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.     

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.     

结核分枝杆菌实验室耐药诊断技术进展

结核病是由结核分枝杆菌感染引起的一种严重危害人类健康的传染病,随着抗结核药物的不断使用,耐药菌株不断增多,使得结核病流行的控制变得越来越严峻。耐药结核患者治疗方案的制定和改变依赖于实验室药敏检测的数据。传统的检测方法由于报告时间太长,无法及时给临床提供可靠的信息。因此,对于快速准确检测技术的需求正越来越迫切,新型的结核菌药敏快速检测技术将有很大的发展前景。     

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.     

胆囊恶性肿瘤合并消化道艾氏小杆线虫感染一例CSCD

患者男,84岁,江苏省无锡市惠山区人。因“间断右上腹隐痛不适2个月余”入院,否认既往特殊慢性病史。病程中患者皮肤瘙痒明显,可见局部散在丘疹。     

胸水γ干扰素特异性效应T细胞检测对结核性胸膜炎诊断的价值评估

目的探讨胸水中γ-干扰素特异性效应T细胞检测(T-SPOT.TB)对结核性胸膜炎的诊断价值。方法实验组:结核性胸膜炎患者35例,对照组:非结核性胸膜炎患者40例,分别检测其外周血及胸水特异性效应T细胞释放的γ干扰素,通过绘制受试者工作特征曲线(ROC曲线),比较其特异度、敏感度。结果胸水T-SPOT.TB试验对于结核性胸膜炎的诊断效果明显高于外周血,特异性,敏感性达90%以上。其ROC曲线下面积大于90%。结论胸水γ-干扰素特异性效应T细胞检测对于结核性胸膜炎的诊断有着较高的敏感性和特异性,有助于结核性胸膜炎的早期诊断和鉴别诊断。     

巨噬细胞移动抑制因子基因-173G/C多态性与社区女性HPV易感性研究

目的 探讨巨噬细胞移动抑制因子（MIF）基因-173G/C多态性与社区女性人乳头瘤病毒（HPV）易感性之间的关系，并分析不同基因型间MIF水平的差异。方法 收集本社区HPV感染者44例作为感染组，同期HPV阴性的健康女性46例作为对照组。采集研究对象宫颈脱落细胞检测HPV核酸，并采集外周血分离血浆检测MIF浓度；提取基因组DNA，采用聚合酶链式反应-限制性片段长度多态性（PCR-RFLP）检测MIF-173G/C基因多态性。频数计数法计算各组基因型和等位基因频率，并对MIF水平进行统计学分析。结果 HPV感染组与对照组MIF基因-173位点均以GG型为主，GG型与GC/CC型（OR=1.118,95%CI:0.485～2.614）、等位基因G与C(OR=1.313,95%CI:0.636～2.628)分布频率均无差异（P>0.05）。感染组与对照组MIF浓度分别为（0.952±1.111）ng/ml、（1.304±1.230）ng/ml，两组间无统计学差异（P>0.05）。GG型MIF浓度在感染组与对照组中分别为（1.270±1.310）ng/ml、（1.479±1.335...