ突变,出现氨基酸替代.结论 主动外排系统MexAB-OprM、MexXY-OprM在多重耐药铜绿假单胞菌中过度表达,是此菌多重耐药机制之一;外排泵MexAB-OprM、MexXY-OprM高表达分别与调控基因mexR、mexZ发生变异有关,同时存在其他调控机制.     

黄芩苷对氟喹诺酮类抗铜绿假单胞菌协同作用的初步报告

目的：研究黄芩苷对氟喹诺酮类药物抗铜绿假单胞菌的协同作用。方法：以二倍稀释法测定药物的体外抗菌活性,筛选耐药菌,然后测定黄芩苷对4种氟喹诺酮类药物（诺氟沙星、环丙沙星、氧氟沙星、左氧氟沙星）抗铜绿假单胞菌的协同作用;以CCCP筛选存在主动外排系统的耐药菌．测定黄芩苷对4种氟喹诺酮类药物抗该类耐药菌的协同作用。结果：临床分离铜绿假单胞菌耐药现象普遍．黄芩苷联合4种氟喹诺酮类药物对耐药铜绿假单饱菌有协同抗菌作用．MIC值下降1-2倍。同时存在主动外排系统的耐药菌也表现出抗菌增敏作用。结论：黄芩苷与氟喹诺酮类药物联合应用对耐药铜绿假单胞菌有协同抗菌,对存在主动外排系统的耐药菌也表现出抗菌增敏作用。     

铜绿假单胞菌随机扩增多态性DNA指纹法基因分型研究

建立了铜绿假单胞菌（ＰＡ）随机扩增多态性ＤＮＡ（ＲＡＰＤ）指纹图基因分型方法，并应用于流行病学相关或不相关的７５个ＰＡ菌株的基因分型。分型率为１００％。３２株新生儿暴发感染菌株，可分成４个血清型和５个ＲＡＰＤ谱型，其中２５株属于同一谱型。在８例患者的垂直追踪菌株中，除有１例其第１和第３个分离株的血清型和ＲＡＰＤ谱型均相同，但与第２个分离株的血清型和ＲＡＰＤ谱型不同外，其余７例前后菌株的谱型相同。１３株散发菌株可分成３个血清型和１０个ＲＡＰＤ谱型；１２株流行病学无关菌株的ＲＡＰＤ谱型均不相同。本研究结果表明，ＲＡＰＤ指纹图基因分型法具有分型率高、分辨力强、比较快速简便等优点，并且不需要已知核酸序列，是在分子水平上对微生物感染的病原学、发病机理及流行病学研究的较理想的方法。     

耐环丙沙星铜绿假单胞菌的耐药性分析

目的 了解本地区铜绿假单胞菌的耐药情况及超广谱β-内酰酶（ESBLS）的检出率，以指导临床合理用药。方法对90株临床分离菌用纸片扩散法（K-B法）进行药物敏感性试验；纸片扩散表型确证法进行ESBLS检侧。结果本地区铜绿假单胞菌对氨苄西林、萘啶酸、哌拉西林、头胞噻肟、头胞吡肟、阿米卡星、环丙沙星、左氧氟沙星、司帕沙星、加替沙星、氨曲南、头胞他啶、亚胺培南、头胞呋辛的耐药率分别是90．0％、83．3％、77．2％、29．4％、5％、8．9％、65．0％、60．0％、62．2％、50．0％、7．2％、18．3％、0、47．2％；ESBLS的检出率为24．4％。结论本地区铜绿假单胞菌耐药情况不容乐观且多重耐药菌株较多；氟喹诺酮药物之间交叉耐药明显；产ESBLS率较高。     

铜绿假单胞菌的GM-PFGE分型的研究

目的研究全基因组ＤＮＡ稀有位点限制性内切酶酶切脉冲电场电泳图谱（ＧＭ－ＰＦＧＥ）在铜绿假单胞菌基因分型中的应用,并与表型分型比较。方法对１个月内来自两个医院的病人及环境的２０株铜绿假单胞菌进行了ＧＭ－ＰＦＧＥ图谱分析,同时进行３０种生化反应的统计分型、２３种药物的敏感性分型、胞外脂多糖的血清抗原分型;并利用数值分类软件包进行相关性比较研究。结果临床致病铜绿假单胞菌的生化表型基本稳定,但其药物抗性的获得与丢失较为明显。血清型、生化性状及药物抗性之间无明显对应关系。当２菌株ＧＭ－ＰＦＧＥ图谱条带相似系数大于８０％时,为同一菌株的不同克隆亚型,当相似系数在２５％～７０％之间时,则为不同菌株。结论ＧＭ－ＰＦＧＥ分析可显示染色体结构的区域多型性,其重复性好,分辨率明显高于表型分型,结果可靠,必将成为铜绿假单胞菌或其它病原微生物分子流行病学研究的有力工具。     

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.     

某综合医院铜绿假单胞菌2005-2009年耐药性的变迁

目的了解某综合医院2005-2009年铜绿假单胞菌临床分离株的耐药性变迁,为合理应用抗菌药物提供依据。方法应用回顾性调查方法,对广东省汕头市某综合医院2005-2009年住院病人送检标本中分离的铜绿假单胞菌的药敏试验结果进行统计分析。结果 5年间共检出铜绿假单胞菌1809株,总的分离率为16.34%;标本分布以痰液来源为主,占84.9%;几年间铜绿假单胞菌对氨苄西林、复方新诺明、呋喃妥因、头孢唑啉的耐药率变化不大,对阿米卡星、庆大霉素、妥布霉素、亚胺培南、头孢他啶、头孢吡肟、环丙沙星、左旋氧氟沙星等抗菌药物的耐药率均在2007年达到高峰,在2008、2009年两年间呈连续下降趋势。从2009年的情况来看,耐药率最低的是妥布霉素,为28.97%（168/580）,其次为阿米卡星和左旋氧氟沙星,分别为32.07%（186/580）和33.97%（197/580）。结论铜绿假单胞菌对大多数抗菌药物的耐药率有所下降,但总体耐药情况还是比较严重。     

Effect of β-lactam antibiotics on the in vitro development of resistance in Pseudomonas aeruginosa

Objective   To investigate whether stepwise selection of resistance mutations may mirror the continued bacterial exposure to antibiotics that occurs in the clinical setting.
Methods   We examined the in vitro development of resistance to a number of commonly used antibiotics (cefepime, cefpirome, ceftazidime, cefotaxime, piperacillin and imipenem) in Pseudomonas aeruginosa , a significant nosocomial pathogen. Stepwise resistance was assessed by serial passage of colonies located nearest to the inhibition zone on antibiotic-containing gradient plates.
Results   The lowest frequencies of spontaneous resistance mutations were found with cefepime and imipenem; these drugs also resulted in the slowest appearance of resistance of spontaneous resistance mutations. In five wild-type P. aeruginosa strains, cefepime-selected isolates required a mean of 30 passages to reach resistance; resistance occurred more rapidly in strains selected with other cephalosporins. P. aeruginosa strains that produced β -lactamase or non-enzymatic resistance generally developed resistance more rapidly than wild-type strains. For most strains, resistance to all antibiotics except imipenem correlated with increased levels of β -lactamase activity. Cross-resistance of cephalosporin-selected resistant mutants to other cephalosporins was common. Cephalosporin-resistant strains retained susceptibility to imipenem and ciprofloxacin.
Conclusions   From our in vitro study, we can conclude that the rate of development of resistance of P. aeruginosa is lower with cefepime compared with other cephalosporines.     

Stimulation of innate immunity by susceptible and multidrug-resistant Pseudomonas aeruginosa: an in vitro and in vivo study

In attempt to investigate the stimulatory effect of Pseudomonas aeruginosa on innate immunity and to correlate it to its level of resistance to antimicrobials, 20 isolates were applied; 8 isolates were susceptible and 12 multidrug-resistant. Genetic diversity was defined by PFGE. Human monocytes of two healthy volunteers were in vitro stimulated by the isolates for the production of pro-inflammatory (TNF-alpha, IL-1beta, IL-6, IL-8 and IL-12) and anti-inflammatory cytokines (IL-10), of malondialdehyde and of procalcitonin. Cytokines were estimated by EIA, malondialdehyde by the thiobarbiturate assay and procalcitonin by an immunochemiluminometric assay. Survival of 48 Wistar rats was recorded after induction of sepsis by the intraperitoneal injection of three susceptible and three multidrug-resistant isolates. To test whether comparative effect of the latter isolates on survival correlates with any difference of monocyte-mediated release of pro-inflammatory mediators, monocytes of two rats were in vitro stimulated for the production of TNF-alpha and of malondialdehyde. In vitro stimulation of human monocytes by the susceptible isolates elicited elevated production of malondiadeheyde, of IL-1beta and of IL-6 compared to stimulation by multidrug-resistant isolates. Similar differences were found for TNF-alpha and IL-8, but they were not statistically significant. Production of IL-10 and IL-12 was not detected after stimulation with any isolate. Levels of procalcitonin were similar after induction with either susceptible or multidrug-resistant isolates. Mean survival of animals was 7.56, 21.80 and 55.20 h, respectively, after challenge by the susceptible isolates and 28.89, 61.8 and more than 120 h, respectively, after challenge by the multidrug-resistant isolates. Differences of survival were accompanied by greater rodent monocyte-release of TNF-alpha and malondialdehyde after stimulation by the susceptible isolates compared to multidrug-resistant ones. It is concluded that considerable differences are encountered on the stimulation of human monocytes by susceptible and resistant isolates of Pseudomonas aeruginosa. These results correlate with in vivo evidence and might influence decision on therapeutics.