儿茶素类化合物增强β-内酰胺类抗生素抗MRSA的作用

目的确定儿茶素类化合物——儿茶素(C)、表儿茶素没食子酸酯(ECG)、表没食子酸儿茶素(EGC)联合增强β-内酰胺类抗生素对耐甲氧西林的金黄色葡萄球菌(MRSA)产生抗菌增敏作用的最佳配伍比并对其可能的抗菌增敏作用的机制进行初步探讨。方法以微量稀释法测定C、ECG、EGC和几种β-内酰胺类抗生素单独对MRSA WHO-2菌株的最小抑菌浓度(MIC),以棋盘法测定C、ECG和EGC联合β-内酰胺类抗生素后对MRSA WHO-2菌株和25株MRSA临床分离株的最小抑菌浓度(MIC);以荧光分光光度计法和激光共聚焦扫描显微镜法观察C、ECG、EGC单独、两两联合、三者联合后对柔红霉素在MRSA WHO-2菌株菌体聚集的影响。结果 C、ECG、EGC三者联用时可以增强苯唑西林抗MRSA WHO-2菌株的能力,其中C、ECG、EGC按照1:1:1的比例配伍后可以取得最佳的抗菌增敏效果,后续实验选用此药物配伍。药物总浓度为16μg/mL的C2E(C+ECG+EGC)与苯唑西林、头孢唑林、氨苄西林联合时对于MRSA WHO-2菌株可产生抗菌增敏作用,对应的FIC指数均为0.38,同样浓度的C2E联合苯唑西林、氨苄西林、头孢唑林、头孢吡肟、泰能后对25株MRSA临床分离株中可产生抗菌增敏作用的菌株数所占比例分别为80%、76%、88%、80%、92%,说明C2E在MRSA临床分离株中同样存在广泛的抗菌增敏作用。经过均为16μg/mL的C、ECG、EGC三者联合处理后,MRSA WHO-2菌株菌体内的柔红霉素在荧光分光光度计下测得的A值为9.26±0.16,大于单独或两两联合处理时的A值(P<0.05),提示三者联合处理后增强了柔红霉素在MRSA WHO-2菌体内的聚集;激光共聚焦扫描显微镜法也显示出相同的结果。结论 C、ECG、EGC三者联合后可显著增强β-内酰胺类抗生素抗MRSA的作用,其最佳配伍比为1:1:1。抗菌增敏作用机制可能与其增加药物在菌体内的聚集有关。     

黄芩总黄酮对金葡菌肺感染模式识别受体TLR2/Nod2及其相关炎性因子表达的影响

目的观察黄芩总黄酮对金葡菌感染后肺脏模式识别受体TLRs和Nods及其相关炎性因子表达的作用,探讨其抗炎药理可能的作用靶点和机制。方法通过构建体外金葡菌感染鼠肺上皮细胞和体内金葡菌急性小鼠肺感染模型,利用RT-PCR技术,通过多种给药形式观察TLR2/Nod2和下游相关炎性因子的mRNA的表达,金葡菌的增殖数量,以探讨其可能的作用靶点和机制。结果黄芩总黄酮不能减少侵入到胞内的活菌数目,但可以下调由于金葡菌侵入导致的TNF-α的大量合成。金葡菌侵入后Nod2表达大幅升高,黄芩总黄酮有明显的抑制作用,并且和TNF-α的变化呈现一定的相关性。TLR2/MyD88的表达无变化。结论黄芩总黄酮的抗炎作用可能与其下调Nod2受体表达进而抑制下游炎性因子表达相关。     

青蒿琥酯对类风湿关节炎滑膜细胞TNF-α分泌的抑制作用及其机制研究

目的研究青蒿素衍生物青蒿琥酯对类风湿关节炎(RA)滑膜细胞(FLS)分泌肿瘤坏死因子(TNF)-α的影响,并进一步探讨其机制。方法来自活动性RA患者的FLS用脂多糖(LPS)刺激和青蒿琥酯处理,TNF-α水平用酶联免疫吸附试验(ELISA)测定,核因子(NF)-κB活性检测采用电泳迁移率改变试验(EMSA)。结果青蒿琥酯显著抑制LPS诱导的TNF-α分泌,并且呈剂量依赖性。青蒿琥酯对LPS刺激的NF-κB活化有明显抑制作用,NF-κB抑制剂二硫代氨基吡咯烷(PDTC)亦显著抑制LPS刺激的滑膜细胞分泌TNF-α。结论青蒿琥酯通过调节NF-κB活性抑制LPS诱导的RA滑膜细胞分泌TNF-α,提示青蒿琥酯可能具有抑制RA滑膜炎症的作用。     

黄芩苷/黄芩素对耐甲氧西林金黄色葡萄球菌抗药性的逆转作用研究

目的:研究黄芩苷/黄芩素对耐甲氧西林金黄色葡萄球菌(MRSA)抗药性的逆转作用,并初步探索其逆转机制。方法:以平皿法测定黄芩苷/黄芩素与苯唑西林对MRSA的协同抗菌效力;以分光光度法测定黄芩苷/黄芩素协同苯唑西林对MRSA生长密度的抑制作用;以青霉素结合蛋白(PBP2a)胶乳凝集试剂盒检测黄芩苷/黄芩素对PBP2a生成的抑制效力。结果:黄芩苷/黄芩素联合苯唑西林对临床分离的MRSA有显著的协同抗菌作用,16μg·mL-1的黄芩素即可显著逆转MRSA对苯唑西林的高度耐药;黄芩苷/黄芩素对MRSA产生PBP2a有显著抑制作用。结论:黄芩苷/黄芩素可通过抑制MRSA产生PBP2a来逆转其对苯唑西林的高度耐药,这一现象从理论上为临床治疗MRSA感染提供了新的方法和思路。     

青蒿琥酯对大鼠佐剂性关节炎的抗炎免疫研究

目的研究青蒿琥酯对佐剂性关节炎大鼠的抗炎免疫机制。方法建立佐剂性关节炎大鼠模型,随机分为空白组、AA模型组、甲氨蝶呤阳性药物对照组和青蒿琥酯大、中、小剂量组(20.0,10.0,2.5 mg.kg.d-1),并灌胃给药。ELISA法检测各组对单核细胞趋化因子(MCP-1)、调节活化T细胞表达和分泌因子(RANTES)和肿瘤坏死因子(TNF-α)的影响。结果青蒿琥酯大、中、小剂量组能显著降低血清TNF-α,与模型组比较差异有统计学意义(P<0.05);青蒿琥酯小剂量组与其大、中剂量组或者与甲氨蝶呤组比较差异有统计学意义(P<0.05);青蒿琥酯各剂量组均能显著降低血清中MCP-l和RANTES的表达,与模型组比较差异有统计学意义(P<0.05);而青蒿琥酯小剂量组与大、中剂量组或者与甲氨蝶呤组比较差异有统计学意义(P<0.05)。结论青蒿琥酯抗炎和免疫调节作用可能是与抑制炎症因子相关。     

耐甲氧西林金黄色葡萄球菌检测方法的比较

目的 对耐甲氧西林金黄色葡萄球菌(MRSA)的检测方法进行比较.方法 分别用mecA基因PCR扩增法、PBP2a胶乳凝集试验法、头孢西丁纸片扩散法和苯唑西林纸片扩散法对临床分离的135株金黄色葡萄球菌进行榆测.结果 mecA基因PCR扩增法显示阳性79株,阴性56株;PBP2a胶乳凝集试验法的敏感性和特异性分别为96.2%和100.0%;头孢西丁纸片扩散法的敏感性和特异性分别为94.9%和96.4%;苯唑西林纸片扩散法的敏感性和特异性分别为94.9%和62.5%.结论 PBP2a胶乳凝集试验法和头抱西丁纸片扩散法与mecA基因PCR扩增法的检测结果高度一致;苯唑西林纸片扩散法特异性低,不再适用于MRSA的检测.     

Modulation of beta-lactam resistance in Staphylococcus aureus by catechins and gallates

Aqueous extracts of Japanese green tea (Camellia sinensis) are able to reverse beta-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). We have attributed the capacity to reverse oxacillin resistance in the homogeneous PBP2a producer BB568 and in EMRSA-16 to (-)-epicatechin gallate (ECG) and (-)-catechin gallate (CG). Minimum inhibitory concentration (MIC) values for oxacillin were reduced from 256 and 512 to 1-4 mg/l, respectively, in the presence of these polyphenols. In addition, (-)-epigallocatechin gallate (EGCG) had a moderate capacity to modulate oxacillin activity against S. aureus BB568, but none against EMRSA-16. ECG, CG and EGCG increased the sensitivity of EMRSA-15 to oxacillin. The gallate moiety was essential for the oxacillin-modulating activity of ECG, as both (-)-epicatechin and (-)-epicatechin-3-cyclohexylcarboxylate were unable to reverse resistance to oxacillin. Gallic acid and three alkyl gallates (methyl gallate, propyl gallate, and octyl gallate) did not modulate beta-lactam resistance in MRSA. Octyl gallate exhibited direct antibacterial activity against S. aureus BB568 (16 mg/l). Modulation of beta-lactam resistance by ECG significantly enhanced the activities of flucloxacillin and the carbapenem antibiotics imipenem and meropenem against 40 MRSA isolates, with MIC(90) values for the antibiotics reduced to the susceptibility breakpoint or below. Consequently, EGCG, CG and, particularly, ECG warrant further investigation as agents to combat beta-lactam resistance in S. aureus.     

脱氧核酶抑制耐药基因mecR1的表达逆转MRSA耐药性

目的探讨脱氧核酶抑制耐甲氧西林金黄色葡萄球菌（MRSA）耐药基因mecR1的表达对MRSA耐药性的影响。方法设计、合成特异性针对耐药基因mecR1 mRNA的硫代脱氧核酶，将其导入细菌，通过半定量RT-PCR观察脱氧核酶对耐药基因mecR1及其下游基因mecA表达的抑制作用，并通过平板克隆形成实验观察脱氧核酶对MRSA耐药性的影响。结果加入脱氧核酶后培养的MRSA，mecR1与mecA的mRNA表达水平较对照组显著降低（P〈0．01），在含苯唑西林（6mg／L）的M-H琼脂培养基表面生长的菌落单元数也低于对照组（P〈0．01）。结论特异性抗mecR1脱氧核酶阻断耐药基因mecR1-mecA的表达，可以部分恢复MRSA对抗生素的敏感性．     

耐甲氧西林金葡菌的头孢西丁检测法与耐药性分析

目的评价头孢西丁纸片扩散法检测耐甲氧西林金葡菌（MRSA）的效果．了解MRSA的耐药状况。方法用头孢西丁及苯唑西林纸片扩散法、mecA基因聚合酶链反应（PCR）检测MRSA．并以mecA基因PCR法为参考方法与头孢西丁及苯唑西林纸片扩散法检测MRSA进行比较分析。按CLSI（原NCCLS）规定的标准测定MRSA对青霉素等10种抗生素的耐药率（KB法）。结果95株金葡菌（SA）中mecA基因阳性株42株。头孢西丁纸片法筛选出40株MRSA，其中39株mecA基因阳性，方法特异性为97．5％，灵敏度92．9％；苯唑西林纸片法筛选出44株MRSA，其中36株mecA基因阳性．方法特异性为81．8％．灵敏度85．7％。MRSA对青霉素、苯唑西林100％耐药，对万古霉素全部敏感．对红霉素、头孢唑林、克林霉素、氨苄西林／舒巴坦、头孢呋辛、左氧氟沙星、奈替米星呈不同程度耐药。结论头孢西丁纸片扩散法检测MRSA特异性和灵敏度优于苯唑西林纸片扩散法。MRSA具有多重耐药性．须加强其对抗生素的耐药性监测。     

耐甲氧西林金黄色葡萄球菌mecA基因原核表达载体的构建

目的对耐甲氧西林的金黄色葡萄球菌(MRSA)mecA基因片段进行扩增表达,纯化及鉴定。方法应用聚合酶链反应(PCR)扩增技术获得编码PBP2a转肽酶区的mecA基因片段,将此目的基因片段与pET-21a(+)载体连接,构建pET-21a(+)-mecA的重组质粒,经双酶切、测序正确后,将重组质粒转入E.coli BL21 DE3中,用IPTG诱导mecA融合蛋白,并对表达产物进行鉴定。结果构建了mecA基因的原核表达载体,并获得高效表达。结论获得了mecA基因编码的PBP2a蛋白,为MRSA的耐药机制、药物治疗等进一步研究奠定了基础。     

Inhibitory effect of polyoxotungstates on the production of penicillin-binding proteins and beta-lactamase against methicillin-resistant Staphylococcus aureus.

In our continuous work on the enhancement of the antibacterial activity of beta-lactam antibiotics against the cells of methicillin-resistant Staphylococcus aureus (MRSA) strains by Keggin-structural polyoxotungstates and their lacunary species, Wells-Dawson, double-Keggin, and Keggin-sandwich polyoxotungstates are also found to be synergistic but highly cytotoxic. The coexistence of polylysine or protamine sulphate decreased the synergistic potency of the polyoxotungstates, due to their electrostatic interaction with negatively charged polyoxotungstates. Inductively coupled plasma atomic emission spectrometry (ICP) analysis of the polyoxotungstate-treated cells indicated that the polyoxotungstates uptaken in the cell are preferentially located at the membrane fraction with intact composition. The polyoxotungstates depressed not only the production of PBP2', but also the production of beta-lactamase which hydrolyzes beta-lactam antibiotics on the membrane. This leads to the synergistic effect of polyoxotungstates against the MRSA cells in the coexistence of beta-lactam antibiotics which have high affinities to PBPs 1-4. MRSA cells which were modified to be susceptible to beta-lactam antibiotics during incubation in the presence of polyoxotungstates recovered their resistance to beta-lactam antibiotics when they were subcultured in the absence of the polyoxotungstate.     

Methicillin-resistant Staphylococcus aureus in the hospitals of central Greece.

A total of 250 consecutive Staphylococcus aureus clinical isolates were collected during the period 1999-2000 from the five major hospitals of the district of Thessaly (Central Greece). Thirty seven (14.8%) of the isolates were mecA-positive (MRSA) in a PCR-based assay; all exhibited resistance to oxacillin (agar dilution MICs > or =4 mg/L) and were also resistant to multiple antibiotics. Most of the MRSA isolates had been collected in the intensive care units and the surgical wards of the participating hospitals in a sporadic fashion. The MRSA incidence found here was significantly lower than reported in previous studies from Greece. Molecular typing by PFGE showed that the MRSA isolates were distributed between three pulsotypes. Evaluation of various conventional methods for assessing methicillin resistance showed that oxacillin agar dilution and immunological detection of PBP2a with the Slidex MRSA Detection kit were the most reliable in this setting. Misclassifications of isolates exhibiting low-level resistance (oxacillin MIC 2-4 mg/L) occurred with the salt agar screen, the oxacillin disk diffusion and the ATB Staph System methods.     

Susceptibility patterns of coagulase-negative staphylococci to several newer antimicrobial agents in comparison with vancomycin and oxacillin

Coagulase-negative staphylococci (CoNS) have emerged as important nosocomial pathogens. CoNS resistance to meticillin and other semisynthetic penicillins is now common. Elevated vancomycin minimal inhibitory concentrations (MICs) have been reported and are associated with worse treatment outcomes. Several newer antibiotics have recently become available for the treatment of Gram-positive infections. The purpose of this study was to assess the in vitro activity of telavancin, daptomycin, linezolid and tigecycline in comparison with oxacillin and vancomycin against 653 non-duplicate clinical isolates of CoNS by the agar dilution method. The greatest variability in MIC was observed for oxacillin. Presence of the mecA gene conferred higher MICs for oxacillin but did not influence MICs to all other antibiotics tested. Telavancin tended to have MICs that were 1-2 dilutions lower than vancomycin. Daptomycin had good activity against all isolates. Staphylococcus haemolyticus, Staphylococcus hominis subsp. novobiosepticus, Staphylococcus saprophyticus, Staphylococcus schleiferi and Staphylococcus simulans were the most daptomycin-susceptible CoNS species tested. The validity of the agar dilution method for daptomycin was confirmed, with >90% isolates having MICs that were within 1 dilution of parallel Etest results. Within-species MIC variation was most restricted for linezolid and tigecycline, with the exception of Staphylococcus epidermidis and Staphylococcus haemolyticus that demonstrated higher overall MICs to tigecycline.     

青蒿琥酯对盲肠结扎穿孔术脓毒症小鼠的保护作用

目的:探讨青蒿琥酯(AS)对盲肠结扎穿孔术(CLP)脓毒症小鼠的保护作用及其可能机制。方法:采用CLP制作小鼠脓毒症模型,术后0、4、24、48h重复肌肉注射AS,观察7d内CLP小鼠的死亡率;检测术后4、8h血浆脂多糖(LPS)、TNF-α、IL-6水平;术后8h小鼠肺脏组织形态的变化。结果:AS明显降低CLP小鼠的死亡率(P<0.05)及血浆LPS、TNF-α和IL-6水平(P<0.05或P<0.01),减轻肺组织的损伤。结论:AS对CLP小鼠具有显著的保护作用,该作用可能与其降低血浆LPS水平、减少促炎细胞因子TNF-α、IL-6的释放和减轻组织脏器的损伤有关。     

耐甲氧西林金黄色葡萄球菌耐药表型分型与耐药基因检测分析研究

目的分析研究耐甲氧西林金黄色葡萄球菌(methicillin-resistant staphylococcus aureus,MRSA)的耐药表型及耐药基因,探索治疗MRSA感染的有效手段。方法对临床分离的耐甲氧西林金黄色葡萄球菌株,依据美国临床实验室标准化研究所(CLSI)标准操作规程进行万古霉素、利奈唑胺和苯唑西林等药物的药物敏感性试验和耐药基因检测,分别采用微量肉汤稀释法和多重聚合酶链反应(PCR)扩增法。结果 MRSA耐药表型分为Ⅰ~Ⅶ型,MRSA对抗生素产生多重耐药,万古霉素、利奈唑胺、氯霉素、复方新诺明、利福平耐药率分别为0、7.0%、40.8%、47.9%、60.6%,β-内酰胺类药物耐药率高达100%;实验菌株mecA、ermA、ermC、tetK、tetM、ratA基因检出率分别为98.6%、60.6%、18.3%、100%、7.0%和0。结论 MRSA对万古霉素、利奈唑胺敏感,其他药物呈多重耐药,需加强MRSA对万古霉素、利奈唑胺、氯霉素、复方磺胺、利福平等常用药物最小抑菌浓度(MIC)监测和临床报告,关注万古霉素对MRSA治疗不佳患者。     

Salvipisone and aethiopinone from Salvia sclarea hairy roots modulate staphylococcal antibiotic resistance and express anti-biofilm activity

Two diterpenoids, salvipisone (Salv) and aethiopinone (Aeth), isolated from hairy roots of Salvia sclarea, were tested with respect to their activity against methicillin-resistant Staphylococcus aureus (MRSA) and S. epidermidis (MRSE) strains, cultured as planktonic cells or as adherent biofilms. The standard CLSI method, MTT reduction assay or confocal laser scanning microscopy (CLSM) were used for this purpose and also applied for testing the susceptibility to oxacillin, vancomycin, linezolid and their potential synergy with diterpenoids (evaluated as a fractional inhibitory concentration (FIC) index). Salv and Aeth were shown to be bactericidal or bacteriostatic against S. aureus and S. epidermidis planktonic cultures. Both diterpenoids, at the concentrations of 1/2 MIC, showed synergy with antibiotics representing the beta-lactam, glycopeptide and oxazolidinone groups. None of the antibiotics used at a high concentration killed the staphylococcal biofilms. On the contrary, Salv and Aeth decreased the number of live biofilm cells by 45.7 - 77.1% and slightly reduced the biofilm inhibitory concentration of oxacillin. Diterpenoids also changed the parameters of biofilm morphology, as shown by the CLSM image processing package (PHLIP). It was concluded that salvipisone and aethiopinone (relatively highly lipophilic, log P respectively = 3.4; 4.8) synergized the action of beta-lactam antibiotics towards MRSA and MRSE probably by alteration of cell surface hydrophobicity and cell wall/membrane permeability, but not by changing penicillin-binding protein, PBP2a expression and penicillinase production or by direct binding to the cell wall peptidoglycan and teichoic acids.     

青蒿琥酯抗肿瘤作用的实验研究

目的：探讨青蒿琥酯的抗肿瘤作用。方法：采用体内试验以荷瘤小鼠实体瘤重、抑瘤率为实验指标评价青蒿琥酯对S180移植性肉瘤的抑制作用；以荷瘤小鼠存活时间为实验指标评价青蒿琥酯对H22移植性腹水癌的抑制作用。结果：青蒿琥酯能明显抑制实体瘤重，并延长荷瘤小鼠存活时间。结论：青蒿琥酯对S180、H22诱发的移植性肿瘤具有一定的抑制作用。     

苯唑西林敏感耐甲氧西林金黄色葡萄球菌(OS-MRSA)研究进展

摘要：金黄色葡萄球菌(Staphylococcus aureus, SA)是导致医院内感染及社区获得性感染的一种常见病原菌，可引起宿主 不同部位的感染，感染类型多样。耐甲氧西林金黄色葡萄球菌(methicillin resistant Staphylococcus aureus, MRSA)呈现对多种类 型抗菌药物的耐药特征，其在不同国家和地区的检出率和流行特征存在明显差异。随着对MRSA耐药性和耐药机制研究的不 断深入，一种具有特殊耐药特征的MRSA亚群逐渐被报道，即苯唑西林敏感的耐甲氧西林金黄色葡萄球菌(oxacillin-susceptible methicillin-resistant Staphylococcus aureus，OS-MRSA)。OS-MRSA典型特征为携带抗药性基因mecA，但其对苯唑西林的MIC值 较低(通常＜4mg/L)。由于目前临床实验室主要采用苯唑西林和或头孢西丁药敏试验方法来区分MRSA及甲氧西林敏感金黄色葡 萄球菌(MSSA)，而很少应用基因检测方法(mecA基因)进行检测或确认，因此OS-MRSA常被错误报告为MSSA，临床依据药敏 报告进行抗感染治疗，常导致治疗失败及患者病死率的增加。此外，OS-MRSA菌株在不同国家和地区人群分离率及其分子流 行病学特征存在较大差异。因此，本文将从分子流行病学、实验室检测方法、耐药机制、毒力特征及临床治疗等5个方面对OSMRSA 的最新研究进展进行综述。