The characteristics of enzyme kinetics of CTX-M-14 type extended-spectrum β-lactamase with Pro167 residue substitution

Objective To analyze and evaluate the characteristics of enzyme kinetics of CTX-M-14 type extended-spectrum β-lactamase(ESBL) with Pro167 residue substitution. Methods By molecular cloning and PCR techniques, CTX-M-14 gene was directionally cloned into plasmid pET28a( + ) from a clinical E. coli isolate and formed an expression vector to transform competent E. coli BL21 (DE3 ). Prol67 residue substitutions of P167G, P167Q, P167S and P167T were introduced to CTX-M-14 by site-directed muta-genesis based on overlap extension PCR with the former recombinant plasmid as PCR template, respectively.The wild-type CTX-M-14, recombinant CTX-M-14 protein and its variants were expressed and purified, then their steady-state kinetic parameters (Kcat, Km and Kcat/Km ) against β-lactam antibiotics were determined.Results The kinetic parameters of wild-type and recombinant CTX-M-14 had no statistically significant differences (P>0.1). The 1/Km, Kcat and Kcat/Km values of P167S variant against ceftazidime were 16-fold, 2.87-fold and 43.6-fold higher than those of recombinant CTX-M-14, respectively, and the Kcat/Km value of P167S variant against penicillin, ampicillin, cefazolin, cefuroxime, ceftriaxone, cefotaxime decreased( < 0.05). Compared with the kinetic parameters of recombinant CTX-M-14, the kinetic parameters of P167T variant against ceftazidime had no significant change, but the Kcat values of P167Q and P167G variants decreased dramatically(P<0.01). Conclusion There was no difference between the enzyme activities of wild-type and recombinant CTX-M-14. P167S variant could not only promote the enzyme affinity of CTX-M-14 to ceftazidime but also improve the conversion rate of enzyme-substrate complex in the ceftazidime hydrolysis. The comparison of the kinetic parameters of CTX-M-14 and its variants with Pro167 residue substitution showed that the increased activity of CTX-M ESBL variants against ceftazidime could not be simply explained with the enlarged cavity in active site that may be caused by the replacement of Pro167 residue by smaller amino acids.     

高白细胞抗凝血放置时间对血糖测定的影响

我们在工作中发现一例慢性粒细胞白血病患者白细胞总数为400×10~9/L 以上,粒细胞92～96%,晨常规抽血查空腹血糖,血液在室温(25℃)放置后,三次空腹血糖分别为2.72mmol/L、0.67mmol/L、1.53mmol/L,但临床无低血糖症状.     

SHV-4型ESBL在pET26b/BL(DE3)中的表达纯化及其特性研究

目的:表达SHV-4型超广谱β-内酰胺酶,对纯化的SHY-4酶进行特性研究.方法:将SHV-4基因克隆到pET26b表达质粒,重组质粒转入E.coli BL21(DE3)构建工程菌,重组工程菌经摇瓶发酵后获得种子菌,种子菌转入发酵罐发酵,当菌体密度OD600为0.8时,加入诱导剂IPTG至终浓度为1.0 mmol/L,于37℃培养6 h.培养液离心收获菌体沉淀,其超声裂解上清液进行Ni2 亲和层析,获取的SHV-4酶进行分子量、纯度、等电点、最适条件、米氏常数、催化常数等特性进行分析.结果:SDS-PAGE电泳,等电聚焦电泳及系列的酶动力学实验表明SHV-4酶分子量约为30 kD,纯度大于95%,等电点为7.9,最适pH值为6.0～8.0,最适温度为37℃,以头孢硝噻吩为底物测定的Km值为2.61×10-6 mol/L,Kcat为258 s,Kcat/Km为9.88×107 mol-1·s-1·vL.结论:重组的SHV-4酶初步满足制成商品酶的条件.     

耐甲氧西林金黄色葡萄球菌体外抗菌活性研究

为给临床合理选用抗生素和有效控制耐甲氧西林金黄色葡萄球菌（ＭＲＳＡ）的感染提供依据，对ＭＲＳＡ的耐药特点进行了研究。测定了１７种抗生素对临床分离的７５株金黄色葡萄球菌的最低抑菌浓度和β－内酰胺酶。结果表明，临床分离菌的６６．７％为ＭＲＳＡ，ＭＲＳＡ中产β－内酰胺酶菌株占９２％。所有ＭＲＳＡ对青霉素Ｇ、氨苄青霉素和洁霉素耐药，但皆对万古霉素敏感，对丁胺卡那霉素、氟哌酸、氟嗪酸、环丙氟哌酸、头孢哌酮等敏感率大于５５％。ＭＲＳＡ耐抗生素种类数为５到１６种不等。对苯唑青霉素耐药水平越高，则耐抗生素种类数也越多，二者呈显著正相关（ｒ＝０．９３５３，Ｐ＜０．００５）。建议治疗ＭＲＳＡ感染首选万古霉素。     

高铁氰化血红蛋白参考标准品及血红蛋白控制物制备方法的改进

血红蛋白(Hb)测定自1964年国际血液学标准化委员会(ICSH)推荐标准化方法和氰化高铁血红蛋白(HiCH)参照品的制法及规格以来,被我国越来越多的实验室所采用,准确性也有所提高.但简便、准确和长期稳定的制备HiCN 参考标准及控制液的方法国     

体外抗菌素后效应的方法学及其对临床金黄色葡萄球菌的研究

本文报告了PAE 方法学及其对临床金葡菌的研究结果.氨苄青霉素、头孢雷定、庆大霉素和四环素对金葡菌ATCC25923的PAE 具有可接受的天内和天间重复性。临床11株β-内酰胺酶阴性金葡菌的测定结果中,将PAE<20min 作无意义处理,则抑制蛋白质和RNA 合成的庆大霉素和四环素之PAE 均值明显高于β-内酰胺类的氨苄青霉素和头孢雷定(P<0.01),而类内种间不具有显著性差异(P>0.05),该结论与传统敏感度无关。PAE<20min 的3株MIC 敏感菌株提示由这些菌株引起的临床感染宜持续给药。PAE 高达72min 的SA11菌株尽管对氨苄青霉素耐药,可是MIC 仅为4.0μg/ml 的临床剂量,故预示良好的疗效,相比之下,对四种药物均具较长PAE的SA1。因MIC 为临床剂量难以达到的高度耐药水平,所以提示了不良的疗效.这些结果均说明:PAE 与MIC 的联合应用对确定给药剂量和方式以及评价抗菌素的全面敏感性具有深远的意义.     

大肠埃希菌16S rRNA甲基化酶基因的接合传递及其与整合子的相关性

目的 对临床分离的多重耐药（MDR）大肠埃希菌株的16S rRNA甲基化酶基因特征与接合传递效率进行研究,探讨其与整合子的相关性。 方法 136株MDR大肠埃希菌经PCR筛检16S rRNA甲基化酶基因armA、rmtA、rmtB、rmtC、rmtD;对阳性菌株作整合酶基因intI1、intI2和intI3检测,并扩增Ⅰ类整合子可变区插入片段,对扩增产物进行测序与鉴定所含耐药基因盒;以阳性菌株为供体菌,耐叠氮化钠大肠埃希菌J53为受体菌进行接合试验,并结合质粒图谱对16S rRNA甲基化酶基因进行初步定位。 结果 在136株多重耐药大肠埃希菌中,共检出16S rRNA甲基化酶阳性菌12株（8.8%）,其中,armA阳性3株（2.2%）,rmtB阳性10株（7.4%）,未检出rmtA、rmtC、rmtD基因。阳性菌株均只含Ⅰ类整合子,对其可变区扩增片段（1 000～2 300 bp）的测序结果显示,该区域含有多种耐药基因盒,但不含16S rRNA甲基化酶基因。接合试验与质粒图谱结果初步表明armA和rmtB编码基因位于约23 000 bp的质粒上,接合试验的耐药质粒传递率高达83.3%(10/12)。 结论 在MDR大肠埃希菌中,armA和rmtB编码基因位于约23 000 bp质粒上,其中,rmtB为优势基因,接合试验和质粒图谱证明该类耐药质粒很容易在同种菌间传播。Ⅰ类整合子与16S rRNA甲基化酶基因虽然存在于同一菌体内和/或同处于一个质粒上,但整合子基因盒对该类基因的捕获率很低或根本不捕获。     

Pro167位点突变型CTX-M-14超广谱β-内酰胺酶的动力学特征

目的 对Pr0167位点突变型CTX-M-14超广谱β-内酰胺酶(ESBL)的动力学特征进行分析与评价.方法 以携带CTX-M-14基因的大肠埃希菌临床株为模板,克隆目的 基因,重组工程菌,并表达CTX-M-14型ESBL.进而采用基于重叠延伸PCR法的定点突变技术,将CTX-M-14型ESBL的167位点Pro(P)分别突变为Gly(G)、Gln(Q)、Ser(S)和Thr(T),重组构建P167G、P167Q、P167S和P167T四株突变型的CTX-M-14工程菌.表达与纯化野生型、重组型和突变型CTX-M-14型ESBL,检测其水解β-内酰胺类抗菌素的酶动力学参数(Kcat、Km和Km).结果 对野生型与重组型CTX-M-14型ESBL的酶动力学参数进行配对t检验,结果显示两者Kcat(t=1.796,P=0.123)、Km(t=0.559,P=0.596)、Kcat/Km(t=0.893,P=0.406)间的差异无统计学意义(P>0.1).与重组CTX-M-14型ESBL相比,P167S突变型酶对头孢他啶的Km值大幅降低,为突变前的1/16(8.39/134.85);Kcat和Km值分别为突变前的2.87倍(1.81/0.63)和43.6倍(0.218/0.005);且对青霉素、氨苄西林、头孢唑啉、头孢呋辛、头孢曲松和头孢噻肟的Kcat/Km值都呈现出显著减小的趋势(P<0.05).与重组CTX-M-14 ESBL相比,P167Q和P167G突变型酶对头孢他啶的Kcat值亦大幅减小(P<0.01),而P167T突变型对头孢他啶的酶动力学参数无明显变化.结论 重组型与野生型CTX-M-14 ESBL之间各项酶动力学参数的差异无统计学意义(P>0.1).而P167S位点的突变,不仅增强了CTX-M-14型ESBL对头孢他啶的亲和力,也加快了酶-底物复合物的转换速率.通过对Pr0167位点4种突变型酶的动力学参数比较,初步说明突变型CTX-M ESBL对头孢他啶所具备的高水解活性机制,不能简单地归结于Pro167位点被小侧链氨基酸残基取代而导致酶活性中心空间扩大的解释.

Abstract：

Objective To analyze and evaluate the characteristics of enzyme kinetics of CTX-M-14 type extended-spectrum β-lactamase(ESBL) with Pro167 residue substitution. Methods By molecular cloning and PCR techniques, CTX-M-14 gene was directionally cloned into plasmid pET28a( + ) from a clinical E. coli isolate and formed an expression vector to transform competent E. coli BL21 (DE3 ). Prol67 residue substitutions of P167G, P167Q, P167S and P167T were introduced to CTX-M-14 by site-directed muta-genesis based on overlap extension PCR with the former recombinant plasmid as PCR template, respectively.The wild-type CTX-M-14, recombinant CTX-M-14 protein and its variants were expressed and purified, then their steady-state kinetic parameters (Kcat, Km and Kcat/Km ) against β-lactam antibiotics were determined.Results The kinetic parameters of wild-type and recombinant CTX-M-14 had no statistically significant differences (P>0.1). The 1/Km, Kcat and Kcat/Km values of P167S variant against ceftazidime were 16-fold, 2.87-fold and 43.6-fold higher than those of recombinant CTX-M-14, respectively, and the Kcat/Km value of P167S variant against penicillin, ampicillin, cefazolin, cefuroxime, ceftriaxone, cefotaxime decreased( < 0.05). Compared with the kinetic parameters of recombinant CTX-M-14, the kinetic parameters of P167T variant against ceftazidime had no significant change, but the Kcat values of P167Q and P167G variants decreased dramatically(P<0.01). Conclusion There was no difference between the enzyme activities of wild-type and recombinant CTX-M-14. P167S variant could not only promote the enzyme affinity of CTX-M-14 to ceftazidime but also improve the conversion rate of enzyme-substrate complex in the ceftazidime hydrolysis. The comparison of the kinetic parameters of CTX-M-14 and its variants with Pro167 residue substitution showed that the increased activity of CTX-M ESBL variants against ceftazidime could not be simply explained with the enlarged cavity in active site that may be caused by the replacement of Pro167 residue by smaller amino acids.     

微量肉汤稀释法测定MIC的评价

采用抗生素最低抑菌浓度（ＭＩＣ）的微量肉汤稀释法，以１５种抗生素组合成适用于革兰阳性菌、革兰阴性菌和非发酵菌的３种微量稀释法药敏板，并介绍了具体的制备方法。本法对３株ＡＴＣＣ质控菌的ＭＩＣ测定值与质控中位数的±１稀释度符合率室内、室间分别为９０．７％和９０．９％，提示良好的方法学性能和准确度。６０株临床分离菌用本法与常量肉汤稀释法作１４４０对次测定，ＭＩＣ±１稀释度的符合率高达９２．４％。对抗生素最低杀菌浓度测定亦获得令人满意的结果。总之，微量法是易为临床实验室接受的一种可靠的ＭＩＣ测定法。