抗vWF与GPIb-IX结合部位的噬菌体呈现型单链抗体制备及功能研究

目的：为了研制新型特异性抗血栓制剂。方法：利用噬菌体展示文库技术筛选与vWF-A1区有高亲合力单链抗体(ScFv)；基因重组的方法构建高效表达载体pET-20b( )-ScFv，在大肠杆菌中诱导表达；夹心ELISA方法鉴定此单抗的抗原结合活性；瑞斯脱霉素诱导的血小板聚集试验(RIPA)测定ScFv对血小板聚集的抑制作用。结果：噬菌体展示技术筛选的ScFv在大肠杆菌中成功地诱导表达，表达的ScFv占菌体总蛋白的41％，以包涵体形式存在；经纯化复性的ScFv可以与。vWF、rvWF-A1、rvWF-A1/／A3结合，但不与rvWF-A3、BSA反应；ScFv具有抑制RIPA功能，抑制率为73．7％。结论：在大肠杆菌中高效表达的噬菌体展示技术筛选的抗vWF-A1区ScFv可特异性与vWF-A1区结合，而抑制瑞斯脱霉素诱导的血小板聚集，显示出很好的应用前景，为研制新型抗栓药物奠定基础。     

重组血小板糖蛋白Ibα活性片段在CHO细胞中表达及其功能研究

目的：建立表达重组糖蛋白Ibα活性片段（rGPIbαH1-V289）细胞株,并且纯化其表达产物,研究其生物学功能.方法：利用脂质体将含有编码GPIbαH1-V289 的DNA质粒pCMV3转染CHO细胞.用亲和层析法纯化重组片段.用SDS-PAGE和Western blot鉴定纯化产品的纯度和免疫学活性.用血小板聚集法检测纯化产品对瑞斯托霉素诱导血小板聚集功能的影响.用ELISA测定纯化产品对vWF与血小板结合能力的影响.结果：每1 L培养上清液可纯化到6.15 mg rGPIbαH1-V289重组产品.SDS-PAGE显示,纯化的重组片段主要在42 kD处显一蛋白区带,Western blot显示,抗GPIbα单抗SZ-2能与重组片段在42 kD处显单一蛋白区带.纯化重组片段能抑制瑞斯托霉素诱导的血小板聚集功能.ELISA测定显示纯化产品抑制血浆vWF与血小板的结合能力.结论：CHO细胞株A1能恒定表达rGPIbαH1-V289,重组片段具有较高的纯度、免疫学活性和生物学活性,有可能开发为有效的抗血栓药物.     

GST-GP302融合蛋白在大肠杆菌中的表达及其抗血清的制备

目的 :在大肠杆菌中表达血小板糖蛋白GPIbα之vWF结合区(GP30 2 )与谷胱甘肽S 转移酶GST的融合蛋白并制备其抗血清。方法 :将GP30 2片断插入GST融合表达载体 pGEX 4T 1,重组载体酶切鉴定后 ,在大肠杆菌中经IPTG诱导表达获得GST GP30 2融合蛋白 ,SDS PAGE分析表达产物。包涵体经变性复性后免疫新西兰白兔 ,制备抗血清 ,ELISA、Westernblot检测重组抗原的免疫活性。结果 :重组质粒酶切鉴定表明 ,GP30 2基因已正确插入到 pGEX 4T 1中 ,经IPTG诱导后 ,表达出相对分子质量 (Mr)约为 5 90 0 0的融合蛋白 ,获得了ELISA效价为 1× 10 -5的多克隆抗体。Westernblot证明所制备的多抗可以与血小板糖蛋白特异性结合。结论 :GP30 2片断在大肠杆菌中的成功表达及制备得到的多克隆抗体 ,为检测血小板糖蛋白GPIbα及其在其他体系中的表达提供了一种检测途径     

RGD-重组葡激酶-人α微球蛋白融合蛋白原核表达、纯化及鉴定

目的构建RGD-重组葡激酶-人α微球蛋白融合蛋白的原核表达质粒,在大肠杆菌中表达融合蛋白,纯化并初步鉴定其生物学活性。方法利用重叠延伸PCR获得目的融合基因片段RGD-r SAK-α1M,插入带有His-GST标签的原核高效可溶性表达载体PEGX-6P-1中。经酶切鉴定后,将重组表达质粒转化大肠杆菌BL21菌株,IPTG诱导目的蛋白表达,经Ni+亲和层析柱纯化后用3C蛋白酶切除重组蛋白的His-GST标签,再利用DEAE离子交换柱和分子筛纯化蛋白。最后应用纤维蛋白平板溶圈法和血小板聚集抑制试验分别测定评价重组融合蛋白的溶栓活性和抗血小板聚集作用。结果 1)成功构建了RGD-重组葡激酶-人α微球蛋白融合蛋白。2)实现了目的融合蛋白在大肠杆菌上清液中的高效表达,并纯化了融合蛋白。3)体外实验表明纯化后的融合蛋白纤溶活性同尿激酶标准品无明显差异。4)融合蛋白抗血小板聚集作用较单纯重组葡激酶明显提高。结论经文献检索确认为首次获得了纯化的RGD-重组葡激酶-人α微球蛋白融合蛋白,并验证了其纤溶活性和尿激酶标准品无明显差异,而抗血小板聚集作用较单纯重组葡激酶增强,为下一步进行融合蛋白免疫原性鉴定奠定了基础。     

谷胱甘肽S-转移酶-KGDX融合蛋白的抗血小板作用

目的观察重组GST-KGDX(谷胱甘肽S-转移酶-赖-甘-天冬-X)融合蛋白对血小板聚集的抑制作用.方法设计合成了两条编码KGDX(赖-甘-天冬-X)的寡核苷酸链,长度为36个碱基对,两端分别为BamHI酶和XcoI酶切位点.将该基因插入原核高效表达载体PGEX-4T-1的tac启动子下游,获得重组质粒PGEX-4T-1KGDX,转化大肠杆菌DH5a,37℃诱导使重组子表达.采用谷胱甘肽-琼脂糖悬珠亲和层析法获得纯化蛋白.用血小板凝聚仪检测活性.结果 GST-KGDX融合蛋白具有可溶性,产量为35mg/升培养基,表达量占菌体总蛋白48.02%,容易从裂解菌中纯化得到.体外实验:GST、GST-KGDX融合蛋白均能抑制ADP诱导的人血小板聚集, GST-KGDX强于GST(p＜0.05或＜0.01),其IC50值分别为4μmol/L、6μmol/L.结论 (1)GST、GST-KGDX融合蛋白在体外均能抑制ADP诱导的人血小板聚集.(2)大肠杆菌中能够表达出较高产量的GST-KGDX融合蛋白,为抗血小板因子提供了一个很好的蛋白质来源.     

重组人白细胞介素7蛋白的表达、复性和纯化

目的表达、复性和纯化重组人白细胞介素7(recombinant human interleukin-7,rhIL-7)蛋白,为其功能研究打下基础。方法在已经对IL-7基因TA克隆的基础上进行亚克隆,插入pET-21b载体中,构建rhIL-7的原核表达载体,阳性克隆经测序成功后转化入大肠杆菌BL21(DE3)中,优化rhIL-7蛋白表达条件,并对表达的蛋白进行复性及纯化,利用免疫印迹鉴定重组蛋白。结果表达的蛋白与预期相对分子质量17 400相符,经优化后确定蛋白表达条件为:诱导温度为37℃、IPTG诱导浓度为1.0 mmol/L、诱导时间为2 h,且rhIL-7的复性效率较好(约50%),纯化后蛋白纯度高达95%以上,并经Western blot证实表达的蛋白为rhIL-7。结论成功获得高纯度的rhIL-7复性蛋白。     

谷胱甘肽S-转移酶-KGDX融合蛋白的抗血小板作用

目的观察重组GST-KGDX(谷胱甘肽S-转移酶-赖-甘-天冬-X)融合蛋白对血小板聚集的抑制作用.方法设计合成了两条编码KGDX(赖-甘-天冬-X)的寡核苷酸链,长度为36个碱基对,两端分别为BamHI酶和XcoI酶切位点.将该基因插入原核高效表达载体PGEX-4T-1的tac启动子下游,获得重组质粒PGEX-4T-1KGDX,转化大肠杆菌DH5a,37℃诱导使重组子表达.采用谷胱甘肽-琼脂糖悬珠亲和层析法获得纯化蛋白.用血小板凝聚仪检测活性.结果GST-KGDX融合蛋白具有可溶性,产量为35mg/升培养基,表达量占菌体总蛋白48.02%,容易从裂解菌中纯化得到.体外实验GST、GST-KGDX融合蛋白均能抑制ADP诱导的人血小板聚集,GST-KGDX强于GST(p＜0.05或＜0.01),其IC50值分别为4μmol/L、6μmol/L.结论(1)GST、GST-KGDX融合蛋白在体外均能抑制ADP诱导的人血小板聚集.(2)大肠杆菌中能够表达出较高产量的GST-KGDX融合蛋白,为抗血小板因子提供了一个很好的蛋白质来源.     

小鼠FcγRⅢ原核表达、纯化及鉴定

目的: 制备纯化重组蛋白mFcγRⅢ,并鉴定其生物学活性.方法: 从克隆载体mRⅢ-T中扩增mFcγRⅢ胞外区,构建原核表达载体pETmRⅢ,转化大肠杆菌BL21(DE3),IPTG诱导重组蛋白表达,SDS-PAGE和Western blot对重组表达蛋白进行鉴定;表达产物通过包涵体纯化后用稀释方法复性;ELISA检测复性蛋白活性.结果: 成功构建了重组表达质粒pETmRⅢ,纯化和复性了重组蛋白mFcγRⅢ;复性的重组蛋白mFcγRⅢ具有较强的体外活性.结论: 原核重组蛋白mFcγRⅢ复性后具有较强的体外结合配体特性,为探索重组蛋白治疗自身免疫病奠定了基础.     

抗血小板糖蛋白VI单链抗体的制备和功能研究

将抗血小板膜表面糖蛋白VI(GPVI)单克隆抗体SZ118构建成单链抗体,以研究开发新型抗血栓药物。采用RT-PCR技术,从分泌SZ118杂交瘤细胞中克隆该抗体的重链可变区(VH)和轻链可变区(VL)基因。经测序鉴定后构建表达质粒pET20b(+)-SZ118scFv,并体外表达SZ118单链抗体(SZ118scFv)。同时测定了SZ118scFv对血小板黏附和聚集功能的影响。结果表明成功克隆了SZ118 VH和VL基因并正确构建了SZ118scFv表达质粒。复性后SZ118scFv保留源抗体与血小板结合能力,显著抑制纤维状胶原和Convulxin诱导的血小板聚集,且呈剂量依赖性,最大抑制率分别为(84.3%±5.6%)和(50.3%±15.5%);SZ118scFv明显抑制高剪切力条件下血小板与纤维状胶原的黏附,抑制率达68.3%。结果提示,成功制备了SZ118scFv,SZ118scFv可阻断血小板与胶原的相互作用,具有潜在的实际应用价值。     

重组人骨形成蛋白－2在大肠杆菌中的表达、纯化和复性

目的:通过对大肠杆菌表达重组人骨形成蛋白－2的复性研究,得到高活性的重组人骨形成蛋白－2。方法:在大肠杆菌中通过温度诱导表达重组人骨形成蛋白－2经过Triton X-100清洗之后,又通过DEAE离子交换层析纯化包涵体,包涵体在8 mol/L 尿素变性溶解,在氧化－还原(还原型和氧化型谷胱甘肽)复性系统中,通过简单的稀释复性,通过肝素亲和层析一步纯化法纯化重组人骨形成蛋白－2,最后通过诱导C2C12细胞产生碱性磷酸酶检测重组人骨形成蛋白－2活性。结果:温度诱导表达重组人骨形成蛋白－2是以包涵体单体的形式存在,经过几步纯化后,得到高纯度的包涵体。重组人骨形成蛋白－2在不同氧化－还原剂比例,和不同小分子化学辅助剂浓度中复性,得到复性的效率也不同。亲和层析纯化后,本实验得到重组人骨形成蛋白－2的生物学活性比商业化的重组人骨形成蛋白－2更高。结论:重组人骨形成蛋白－2属于转化因子－β家族,此复性方法可能应用于此家族的其他成员同时得到成本低、产量高的重组人骨形成蛋白－2,可能为临床应用创造了良好的条件。     

Effect of statins on platelet function in patients with hyperlipidemia

Introduction

It is generally assumed that cholesterol reduction by statins is the predominant therapeutic result underlying their beneficial effects in cardiovascular disease. However, the action of statins may be partially independent of their effects on plasma cholesterol levels, as they combine lipid lowering with positive effects on hemorheological conditions and endothelial function. We evaluated the impact of statin treatment on platelet adhesion to fibrinogen (spontaneous and ADP-activated), along with ADP, collagen or ristocetin-induced aggregation in type II hyperlipidemic patients.

Material and methods

The study group included 70 persons: 50 patients affected by type II hyperlipidemia without concomitant diseases and 20 healthy volunteers. The effects of 8-week statin treatment (atorvastatin 10 mg/day, simvastatin 20 mg/day, or pravastatin 20 mg/day) on platelet activation were evaluated.

Results

Regardless of the type of statin, a significant decrease in ADP-induced platelet aggregation was observed: for atorvastatin 50.6 ±12.8% vs. 41.1 ±15.8% (p < 0.05), for simvastatin 57.2 ±18.0% vs. 44.7 ±22.1% (p = 0.05), and for pravastatin 55.8 ±19.5% vs. 38.8 ±23.3% (p < 0.05). There was no significant effect of statins on collagen or ristocetin-induced platelet aggregation and adhesion.

Conclusions

Therapy with statins beneficially modifies ADP-induced platelet aggregation in patients with hyperlipidemia and does not affect spontaneous or ADP-induced platelet adhesion to fibrinogen and platelet aggregation induced by collagen or ristocetin.     

Cloning,expression, and characterization of salivary apyrase from <Emphasis Type="Italic">Aedes albopictus</Emphasis>

Apyrases (ATP diphosphohydrolase) hydrolyze the phosphodiester bonds of nucleoside tri- and diphosphates to orthophosphate and mononucleodides. They can inhibit platelet activation by depletion of adenosine diphosphate. In the current study, the Escherichia coli expression vector pET-19b equipped with an N-terminal histidine tag was applied to express the apyrase of Aedes albopictus. The gene-coding mature apyrase protein was amplified by RT-PCR and cloned into pET-19b. Soluble apyrase protein with high purity was successfully obtained by utilization of the suitable renaturation approach and Ni-NTA purification column. Four monoclonal antibodies to apyrase from A. albopictus were produced in male BALB/c mice immunized with the renatured apyrase. Using immunofluorescence assay and immunoblotting analysis, recombinant apyrase showed fine consistency with native apyrase. From kinetic analysis, it had a K _m of 11.6 μM and V _max of 1.02 nM/S/μg protein for adenosine triphosphate. Adenosine diphosphate-induced platelet aggregation was inhibited by approximately 6% when 0.4 μM recombinant apyrase was added and by about 9.5% when the concentration of recombinant apyrase was 0.8 μM. The effect on platelet aggregation was dose dependent. In conclusion, the apyrase of A. albopictus was cloned and expressed highly in the E. coli expression system. Recombinant apyrase protein showed biological activity, and anti-apyrase monoclonal antibody was also prepared.     

肾上腺功能水平对脑缺血再灌注大鼠海马bax和bcl-2基因表达的影响

目的：观察肾上腺功能水平对大鼠脑缺血再灌注后海马bax和bcl-2基因表达的影响。方法： 成年雄性Wistar大鼠36只随机分为单侧肾上腺切除组（ADX）、单侧肾上腺切除+注射糖皮质激素组（GC）和假手术对照组（sham）。所有大鼠在单侧肾上腺切除或假手术14 d后行全脑再灌注手术。每组大鼠分别于再灌注后1 h、4 h、8 h、24 h随机取3只断颈处死，取海马组织提取总RNA，RT-PCR半定量检测c-fos、bax和bcl-2的表达情况。另设3只大鼠为正常组（normal）。结果： c-fos、bax表达水平在ADX、GC、sham 3组之间无统计学差异（P>0.05）。Sham组bcl-2表达水平明显高于ADX组和GC组(P<0.05)，而ADX组与GC组bcl-2表达无统计学差异(P>0.05)。Sham组bax/bcl-2明显低于ADX组和GC组(P<0.05)，而ADX组与GC组间bax/bcl-2无统计学差异(P>0.05)。结论: 肾上腺功能水平对脑缺血再灌注后大鼠海马组织c-fos和bax基因表达无影响，但肾上腺功能低下会导致脑缺血再灌注后大鼠海马组织bcl-2基因表达下调，bax/bcl-2比值升高，可能促进海马组织细胞发生凋亡，补充糖皮质激素对此bcl-2基因表达下调无纠正作用，表明还有其它机制存在。     

Glucosamine,a naturally occurring amino monosaccharide,suppresses the ADP-mediated platelet activation in humans

Objective: To evaluate the anti-thrombotic action of glucosamine, a naturally occurring amino monosaccharide, platelets were stimulated with ADP in the presence of glucosamine, and its effects on platelet functions were examined.Materials and Methods: Human platelet-rich plasma was stimulated with 2.5 M ADP in the presence of glucosamine (0.01 ~ 1 mM) or other aminosugars (N-acetyl-glucosamine, galactosamine or N-acetyl-galactosamine, 1 mM), and platelet aggregation was monitored. Furthermore, the effects of glucosamine on the thromboxane A2 production, release of granule contents, intracellular calcium mobilization and phosphorylation of Syk (a 72 kD protein tyrosine kinase) were evaluated following ADP-stimulation. In addition, the binding of [³H] ADP to its receptors was examined.Results: Glucosamine (>0.01 mM) dose-dependently suppressed platelet aggregation in response to ADP (p < 0.05), whereas N-acetyl-glucosamine, galactosamine or N-acetyl-galactosamine (1 mM) did not affect the ADP-induced platelet aggregation. Furthermore, glucosamine (>0.1 mM) inhibited the extracellular release of granule contents (ATP and platelet factor 4) and production of thromboxane A₂ from ADP-stimulated platelets (p < 0.05). Moreover, glucosamine significantly repressed the intracellular calcium mobilization at >0.1 mM and phosphorylation of Syk at >0.01 mM upon ADP-stimulation (p < 0.05). In addition, glucosamine (>0.1 mM) inhibited the binding of ADP to its receptors (p < 0.05).Conclusion: Glucosamine is able to suppress platelet aggregation, release of granule constituents, thromboxane A₂ production, calcium mobilization and phosphorylation of Syk possibly via the inhibition of ADP-binding to the receptors. Glucosamine could be expected as a novel anti-platelet agent for thrombotic disorders due to its suppressive actions on platelets.Received 18 June 2004; returned for revision 11 July 2004; accepted by M. Katori 9 August 2004     

狼疮肾炎患者外周血单个核细胞IL-17信号转导途径的初步研究

目的:了解IL-17及其信号转导成分JNK活化状态在狼疮肾炎(LN)发病机制中作用。方法:活动期LN病人15例,分离、培养外周血单个核细胞(PBMC);ELISA法检测上清液IL-6蛋白水平;逆转录多聚酶链反应法检测IL-6mRNA水平;蛋白印迹法检测JNK活性。结果:IL-17刺激下,LN患者PBMC反应性明显增强,各浓度点IL-6蛋白水平明显高于正常对照(均P<0.05)。LN患者PBMCIL-6mRNA表达水平明显高于对照组(无刺激培养:1.80±0.11vs0.36±0.07,P<0.01;刺激培养:3.21±0.24vs1.30±0.14,P<0.05);正常对照或LN患者刺激培养组PBMCIL-6mRNA表达水平均明显高于其无刺激培养组(对照:1.30±0.14vs0.36±0.07,P<0.05;LN患者:3.21±0.24vs1.80±0.11,P<0.01)。LN患者PBMCJNK活性水平明显高于对照组(无刺激培养:3.21±0.32vs1.00,P<0.01;刺激培养:4.82±0.39vs1.21±0.35,P<0.01);LN患者的刺激培养组PBMCJNK活性水平明显高于LN对照培养组(4.82±0.39vs3.21±0.32,P<0.01)。活动性LN病人PBMCJNK活性高低与其SLEDAI和IL-6mRNA水平均呈显著正相关。结论:IL-17介导LN患者PBMC过度表达、分泌IL-6可能是其参与LN的发病机制之一,JNK的活化在IL-17信号转导中具有重要作用。     

Benextramine逆转神经肽Y下调人血管平滑肌细胞LDL-R的表达

目的:探讨benextramine对神经肽Y(NPY)人血管平滑肌细胞(hVSMC)低密度脂蛋白受体(LDL-R)表达影响的逆转作用。方法:应用荧光免疫组化和激光扫描共聚焦显微技术,定量检测hVSMC的LDL-R表达的变化。Benextramine(B组)浓度设为10^-5mol/L,NPY设10^-8mol/L(N1组)、10^-7mol/L(N2组)、10^-6mol/L(N3组)和10^-5mol/L(N4组)4个浓度,培养时间分别为6、12、24和48h。结果:对照组LDL-R表达的平均荧光值较高,介于2564-2649之间,且各时段间均无显著差异(P>0.05)。各NPY组LDL-R表达的平均荧光值均明显低于对照组和各benextramine组(P<0.05),介于1639-2512之间,其荧光值随NPY浓度增加而下降,N1-N4组分别平均下降15.64%、20.31%、22.58%和27.42%,呈现一定的剂量依赖效应;同时其荧光值也随NPY作用时间的延长而下降,在6、12、24和48h时间段分别平均下降11.24%、21.29%、23.04%和30.38%,亦呈现一定的时间依赖效应。各benextramine组(包括B组、B+N1组、B+N2组、B+N3组及B+N4组)的LDL-R表达荧光值介于2554-2631之间,与对照组间差异无显著(P>0.05),且与NPY作用浓度及时间无关(P>0.05)。结论:NPY能导致hVSMC的LDL-R表达下降,benextramine可逆转该作用。     

Immunodiagnosis of Citrus leprosis virus C using a polyclonal antibody to an expressed putative coat protein

Citrus leprosis virus C (CiLV-C), a causal agent for citrus leprosis disease, is present in South and Central America and is a threat for introduction into the U.S. citrus industry. A specific, inexpensive and reliable antibody based detection system is needed for the rapid identification of CiLV-C. The CiLV-C is very labile and has not been purified in sufficient amount for antibody production. The p29 gene of CiLV-C genome that codes for the putative coat protein (PCP) was codon optimized for expression in Escherichia coli and synthesized in vitro. The optimized gene was sub-cloned into the bacterial expression vector pDEST17 and transferred into E. coli BL21AI competent cells. The expression of PCP containing N-terminal His-tag was optimized by induction with l-arabinose. Induced cells were disrupted by sonication and expressed PCP was purified by affinity chromatography using Ni-NTA agarose. The purified expressed PCP was then used as an immunogen for injections into rabbits to produce polyclonal antibody (PAb). The PAb specific to the expressed PCP was identified using Western blotting. The antibody was successfully used to detect CiLV-C in the symptomatic CiLV-C infected tissues using double antibody sandwich-enzyme-linked-immunosorbent (DAS-ELISA), indirect ELISA and dot-blot immunoassay (DBIA) formats.     

Comparative assessment of inoculum effects on the antimicrobial activity of amoxycillin-clavulanate and piperacillin-tazobactam with extended-spectrum β-lactamase-producing and extended-spectrum β-lactamase-non-producing Escherichia coli isolates.

A significant inoculum-size effect has been observed with piperacillin-tazobactam, and has been associated with β-lactamase production in extended-spectrum β-lactamase (ESBL) producers. This association has not been previously studied in the case of amoxycillin-clavulanate. Piperacillin-tazobactam and amoxycillin-clavulanate were compared, using high inocula of susceptible strains either harbouring ESBLs or not. Two non-ESBL-producing and 15 amoxycillin-clavulanate-susceptible and piperacillin-tazobactam-susceptible ESBL-producing Escherichia coli isolates, and their respective transconjugants, were tested in dilution susceptibility tests using standard and 100-fold higher inocula. Three ESBL-producing strains and E. coli ATCC 25922 were selected for time-kill studies using standard and high initial inocula. At high inocula, MICs of piperacillin increased >eight-fold for non-ESBL-producing strains, and MICs of piperacillin-tazobactam (8 : 1 ratio or with tazobactam fixed at 4 mg/L) increased>eight-fold for all ESBL-producing strains. However, amoxycillin MICs were not affected by a high inoculum with non-ESBL-producing strains, whereas the MICs of amoxycillin-clavulanate (2 : 1 and 4 : 1) increased ≤four-fold for ESBL producers, using the broth and agar dilution methods. In kinetic studies at a high inoculum, amoxycillin and amoxycillin-clavulanate were bactericidal against E. coli ATCC 25922, whereas piperacillin and piperacillin-tazobactam yielded decreases of <1 log₁₀ CFU/mL. Similarly, at a high inoculum, only amoxycillin-clavulanate was able to maintain bactericidal rates of killing over 24 h against the ESBL-positive E. coli isolates. The stability of amoxycillin-clavulanate and the contrasting results obtained with piperacillin-tazobactam against high inocula of ESBL-non-producing and ESBL-producing E. coli strains appear to be related to aspects other than the amount of β-lactamase production.