TEM-116型ESBL天然酶与重组酶的动力学特性研究

目的 研究TEM-116型ESBL天然酶与重组酶的动力学特性，并比较它们的差异。方法 采用紫外分光光度法检测酶促抗生素水解反应，以Lee-Wilson改良双倒数方程数据处理法进行数据处理，测定天然酶与重组酶的Km、Vmax及kcat，观察温度和pH对酶促反应的影响。结果 以Lee-Wilson改良双倒数方程数据处理法进行数据处理方便而准确地测定了天然酶与重组酶的Km与Vmax并计算出kcat,温度和pH对天然酶与重组酶酶促反应效应相似。天然酶与重组酶最优先的底物均为头孢哌酮，其次是头孢氨苄；对氨苄西林、阿莫西林、青霉素和哌拉西林有最高的催化效率。结论 天然酶与重组酶的动力学参数无显著差异。     

ESBL的检测、分型及产ESBL菌药敏分析

彩和双纸片试验,等电聚焦电泳及微量稀释法对临床标本中分离出的肺炎克雷伯氏菌,大肠埃杀氏菌中的产ＥＳＢＬ。菌的流行及产ＥＳＢＬ菌对１２种抗菌药物敏感情况进行。１９９８年３月－１９９８年１０月共分离出肺炎克雷伯氏菌２１６株,大肠埃工菌３１８株,从中筛选出产ＥＳＢＬ菌７７株,其中肺炎克雷伯氏菌５９株（２７．３％）,大肠埃希氏菌１８析。产ＥＳＢＬ菌所产β－内酰胺酶以ＰＩ７．６的酶为主,其次是ＰＩ８．２的酶     

肠杆菌科细菌的分布及其产ESBL菌株的耐药性变迁

目的:分析肠杆菌科细菌的分布及其产超广谱β-内酰胺酶(ESBL)菌株的耐药趋势,为临床合理选择抗菌药物提供依据。方法:对普宁市人民医院2013年临床标本中分离的926株肠杆菌科细菌及其产ESBL情况进行统计、分析。结果:该院肠杆菌科细菌分离率均在50%以上,尤以大肠杆菌和肺炎克雷伯菌感染分离率最高;肺炎克雷伯菌和大肠杆菌是产ESBL的代表菌种;肠杆菌科细菌对氨苄西林及头孢曲松的耐药率普遍保持在较高水平,但对亚胺培南和哌拉西林/他唑巴坦的耐药率较低;应用的抗菌药物主要为加酶抑制剂和第三代头孢菌素。结论:该院肠杆菌科产ESBL菌的耐药性在四个季度间无明显差异;ESBL介导的细菌耐药性复杂而难治,必须长期严密地监测产ESBL菌情况,合理采取干预措施,遏制产ESBL菌的流行。     

同时产CTX-M-22及TEM-1型β-内酰胺酶的阴沟肠杆菌耐药性分析

目的 了解临床分离阴沟肠杆菌株EC003产β-内酰胺酶的耐药特征和基因型。方法采用琼脂二倍稀释法对阴沟肠杆菌EC003进行MIC检测，酶提取物三维实验检测AmpC酶，双纸片法筛选及确证实验检测超广谱β-内酰胺酶（ESBLs），等电聚焦（IEF）电泳测定其等电点。以耐药质粒为模板进行PCR扩增，将β-内酰胺酶全编码基因克隆、表达，并对其扩增产物进行DNA序列测定和同源性分析。结果阴沟肠杆菌株EC003对所试多数β-内酰胺类抗生素耐药，表型检测AmpC酶为阴性。ESBLs阳性，IEF显示该菌产两种p1分别为8．7及5．4的β-内酰胺酶。PCR扩增产物DNA测序证实为CTX-M-22、TEM-1型β-内酰胺酶全编码基因，产TEM-1的克隆菌株仅对青霉素类耐药，产CTX—M-22的克隆菌株对所试多数β-内酰胺类抗生素耐药。对头孢噻肟的水解程度明显强于头孢他啶。结论临床分离阴沟肠杆菌株EC003同时产CTX—M-22和TEM-1型两种β-内酰胺酶，可导致对多数β-内酰胺类抗生素耐药。     

应用哌拉西林/他唑巴坦控制产ESBL菌株寄殖

目的 研究哌拉西林/他唑巴坦部分替代三代头孢菌素应用于临床血液病患者,防治院内产超广谱β-内酰胺酶(ESBL)的大肠埃希菌、肺炎克雷伯菌和产酸克雷伯菌的价值.方法 在哌拉西林/他唑巴坦临床干预前后,用美国临床实验室标准化委员会(NCCLS)推荐的方法,检测患者直肠拭子中是否存在上述3种产ESBL菌,比较前后两阶段的寄殖率.结果 哌拉西林/他唑巴坦干预后,3种ESBL菌寄殖率从32.1%降至19.6%(P＜0.05).结论 哌拉西林/他唑巴坦部分替代三代头孢菌素应用于临床血液病患者抗感染治疗,能显著减少院内产ESBL的大肠埃希菌、肺炎克雷伯菌和产酸克雷伯菌在肠道的寄殖.     

产"超-超广谱"β-内酰胺酶革兰阴性杆菌的耐药表型及基因分析

目的 首次分析本地区产超-超广谱β-内酰胺酶(super-spectrum β-lactamase,SSBLs)菌株的耐药性及分子流行病学现状.方法 收集自2008年1月～2009年6月本院从临床分离的对头孢西丁和头孢他啶耐药的革兰阴性杆菌105株,采用改良三维试验与质粒结合实验筛选产SSBLs菌株,通过药敏试验鉴定其耐药表型,运用PCR扩增检测其ESBLs基因型与AmpC酶基因型.结果 筛选出的2株产SSBLs菌株(1株阴沟肠杆菌和1株大肠埃希菌)均表现为多重耐药,仅对亚胺培南敏感.阴沟肠杆菌质粒编码的ESBLs基因型为TEM、CTX-M,AmpC酶基因型为ACT;大肠埃希菌质粒编码的ESBLs基因型为SHV、CTX-M,AmpC酶基因型为ACT.结论 本院存在产SSBLs革兰阴性杆菌引起的感染,但此类菌株尚未在本院传播流行,ACT型是SSBLs菌株质粒AmpC酶主要基因型.     

ChromID ESBL产色培养基检测大肠埃希菌ESBLs效果探讨

目的分析ChromID ESBL产色培养基对检测产超广谱β-内酰胺酶（ESBL）大肠埃希菌的灵敏度和特异度,为临床尽早隔离携带者,合理选择抗生素提供依据,对控制耐药菌株的播散和流行具有十分重要的意义。方法对临床分离大肠埃希菌菌株451株分别用ChromID ESBL产色培养基筛查ESBLs法和双纸片确证法进行对比试验。结果上述两检测方法无显著性差异（P〉0.05）,ChromID ESBL法的灵敏度=94.2%,特异度=85.8%。结论ChromID ESBL法能较好地筛查产ESBLs的大肠埃希菌。     

产ESBL菌株的检测及临床研究

目的了解产超广谱β-内酰胺酶(ESBL)菌株在医院的分离、分布的情况,以利于对产ESBL菌株的监控与治疗.方法采用Vitek-32全自动细菌仪鉴定和双纸片协同法进行ESBL检测.结果我院有22个科室检出ESBL菌株,大肠埃希氏菌及肺炎克雷伯氏菌的ESBL( )检出率分别达到36.3%和37.7%;目前没有发现对亚胺培南耐药的ESBL菌株.结论合理使用及严格限定抗生素的应用指征,对延缓细菌耐药性的产生至关重要.亚胺培南治疗对产ESBL细菌引起的感染效果最好.     

头孢吡肟对产ESBL大肠埃希菌所致泌尿道感染的临床疗效观察

目的了解头孢吡肟治疗泌尿道感染的疗效以及针对大肠杆菌所致泌尿道感染的疗效,探讨头孢吡肟对产ESBL大肠杆菌泌尿道感染的临床使用及其疗效。方法收集临床诊断之泌尿道感染病人,使用头孢吡肟2.0 ivgtt q12h治疗,疗程7~14d;对临床分离的大肠埃希菌按照CLSI标准用纸片扩散法进行确证实验了解产超广谱β-内酰胺酶情况,并测定其MIC值。分析其所致感染用头孢吡肟治疗的临床疗效。结果共纳入泌尿系感染病人168例,疗程结束痊愈率70.83%,有效率85.12%。临床共分离大肠埃希菌81株,其中产超广谱β-内酰胺酶22株。体外对头孢吡肟的敏感率77.27%(17/22),临床治疗有效率81.82%(18/22),体外药敏与临床疗效具有一致性。结论头孢吡肟治疗泌尿道感染临床疗效好。针对体外敏感的产ESBL大肠埃希菌感染也存在确实疗效,是除碳青霉烯和头孢菌素/酶抑制剂外的另一选择。     

超广谱β-内酰胺酶与碳青酶烯类抗生素

近年来，随着第三代头孢菌素的广泛应用，产超广谱β-内酰胺酶(ESBLs)导致的多重耐药越来越严重。ESBLs是革兰氏阴性杆菌的产物，主要由肠杆菌属中的肺炎克雷伯菌和大肠杆菌产生。该酶能水解窄谱青霉素类、头孢菌素类及单环β-内酰胺酶类抗生素，使三代头孢菌素和单酰胺类抗生素敏感性下降，而且对氨基糖苷类、喹喏酮类交叉耐药。     

葡萄糖氧化酶电极中酶固定化的研究

研究了以丝纤维为载体，三聚氯氰为交联剂固定化葡萄糖氧化酶膜的方法和条件。实验表明，用二氧六环－二甲苯（质量比１１）为三聚氯氰的溶剂，固定化酶膜效果较好。酶的偶联条件以温度２５℃、ｐＨ＝９、酶液浓度４ｍｇ／ｍｌ为最佳，此时固定化酶膜稳定性好，反复使用３０次酶膜活性保留９０％以上，贮存在离子强度较高的溶液中，酶膜的半衰期为４０～５０周。     

Effects of the characteristics of chitosan on controlling drug release of chitosan coated PLLA microspheres

Chitosan has been shown to be a biomaterial with good biocompatibility, and is highly biodegradable. This study investigated the effect of post-coating PLLA microspheres with different chitosans on the initial burst and controlling the drug release of the microspheres. Without chitosan, 19.2% of encapsulated lidocaine would release from PLLA microspheres within the first hour (R₁), and the time of 50% release (T₅₀     

Nasal delivery of insulin using chitosan microspheres

Nasal delivery of insulin is an alternative route for administration of this drug. The objective of this study was preparation of chitosan microspheres for insulin nasal delivery. After preparation of insulin chitosan microspheres by emulsification-cross linking process, the effect of chitosan quantity (200–400?mg), cross-linker type (ascorbic acid or ascorbyl palmitate) and amount (70–140?mg) were studied on the morphology, particle size, loading efficiency, flow and release of insulin from the microspheres by a factorial design. Optimized formulation was administered nasally in four groups of diabetic rats and their serum insulin levels were analysed by the insulin enzyme immunoassay kit and the serum glucose by the glucose oxidase kits. Insulin loading in microspheres was between 4.7–6.4% w/w, preparation efficiency more than 65% and mean particle size was 20–45?µm. In most cases, drug released followed a Higuchi model. Ascorbic acid caused an increase in stability, particle size and T_50% while decreased the loading efficiency and production efficiency. Increasing the chitosan content, increased particle size, flow and insulin release rate form the microspheres. The increase of cross-linking percentage decreased the flow and size of the microspheres while increase of cross-linking percentage promoted the stability and decreased DE₈% of insulin. Microspheres containing 400?mg of chitosan and 70?mg ascorbyl palmitate caused a 67% reduction of blood glucose compared to i.v. route and absolute bioavaliability of insulin was 44%. The results showed that chitosan microspheres of insulin are absorbable from nasal route.     

固定化超氧化物歧化酶的制备

目的 研究固定化超氧化物歧化酶(SOD)的制备方法。方法 分别以不同方法对铜锌超氧化物歧化酶(CuZnSOD)进行固定并比较其活力,对固定化方法进行相应的考察和优化。结果 以壳聚糖为载体,戊二醛交联法制备固定化SOD酶,酶活力和酶活回收率均较理想,固定化过程中的pH在6.0-8.2范围内,对固定化效果无明显影响;优化条件下制备的固定化铜锌超氧化物歧化酶,所得壳聚糖酶粉活力可达141 U·g-1,酶活回收率大于60％。结论 壳聚糖-戊二醛交联法可用于固定化超氧化物歧化酶粉的制备。     

Dissemination and persistence of a plasmid-mediated TEM-3-like beta-lactamase, TEM-139, among Enterobacteriaceae in Bulgaria

During a survey of extended-spectrum β-lactamases (ESBLs) in Bulgaria from 1996 to 2003, a TEM-3-like ESBL was detected in strains of Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii and Klebsiella oxytoca from three centres in three different towns. The nucleotide sequence of the cloned gene was identical to that of TEM-3, except for one substitution (C347A) causing an amino acid exchange at position 49 from leucine to methionine. This TEM-3 variant with both a unique nucleotide and amino acid sequence was designated TEM-139. Transformants producing TEM-3 or TEM-139 expressed identical β-lactam resistance phenotypes. TEM-139 was the only TEM-type ESBL detected in the surveyed hospitals (seven centres in three towns). TEM-139 is a natural variant of TEM-3 with an amino acid exchange without informational content, detectable only by molecular procedures, e.g. a nucleotide-specific polymerase chain reaction.     

5-Fluorouracil loaded chitosan microspheres for chemoembolization

In this study, chitosan microspheres were prepared by a suspension crosslinking technique. A petroleum ether/mineral oil mixture was used as the suspension medium which includes an emulsifier, e.g. Tween-80. Glutaraldehyde was used as the cross-linker. 5-Fluorouracil was incorporated in the matrix for the possible use of the microspheres in chemoembolization. The size andsize distributionof thechitosanmicrospheres variedinthesizerangeof 100-200mum, by changing the emulsifier concentration, stirring rate, chitosan/ solvent ratio and drug/chitosan solution ratio. In summary, the size and size distribution of the microspheres decreased when the emulsifier concentration and stirring rate were increased. Smaller microspheres with narrower size distributions were obtained when the chitosan/solvent ratio and drug/chitosan ratiowerelower. Itwas possibletoload thechitosanmicrospheres with 5-FU to a concentration of 10.4mg 5-FU/g chitosan. Around 60%of the loaded drug was released within the first 24h, then the release rate became much slower.     

壳聚糖对三草汤的絮凝工艺研究

目的研究壳聚糖对三草汤的絮凝工艺。方法采用壳聚糖絮凝法。以得膏率及总黄酮得率为指标。利用紫外分光光度法测定总黄酮含量,先通过单因素实验考察壳聚糖用量、絮凝温度、药液浓缩比三因素对絮凝效果的影响,确定因素范围,再利用正交试验确定絮凝的最佳工艺条件。结果优选工艺为絮凝剂加入量1．50g·L^-1,絮凝温度40℃。药液浓缩比1：3。结论壳聚糖絮凝法纯化三草汤的工艺简单易行．纯化效果好。安全性高。     

芹菜素壳聚糖微球的制备及体外释药研究

目的以芹菜素为模型药物、脱乙酰壳聚糖为药物载体,制备芹菜素壳聚糖微球,并测定微球中芹菜素的体外释放度。方法采用复乳-乳化化学交联法制备微球,正交试验优化微球制备的工艺,高效液相色谱法检测芹菜素含量。结果最佳工艺制备4批微球,形态良好,微球圆整,平均载药量为8.54%,平均包封率为69.69%,平均粒径为84.33μm。微球在pH 6.8和pH 7.4的磷酸盐缓冲液中释放36 h。结论所选制备工艺稳定,适用于芹菜素壳聚糖微球的制备,体外药物释放结果显示,微球具有良好的缓释效果。     

Synthesis and characterization of cross-linked chitosan microspheres for drug delivery applications

The present study deals with the synthesis and characterization of cross-linked chitosan microspheres containing an hydrophilic drug, hydroquinone. The microspheres were prepared by the suspension cross-linking method using glutaraldehyde as the cross-linking agent of the polymer matrix. Perfectly spherical cross-linked hydrogel microspheres loaded with hydroquinone were obtained in the size range of 20–100 μm. The effect of the degree of polymer cross-linking, chitosan molecular weight, chitosan concentration and amount of the encapsulated drug on the hydroquinone release kinetics was extensively investigated. It was found that slower drug release rates were obtained from microspheres prepared by using a higher initial concentration of chitosan, a higher molecular weight of chitosan or/and a lower drug concentration. Most importantly, it was shown that the release rate of hydroquinone was mainly controlled by the polymer cross-linking density and, thus, by the degree of swelling of the hydrogel matrix.