TEM-116型超广谱β-内酰胺酶对牛奶中抗生素的清除

目的利用TEM-116型超广谱β-内酰胺酶能降解具有β-内酰胺环的抗生素的特性清除牛奶中的此类抗生素。方法DelvotestSP试剂盒检测三个温度条件下所需的TEM-116型酶的剂量。结果37℃，反应0．5h，1．032IU／L的TEM-116型酶能把牛奶中青霉素G清除；23℃，0．5h，1．29IU／L的TEM-116酶能把牛奶中的青霉素G清除；4：C，8～12h，0．22IU／L的TEM-116酶能清除牛奶中的青霉素G。结论TEM-116型超广谱β-内酰胺酶在不苛刻的条件下很容易清除牛奶中残留的青霉素G。     

交联壳聚糖微球偶联胰蛋白酶研究

采用反相悬浮交联法,制备交联壳聚糖微球(CCTS),再以戊二醛为偶联剂,偶联胰蛋白酶.研究了胰蛋白酶偶联的最佳条件和偶联后酶的理化性质.结果表明:用终浓度为0.6%的戊二醛,在50℃下,先处理CCTS 6 h,再置冰浴加酶为12 mg/g,搅拌24 h,固定化酶活性回收率达63.4%.胰蛋白酶与壳聚糖微球偶联的最适温度为80℃、最适pH值为7、表观米氏常数(Km)为3.16 mmol/L.与壳聚糖载体相比,CCTS具有机械强度好、粒度均匀、耐酸性能好、对牛血清白蛋白非特异性吸附较弱的优点.     

星点设计-效应面法优化壳聚糖微球对凝血酶的固定化作用研究

目的 考察壳聚糖微球对凝血酶的固定化作用。方法 本研究以壳聚糖微球为载体,以戊二醛为交联剂将凝血酶固定于空白微球上,以固定化凝血酶的活性回收率为指标,采用星点设计-效应面法优化固定凝血酶的条件。结果 试验结果表明,壳聚糖微球固定凝血酶的最优条件是：凝血酶浓度69.32 U·mL^-1、戊二醛浓度0.18%、固定化时间1.88 h、固定化pH 7.08,凝血酶的活性回收率为81.55 %。结论 壳聚糖微球对凝血酶的固定化效果良好。     

环氧化磁性羟基微球的制备及在固定化青霉素G酰化酶中的应用

采用悬浮聚合法合成了一系列磁性羟基微球,以环氧氯丙烷活化后用于固定化青霉素G酰化酶.交联度为30%的微球固定化酶效果最优,并得到最适固定化条件为:给酶量2000u/g,反应时间24h,反应温度35℃,pH 8.0.制得固定化酶酶活力为782.2u/g.水解青霉素G钾盐的最适pH 8.5,最适温度45℃.     

单宁酶的固定化及性质研究

比较几种固定化载体，确定以壳聚糖为载体，用戊二醛作交联剂制得固定化单宁酶。壳聚糖用量0．1g，用3％戊二醛5ml交联4h，然后加入酶58．4u，于4℃反应4h，固定化酶活回收率可达73％。单宁酶经固定化后，热稳定性、pH稳定性及最适温度均有所提高，最适pH降低。     

壳聚糖固定化酵母蔗糖酶的研究

用壳聚糖作吸附剂、戊二醛作交联剂,对酵母蔗糖酶进行固定化研究,同时用琼脂糖固定化的蔗糖酶和游离酶与其进行比较。结果表明,壳聚糖固定化蔗糖酶贮藏稳定性、对变性剂以及对温度的耐受性均明显优越,而Km值与琼脂糖固定化酶相当,但比游离酶明显增高,酶的最适pH三者相近。研究表明,壳聚糖固定化酶所需的戊二醛浓度为0.6%,交联时间不低于6 h。固定化蔗糖酶对变性剂(乙醇、脲)的耐受力明显高于游离酶。     

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。     

壳聚糖固定谷氨酸脱羧酶的研究

以壳聚糖为载体，戊二醛为交联剂。制备固定化谷氨酸脱羧酶(GDC)，研究固定化反应中pH值、戊二醛浓度、给酶量以及交联反应搅拌时间对固定化GDC活力的影响以及GDC固定化酶和自由酶的酶学特性。实验结果表明：壳聚糖固定化GDC的最适pH4．4．最适温度55℃。与自由酶相比。具有较好的热稳定性、贮存稳定性及可操作性；在米胚芽生产制备γ-氮基丁酸中具有实用价值。     

青霉素G酰化酶在γ—氧化铝上的吸附交联固定化

以γ-氧化铝为载体,利用戊二醛对巨大芽孢杆菌(Bacillus megaterium)Bp931胞外青霉素G酰化酶进行吸附交联固定化.酶活力回收为31%,在37 C、pH8.0的条件下,固定化酶对青霉素G的活力为304IU/g;作用于青霉素G的表观米氏常数Km值为3.23×10-2M.该固定化酶可耐高离子强度洗涤,连续合成头孢拉定(cephradine)10批次,酶活力剩余82%.     

壳聚糖珠固定化乳糖酶的条件及特性研究

目的研究乳糖酶的固定化。方法以壳聚糖珠(2.0 g)为载体,0.02%戊二醛为交联剂,考察固定化乳糖酶的条件及其特性。结果固定化条件:室温状态下,在pH 6.0、酶量0.2 mg/mL的酶液中固定12 h,固定化乳糖酶活力回收率为31.5%。乳糖酶、固定化乳糖酶的最适pH值分别为5.0~5.5,5.5~6.5;最适温度分别为40℃,50℃。结论固定化乳糖酶最适温度和pH值比溶乳糖高。     

Intra- and inter-generic plasmid-mediated spread of cephalosporin and aminoglycoside resistance amongst Klebsiella aerogenes K41 and other enterobacteria

Klebsiella aerogenes K41, resistant to third generation cephalosporins and aminoglycosides, was isolated from clinical samples of 153 in-patients. Blood cultures accounted for 24 (15.7%) of isolates. The MIC(90) of ceftazidime for the isolates of 84 patients was >512 mg/l and was reduced to 2.0 by 4 mg/l of clavulanic acid, but only to 64 by 4 mg/l of sulbactam. Isolates of K. aerogenes K41 produced extended-spectrum beta-lactamase (ESBL) SHV-5 and TEM-1, identified by isoelectric focusing. Plasmid profiles showed that co-dissemination of cephalosporin and aminoglycoside resistance, plus ESBL production, coincided with the acquisition of a 116-kb plasmid. This plasmid was transferable in vitro from K. aerogenes K41 to other serotypes and genera of the Enterobacteriaceae.     

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.     

Antimicrobial susceptibility of clinical isolates of Enterobacteriaceae producing complex beta-lactamase patterns including extended-spectrum enzymes

The antimicrobial susceptibility of 103 clinical isolates of Enterobacteriaceae to 11 antibiotics, was investigated, using a conventional inoculum size (5 x 10(5) CFU) and a higher inoculum size (5 x 10(8) CFU). All the isolates produced complex beta-lactamase patterns, including an extended-spectrum beta-lactamase (ESBL) of the TEM- or SHV-type plus other enzymes (a TEM-type or an SHV-type non-ESBL and/or a class C enzyme). The following repertoire of ESBLs was produced by the isolates: TEM-15, TEM-19, TEM-26, TEM-52, TEM-72, TEM-87, TEM-92, SHV-2a, SHV-5 and SHV-12, as assessed by sequencing. Production of the other enzymes was showed by analytical isoelectric focusing. Overall, meropenem was the most active agent and less influenced by inoculum size, while other beta-lactams showed a lower activity and a significant inoculum size effect. In conclusion, from its in vitro performance, meropenem could be considered as the last resource drug against strains producing complex beta-lactamase patterns including an ESBL.     

肺炎克雷伯菌产超广谱β-内酰胺酶基因分型研究

目的调查广州地区下呼吸道感染肺炎克雷伯菌产超广谱β-内酰胺酶(ESBL)的分离率及各种基因型的流行病学分布。方法收集广州地区13家医院2001-10～2002-12临床分离的肺炎克雷伯菌511株,采用美国临床实验室标准化委员会规定的ESBL表型筛选和确证试验确定ESBL的发生率、PCR扩增对产ESBL菌初步分型,然后对部分PCR扩增阳性产物测序,序列分析进一步确定基因型。结果肺炎克雷伯菌产ESBL分离率为40.1%。TEM型49株占21.9%,均为TEM-1型;CTX-M型59株占28.8%;SHV型96株占46.8%。结论SHV型是广州地区下呼吸道感染肺炎克雷伯菌产ESBL流行的常见基因型。     

Bactericidal activity of oral beta-lactam antibiotics in plasma and urine versus isogenic Escherichia coli strains producing broad- and extended-spectrum beta-lactamases

Bacteria harbouring extended-spectrum β-lactamases (ESBLs), derived by mutation from TEM-1, TEM-2 or SHV-1 β-lactamases, have been described world-wide. The in vitro activities of these enzymes against β-lactam antibiotics, including oral cephalosporins, are well recognised. The aim of this investigation was to assess the bactericidal activity of oral β-lactam antibiotics available in Croatia (amoxicillin/clavulanate, cephalexin, cefuroxime, cefadroxil and ceftibuten), in biological fluids against isogenic Escherichia coli strains producing broad-spectrum (TEM-1, TEM-2 and SHV-1) and extended-spectrum β-lactamases (SHV-2, SHV-3, SHV-4, SHV-5, SHV-12). Bactericidal activity of oral β-lactams in plasma and urine was tested in time-kill experiments and by determining bactericidal titres at different time intervals post-dose. The killing rate of antibiotics in urine was slower than in plasma, but faster than in Mueller–Hinton broth. High bactericidal titres in urine were only maintained throughout the whole dosing interval by ceftibuten against strains producing broad-, SHV-2 and SHV-3 β-lactamases. The older generation cephalosporins can be considered for the therapy of urinary tract infections caused by E. coli harbouring TEM-1, TEM-2 and SHV-1 β-lactamases but a shorter dosing interval is needed. Ceftibuten can be recommended with caution in ESBL producing E. coli except those producing SHV-4, SHV-5 and SHV-12 that confer resistance to it. If these enzymes are produced, fluoroquinolones or carbapenems could be considered.     

CTX-M-37 in Salmonella enterica serotype Isangi from Durban, South Africa

Beta-lactamase-mediated resistance was investigated in 59 putative extended-spectrum beta-lactamase (ESBL)-positive Salmonella spp. from the paediatric ward of a tertiary hospital in Durban, South Africa. Three Salmonella enterica serotype Isangi cultured from stool samples were multidrug resistant, with susceptibility only to meropenem, piperacillin/tazobactam and cefoxitin. Isoelectric focusing revealed beta-lactamases with isoelectric points of pI 5.8, 6.8 and 7.2. Sequencing identified beta-lactamases CTX-M-37 and TEM-1. To our knowledge, this is the first report of CTX-M-37 from S. enterica serotype Isangi in South Africa.     

Molecular epidemiology of Enterobacteriaceae producing extended-spectrum beta-lactamase in a French university-affiliated hospital

We conducted a retrospective study to investigate the epidemiology of Enterobacteriaceae producing extended-spectrum beta-lactamase (ESBLE) in our hospital. We determined the occurrence of extended-spectrum beta-lactamase (ESBL) in Enterobacteriaceae over a 2-year period. We also characterised ESBLs by isoelectric focusing (IEF) and investigated the epidemiological relatedness of EBLSE by pulsed field gel electrophoresis (PFGE). During this period, 70 patients were colonised/infected with one or several strains of EBLSE, giving a crude incidence of 0.095 per 1000 patient-days. We found that ESBL-producing Enterobacter aerogenes were the main source of ESBLE dissemination. Indeed, 59.5% of ESBLE were E. aerogenes and 21.9% of the other ESBLE resulted from a plasmid transfer originating from E. aerogenes. IEF and PFGE analysis demonstrated that the dissemination of ESBL from E. aerogenes in our hospital was due to a single clone that always harbours TEM-24. This emphasises the importance of standard contact isolation precautions and the early detection of ESBLE-colonised patients in high risk departments like intensive care units.     

肺炎克雷伯菌超广谱β-内酰胺酶基因型研究

目的探讨我院肺炎克雷伯菌中超广谱β-内酰胺酶（ESBLs）基因分布规律。方法对20株经双纸片试验确证为ESBLs表型阳性的肺炎克雷伯菌进行bla TEM-1、blaSHV-1、CTX—M-1组、TOHO-1组等4种基因PCR扩增，并对16株blaSHV-1基因PCR扩增阳性的菌株进行基因序列测定，在Internet网上与GenBank中的已知序列进行核苷酸相似性分析，并进行编码基因对位和氨基酸序列对比分析。结果产ESBLs肺炎克雷伯菌中blaTEM-1，blaSHV-1，CTX—M-1组等3种基因扩增阳性率分别是50．0％、95．0％、20．0％。16株肺炎克雷伯菌中有4株序列与SHV—1a（序列号：X98101，74→934）氨基酸序列完全相同；有3株序列与SHV-2（序列号：AY570959，42→812）100％相同；有2株序列与SHV-11（序列号：AY293069，41→817）100％相同；有4株序列与SHV-27（序列号：AF293345，2→821）氨基酸序列完全相同；有1株序列与SHV-28（序列号：AF538324，12→823）100％相同；有2株序列在GenBank中未找到与之完全相同的序列。结论本地肺炎克雷伯菌中有SHV—1a、SHV-11、SHV-28广谱β-内酰胺酶基因和SHV-2、SHV-27 ESBLs基因存在。     

Carriage of genes for various extended-spectrum β-lactamases: a novel resistance strategy of Klebsiella pneumoniae in Poland

Among 110 randomly sampled strains from a collection of 247 extended-spectrum β-lactamase (ESBL)-producing clinical isolates of Klebsiella pneumoniae collected from hospitalised children in three paediatric hospitals in Poland, 64 strains (58.2%) with multiple ESBLs were found, including five non-clonal strains (4.5%) harbouring bla genes for ESBLs of three families (CTX-M, SHV and TEM). This is the first report of the emergence of triple ESBL-producing K. pneumoniae in Poland. In addition, K. pneumoniae strains harbouring bla genes for TEM-130 and TEM-132 ESBLs were detected in Poland for the first time. Epidemiological analysis of the multiple ESBL-producing K. pneumoniae isolates by pulsed-field gel electrophoresis (PFGE) revealed a relatively high genetic diversity between isolates producing the same combination of enzymes. Clonally related strains were uncommon.