介导对氟喹诺酮类药物耐药的金黄色葡萄球菌norA基因的研究

目的研究导致对氟喹诺酮类药物耐药的金葡菌norA基因的介导机制。方法 K-B纸片扩散法测定细菌敏感性(Kirby-Bauer法),研究两种FQNS在菌体内的蓄积量,通过Real-time PCR方法检测金黄色葡萄球菌norA基因的表达。结果两种药物在敏感菌菌体内的稳态蓄积浓度明显高于耐药菌,所有实验菌株中均检测到norA基因的存在,经RealTime PCR扩增后,检测到高度耐药菌株的norA基因表达量较敏感菌菌株平均norA基因表达量高0～8倍;中度耐药菌株的norA基因表达量较敏感菌株高5～17倍。结论推测norA基因表达增加是菌体内药物蓄积量减少的主要原因之一;部分菌株的耐药可能没有norA基因参与,即使有norA基因参与,起作用也不相同;可能有其他外排泵共同参与金葡菌的耐药。     

金黄色葡萄球菌NorA蛋白免疫检测方法的建立

目的建立金黄色葡萄球菌耐药转运蛋白NorA免疫检测方法。方法利用琼脂稀释法测定诺氟沙星（NFLX）对临床分离的6株金葡菌和标准菌株ATCC25923的MIC。对norA基因进行克隆、表达，提取表达产物包涵体，并进行了纯化。将纯化的表达蛋白作为免疫原免疫家兔，获取NorA蛋白的抗体血清。利用制得的抗体通过Western blotting检测临床分离的6株金葡菌的NorA蛋白表达水平。结果氟喹诺酮类药物耐药水平较高的临床分离金葡菌菌株的NorA蛋白表达水平一般较高，敏感菌株的耐药水平较低且接近。结论免疫检测方法可用于检测金葡萄菌耐药转运蛋白NorA的表达水平。     

金葡菌细胞壁变化与对万古霉素耐药的关系

目的了解金葡菌细胞壁变化与万古霉素耐药的关系，探讨耐药机制。方法将1株从临床标本中分离的异质性耐万古霉素的金葡菌（h-VRSA）在含有不同浓度的万古霉素培养基上连续诱导转种，用微量肉汤稀释法和菌谱分析法检测其对万古霉素的敏感性；同时采用扫描电镜和透射电镜分别对细胞壁的表面结构和厚度进行摄片观察；用PCR方法检测万古霉素耐药基因（vanA、vanB和vanC）。结果随万古霉素诱导浓度的增加，金葡菌对万古霉素的MIC逐渐增加；菌谱分析显示耐药亚群逐渐增加；电镜摄片可见细胞壁增厚，细胞壁粗糙，有结节状凸起；van基因检测为阴性。结论在万古霉素的选择性压力下金葡菌的耐药性可逐渐增加，细胞壁增厚是金葡菌对万古霉素耐药的重要机制。     

金黄色葡萄球菌耐消毒剂基因qacA的研究

目的 检测我院近年来临床分离的金葡菌耐消毒剂的qacA基因水平，为动态监测细菌耐药性的变化趋势、有效控制细菌耐药性的传播提供对策。方法 收集我科临床分离的金葡菌共252株，并经本实验室重新鉴定确认。参照美国CLSI／NCCLS标准，用6μg／ml苯唑西林和4％NaCl平板，筛选出耐苯唑西林金葡菌和对苯唑西林敏感的金葡菌。应用PCR基因扩增试验检测金葡菌中耐消毒剂的qacA基因水平。随机抽取一株MRSA阳性株进行基因测序，作qacA基因验证。作qacA基因阳性株的MIC检测，了解阳性株是否有耐药性的表达。结果 共检测金葡菌252株，其中MRSA84株（带有qacA基因4株），MSSA168株（带有qacA基因1株）。MRSA阳性菌株对消毒剂的MIC值明显高于MSSA菌株。结论 携带含有耐消毒剂基因qacA的金葡菌未在我院流行．     

3294株病原菌分布特征及四种细菌耐药情况分析

目的 了解某院患者感染的病原菌分布特征及相关的四种细菌耐药情况.方法 使用上海复星佰路生物技术有限公司的BioFosun微生物鉴定及药敏分析系统,对送检验科的样本进行菌种鉴定,同时对肺炎克雷伯菌、大肠埃希菌、金葡菌、肠球菌四种细菌进行耐药评价.结果 检出菌株的标本总例数为3294例,其中检出细菌的为2829例,占85.78％；检出真菌的为465例,占14.12％.肺炎克雷伯菌、大肠埃希菌、金葡菌、肠球菌四种细菌呈现不同程度的耐药.结论 病原菌分布以细菌为主,而细菌又以革兰氏阴性菌为主.检测的四种菌株耐药严重.     

金葡菌多重耐药基因norA的原核表达及其抗体制备

按金葡菌多重耐药转运蛋白NorA的编码序列设计引物，以金葡菌基因组DNA为模板．扩增出基因norA中约1155bp的cDNA片段，将所得片段与pMD18-T载体连接，转化到感受态大肠埃希菌JM109中．成功地筛选到阳性克隆，其质粒测序结果与文献报道一致。从阳性克隆中提取质粒．经NdeⅠ和XhoⅠ酶切，回收1155bp的目的片段．定向克隆到pET-28a（＋）表达载体中，转化至感受态大肠埃希菌DH5a中，提取质粒，再次转化到BL21（DE3）中，成功筛选到阳性克隆。经IPTG诱导阳性菌，通过SDS—PAGE检测出norA部分基因的表达。利用得到的纯化蛋白免疫家兔，并通过间接ELISA法检测抗体效价．证明得到相应抗体。     

金黄色葡萄球菌的多重耐药转运蛋白NorA

金黄色葡萄球菌的NorA蛋白是一种跨膜多重药物外排蛋白,介导了金黄色葡萄球菌对氟喹诺酮类药物的耐药,NorA蛋白属于细菌转运系统中的主要易化族类,由位于染色体上的norA基因编码,norA基因大小约为1．8kb,编码388个氨基酸构成的多肽,NorA蛋白富含疏水性氨基酸,可能构建了亲水性氟喹 诺酮类药物的膜相关性活性外排泵,主要介导了亲水性氟喹诺酮类药物从细胞中主动排出,研究认为主要是由于膜上NorA蛋白数量的增加造成的,目前norA超水平表达的调控机制尚不清楚,部分人认为是norA基因多处位点或（和）启动子区域发生突变,影响了norA的表达,已证明利血平,维拉帕米和5-methoxyhydnocarpin等是NorA外排蛋白的抑制剂,能增强亲水性氟喹诺酮类药物的抗菌活性,降低耐药率。     

痰标本中质粒介导喹诺酮耐药基因qnr在肺炎克雷伯菌中的分布特征及耐药分析

目的:了解从痰标本中分离出的肺炎克雷伯菌对16种抗菌药物的耐药性,以及研究由质粒介导的喹诺酮类耐药基因qnr在肺炎克雷伯菌中的存在情况。方法:用PCR及直接测序的方法对135株肺炎克雷伯菌进行qnr基因检测,并用K-B纸片法检测其对16种抗菌药物的体外抗菌活性。另外,用琼脂平皿二倍稀释法检测阳性菌株对左氧氟沙星的MIC值。结果:135株肺炎克雷伯菌中,9株(6.6%)检出qnr基因。阳性菌株均对亚胺培南敏感且对多种抗生素耐药,其中2株qnr阳性菌株对左氧氟沙星敏感。结论:肺炎克雷伯菌中存在质粒介导喹诺酮类耐药基因qnr基因,qnr阳性菌株呈现多重耐药。临床工作中,应加强对耐药基因的监测,降低细菌耐药的发生。     

金葡菌对喹诺酮类药物的耐药机制

喹诺酮类药物是一类以1,4-二氢-4-氧- 3喹啉羧酸为基本结构的化学合成类抗菌药物.因具抗菌谱广、抗菌活性强、胞内渗透力强及不良反应少等特点,故得以迅速发展,近十年一系列新合成药物相继问世.然而,随着临床的广泛应用,其耐药菌株尤其是耐药葡萄球菌明显增加.1987年上海地区临床分离的甲氧西林耐药金葡菌(MRSA)对氟喹诺酮药物的敏感率为98.l%,而1992年其耐药率已上升至45%～66%.近年来,国内外对其耐药机制进行了大量研究,发现金葡菌对喹诺酮药物的耐药机制包括以下四个方面:(1)GyrA的改变导致喹诺酮药物抑制DNA旋转酶的活性降低,金葡菌GyrA上的点突变与大肠杆菌的点突变极相似;(2)norA介导的膜蛋白黄花系统改变,通过该机制使细菌对诺氟沙星、氧氟沙星及环丙沙星等亲水性药物的耐药性高于司帕沙星等疏水性药物;(3)cfxB-ofxC(flqA)可引起金葡菌对喹诺酮类药物耐药,此机制目前尚不清楚;(4)GyrB上的点突变与喹诺酮耐药亦有关.本文对上述耐药机制作一综述.     

二重实时PCR快速检测耐甲氧西林金黄色葡萄球菌

目的 建立二重实时聚合酶链反应（duplexreal-timePCR）快速检测耐甲氧西林金黄色葡萄球菌（MRSA）。方法 采用二重SYBR Green实时PCR快速检测MRSA的决定基因mecA和金葡菌的种特异性基因nuc，经熔解曲线分析鉴定产物。结果 所有MRSA菌株的熔解曲线均呈现mecA、nuc基因特异性的峰，甲氧西林敏感的金葡菌仅有nuc峰，耐甲氧西林的表葡菌仅有mecA峰，甲氧西林敏感的表葡菌与其他菌种的菌株无特异峰出现；当MRSA菌浓度达10^2cfu／ml时就可检出。在131株金葡菌中，30．53％（40／131）耐药；二重实时PCR扩增mecA基因29．01％（38／131）阳性，nuc基因100％阳性。单引物与二重实时PCR两者阳性扩增符合率为100％。结论 对mecA、nuc基因进行二重实时PCR能准确、快速地鉴定耐甲氧西林的金葡菌和凝固酶阴性的葡萄球菌。     

Characterisation of laboratory-generated vancomycin intermediate resistant Staphylococcus aureus strains

Vancomycin has been the drug of choice for 30 years for the treatment of methicillin-resistant Staphylococcus aureus (MRSA). Emergence of decreased vancomycin susceptibility in MRSA strains presents a significant clinical problem with few therapeutic options. This study was performed to generate and characterise S. aureus strains with reduced susceptibility to vancomycin. Eighteen S. aureus strains were subjected to serial passaging on vancomycin to generate vancomycin intermediate resistant S. aureus (VISA) strains. Minimum inhibitory concentration (MIC) determination was performed for the parent and the passaged cultures with 13 different antibiotics. The strains were tested by the following five methods: simplified population analysis; CDC method; modified vancomycin agar screen; population analysis profile (PAP); and modified population analysis (PAP-area under the curve (AUC) ratio). Phenotypic changes such as doubling time, synergy with beta-lactam antibiotics and effect on norA efflux pumps were also studied for these strains. The result indicated that 8 VISA mutants (vancomycin MICs, 8-16 microg/mL) were generated in vitro from the 18 S. aureus strains. The CDC and modified agar methods proved to be the most sensitive and specific methods for detection of VISA strains. The PAP for all the VISA strains ranged from 12 microg/mL to > 16 microg/mL, with a PAP-AUC ratio of > 1.3. All mutants showed increased doubling time compared with their parent isolate. Synergism of the vancomycin and beta-lactam combinations was observed for all methicillin-resistant mutants. Upon acquisition of vancomycin resistance, a few mutants showed decreased oxacillin resistance. Two VISA strains were chosen for molecular characterisation of the mecA gene and one mutant showed genotypic changes with deletion of mecA. Loss of norA efflux pumps leading to fluoroquinolone sensitivity was also observed in four mutants.     

Frequency and genetic characterization of multidrug-resistant mutants of Staphylococcus aureus after selection with individual antiseptics and fluoroquinolones

The frequency of generation of multidrug-resistant mutants of Staphylococcus aureus was examined by selection with various antiseptics and fluoroquinolones and the mutated genes were characterized. Multidrug-resistant mutants with mutation of the region between the promoter and structural regions of the norA gene were generated more frequently during selective pressure with antiseptics than during selective pressure with FQs. We also obtained evidence for the existence of at least one other gene related to resistance to antiseptics and fluoroquinolones on the chromosome of S. aureus in addition to norA gene.     

Expression of multidrug resistance efflux pump genes in clinical and environmental isolates of Staphylococcus aureus

Increased expression of multidrug resistance efflux pump (MDR-EP) genes in clinical isolates of Staphylococcus aureus occurs frequently, but its temporal and geographic variability is unknown. Such strains may contaminate the hospital environment, posing an infection control problem. Nearly 700 clinical isolates from different geographic locales as well as 91 environmental isolates recovered from two Detroit hospitals were studied. Ethidium bromide (EtBr) minimum inhibitory concentration (MIC), quantitative expression of all characterised chromosomal MDR-EP genes, and the presence of qacA/B and smr were determined for all strains. In addition, for norA- and/or mepA-overexpressing strains, the spa type was established. MDR-EP gene overexpression varied temporally and geographically, and overexpressing strains were present in the hospital environment. Increased expression of norA was associated with meticillin resistance and spa type t002, a rare type among control strains, consistent with widespread dissemination of a norA-overexpressing, meticillin-resistant S. aureus (MRSA) clone. Clonal spread also played a role for spa type t008, mepA-overexpressing, meticillin-susceptible strains. An EtBr MIC of ≤12.5μg/mL was highly specific (>90%) in identifying strains lacking MDR-EP gene overexpression.     

41株金黄色葡萄球菌耐药性分析及PVL基因检测

目的了解临床分离金葡菌对常用抗生素的耐药性和杀白细胞毒素(PVL)基因携带情况。方法采用头孢西丁纸片扩散法检测耐甲氧西林金黄色葡萄球菌(MRSA),K-B法检测临床分离的41株金葡菌对常用抗生素的敏感性,PCR法检测mecA基因和PVL基因携带情况。结果 41株金葡菌中MRSA占51.2%,MRSA对克林霉素、红霉素、氨基糖苷类和喹诺酮类耐药率明显高于甲氧西林敏感金黄色葡萄球菌(MSSA),MRSA和MSSA对复方新诺明敏感率分别为90.5%和95%,未发现对万古霉素、替考拉林和利奈唑胺耐药株。21株MRSA mecA基因均阳性,41株金葡菌中共检出2株携带PVL基因,MRSA与MSSA各占1株,均分离自骨科病房。结论该院临床分离MRSA耐药率高,应规范临床用药,加强MRSA耐药性监测,加强PVL基因的检测,防止此类菌株的播散。     

Evolution from a natural flavones nucleus to obtain 2-(4-Propoxyphenyl)quinoline derivatives as potent inhibitors of the S. aureus NorA efflux pump

Overexpression of efflux pumps is an important mechanism by which bacteria evade the effects of substrate antimicrobial agents. Inhibition of such pumps is a promising strategy to circumvent this resistance mechanism. NorA is a Staphylococcus aureus efflux pump that confers reduced susceptibility to many structurally unrelated agents, including fluoroquinolones, resulting in a multidrug resistant phenotype. In this work, a series of 2-phenyl-4(1H)-quinolone and 2-phenyl-4-hydroxyquinoline derivatives, obtained by modifying the flavone nucleus of known efflux pump inhibitors (EPIs), were synthesized in an effort to identify more potent S. aureus NorA EPIs. The 2-phenyl-4-hydroxyquinoline derivatives 28f and 29f display potent EPI activity against SA-1199B, a strain that overexpresses norA, in an ethidium bromide efflux inhibition assay. The same compounds, in combination with ciprofloxacin, were able to completely restore its antibacterial activity against both S. aureus SA-K2378 and SA-1199B, norA-overexpressing strains.     

耐万古霉素微生物筛选模型

为获得耐万古霉素的肠球菌和金葡球菌，利用紫外线对Enterococcus faecalis458和Staphylococcus aureus9918进行诱变，确定在不同处理中以紫外线照射30s为最佳。最后获得对万古霉素中敏的E．faecalis458 V20和S．aureus9918 V16，药物敏感性试验表明前者对青霉素G钾和红霉素耐药，而后者对青霉素G钾、红霉素、苯唑青霉素钠和氨苄青霉素钠耐药。两者对万古霉素的耐药性在无药培养基上传10代后略微下降。     

2006～2009年苏州地区儿童呼吸道感染金黄色葡萄球菌耐药性分析

目的:研究苏州大学附属儿童医院儿童呼吸道金黄色葡萄球菌(Staphylococcus aureus,SA)的感染情况及其耐药性。方法:对苏州大学附属儿童医院2006年1月至2009年12月间住院的6 404例急性呼吸道感染患儿采用无菌负压吸引法采集新鲜痰液,进行SA培养、分离,观察并总结其呼吸道SA感染情况及相关因素,采用纸片扩散法(K-B法)进行药敏试验,头孢西丁纸片法检测耐甲氧西林金黄色葡萄球菌(MRSA)。结果:6 404例呼吸道感染标本中分离到301株SA,总分离率4.7%,其中MRSA为33株占9.6%。2006、2007、2008、2009年SA年度检出率分别为3.7%(59/1 597)、4.1%(64/1 570)、5.1%(87/1 698)、5.9%(91/1 539),4年年度检出率差异无统计学意义(χ2=0.99,P>0.05)。<1岁组检出率最高。SA对常用抗菌药物耐药率分别为:苯唑西林6.8%～13.2%、青霉素86.4%～96.9%、红霉素52.5%～72.9%、克林霉素15.3%～28.5%、复方新诺明11.5%～16.9%、万古霉素0%、庆大霉素0%～3.4%、环丙沙星0%～3.1%、利福平0%～6.8%。结论:SA是苏州地区儿童呼吸道感染重要的细菌病原之一,婴儿感染多见。分离株对青霉素和红霉素的耐药率高,而对苯唑西林、万古霉素、环丙沙星、庆大霉素和利福平有相对较高的敏感性。     

金黄色葡萄球菌对夫西地酸的体外耐药现状及耐药性改变研究

目的了解金黄色葡萄球菌对夫西地酸的耐药现状及耐药性改变。方法通过琼脂稀释法测定2010年连续收集的260株和2007年收集的220株金黄色葡萄球菌对夫西地酸的MIC值,用K-B纸片法测定这些菌株对万古霉素等7种抗生素的抑菌圈直径。结果 480株金黄色葡萄球菌中对夫西地酸耐药的只有4株,耐药率为0.83%,2007年和2010年收集的菌株对夫西地酸的MIC50均为0.25mg/L,MIC90均为0.5mg/L,纸片法测定结果：所有菌株对万古霉素敏感,2007年的菌株57.8%耐甲氧西林,2010年的菌株76.9%耐甲氧西林。结论夫西地酸对金黄色葡萄球菌的体外抗菌活性很强,而且2007年和2010年菌株的抗菌活性无多大改变。     

Ⅰ类切口处皮肤金黄色葡萄球菌的带菌率和耐药性调查

目的:了解Ⅰ类切口处皮肤上的金黄色葡萄球菌(SA)带菌率及耐药情况,为临床合理使用抗菌药物提供参考。方法:用皮肤拭子采集Ⅰ类切口处皮肤标本,对培养出的SA按K-B法进行药敏试验,遵照《临床微生物检验手册》进行操作。结果:共采集样本991份,检出23株SA,检出率为2.32%;其中17株对苯唑西林敏感,6株对苯唑西林耐药,耐甲氧西林金黄色葡萄球菌(MRSA)带菌率为0.61%,耐药率为26.09%。结论:我院Ⅰ类切口患者皮肤SA的检出率相对较低,耐药率较高,需加强感染控制管理,采取有效措施,防止MRSA的院内传播。