CTX-M-38型超广谱β-内酰胺酶多克隆抗体的制备及应用
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摘要
广谱抗菌药物,尤其是第三、四代头孢菌素的应用,为临床抗感染治疗作出贡献的同时,也逐步使其面临着严峻的考验。抗菌药物的不合理使用,对病原菌可形成选择压力并筛选出多重耐药菌株；多种基因转移元件的存在及其菌间频繁传递,可致多重耐药性的传播与流行。临床抗感染治疗时常面对无药可用的境地。探究菌株多重耐药机制,涉及产生水解酶与钝化酶、主动外排、药物作用靶位改变以及胞膜渗透性改变等,而最为关键的是产生了超广谱β-内酰胺酶。
自1983年首例产超广谱β-内酰胺酶菌株发现以来,产酶菌株呈世界范围内流行。菌株种类涵盖肠杆菌科、非发酵菌科细菌等；单菌株携带酶型存在地域性分布特征；各型别酶抗药活性间存在一定差异。明确各地区超广谱β-内酰胺酶流行型别抗药活性,早期完成产酶菌株分离、鉴定,对于正确指导临床抗感染治疗,最终降低产酶菌株的产生几率及阻断产酶菌株流行意义重大。
抗菌药物应用习惯差异,造成各地域间超广谱β-内酰胺酶流行型别不同。在我国大部分地区临床分离菌株所产酶型中,CTX-M型超广谱β-内酰胺酶占有相当比例。该型酶由德国学者Bauemfeind首先发现(CTX-M-1),目前已有数十种亚型,其分布地域及宿主菌谱极为广泛。CTX-M型酶水解底物为青霉素类、第一、二代头孢菌素,部分型别可水解头孢吡肟。基于酶蛋白结构构象特征以及药物自身空间位阻差异,CTX-M型酶对头孢噻肟的水解能力显著高于头孢他啶,部分菌株体外试验对头孢他啶显示高度敏感。根据氨基酸序列同源性分析结果,可将CTX-M型超广谱β-内酰胺酶分为5组,各组间氨基酸序列同源性小于90%,组内性大于94%。各组间DNA突变位点可位于编码基因的任一部位。进一步研究表明,尽管CTX-M型酶各亚型间核苷酸序列差异较大,但从酶蛋白结构上看,各突变位点多远离酶活性中心,对于酶蛋白抗药活性影响不大。故各亚型酶均具有CTX-M型酶的共有特性。基于此,明确一种型别CTX-M型酶的耐药谱,对于其他型别抗药活性的抑制具有指导价值。
目前,临床实验室对于产酶菌株的鉴定主要采用三种方式,采用双纸片协同试验初步筛选阳性菌株；采用酶抑制剂增强试验/E-test试验进行确证；采用分子生物学试验进行快速特异性鉴定。前两者均需完成菌株的分离培养,耗时较长,难以满足临床简单快速的要求。后者试验费用较高,且实验软、硬件要求高,一般实验室难以开展。
为了解决上述问题,作者采用双纸片协同及酶抑制剂增强试验筛选、收集46株产超广谱β-内酰胺酶大肠埃希菌；采用PCR技术克隆CTX-M型酶表达基因；采用基因重组技术构建CTX-M表达载体并表达；采用液体稀释法完成抗药活性检测。同时,通过免疫小鼠获得超广谱β-内酰胺酶特异性抗体；设计完善酶联免疫吸附试验,建立CTX-M型超广谱β-内酰胺酶的快速鉴定方法。通过上述试验,在明确CTX-M型酶抗药活性的同时,可获得其多克隆抗体,用于快速鉴定,乃至于被动免疫治疗。
本研究包括以下三个部分。
第一部分CTX-M-38型超广谱p-内酰胺酶的表达、定位及其抗药活性研究
方法
1.采用双纸片协同试验及酶抑制剂增强试验,对46株临床分离多重耐药大肠埃希菌进行超广谱β-内酰胺酶筛选与确证。
2.依据CTX-M-38型超广谱β-内酰胺酶核苷酸序列特征,采用DNAstar分析软件,完成特异性PCR扩增引物设计。
3.采用PCR技术,完成CTX-M-38型超广谱β-内酰胺酶全长编码基因的克隆。
4.采用基因重组技术,完成pET-28a-CTX-M-38表达质粒的构建。
5.采用氯化钙转化法,完成pET-28a-CTX-M-38表达质粒在BL21大肠埃希菌中的转化。
6.采用卡那霉素抗性试验,检测BL21-pET-28a-CTX-M-38转化子构建效果。
7．采用双脱氧链终止法对质粒插入片段进行核苷酸序列分析,检测所克隆CTX-M-38型编码基因序列突变状况。
8.采用液体稀释法,检测BL21-pET-28a-CTX-M-38转化子抗药活性。
9.采用K-B法药物敏感试验,检测培养液上清及菌体裂解液中CTX-M-38型超广谱β-内酰胺酶活性,初步进行酶蛋白定位研究。
结果
1.阿莫西林表现耐药,阿莫西林/克拉维酸表现敏感,两者交界处有协同现象,抑菌环直径明显扩大,产超广谱β-内酰胺酶菌株筛选试验阳性。
2.头孢他啶抑菌环直径为8mm,头孢他啶/克拉维酸抑菌环直径为26mm,两者之差显著大于5mm,表明酶抑制剂增强试验阳性,产超广谱β-内酰胺酶菌株确证试验阳性。
3.琼脂糖凝胶电泳显示,PCR扩增阳性产物大小与设计结果相一致。
4.以转化子携带质粒为模板进行PCR扩增,约900bp位置处有目的条带,提示重组质粒构建成功。
5.转化子在含卡那霉素液体培养基中生长良好,表明转化子构建成功。
6.目的基因核苷酸序列与Bla CTX-M-38基因序列比对结果显示,两者在663位点处存在突变(T→A),但氨基酸序列上,两者221位均为丙氨酸,提示此处突变为同义突变。
7.K-B法药敏试验中,滴加上清及菌体裂解液的头孢噻肟纸片均显示耐药,但后者抗药活性较为显著,单纯头孢噻肟纸片显示敏感,提示表达产物主要分布于菌体中。
8.转化子对青霉素类药物包括哌拉西林、氨苄西林显著耐药(MIC>32μg/ml),对于头孢唑啉、头孢吡肟、头孢呋辛及头孢克洛MIC值>16μg/ml,表现为耐药。头孢噻肟及头孢曲松酶水解活性较强(MIC值均>32μg/ml)。头孢他啶MIC值为8μg/ml,表现为体外敏感。转化子对亚胺培南稳定敏感,MIC≤4μg/ml。酶抑制剂复合制剂中,哌拉西林/三唑巴坦敏感(MIC≤16/4μg/ml),而阿莫西林/棒酸、氨苄西林/舒巴坦均表现耐药(MIC>32/16μg/ml)。头孢哌酮／舒巴坦MIC为32/16μg/ml,表现为中介。氨曲南表现为敏感(MIC≤8μg/ml)。非内酰胺类抗菌药物中,庆大霉素、四环素、环丙沙星、左氧氟沙星及美满霉素均表现为耐药。
第二部分CTX-M-38型超广谱β-内酰胺酶多克隆抗体的制备
方法
1.采用IPTG诱导BL21-pET-28a-CTX-M-38转化子表达酶蛋白。
2.采用水煮裂解法,完成全菌蛋白提取。
3.采用超声破碎法,完成包涵体蛋白提取。
4.采用SDS-PAGE蛋白电泳技术,检测BL21-pET-28a-CTX-M-38转化子表达产物。
5.采用扩散洗脱法,回收与纯化CTX-M-38型超广谱β-内酰胺酶蛋白。
6.采用传统多克隆抗体制备方法,制备CTX-M-38型超广谱β-内酰胺酶蛋白特异性抗体。
7.采用间接酶联免疫吸附试验,检测所制备多克隆抗体效价。
8.采用辛酸沉淀法,对所制备的多克隆抗体进行纯化。
9.采用Western blot印迹杂交技术,进行多克隆抗体的特异性鉴定。
结果
1. SDS-PAGE蛋白电泳结果显示,BL21-pET-28a-CTX-M-38转化子培养液上清、全菌蛋白、包涵体蛋白泳道,在约30kDa处出现明显蛋白条带,目的酶蛋白分子量大小与设计相同,表明在IPTG诱导作用下,转化子可显著表达酶蛋白。
2. SDS-PAGE蛋白电泳后,各泳道酶蛋白量多少依次为包涵体蛋白、全菌蛋白以及培养液上清,提示转化子表达产物主要存在于包涵体内。
3.间接酶联免疫吸附试验结果显示,多克隆抗体1：1000至1：16000梯度稀释度检测结果均呈阳性。1：32000以后稀释度时无阳性结果出现,表明多克隆抗体效价为1：16000,也表明多克隆抗体制备成功。
4. Western blot印迹杂交技术检测结果显示,不同稀释度的抗体均与同一蛋白条带(约30kDa)反应,此条带与CTX-M-38型超广谱β-内酰胺酶蛋白大小相一致。提示研究中所制备多克隆抗体特异性较好。
第三部分CTX-M-38型超广谱β-内酰胺酶多克隆抗体的应用研究
方法
1.采用梅花形双向琼脂扩散试验,完成CTX-M-38型超广谱β-内酰胺酶蛋白与多克隆抗体反应最适比检测。其中选择4h培养物作为试验抗原浓度,系列抗体滴度采用倍比稀释法制备。
2.采用K-B法药物敏感试验,进行多克隆抗体抑制酶蛋白活性研究。
3.采用改良过碘酸钠法,进行酶标多克隆抗体的制备。
4.采用50%饱和硫酸铵沉淀法,进行酶标多克隆抗体的纯化。
5.依据双抗体夹心法原理,设计制备酶联免疫吸附试验检测试剂盒。
6.针对BL21-pET-28a-CTX-M-38转化子表达产物进行平行试验,检测批内变异,评价实验重复性。
7.与PCR技术进行平行对照试验,完成临床分离产酶菌株检测,评价方法特异性。
8.对27株产超广谱β-内酰胺酶大肠埃希菌(不携带CTX-M-38型超广谱β-内酰胺酶)进行检测,评价抗体交叉反应状况。
9.对产CTX-M-38型超广谱β-内酰胺酶菌株不同时间段培养液进行检测,明确超广谱β-内酰胺酶最适检测时间。
结果
1.结果显示,抗体效价为1∶64时,沉淀线出现在两反应孔中间位置,表明抗体与4h培养物反应的最适效价为1∶64。
2.K-B法药敏试验结果显示,抗体组抗菌药物抑菌环直径与对照组比较,p值小于0.01,两者结果有差异。表明CTX-M-38型超广谱β-内酰胺酶蛋白与多克隆抗体结合后,其活性受到一定程度的抑制。
3.多克隆抗体抑制效应表现为阿莫西林、头孢他啶、头孢吡肟、氨曲南等药物的抑菌环直径增大,菌株由耐药转变为敏感或MIC进一步减小。含酶抑制剂复合药物两组结果基本相同。头孢噻肟抑菌环两组结果均无变化。
4.对转化子表达产物30次平行试验结果显示,CV值为1.55%,提示本法批内变异小,具有较好的重复性。
5.146株产超广谱β-内酰胺酶大肠埃希菌中,PCR技术检测阳性率为4.11%,本法为7.53%,x2检测可知P>0.05,表明两种检测方法检测结果无差异,提示本法具有一定的特异性。
6.交叉反应试验中,27株临床分离产酶菌株中,有1株检测结果为阳性。表明所制备抗体具有交叉反应,实验结果存在假阳性现象。
7.超广谱β-内酰胺酶最适检测时间结果显示,宿主菌在培养2h后,即有酶蛋白产生。4h时酶量显著增多,8h、18h及24h酶蛋白量已超过检测线性范围,故吸光度改变不大。结合检验流程,本研究确定超广谱β-内酰胺酶最适检测时间为菌株培养4h。
结论
1. CTX-M-38型超广谱β-内酰胺酶主要定位于宿主菌包涵体内；转化子具有广泛抗药活性,对部分抗菌药物抗药活性与其他型别存在差异。
2.完成了CTX-M-38型超广谱β-内酰胺酶多克隆抗体的制备、纯化与鉴定,所制备多克隆抗体效价为1∶16000；抗体与CTX-M-38型超广谱β-内酰胺酶蛋白反应特异性较高。
3.多克隆抗体可抑制酶蛋白抗药活性；双抗体夹心酶联免疫吸附试验检测酶蛋白具有较好的重复性、特异性；临床分离菌株产超广谱β-内酰胺酶最佳检测时间为菌株培养4h。
With the usage of broad spectrum antibiotics, especially the third generation cephalosporins application, more infectious diseases were controlled and obtained better therapeutic efficacy. On the other hand, the overuse or abuse of antibiotics had made the clinic face a severe test gradually. Because selection pressure was formed, more and more multiple antibiotic resistant strains emerged recently. Many genetic transfer elements which contained resistant genes can transfer among the strains isolated from the clinic frequently, as result, the antibiotics resistance was spreaded at the same time. Without effective antibiotics, The clinic had no available project to treat the infectious disease sometimes. According to the results of latest research, we found that the antibiotic resistant mechanisms of the multiple antibiotic resistant strains involved in producing inactive and modification enzyme、changing membrane permeability and forming efflux pump. However, the most important things, especially for the Gram-negative bacterial, is producing extended-spectrumβ-lactamases (ESBLs).
ESBL-producing strain was first reported in 1983. And nowdays, it has become an increasing problem in daily clinical life worldwide. The ESBLs-producing strains include Enterobacteriaceae, non-fermentative bacilli and so on; the ESBLs-types located in the single strain reveal diversity according to districts; different ESBLs type has different antibiotics resistance. So, it is very important for us to confirm the prevailing ESBLs types and its antibiotics resistance. And we must detect the ESBLs-producing strains early if we want to provide a correct treatment theory for the clinic.when all of these works are finished successfully, we can decrease the ESBLs-producing strains emerging rate and intercept the ESBLs-producing strains spreading indeed.
Due to different antibacterials are used to treat infectious disease in different district, the prevalence of ESBL-types differs from country to country and from laboratory to laboratory.In most parts of china, the isolating rate of CTX-M type ESBLs was at a high level. The CTX-M-1 type ESBLs was first confirmed by Bauernfeind in German. Now, there were more subtypes were reported all over the world. The CTX-M type ESBLs may locate in more kinds of strains separated from different laboratory. The hydrolytic substrates of CTX-M type ESBLs include penicillins、the first and second generation cephalosporins. Some subtypes can hydrolyze cefecime. Because of the difference of stereospecific blockade between Cefotaxime and ceftazidime, the hydrolytic activity of enzyme to Cefotaxime is more stronger than to ceftazidime. And some strains producing CTX-M type ESBLs are noticeable sensitive in vitro. According to whole genome sequence nucleotide homology, the CTX-M type ESBLs can be divided into 5 clusters. The nucleotide homology is less than 90%among subtypes which belong to the different cluster, and more than 94%among subtypes which belong to in same group. The mutational sites among groups may locate at anywhere in the DNA sequences. The further researchs show that all of the DNA mutational sites are away from the enzyme active center, as result, all the CTX-M genotypes possess some common properties. Confirming the common properties is useful to inhibit the resistent activity of enzyme of CTX-M type ESBLs.
Separating and identifying the ESBLs-producing strains in time is very important for the clinic to treat the infectious disease early and inhibit the resistent isolates spread effectively. Nowdays, there are three kinds of methods to identify the enzyme-producing strains in the clinical laboratory. The ESBLs-postive isolates are selected by double-disc synergy test and are confirmed by enzymes inhibitor enhancing test or E-test. Both of these methods can be performed base on the isolates separating and would consume a long time. The most available measure is the molecular biology technique which can detect the ESBLs-postive strains specificly and quickly. However, due to the high cost and the sophisticated laboratory requirement, molecular biology technique is inapplicable to be performed in clinical laboratory.
In order to solve the problem discussed above,46 nonduplicate ESBLs-positive E.coli isolates were collected from the first affiliated hospital. ESBLs-Producing isolates were confirmed by double-disc synergy test and enzymes inhibitor enhancing test; The enzyme encoding genes were amplified by PCR; The construction and expression of the vecter of pET-28-CTX-M were preformed by gene recombination technique; The antibiotic resistence was detected by liquid dilution; we prepared and purified the polyclonal antibody through classical immunology methods; we established a quickly detective method of double antibody sandwich ELISA to confirm CTX-M type ESBLs. At last, we inhibited the enzyme abilities by Kirby-Bauer disc agar diffusion test to explore the application of the polyclonal antibody in seroimmunity.
The experiment is divided into three parts.
Part I:The analysis of expression and antibiotic susceptibilities of CTX-M-38 type extended-spectrum-lactamase
Methods:
1.1.46 Multiple antibiotic resistant E.coli isolates were detected corresponding to the phenotype of producing ESBL detected by double-disc synergy screening test, and confirmation for producing ESBL was carried out by enzymes inhibitor enhancing test.
2. Acorrding to nucleotide homology diversity of CTX-M-38 type ESBLs, specific primers were designed by DNAstar analysing software.
3. CTX-M-38 type ESBLs encoding gene was amplified by PCR.
4. The expressing vecter of pET-28-CTX-M-38 was constructed by gene recombination technique.
5. Using CaCl2 transforming method to introduce the expressing vector of pET-28a-CTX-M-38 into E.coli BL21.
6. Selecting the positive strains by kanamycin resistance and PCR.
7. The insert element of pET-28-CTX-M-38 was detected by DNA sequencing to identified the mutation of CTX-M-38 type encoding gene.
8. The antibiotic susceptibilities of the transformant of BL21-CTX-M-38 was carried out by liquid dilution test.
9. The enzyme activities of culture supernatant and bacteria sonicate were tested by Kirby-Bauer disc agar diffusion to reflect its distribution.
Results:
1. The isolates was resistant to Amoxicillin and sensitive to Amoxicillin/clavulanic acid. There was synergy phenomenon in the juncture. All of these indicated that the screening test was positive.
2. The diameter of Ceftazidime was 8mm, and the diameter of Ceftazidime/ clavulanic acid was 26mm, the difference between them was larger than 5mm. All of these indicated that the confirmation test was positive.
3. According to the agarose gel electrophoresis, the molecular weight of amplifying products corresponded to the design.
4. Using PCR to detect the plasmids derived from transformants, the products located at 900bp in the agarose gel electrophoresis. The result indicated that the expressing vectors were constructed successfully.
5. the transformant of BL21-CTX-M-38 could grow in the LB liquid medium (100 u g/ml kanamycin), the result indicated that the transformant of BL21-CTX-M-38 was constructed successfully.
6. The mutation point in DNA sequence between insert element and Bla CTX-M-38 located at 663, the thymine was replaced by adenine. According to the analysis of amino acid sequence, however, the mutation was samesense mutation, the codon 221 of them all was alanine.
7. According to the Kirby-Bauer disc agar diffusion test, the enzyme activities from bacteria sonicate was stronger than culture supernatant. The result showed that the product mainly lied inside the bacteria.
8. The CTX-M-38 transformant was obviously resisitant to ampicillin and piperacillin, and their MICs were more than 32μg/ml. The transformant was noticeable resistant to the first、second and third generation cephalosporins tested in the study too. The MICs of cephazoline、cefuroxime、cefepime and cefaclor were all more than 16μg/ml, and the MIC of ceftriaxone was 32μg/ml. At the same time, the hydrolytic activity of enzyme to Cefotaxime was 4 times as much as it to the ceftazidime, the former was resistant and its MIC was more than 32 u g/ml, and the latter was sensitive in vitro and its MIC was 8μg/ml. On the other hand, The CTX-M-38 transformant was stably sensitive to imipenemwas and its MIC was less than 4μg/ml. Different from other CTX-M type ESBLs, the MIC of aztreonam was less than 8μg/ml, the result indicated that the transformant was sensitive to aztreonam. The transformant was sensitive to piperacillin/ tazobactam and its MIC was less than 16/4μg/ml. To the other antibiotics including beta-lactamase inhibitors including Amoxicillin/clavulanic and ampicillin/sulbactam, however, the transformant was resistance and their MIC were more than 32/16μg/ml. The MIC of cefoperazone/sulbactam was equal to 32/16μg/ml, which indicated that the transformant was intermediate to the antibiotic. The transformant was also resistance to gentamicin, minocycline, ciprofloxacin and levofloxacin.
Part II:the polyclonal antibody preparation of CTX-M-38 type ESBLs
Methods:
1. The CX-M-38 transformant expressed the extended-spectrum beta-lactamases in BL21 E.coli with IPTG derivation.
2. The bacterial protein was extracted by water boiling extraction technique.
3. The inclusion body protein was extracted by sonication method.
4. The expressing products of CTX-M-38 transformant was detected by SDS-PAGE electrophoresis technique.
5. The CTX-M-38 type ESBLs was reclaimed and purified by elution diffusion method.
6. The polyclonal antibody of CTX-M-38 type ESBLs was preparation by classical immunology methods.
7. The antibody titer of polyclonal antibody was detected by indirect enzyme-linked immunosorbent assay.
8. The polyclonal antibody prepared in research was purified by Octanoic acid precipitation method.
9. The specificity of the polyclonal antibody was confirmed by Western blot.
Results:
1. The SDS-PAGE electrophoresis results showed that there was a noticeable protein band in the supernatant, whole bacterial proteins and inclusion body protein lane respectively, and the molecular weight was about 30kDa, which correspond to the design.All of these indicated that the transformants could express enzyme protein significantly with IPTG induction.
2. The SDS-PAGE electrophoresis results showed that the expressing products quantity was supernatant, whole bacterial proteins and inclusion body protein in turn. All these results indicated that the expressing products mainly lie in the inclusion body.
3. According to the results of indirect enzyme-linked immunosorbent assay, we found that the experimental result was positive when the polyclonal antibody was diluted from 1:1000 to 1:16000, and the experimental result was negative when the antibody was diluted more than 1:32000. we could conclude that the antibody titer was 1:16000 and the polyclonal antibody was prepared successfully.
4. The Western blot results showed that different dilutions of antibodies reacted to the same protein band (about 30kDa), which molecular weight was correspond to the CTX-M-38 type ESBLs. All of these results suggested that the specificity of polyclonal antibodies was better.
PartⅢ:Study on the application of polyclonal antibody of CTX-M-38 type ESBLs
Methods:
1. The reaction optimal ratio between CTX-M-38 type ESBLs and polyclonal antibody was confirmed by double agar diffusion test. Selectinig 4h culture medium as the experimental antigen concentration,Series of antibody titers were prepared by fold dilution method.
2. The Kirby-Bauer disc agar diffusion was carried out to explore the Inhibitory effect of enzyme activity by polyclonal antibody.
3. The enzyme labelled antibody was prepared by modified heptaiodic acid sodium method.
4. The enzyme labelled antibody was purified by 50%Saturated ammonium sulfate precipitation method.
5. Based on double-antibody sandwich principle, we designed a quick detection method of enzyme-linked immunosorbent assay for CTX-M-38 type ESBLs.
6. The parallel testing was carried out to detect the products of CTX-M-38 transformant. According the result, we obtained the information of experimental repeatability and intraasay variation.
7.146 ESBLs-producing E.coli strains were detected by PCR and double-antibody sandwich ELISA respectively.Comparing with the results of the two groups, we evaluated the experimental specificity.
8. Using double-antibody sandwich ELISA to detect 27 isolates of ESBL-producing E.coli strains(not carrying CTX-M-38 type ESBLs). According to the result, we evaluated the experimental cross-reactivity.
9. The culture medium of CTX-M-38 type ESBLs-producing E.coli was detected at various time, Aaccording to the result, we confirmed the optimal detective time of ESBLs.
Result:
1. According to the double agar diffusion test result, we found that the Precipitation line located at the middle of two reactions when the antibody titer was 1:64. all of the results indicated that the optimal experimental ratio was 1:64.
2. The antibiotic diameter of antibody group was different from the control group(p<0.01) in K-B disc agar diffusion, the result indicated that the polyclonal antibody could inhibit the enzyme activity to some degrees.
3. The inhibition effect of polyclonal antibody to amoxicillin, ceftazidime, cefepime and aztreonam was obvious. The diameter of these four antibiotics of antibody group turned larger than that of control group, which indicated that the CTX-M-38 transformant turn sensitive or the MIC of them decreased to some extent. It looked like that the anbibody had no effect on the cefotaxime and antibiotics including beta-lactamase inhibitors, the diameters of them were similar between the antibody group and control group.
4. According the results of the parallel testing, the CV was 1.55%, which indicated that the intraasay variation was small and the experimental repeatability was better.
5. There were 6 positive isolates detected by PCR among 146 strains, and 11 isolates of double-antibody sandwich ELISA. According to the statistics,p>0.05, we could conclude that there was no difference between the two groups and double-antibody sandwich ELISA had better Specificity.
6. There was 1 positive strains detected by double-antibody sandwich ELISA among 27 strains, which indicated that the antibody possessed cross-reactivity and the method may have the phenomenon of false-positive.
7. The enzyme protein could be confirmed after the host bacteria was cultured 2h, and the result became significant after 4h. The amount of enzyme protein exceed the linear range of detection when cultured 8h,18h and 24h.As result, there was no change in absorbance. Combination of test procedures, this study was to determine the optimal detection time for ESBLs was culture 4h.
Conclusion:
1. The expressing products mainly lies inside the inclusion body, The transformant is obviously resistant to most of antibiotics.And there are some differences between the CTX-M-38 and other ESBLs types.
2. The polyclonal antibody of CTX-M-38 type ESBLs is prepared, purified and identified. The polyclonal titer is 1:16000, the reaction between the antibody and enzyme protein possess higher specificity.
3. Polyclonal antibody can inhibit the enzyme activity to some extent. Double-antibody sandwich enzyme-linked immunosorbent assay establish in research possesse better repeatability and specificity. The optimal detection time for ESBLs is culture for 4h.
自1983年首例产超广谱β-内酰胺酶菌株发现以来,产酶菌株呈世界范围内流行。菌株种类涵盖肠杆菌科、非发酵菌科细菌等；单菌株携带酶型存在地域性分布特征；各型别酶抗药活性间存在一定差异。明确各地区超广谱β-内酰胺酶流行型别抗药活性,早期完成产酶菌株分离、鉴定,对于正确指导临床抗感染治疗,最终降低产酶菌株的产生几率及阻断产酶菌株流行意义重大。
抗菌药物应用习惯差异,造成各地域间超广谱β-内酰胺酶流行型别不同。在我国大部分地区临床分离菌株所产酶型中,CTX-M型超广谱β-内酰胺酶占有相当比例。该型酶由德国学者Bauemfeind首先发现(CTX-M-1),目前已有数十种亚型,其分布地域及宿主菌谱极为广泛。CTX-M型酶水解底物为青霉素类、第一、二代头孢菌素,部分型别可水解头孢吡肟。基于酶蛋白结构构象特征以及药物自身空间位阻差异,CTX-M型酶对头孢噻肟的水解能力显著高于头孢他啶,部分菌株体外试验对头孢他啶显示高度敏感。根据氨基酸序列同源性分析结果,可将CTX-M型超广谱β-内酰胺酶分为5组,各组间氨基酸序列同源性小于90%,组内性大于94%。各组间DNA突变位点可位于编码基因的任一部位。进一步研究表明,尽管CTX-M型酶各亚型间核苷酸序列差异较大,但从酶蛋白结构上看,各突变位点多远离酶活性中心,对于酶蛋白抗药活性影响不大。故各亚型酶均具有CTX-M型酶的共有特性。基于此,明确一种型别CTX-M型酶的耐药谱,对于其他型别抗药活性的抑制具有指导价值。
目前,临床实验室对于产酶菌株的鉴定主要采用三种方式,采用双纸片协同试验初步筛选阳性菌株；采用酶抑制剂增强试验/E-test试验进行确证；采用分子生物学试验进行快速特异性鉴定。前两者均需完成菌株的分离培养,耗时较长,难以满足临床简单快速的要求。后者试验费用较高,且实验软、硬件要求高,一般实验室难以开展。
为了解决上述问题,作者采用双纸片协同及酶抑制剂增强试验筛选、收集46株产超广谱β-内酰胺酶大肠埃希菌；采用PCR技术克隆CTX-M型酶表达基因；采用基因重组技术构建CTX-M表达载体并表达；采用液体稀释法完成抗药活性检测。同时,通过免疫小鼠获得超广谱β-内酰胺酶特异性抗体；设计完善酶联免疫吸附试验,建立CTX-M型超广谱β-内酰胺酶的快速鉴定方法。通过上述试验,在明确CTX-M型酶抗药活性的同时,可获得其多克隆抗体,用于快速鉴定,乃至于被动免疫治疗。
本研究包括以下三个部分。
第一部分CTX-M-38型超广谱p-内酰胺酶的表达、定位及其抗药活性研究
方法
1.采用双纸片协同试验及酶抑制剂增强试验,对46株临床分离多重耐药大肠埃希菌进行超广谱β-内酰胺酶筛选与确证。
2.依据CTX-M-38型超广谱β-内酰胺酶核苷酸序列特征,采用DNAstar分析软件,完成特异性PCR扩增引物设计。
3.采用PCR技术,完成CTX-M-38型超广谱β-内酰胺酶全长编码基因的克隆。
4.采用基因重组技术,完成pET-28a-CTX-M-38表达质粒的构建。
5.采用氯化钙转化法,完成pET-28a-CTX-M-38表达质粒在BL21大肠埃希菌中的转化。
6.采用卡那霉素抗性试验,检测BL21-pET-28a-CTX-M-38转化子构建效果。
7．采用双脱氧链终止法对质粒插入片段进行核苷酸序列分析,检测所克隆CTX-M-38型编码基因序列突变状况。
8.采用液体稀释法,检测BL21-pET-28a-CTX-M-38转化子抗药活性。
9.采用K-B法药物敏感试验,检测培养液上清及菌体裂解液中CTX-M-38型超广谱β-内酰胺酶活性,初步进行酶蛋白定位研究。
结果
1.阿莫西林表现耐药,阿莫西林/克拉维酸表现敏感,两者交界处有协同现象,抑菌环直径明显扩大,产超广谱β-内酰胺酶菌株筛选试验阳性。
2.头孢他啶抑菌环直径为8mm,头孢他啶/克拉维酸抑菌环直径为26mm,两者之差显著大于5mm,表明酶抑制剂增强试验阳性,产超广谱β-内酰胺酶菌株确证试验阳性。
3.琼脂糖凝胶电泳显示,PCR扩增阳性产物大小与设计结果相一致。
4.以转化子携带质粒为模板进行PCR扩增,约900bp位置处有目的条带,提示重组质粒构建成功。
5.转化子在含卡那霉素液体培养基中生长良好,表明转化子构建成功。
6.目的基因核苷酸序列与Bla CTX-M-38基因序列比对结果显示,两者在663位点处存在突变(T→A),但氨基酸序列上,两者221位均为丙氨酸,提示此处突变为同义突变。
7.K-B法药敏试验中,滴加上清及菌体裂解液的头孢噻肟纸片均显示耐药,但后者抗药活性较为显著,单纯头孢噻肟纸片显示敏感,提示表达产物主要分布于菌体中。
8.转化子对青霉素类药物包括哌拉西林、氨苄西林显著耐药(MIC>32μg/ml),对于头孢唑啉、头孢吡肟、头孢呋辛及头孢克洛MIC值>16μg/ml,表现为耐药。头孢噻肟及头孢曲松酶水解活性较强(MIC值均>32μg/ml)。头孢他啶MIC值为8μg/ml,表现为体外敏感。转化子对亚胺培南稳定敏感,MIC≤4μg/ml。酶抑制剂复合制剂中,哌拉西林/三唑巴坦敏感(MIC≤16/4μg/ml),而阿莫西林/棒酸、氨苄西林/舒巴坦均表现耐药(MIC>32/16μg/ml)。头孢哌酮／舒巴坦MIC为32/16μg/ml,表现为中介。氨曲南表现为敏感(MIC≤8μg/ml)。非内酰胺类抗菌药物中,庆大霉素、四环素、环丙沙星、左氧氟沙星及美满霉素均表现为耐药。
第二部分CTX-M-38型超广谱β-内酰胺酶多克隆抗体的制备
方法
1.采用IPTG诱导BL21-pET-28a-CTX-M-38转化子表达酶蛋白。
2.采用水煮裂解法,完成全菌蛋白提取。
3.采用超声破碎法,完成包涵体蛋白提取。
4.采用SDS-PAGE蛋白电泳技术,检测BL21-pET-28a-CTX-M-38转化子表达产物。
5.采用扩散洗脱法,回收与纯化CTX-M-38型超广谱β-内酰胺酶蛋白。
6.采用传统多克隆抗体制备方法,制备CTX-M-38型超广谱β-内酰胺酶蛋白特异性抗体。
7.采用间接酶联免疫吸附试验,检测所制备多克隆抗体效价。
8.采用辛酸沉淀法,对所制备的多克隆抗体进行纯化。
9.采用Western blot印迹杂交技术,进行多克隆抗体的特异性鉴定。
结果
1. SDS-PAGE蛋白电泳结果显示,BL21-pET-28a-CTX-M-38转化子培养液上清、全菌蛋白、包涵体蛋白泳道,在约30kDa处出现明显蛋白条带,目的酶蛋白分子量大小与设计相同,表明在IPTG诱导作用下,转化子可显著表达酶蛋白。
2. SDS-PAGE蛋白电泳后,各泳道酶蛋白量多少依次为包涵体蛋白、全菌蛋白以及培养液上清,提示转化子表达产物主要存在于包涵体内。
3.间接酶联免疫吸附试验结果显示,多克隆抗体1：1000至1：16000梯度稀释度检测结果均呈阳性。1：32000以后稀释度时无阳性结果出现,表明多克隆抗体效价为1：16000,也表明多克隆抗体制备成功。
4. Western blot印迹杂交技术检测结果显示,不同稀释度的抗体均与同一蛋白条带(约30kDa)反应,此条带与CTX-M-38型超广谱β-内酰胺酶蛋白大小相一致。提示研究中所制备多克隆抗体特异性较好。
第三部分CTX-M-38型超广谱β-内酰胺酶多克隆抗体的应用研究
方法
1.采用梅花形双向琼脂扩散试验,完成CTX-M-38型超广谱β-内酰胺酶蛋白与多克隆抗体反应最适比检测。其中选择4h培养物作为试验抗原浓度,系列抗体滴度采用倍比稀释法制备。
2.采用K-B法药物敏感试验,进行多克隆抗体抑制酶蛋白活性研究。
3.采用改良过碘酸钠法,进行酶标多克隆抗体的制备。
4.采用50%饱和硫酸铵沉淀法,进行酶标多克隆抗体的纯化。
5.依据双抗体夹心法原理,设计制备酶联免疫吸附试验检测试剂盒。
6.针对BL21-pET-28a-CTX-M-38转化子表达产物进行平行试验,检测批内变异,评价实验重复性。
7.与PCR技术进行平行对照试验,完成临床分离产酶菌株检测,评价方法特异性。
8.对27株产超广谱β-内酰胺酶大肠埃希菌(不携带CTX-M-38型超广谱β-内酰胺酶)进行检测,评价抗体交叉反应状况。
9.对产CTX-M-38型超广谱β-内酰胺酶菌株不同时间段培养液进行检测,明确超广谱β-内酰胺酶最适检测时间。
结果
1.结果显示,抗体效价为1∶64时,沉淀线出现在两反应孔中间位置,表明抗体与4h培养物反应的最适效价为1∶64。
2.K-B法药敏试验结果显示,抗体组抗菌药物抑菌环直径与对照组比较,p值小于0.01,两者结果有差异。表明CTX-M-38型超广谱β-内酰胺酶蛋白与多克隆抗体结合后,其活性受到一定程度的抑制。
3.多克隆抗体抑制效应表现为阿莫西林、头孢他啶、头孢吡肟、氨曲南等药物的抑菌环直径增大,菌株由耐药转变为敏感或MIC进一步减小。含酶抑制剂复合药物两组结果基本相同。头孢噻肟抑菌环两组结果均无变化。
4.对转化子表达产物30次平行试验结果显示,CV值为1.55%,提示本法批内变异小,具有较好的重复性。
5.146株产超广谱β-内酰胺酶大肠埃希菌中,PCR技术检测阳性率为4.11%,本法为7.53%,x2检测可知P>0.05,表明两种检测方法检测结果无差异,提示本法具有一定的特异性。
6.交叉反应试验中,27株临床分离产酶菌株中,有1株检测结果为阳性。表明所制备抗体具有交叉反应,实验结果存在假阳性现象。
7.超广谱β-内酰胺酶最适检测时间结果显示,宿主菌在培养2h后,即有酶蛋白产生。4h时酶量显著增多,8h、18h及24h酶蛋白量已超过检测线性范围,故吸光度改变不大。结合检验流程,本研究确定超广谱β-内酰胺酶最适检测时间为菌株培养4h。
结论
1. CTX-M-38型超广谱β-内酰胺酶主要定位于宿主菌包涵体内；转化子具有广泛抗药活性,对部分抗菌药物抗药活性与其他型别存在差异。
2.完成了CTX-M-38型超广谱β-内酰胺酶多克隆抗体的制备、纯化与鉴定,所制备多克隆抗体效价为1∶16000；抗体与CTX-M-38型超广谱β-内酰胺酶蛋白反应特异性较高。
3.多克隆抗体可抑制酶蛋白抗药活性；双抗体夹心酶联免疫吸附试验检测酶蛋白具有较好的重复性、特异性；临床分离菌株产超广谱β-内酰胺酶最佳检测时间为菌株培养4h。
With the usage of broad spectrum antibiotics, especially the third generation cephalosporins application, more infectious diseases were controlled and obtained better therapeutic efficacy. On the other hand, the overuse or abuse of antibiotics had made the clinic face a severe test gradually. Because selection pressure was formed, more and more multiple antibiotic resistant strains emerged recently. Many genetic transfer elements which contained resistant genes can transfer among the strains isolated from the clinic frequently, as result, the antibiotics resistance was spreaded at the same time. Without effective antibiotics, The clinic had no available project to treat the infectious disease sometimes. According to the results of latest research, we found that the antibiotic resistant mechanisms of the multiple antibiotic resistant strains involved in producing inactive and modification enzyme、changing membrane permeability and forming efflux pump. However, the most important things, especially for the Gram-negative bacterial, is producing extended-spectrumβ-lactamases (ESBLs).
ESBL-producing strain was first reported in 1983. And nowdays, it has become an increasing problem in daily clinical life worldwide. The ESBLs-producing strains include Enterobacteriaceae, non-fermentative bacilli and so on; the ESBLs-types located in the single strain reveal diversity according to districts; different ESBLs type has different antibiotics resistance. So, it is very important for us to confirm the prevailing ESBLs types and its antibiotics resistance. And we must detect the ESBLs-producing strains early if we want to provide a correct treatment theory for the clinic.when all of these works are finished successfully, we can decrease the ESBLs-producing strains emerging rate and intercept the ESBLs-producing strains spreading indeed.
Due to different antibacterials are used to treat infectious disease in different district, the prevalence of ESBL-types differs from country to country and from laboratory to laboratory.In most parts of china, the isolating rate of CTX-M type ESBLs was at a high level. The CTX-M-1 type ESBLs was first confirmed by Bauernfeind in German. Now, there were more subtypes were reported all over the world. The CTX-M type ESBLs may locate in more kinds of strains separated from different laboratory. The hydrolytic substrates of CTX-M type ESBLs include penicillins、the first and second generation cephalosporins. Some subtypes can hydrolyze cefecime. Because of the difference of stereospecific blockade between Cefotaxime and ceftazidime, the hydrolytic activity of enzyme to Cefotaxime is more stronger than to ceftazidime. And some strains producing CTX-M type ESBLs are noticeable sensitive in vitro. According to whole genome sequence nucleotide homology, the CTX-M type ESBLs can be divided into 5 clusters. The nucleotide homology is less than 90%among subtypes which belong to the different cluster, and more than 94%among subtypes which belong to in same group. The mutational sites among groups may locate at anywhere in the DNA sequences. The further researchs show that all of the DNA mutational sites are away from the enzyme active center, as result, all the CTX-M genotypes possess some common properties. Confirming the common properties is useful to inhibit the resistent activity of enzyme of CTX-M type ESBLs.
Separating and identifying the ESBLs-producing strains in time is very important for the clinic to treat the infectious disease early and inhibit the resistent isolates spread effectively. Nowdays, there are three kinds of methods to identify the enzyme-producing strains in the clinical laboratory. The ESBLs-postive isolates are selected by double-disc synergy test and are confirmed by enzymes inhibitor enhancing test or E-test. Both of these methods can be performed base on the isolates separating and would consume a long time. The most available measure is the molecular biology technique which can detect the ESBLs-postive strains specificly and quickly. However, due to the high cost and the sophisticated laboratory requirement, molecular biology technique is inapplicable to be performed in clinical laboratory.
In order to solve the problem discussed above,46 nonduplicate ESBLs-positive E.coli isolates were collected from the first affiliated hospital. ESBLs-Producing isolates were confirmed by double-disc synergy test and enzymes inhibitor enhancing test; The enzyme encoding genes were amplified by PCR; The construction and expression of the vecter of pET-28-CTX-M were preformed by gene recombination technique; The antibiotic resistence was detected by liquid dilution; we prepared and purified the polyclonal antibody through classical immunology methods; we established a quickly detective method of double antibody sandwich ELISA to confirm CTX-M type ESBLs. At last, we inhibited the enzyme abilities by Kirby-Bauer disc agar diffusion test to explore the application of the polyclonal antibody in seroimmunity.
The experiment is divided into three parts.
Part I:The analysis of expression and antibiotic susceptibilities of CTX-M-38 type extended-spectrum-lactamase
Methods:
1.1.46 Multiple antibiotic resistant E.coli isolates were detected corresponding to the phenotype of producing ESBL detected by double-disc synergy screening test, and confirmation for producing ESBL was carried out by enzymes inhibitor enhancing test.
2. Acorrding to nucleotide homology diversity of CTX-M-38 type ESBLs, specific primers were designed by DNAstar analysing software.
3. CTX-M-38 type ESBLs encoding gene was amplified by PCR.
4. The expressing vecter of pET-28-CTX-M-38 was constructed by gene recombination technique.
5. Using CaCl2 transforming method to introduce the expressing vector of pET-28a-CTX-M-38 into E.coli BL21.
6. Selecting the positive strains by kanamycin resistance and PCR.
7. The insert element of pET-28-CTX-M-38 was detected by DNA sequencing to identified the mutation of CTX-M-38 type encoding gene.
8. The antibiotic susceptibilities of the transformant of BL21-CTX-M-38 was carried out by liquid dilution test.
9. The enzyme activities of culture supernatant and bacteria sonicate were tested by Kirby-Bauer disc agar diffusion to reflect its distribution.
Results:
1. The isolates was resistant to Amoxicillin and sensitive to Amoxicillin/clavulanic acid. There was synergy phenomenon in the juncture. All of these indicated that the screening test was positive.
2. The diameter of Ceftazidime was 8mm, and the diameter of Ceftazidime/ clavulanic acid was 26mm, the difference between them was larger than 5mm. All of these indicated that the confirmation test was positive.
3. According to the agarose gel electrophoresis, the molecular weight of amplifying products corresponded to the design.
4. Using PCR to detect the plasmids derived from transformants, the products located at 900bp in the agarose gel electrophoresis. The result indicated that the expressing vectors were constructed successfully.
5. the transformant of BL21-CTX-M-38 could grow in the LB liquid medium (100 u g/ml kanamycin), the result indicated that the transformant of BL21-CTX-M-38 was constructed successfully.
6. The mutation point in DNA sequence between insert element and Bla CTX-M-38 located at 663, the thymine was replaced by adenine. According to the analysis of amino acid sequence, however, the mutation was samesense mutation, the codon 221 of them all was alanine.
7. According to the Kirby-Bauer disc agar diffusion test, the enzyme activities from bacteria sonicate was stronger than culture supernatant. The result showed that the product mainly lied inside the bacteria.
8. The CTX-M-38 transformant was obviously resisitant to ampicillin and piperacillin, and their MICs were more than 32μg/ml. The transformant was noticeable resistant to the first、second and third generation cephalosporins tested in the study too. The MICs of cephazoline、cefuroxime、cefepime and cefaclor were all more than 16μg/ml, and the MIC of ceftriaxone was 32μg/ml. At the same time, the hydrolytic activity of enzyme to Cefotaxime was 4 times as much as it to the ceftazidime, the former was resistant and its MIC was more than 32 u g/ml, and the latter was sensitive in vitro and its MIC was 8μg/ml. On the other hand, The CTX-M-38 transformant was stably sensitive to imipenemwas and its MIC was less than 4μg/ml. Different from other CTX-M type ESBLs, the MIC of aztreonam was less than 8μg/ml, the result indicated that the transformant was sensitive to aztreonam. The transformant was sensitive to piperacillin/ tazobactam and its MIC was less than 16/4μg/ml. To the other antibiotics including beta-lactamase inhibitors including Amoxicillin/clavulanic and ampicillin/sulbactam, however, the transformant was resistance and their MIC were more than 32/16μg/ml. The MIC of cefoperazone/sulbactam was equal to 32/16μg/ml, which indicated that the transformant was intermediate to the antibiotic. The transformant was also resistance to gentamicin, minocycline, ciprofloxacin and levofloxacin.
Part II:the polyclonal antibody preparation of CTX-M-38 type ESBLs
Methods:
1. The CX-M-38 transformant expressed the extended-spectrum beta-lactamases in BL21 E.coli with IPTG derivation.
2. The bacterial protein was extracted by water boiling extraction technique.
3. The inclusion body protein was extracted by sonication method.
4. The expressing products of CTX-M-38 transformant was detected by SDS-PAGE electrophoresis technique.
5. The CTX-M-38 type ESBLs was reclaimed and purified by elution diffusion method.
6. The polyclonal antibody of CTX-M-38 type ESBLs was preparation by classical immunology methods.
7. The antibody titer of polyclonal antibody was detected by indirect enzyme-linked immunosorbent assay.
8. The polyclonal antibody prepared in research was purified by Octanoic acid precipitation method.
9. The specificity of the polyclonal antibody was confirmed by Western blot.
Results:
1. The SDS-PAGE electrophoresis results showed that there was a noticeable protein band in the supernatant, whole bacterial proteins and inclusion body protein lane respectively, and the molecular weight was about 30kDa, which correspond to the design.All of these indicated that the transformants could express enzyme protein significantly with IPTG induction.
2. The SDS-PAGE electrophoresis results showed that the expressing products quantity was supernatant, whole bacterial proteins and inclusion body protein in turn. All these results indicated that the expressing products mainly lie in the inclusion body.
3. According to the results of indirect enzyme-linked immunosorbent assay, we found that the experimental result was positive when the polyclonal antibody was diluted from 1:1000 to 1:16000, and the experimental result was negative when the antibody was diluted more than 1:32000. we could conclude that the antibody titer was 1:16000 and the polyclonal antibody was prepared successfully.
4. The Western blot results showed that different dilutions of antibodies reacted to the same protein band (about 30kDa), which molecular weight was correspond to the CTX-M-38 type ESBLs. All of these results suggested that the specificity of polyclonal antibodies was better.
PartⅢ:Study on the application of polyclonal antibody of CTX-M-38 type ESBLs
Methods:
1. The reaction optimal ratio between CTX-M-38 type ESBLs and polyclonal antibody was confirmed by double agar diffusion test. Selectinig 4h culture medium as the experimental antigen concentration,Series of antibody titers were prepared by fold dilution method.
2. The Kirby-Bauer disc agar diffusion was carried out to explore the Inhibitory effect of enzyme activity by polyclonal antibody.
3. The enzyme labelled antibody was prepared by modified heptaiodic acid sodium method.
4. The enzyme labelled antibody was purified by 50%Saturated ammonium sulfate precipitation method.
5. Based on double-antibody sandwich principle, we designed a quick detection method of enzyme-linked immunosorbent assay for CTX-M-38 type ESBLs.
6. The parallel testing was carried out to detect the products of CTX-M-38 transformant. According the result, we obtained the information of experimental repeatability and intraasay variation.
7.146 ESBLs-producing E.coli strains were detected by PCR and double-antibody sandwich ELISA respectively.Comparing with the results of the two groups, we evaluated the experimental specificity.
8. Using double-antibody sandwich ELISA to detect 27 isolates of ESBL-producing E.coli strains(not carrying CTX-M-38 type ESBLs). According to the result, we evaluated the experimental cross-reactivity.
9. The culture medium of CTX-M-38 type ESBLs-producing E.coli was detected at various time, Aaccording to the result, we confirmed the optimal detective time of ESBLs.
Result:
1. According to the double agar diffusion test result, we found that the Precipitation line located at the middle of two reactions when the antibody titer was 1:64. all of the results indicated that the optimal experimental ratio was 1:64.
2. The antibiotic diameter of antibody group was different from the control group(p<0.01) in K-B disc agar diffusion, the result indicated that the polyclonal antibody could inhibit the enzyme activity to some degrees.
3. The inhibition effect of polyclonal antibody to amoxicillin, ceftazidime, cefepime and aztreonam was obvious. The diameter of these four antibiotics of antibody group turned larger than that of control group, which indicated that the CTX-M-38 transformant turn sensitive or the MIC of them decreased to some extent. It looked like that the anbibody had no effect on the cefotaxime and antibiotics including beta-lactamase inhibitors, the diameters of them were similar between the antibody group and control group.
4. According the results of the parallel testing, the CV was 1.55%, which indicated that the intraasay variation was small and the experimental repeatability was better.
5. There were 6 positive isolates detected by PCR among 146 strains, and 11 isolates of double-antibody sandwich ELISA. According to the statistics,p>0.05, we could conclude that there was no difference between the two groups and double-antibody sandwich ELISA had better Specificity.
6. There was 1 positive strains detected by double-antibody sandwich ELISA among 27 strains, which indicated that the antibody possessed cross-reactivity and the method may have the phenomenon of false-positive.
7. The enzyme protein could be confirmed after the host bacteria was cultured 2h, and the result became significant after 4h. The amount of enzyme protein exceed the linear range of detection when cultured 8h,18h and 24h.As result, there was no change in absorbance. Combination of test procedures, this study was to determine the optimal detection time for ESBLs was culture 4h.
Conclusion:
1. The expressing products mainly lies inside the inclusion body, The transformant is obviously resistant to most of antibiotics.And there are some differences between the CTX-M-38 and other ESBLs types.
2. The polyclonal antibody of CTX-M-38 type ESBLs is prepared, purified and identified. The polyclonal titer is 1:16000, the reaction between the antibody and enzyme protein possess higher specificity.
3. Polyclonal antibody can inhibit the enzyme activity to some extent. Double-antibody sandwich enzyme-linked immunosorbent assay establish in research possesse better repeatability and specificity. The optimal detection time for ESBLs is culture for 4h.
引文
[1]俞云松.超广谱β-内酰胺酶研究进展[J].中华医学杂志,2006,86(9)：641-644
[2]张阳根,张海艳,江先海,等.超广谱内酰胺酶研究进展[J].江西医学检验,2007,25(3)：254～255
[3]郭小兵,张志坚,阎志勇,等.CTX-M-14型超广谱β-内酰胺酶原核表达载体的构建及其抗药活性分析[J].山东医药,2009,49(11)：21～23
[4]李金钟,刘利平.CTX-M型超广谱β-内酰胺酶的研究进展[J].国际检验医学杂志.2007,28(6)：543～545
[5]张盛斌,刘朝辉.CTX-M型ESBLs的研究进展[J].中国感染与化疗杂志,2006,6(3)：212～215
[6]Bonnet R, Growing group of extended-spectrum beta-lactamases:the CTX-M enzymes [J].Antimicrob Agents Chemother,2004,48 (1):1-14
[7]Humeniuk C, Arlet G, Gautier V, et al.Beta-lactamases of Kluyvera ascorbata, probable progenitors of some plasmid-encoded CTX-M types[J].Antimicrob Agents Chemother, 2002,46(9):3045-3049
[8]陆坚,唐英春,吴本权,等.华南地区质粒介导超广谱β-内酰胺酶的基因分型研究[J].中华微生物学和免疫学杂志,2002,22(6)：638～643
[9]Yan JJ, Ko WC, Tsai SH, et al.Dissemination of CTX-M-3 and CMY-2 beta-lactamases among clinical isolates of Escherichia coli in southern Taiwan[J].Clin Microbiol,2000, 38(12):4320-4325
[10]Chanawong A, M'zali FH, Heritage J, et al. Three cefotaximases, CTX-M-9, CTX-M-13, and CTX-M-14, among Enterobacteriaceae in the People's republic of China[J].Antimicrob Agents Chemother,2002,46(3):630-637
[11]张阮章,卢月梅,胡玉华,等．一种新型β内酰胺酶CTX-M-38的发现[J].中国感染与化疗杂志,2009,9(3)：216～218
[12]赵玉莲,张润梅,汪军荣.产超广谱β-内酰胺酶细菌的耐药性监测[J].中国药物与临床,2006,6(4)：266～268
[13]孙长贵,陈汉美.超广谱β-内酰胺酶及其实验室筛检[J].临床检验杂志,2000,18(1)：52～55
[14]张阮章,王沙燕,卢月梅,等.产超广谱β-内酰胺酶大肠埃希菌和肺炎克雷伯菌bla-CTX-M基因的检测[J].中国微生态杂志,2004,16(6)：342～344
[15]Boyd DA, Tyler S, Christianson S, et al. Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum beta-lactamase involved in an outbreak in long-term care facilities in Toronto, Canada[J]. Antimicrob Agents Chemother, 2004,48 (10):3758-3764
[16]Ibuka A, Taguchi A, Ishiguro M, et al.Crystal structure of the E166A mutant of extended-spectrum beta-lactamase Toho-1 at 1.8A resolution[J].J Mol Biol,1999,285(5): 2079～2087
[17]Bonnet R, Sampaio JL, Labia R, et al.A novel CTX-M β-lactamase (CTX-M-8) in cefotaxime-resistant enterobacteriaceae isolated in brazil[J].Antimicrob Agents Chemother, 2000,44 (7):1936-1942
[18]郭小兵,张志坚,阎志勇,等.CTX-M-38型超广谱β-内酰胺酶的表达、定位及抗抗药活性分析[J].天津医药,2009,37(12)：1032～1034
[19]李霜,李咏梅.β-内酰胺酶及超广谱β-内酰胺酶的研究进展[J].吉林医药学院学报,2009.27(3)：171～174
[20]袁燕,林果为.β-内酰胺酶及超广谱β内酰胺酶的研究新进展[J].英国医学杂志中文版,2004,7(4)：220～224
[21]秦东春,任丽娟,闫志勇,等.铜绿假单胞菌超广谱β-内酰胺酶编码基因OXA-1表达产物的纯化及复性分析[J].中国现代医学杂志,2007,17(19)：2341～2344
[22]萨母布鲁克,拉塞尔著.分子克隆实验指南：上册.第三版[M].黄培堂译.北京：科学出版社,2002
[23]赵春丽,范秀军,赵冰,等.IPTG诱导浓度对重组人肝再生增强因子基因表达的影响[J].东北农业大学学报,2004,35(4)：441～445
[24]吉清,何凤田.包涵体复性的研究进展[J].国外医学临床生物化学与检验学分册,2004,25(6)：516～518
[25]张庶民,祁自柏,李河民.基因工程表达蛋白包涵体的形成于纯化[J].微生物学免疫学进展,1995,23(1)：52～54
[1]zhijian zhang, xiaobing guo, qinxian zhang.Prevalence characterization of Extended-Spectrum β-Lactamases among Escherichia coli isolates collected in zhengzhou[J]. Journal of clinical laboratory analusis,2009,23 (6):404～407
[2]Mamlouk K, Boutiba-Ben Boubaker I, Gautier V, Vimont S, Picard B, et al. Emergence and outbreaks of CTX-M β-lactamase-producing Escherichia coli and Klebsiella pneumoniae strains in a Tunisian hospital[J]. J Clin Microbiol,2006,44 (11):4049～4056
[3]Yu WL, Chuang YC, Walther-Rasmussen J. Extended-spectrum beta-lactamases in Taiwan: epidemiology, detection, treatment, and infection control[J]. J. Microbiol. Immunol. Infect. 2006,39 (4):264～277
[4]张志坚,郭小兵,阎志勇,等.郑州地区大肠埃希菌中CTX-M型超广谱β-内酰胺酶的基因型分布[J].中华医院感染学杂志,2009,19(18)：2382～2384
[5]阎志勇,高晓群,郭小兵.郑州地区ESBLs基因型分布研究[J].医药论坛杂志,2008,29(2)：7-9
[6]Marra AR, Pemira CA, Castelo A, et al. Health and economic out-comes of the detection of Klebsiella pneumoniae-produced extended-spectrum beta-lactamase (ESBL) in a hospital with high prevalence of this infection[J].Int J Infect Dis,2006,10(1):56～60.
[7]James H, Jorgensen M L, Me Elmeel. Detection of CTX-M-Type Extended-Spectrum Beta-Lactamase (ESBLs) by Testing with MicroScan Overnight and ESBL Confirmation Panels[J]. Clinical Microbiology,2010,48(1):120～123
[8]Mena A, Plasencia V, Garcia L, et al. Characterization of a large outbreak by CTX-M-1-producing Klebsiella pneumoniae and mechanisms leading to in vivo carbapenem resistance development[J]. Clin Microbiol,2006,44 (8):2831～2837
[9]Paterson DL, Bonnamo RA. Extend-spectum beta-lactmases:a clinical update[J].Clin Microbiol Rev,2005,18(4):657～686
[10]李菁,林彤,宋帅,等.基因工程抗体研究进展[J].生物技术通报,2009,20(10)：40～44
[11]王秀红,周素芳.基因工程抗体研究进展[J].生物技术通讯,2007,18(2)：304～306
[12]De Bernardez Clark E, Schwarz E, Rudolph R.Inhibition of aggregation side reactions during in vitro protein folding[J].Methods Enzymol,1999,309:217～236
[13]Ejima D, Watanabe M, Sato Y, et al.High yield refolding and punification process for recombinant human interleukin-6 expressed in Escherichia coli[J].Biotechnol bioeng, 1999,62 (3):301～310
[14]唐玉钗,刘丽霞.应用辛酸法提取血清和腹水中的免疫球蛋白[J].上海免疫学杂志,1989,9(6)：370～371
[15]吴爱琴,元刚,吴昭怡,等CTX-M型超广谱β-内酰胺酶的研究进展[J].检验医学与临床,2009,6(6)：450～452
[16]朱戌冬,吴伟元,陈民钧.超广谱β-内酰胺酶CTX-M型新进展[J].中国抗感染化疗杂志,2001,1(2)：109～110
[1]Shu JC, Chia JH, Kuo AJ, et al. A 7-year surveillance for ESBL-producing Escherichia coli and Klebsiella pneumoniae at a university hospital in Taiwan:the increase of CTX-M-15 in the ICU[J]. Epidemiol Infect,2010,138 (2):253～263
[2]Jones CH, Tuckman M, Keeney D, et al. Characterization and sequence analysis of extended-spectrum-{beta}-lactamase-encoding genes from Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates collected during tigecycline phase 3 clinical trials[J]. Antimicrob Agents Chemother,2009,53 (2):465～475
[3]沈关心,周汝麟.现代免疫学实验技术.第二版[M].武汉：湖北科学技术出版社,2002,102～106
[4]Burgess DS, Hall RG 2nd, Lewis JS 2nd, et al. Clinical and microbiologic analysis of a hospital's extended-spectrum beta-lactamase-producing isolates over a 2-year period[J]. Pharmacotherapy,2003,23 (10):1232～1237
[5]徐敏超,王晶,严群,等.宽噬噬菌体治疗实验性小鼠产超广谱β-内酰胺酶大肠埃希菌感染的研究[J].中华医院感染学杂志,2006,16(3)：252～255
[6]陈民钧.美国临床实验室标准化研究所2005年版有关药敏试验标准化更新要点[J]．中华检验医学杂志,2005,28(4)：449～451
[7]孙长贵,陈汉美.超广谱β-内酰胺酶及其实验室筛检[J].临床检验杂志,2000,18(1)：52～55
[8]李巧菊,韩明,张再伟.医院感染ESBLs菌株的分子生物学测定[J].现代预防医学,2006,33(12)：2425～2427
[9]吕世静.临床免疫学检验.第一版[M].北京：中国医药科技出版社,2004,173～174
[10]吕世静.临床免疫学检验.第一版[M].北京：中国医药科技出版社,2004,207～209
[11]Rowe Magnus DA, Mazel D. The role of integrons in antibiotic resistance gene capture[J]. Int Med Microbiol,2002,292 (2):115～125
[12]Lavigne JP, Gaillard JB, Bourg G, et al.Extended-spectrum beta-lactamases-producing Stenotrophomonas maltophilia strains:CTX-M enzymes detection and virulence study[J]. Pathol Biol (Paris),2008,56 (7-8):447～453
[13]Yu WL, Chuang YC. Wahher-Rasmussen J. Extended-spectrum beta-lactamases in Taiwan: epidemiology, detection, treatment and infection control[J].Microbiol Immunol Infect, 2006,39(4):264～277
[14]郭小兵,张志坚,阎志勇,等.CTX-M-14型β-内酰胺酶原核表达载体的构建及其抗药活性分析[J].山东医药,2009,49(11)：21～23
[15]张志坚,郭小兵,阎志勇,等.郑州地区大肠埃希菌中CTX-M型超广谱β-内酰胺酶的基因型分布[J].中华医院感染学杂志,2009,19(18)：2382～2384
[16]杜艳霞.酶联免疫吸附试验测定操作中的体会[J].中国实用医药,2009,4(27)：139～140
[17]孙桂梅,韩金玲,赵莉,等.免疫诊断(主要是ELISA)试验操作的几点体会[J].中国现代药物应用,2009,3(3)：161～162
[1]Jacoby GA, Munoz-Price LS. The new beta-lactamases[J]. N Engl J Med,2005,352(4): 380～391
[2]Bradford PA. Extended-spectrum β-lactamases in the 21st century:characterization, epidemiology, and detection of this important resistance threat[J]. Clin Microbiol Rev 2001,14 (4):933～951
[3]Aranzazu Valverde, Teresa M. Coquel, et al.Complex molecular epidemiology of extended-spectrumβ-lactamases in Klebsiella pneumoniae:a long-term perspective from a single institution in Madrid[J]. Antimicrobial Chemotherapy,2008,61 (1):64～72
[4]Livermorel DM, Canton R, Gniadkowski M, et al. CTX-M:changing the face of ESBLs in Europe[J]. Antimicrobial Chemotherapy,2007,59 (2):165～174
[5]Ben-Hamouda T, Foulon T, Ben-Mahrez K. Involvement of SHV-12 and SHV-2a encoding plasmids in outbreaks of extended-spectrum β-lactamase-producing Klebsiella pneumoniae in a Tunisian neonatal ward[J]. Microb Drug Resist,2004,10(2):132～138.
[6]Gangoue-Pieboji J, Bedenic B, Koulla-Shiro S, et al. Extended spectrum-β-lactamase-producing Enterobacteriaceae in YaoundeCameroon[J]. Clin Microbiol,2005,43 (7): 3273～3277
[7]Gangoue-Pieboji J, Miriagou V, Vourli S, et al. Emergence of CTX-M-15-producing enterobacteria in Cameroon and characterization of a blaCTX-M-15-carrying element[J]. Antimicrob Agents Chemother,2005,49 (1):441～443
[8]Blomberg B, Jureen R, Manji KP, et al. High rate of fatal cases of pediatric septicemia caused by gram-negative bacteria with extendedspectrum β-lactamases in Dar es Salaam, Tanzania[J]. Clin Microbiol,2005,43 (2):745～749
[9]Gray KJ, Wilson LK, Phiri A, et al.Identification and characterization of ceftriaxone resistance and extended-spectrum β-lactamases in Malawian bacteraemic Enterobacteriaceae[J].Antimicrob Agents Chemother,2006,57 (4),661～667
[10]Perilli M, Dell'Amico E, Segarore B, et al. Molecular characterization of extended-spectrum beta-lactamases produced by nosocomial isolates of Enterobacteriaceae from an Italian national wide survey [J].Microbiol,2002,40(2):611～614
[11]Coque TM, Oliver A, PerezDiaz JC, et al.Genes encoding TEM-4, SHV-2 and CTX-M-10 extended-spectrum beta-lactamases are carried by multiple Klebsiella pneumoniae clones in a single hospital(Madrid,1989 to 2000) [J].Antimicrob Agents Chemother,2002,46(2): 500～510
[12]Yu WL, Chuang YC Wahher-Rasmussen J. Extended-spectrum beta-lactamases in Taiwan: epidemiology, detection, treatment and infection control [J].Microbiol Immunol Infect, 2006,39(4):264～277
[13]Ho PL, shek RH, chow KH, et al. Detection and characterization of extended-spectrum (3-lactamases among bloodstream isolates of Enterobacter spp. in Hong Kong, 2000-2002[J]. Antimicrobial Chemotherapy,2005,55(3):326～332
[14]俞云松.超广谱β-内酰胺酶研究进展[J].中华医学杂志,2006,86(9)：641～644
[15]Severin JA, Mertaniasih NM, Kuntaman K,et al.Molecular characterization of extended-spectrum beta-lactamases in clinical Escherichia coli and Klebsiella pneumoniae isolates from Surabaya, Indonesia[J]. J Antimicrob Chemother,2010,65(3):465-469
[16]Blanco M, Alonso MP, Nicolas-Chanoine MH,et al. Molecular epidemiology of Escherichia coli producing extended-spectrum{beta}-lactamases in Lugo (Spain): dissemination of clone O25b:H4-ST131 producing CTX-M-15[J]. J Antimicrob Chemother, 2009,63(6):1135～1141
[17]Yu WL, Chuang YC, Walther-Rasmussen J. Extended-spectrum beta-lactamases in Taiwan: epidemiology, detection, treatment and infection control[J]. J Microbiol Immunol Infect, 2006,39 (4):264～277
[18]Empel J, Baraniak A, Literacka E, et al. Molecular survey of beta-lactamases conferring resistance to newer beta-lactams in Enterobacteriaceae isolates from Polish hospitals[J]. Antimicrob Agents Chemother,2008,52 (7):2449～2454
[19]Oliveira CF, Dal Forno NL, Alves IA, et al. Prevalence of the TEM, SHV and CTX-M families of extended-spectrum beta-lactamases in Escherichia coli and Klebsiella spp at the University Hospital of Santa Maria, State of Rio Grande do Sul[J].Rev Soc Bras Med Trop,2009,42 (5):556～560
[20]Doublet B, Granier SA, Robin F, et al.Novel plasmid-encoded ceftazidime-hydrolyzing CTX-M-53 extended-spectrum beta-lactamase from Salmonella enterica serotypes Westhampton and Senftenberg[J]. Antimicrob Agents Chemother,2009,53(5):1944～1951
[21]陈民钧.美国临床实验室标准化研究所2005年版有关药敏试验标准化更新要点[J].中华检验医学杂志,2005,28(4)：449～451
[22]陈民钧,俞云松,王辉,等.改进药敏折点标准指导临床合理选择抗菌药物[J]．中华检验医学杂志,2005,28(6)：565～568
[23]Steward CD, Rasheed JK, Hubert SK, et al. Characterization of clinical isolates of Klebsiella pneumoniae from 19 laboratories using the National Committee for Clinical Laboratory Standards extended-spectrum beta-lactamase detection methods[J].J Clin Microbiol,2001,39(8):2864～2872
[24]Queenan AM, Foleno B, Gownley C, et al. Effects of inoculum and be-ta-laetamase activity in AmpC-and extended-spectrum beta-lactamlase(ESBL)-producing Escherichia coli and Klebsiella pneumoniae clinical isolates tested by using National Committee for Clinical Laboratory Standards ESBL methodology[J].J Clin Microbiol,2004,42(1): 269～275
[25]Cagatay AA, Kocagez T, Emksoy H.Dio-Sensimedia:a novel culturemediam for rapid detection of extended spectrum beta-lactamases. BMC Infect Dis,2003,25(3):22
[26]Menon T, Bindu D, Kumar CP, et al. Comparison of double disc and three dimensional methods to screen for ESBL producers in a tertiary care hospital[J].Indian J Med Microbiol, 2006,24(2):117～120
[27]Marra AR, Pemira CA, Castelo A, et al. Health and economic out-comes of the detection of Klebsiella pneumoniae-produced extended-spectrum beta-lactamase (ESBL) in a hospital with high prevalence of this infection[J].Int J Infect Dis,2006,10(1):56～60.
[28]Leverstein-vail Hall MA, Fluit AC, Paauw A, et al. Evaluation of the E-test ESBL and the BD Phoenix, VITEK 1, and VITEK 2 Automated Instruments for detection of extended-spectrum beta-lactamase-in multi-resistant Eschefichia coli and Klebaiella spp[J].J Clin Microbiol,2002,40(10):3703～3711
[29]Dashti AA, west P, Paton R, etal. Characterization of extended-spectrum beta-lactamase (ESBL)-producing Kuwait and UK strains identified by the Vitek system, and subsequent comparison of the Vitek system with other commercial ESBL-testing systems using these strains[J].J Med Microbiol,2006,55(Pt4):417～421
[30]Skippen I, Shemko M, Palmer C, etal Laboratory diagnosis of bloodstream infections caused by extended-spectrum beta-lactamase-producing E coli and Klebaiella species[J]. Br J Biomed Sci,2006,63(1):1-4
[31]Spanu T, Sanguinetti M, Tumbarello M, et al. Evaluation of the new VITEK 2 extended-spectrum beta-lactamase (ESBL)test for rapid detection of ESBL production in Enterobacteriaceae isolates[J].J Clin Microbiol,2006,44(9):3257～3262
[32]Schwaber MJ, Navon-Venezia S, Chmelnitsky I, et al Utility of the VITEK 2 Advanced Expert System for identification of extended-spectrum beta-lactamase production in Enterobacter spp[J].J Clin Microbiol,2006,44(1):241～243
[33]Limcott AJ, Brown WJ. Evaluation of four commercially available extended-spectrum beta-lactamase phenotypie confirmation tests[J].J Chn Microbiol,2005,43(3):1081～1085
[34]James H, Jorgensen M L, Mc Elmeel. Detection of CTX-M-Type Extended-Spectrum Beta-Lactamase (ESBLs) by Testing with MicroScan Overnight and ESBL Confirmation Panels[J]. Clinical Microbiology,2010,48(1):120～123
[35]Bagattini M, Crivaro V, Di Popolo A, et al. Molecular epidemiology of extended-spectrum β-lactamase-producing Klebsiella pneumoniae in a neonatal intensive care unit[J]. Antimicrob Chemother,2006,57(5):979～982
[36]Romero L, Lopez L, Rodn'guez-Bano J, et al. Long-term study of the frequency of Escherichia coli and Klebsiella pneumoniae isolates producing extended-spectrum β-lactamases[J]. Clin Microbiol Infect,2005,11(8):625～631
[37]Garci'a San Miguel L, Cobo J, Valverde A, et al. Clinical variables associated with the isolation of Klebsiella pneumoniae expressing different extended-spectrum β-lactamases[J]. Clin Microbiol Infect,2007,13(5):532～538
[38]Mena A, Plasencia V, Garcia L, et al. Characterization of a large outbreak by CTX-M-1-producing Klebsiella pneumoniae and mechanisms leading to in vivo carbapenem resistance development[J]. Clin Microbiol,2006,44(8):2831～2837
[39]Yan JJ, Ko WC, Wu HM, et al. Complexity of Klebsiella pneumoniae isolates resistant to both cephamycins and extended-spectrum cephalosporins at a teaching hospital in Taiwan[J]. Clin Microbiol,2004,42(11):5337-5340
[40]Lehner S, Grabein B, Pfaller P, et al. Relevance of ESBL-producing pathogens for clinical surgery:diagnostics, therapy, and prevention[J]. Chirurg,2009,80(6):527-536.
[41]J. Farber, KA. Moder, F Layer, et al. Extended-Spectrum Beta-Lactamase Detection with Different Panels for Automated Susceptibility Testing and with a Chromogenic Medium[J]. Clin Microbiol,2008,46(11):3721-3727
[42]Paterson DL, Bonnamo RA. Extend-spectum beta-lactmases:a clinical update[J].Clin Microbiol Rev,2005,18(4):657-686
[43]Tenover FC, Raney PM, Williams PP, et al. Evaluation of the NCCLS extended-spectrum beta-lactamase confirmation methods for Escherichia coli with isolates collected during Project ICARE[J].J Clin Microbiol Rev,2003,41(7):3142-3146
[44]Jemima SA, Verghese Susan. Multiplex PCR for blaCTX-M & blaSHV in the extended spectrum beta lactamase (ESBL) producing Gram-negative isolates[J]. Indian Med Res, 2008,128(3):313～317
[45]Cynthia O, Ayla E, Thierry N, et al. Rapid detection of CTX-M-producing Enterobacteriaceae in urine samples[J]. Antimicrob Chemother.2009,64(5):986～989
[46]Barlaan EA, Sugimofi M, Furukawa S, et al. Profiling and monitoring of microbial populations by denaturing high-performance liquid chromatography[J].J Microbiol Methods,2005,61(3):399～412.
[47]刘朝晖,王汉平,陈劲龙,等.运用变性高效液相色谱对肺炎克雷伯菌产ESBL进行基因分型[J].中华微生物学和免疫学杂志,2005,25(9)：764～767
[48]张盛斌,刘朝晖,杨银梅,等.运用变性高效液相色谱技术快速检测肺炎克雷伯菌耐药性[J]中华医院感染学杂志,2006,16(9)：1075～1077
[49]刘朝晖,张盛斌,王汉平,等.运用变性高效液相色谱快速检测CTX-M型超广谱β内酰胺酶[J].中国感染与化疗杂志,2006,6(6)：384～388
[50]Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for β-lactamases and its correlation with molecular structure[J]. Antimicrob Chemother,1995,19(6): 1211～1233
[51]Paterson DL, Ko WC, Von Gottberg A, et al. Antibiotic therapy for Klebsiella pneumoniae bacteremia:implications of production of extended-spectrum β-lactamases[J].Clin Infect Dis,2004,39 (1):31～37
[52]Sohei Harada MD, Yoshikazu Ishii M.D, Keizo Yamaguchi MD.Extended-spectrum β-Lactamases:Implications for the Clinical Laboratory and Therapy[J]. Korean Lab Med, 2008,28 (6):401～412
[53]Vora S, Auckenthaler R. What is the significance of extended spectrum betalactamases in clinical practice. Rev Med Suisse[J].2009,5(220):1991～1994
[54]Isturiz R.Global resistance trends and the potential impact on empirical therapy[J]. Antimicrob Agents.,2008,32, Suppl(4):201～206
[55]Zahar JR, Lortholary O, Martin C, et al. Addressing the challenge of extended-spectrum beta-lactamases[J].Curr Opin Investing Drugs,2009,10(2):172～180
[56]Shahid M, Sobia F, Singh A, et al. Beta-lactams and beta-lactamase-inhibitors in current-or potential-clinical practice:a comprehensive update[J]. Crit Rev Microbiol,2009,35(2): 81～108
[57]Bassetti M, Righi E, Viscoli C. Novel beta-lactam antibiotics and inhibitor combinations[J]. Expert Opin Investig Drugs,2008,17(3):285～296
[58]徐敏超,王晶,严群,等.宽噬噬菌体治疗实验性小鼠产超广谱β-内酰胺酶大肠埃希菌感染的研究[J].中华医院感染学杂志,2006,16(3)：252～255
[59]Ren N, Wang F, Niu JQ. A study of 10-23 DNAzyme inhibiting the expression of hepatitis B virus genes[J].Zhonghua Gan Zang Bing Za Zhi.2005,13(10):745～748
[60]洪洁华,刘晓波,吕小迅.“10-23”型脱氧核酶的研究进展[J].国外医学药学分册,2007,34(2)：99～102
[61]Zaborowska Z, Furste JP, Erdmann VA, et al.Sequence requirements in the catalytic core of the "10-23" DNA enzyme[J].J Biol Chem,2002,277 (43):40617-60622
[62]侯征,孟静茹,扈本荃,等.脱氧核酶抑制耐药基因mecRl的表达逆转MRSA耐药性[J].中国抗生素杂志,2006,31(3)：144～148
[63]谢勇恩,凌保东,余娴,等.10-23脱氧核酶抑制细菌β-内酰胺酶基因表达的实验研究[J].中国病理生理学杂志,2006,22(4)：652～655
[64]刘刚,凌保东,谢勇恩,等.脱氧核酶抑制细菌TEM-1β-内酰胺酶基因表达的实验研究[J].中国抗生素杂志,2007,32(11)：693～696
[65]Lai R, Liu H, Hui Lee, et al. An anionic antimicrobial peptide from toad Bombina maxima [J]. Biochem Biophys Res Commun,2002,295 (4):796～799
[66]Brogden, K A, De Lucca AJ, Bland J, et al.Isolation of an ovine pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica[J]. Proc Natl Acad Sci U S A,1996,93 (1):412-416
[67]Schittek B, Hipfel R, Sauer B, et al. Dermcidin:a novel human antibiotic peptide secreted by sweat glands[J]. Nat Immunol,2001,2 (12):1133-1137
[68]Andersson M, Boman A, Boman H G Ascaris nematodes from pig and human make three antibacterial peptides:isolation of cecropin PI and two ASABF peptides[J]. Cell Mol Life Sci,2003,60 (3):599～606
[69]Otvos LJr. The short proline-rich antibacterial peptide family[J].Cell Mol Life Sci,2002, 59 (7):1138～1150
[70]Shamova O, Brogden K A, Zhao C, et al. Purification and properties of proline-rich antimicrobial peptides from sheep and goat leukocytes [J]. Infect Immun,1999,67 (8): 4106～4111
[71]Park Y, Lec DG, Jang SH, et al. A leu lys rich antimicrobial peptide:activity and mechanism[J].Biochim Biophys Acta,2003,1645 (2):172～182
[72]De Zoysa M, Whang I, Lee S, et al. Defensin from disk abalone Haliotis discus discus: molecular cloning, sequence characterization and immune response against bacterial infection[J]. Fish Shellfish Immunol,2010,28 (2):261～266
[73]Vaara M.New approaches in peptide antibiotics[J].Curr Opin Pharmacol,2009,9(5):571～ 576
[74]Juretic D, Vukicevic D, Ilic N, et al. Computational design of highly selective antimicrobial peptides[J]. J Chem Inf Model,2009,49 (12):2873～2882
[75]Chou HT, Kuo TY, Chiang JC, et al. Design and synthesis of cationic antimicrobial peptides with improved activity and selectivity against Vibrio spp[J]. Int J Antimicrob Agents,2008,32 (2):130～138
[2]张阳根,张海艳,江先海,等.超广谱内酰胺酶研究进展[J].江西医学检验,2007,25(3)：254～255
[3]郭小兵,张志坚,阎志勇,等.CTX-M-14型超广谱β-内酰胺酶原核表达载体的构建及其抗药活性分析[J].山东医药,2009,49(11)：21～23
[4]李金钟,刘利平.CTX-M型超广谱β-内酰胺酶的研究进展[J].国际检验医学杂志.2007,28(6)：543～545
[5]张盛斌,刘朝辉.CTX-M型ESBLs的研究进展[J].中国感染与化疗杂志,2006,6(3)：212～215
[6]Bonnet R, Growing group of extended-spectrum beta-lactamases:the CTX-M enzymes [J].Antimicrob Agents Chemother,2004,48 (1):1-14
[7]Humeniuk C, Arlet G, Gautier V, et al.Beta-lactamases of Kluyvera ascorbata, probable progenitors of some plasmid-encoded CTX-M types[J].Antimicrob Agents Chemother, 2002,46(9):3045-3049
[8]陆坚,唐英春,吴本权,等.华南地区质粒介导超广谱β-内酰胺酶的基因分型研究[J].中华微生物学和免疫学杂志,2002,22(6)：638～643
[9]Yan JJ, Ko WC, Tsai SH, et al.Dissemination of CTX-M-3 and CMY-2 beta-lactamases among clinical isolates of Escherichia coli in southern Taiwan[J].Clin Microbiol,2000, 38(12):4320-4325
[10]Chanawong A, M'zali FH, Heritage J, et al. Three cefotaximases, CTX-M-9, CTX-M-13, and CTX-M-14, among Enterobacteriaceae in the People's republic of China[J].Antimicrob Agents Chemother,2002,46(3):630-637
[11]张阮章,卢月梅,胡玉华,等．一种新型β内酰胺酶CTX-M-38的发现[J].中国感染与化疗杂志,2009,9(3)：216～218
[12]赵玉莲,张润梅,汪军荣.产超广谱β-内酰胺酶细菌的耐药性监测[J].中国药物与临床,2006,6(4)：266～268
[13]孙长贵,陈汉美.超广谱β-内酰胺酶及其实验室筛检[J].临床检验杂志,2000,18(1)：52～55
[14]张阮章,王沙燕,卢月梅,等.产超广谱β-内酰胺酶大肠埃希菌和肺炎克雷伯菌bla-CTX-M基因的检测[J].中国微生态杂志,2004,16(6)：342～344
[15]Boyd DA, Tyler S, Christianson S, et al. Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum beta-lactamase involved in an outbreak in long-term care facilities in Toronto, Canada[J]. Antimicrob Agents Chemother, 2004,48 (10):3758-3764
[16]Ibuka A, Taguchi A, Ishiguro M, et al.Crystal structure of the E166A mutant of extended-spectrum beta-lactamase Toho-1 at 1.8A resolution[J].J Mol Biol,1999,285(5): 2079～2087
[17]Bonnet R, Sampaio JL, Labia R, et al.A novel CTX-M β-lactamase (CTX-M-8) in cefotaxime-resistant enterobacteriaceae isolated in brazil[J].Antimicrob Agents Chemother, 2000,44 (7):1936-1942
[18]郭小兵,张志坚,阎志勇,等.CTX-M-38型超广谱β-内酰胺酶的表达、定位及抗抗药活性分析[J].天津医药,2009,37(12)：1032～1034
[19]李霜,李咏梅.β-内酰胺酶及超广谱β-内酰胺酶的研究进展[J].吉林医药学院学报,2009.27(3)：171～174
[20]袁燕,林果为.β-内酰胺酶及超广谱β内酰胺酶的研究新进展[J].英国医学杂志中文版,2004,7(4)：220～224
[21]秦东春,任丽娟,闫志勇,等.铜绿假单胞菌超广谱β-内酰胺酶编码基因OXA-1表达产物的纯化及复性分析[J].中国现代医学杂志,2007,17(19)：2341～2344
[22]萨母布鲁克,拉塞尔著.分子克隆实验指南：上册.第三版[M].黄培堂译.北京：科学出版社,2002
[23]赵春丽,范秀军,赵冰,等.IPTG诱导浓度对重组人肝再生增强因子基因表达的影响[J].东北农业大学学报,2004,35(4)：441～445
[24]吉清,何凤田.包涵体复性的研究进展[J].国外医学临床生物化学与检验学分册,2004,25(6)：516～518
[25]张庶民,祁自柏,李河民.基因工程表达蛋白包涵体的形成于纯化[J].微生物学免疫学进展,1995,23(1)：52～54
[1]zhijian zhang, xiaobing guo, qinxian zhang.Prevalence characterization of Extended-Spectrum β-Lactamases among Escherichia coli isolates collected in zhengzhou[J]. Journal of clinical laboratory analusis,2009,23 (6):404～407
[2]Mamlouk K, Boutiba-Ben Boubaker I, Gautier V, Vimont S, Picard B, et al. Emergence and outbreaks of CTX-M β-lactamase-producing Escherichia coli and Klebsiella pneumoniae strains in a Tunisian hospital[J]. J Clin Microbiol,2006,44 (11):4049～4056
[3]Yu WL, Chuang YC, Walther-Rasmussen J. Extended-spectrum beta-lactamases in Taiwan: epidemiology, detection, treatment, and infection control[J]. J. Microbiol. Immunol. Infect. 2006,39 (4):264～277
[4]张志坚,郭小兵,阎志勇,等.郑州地区大肠埃希菌中CTX-M型超广谱β-内酰胺酶的基因型分布[J].中华医院感染学杂志,2009,19(18)：2382～2384
[5]阎志勇,高晓群,郭小兵.郑州地区ESBLs基因型分布研究[J].医药论坛杂志,2008,29(2)：7-9
[6]Marra AR, Pemira CA, Castelo A, et al. Health and economic out-comes of the detection of Klebsiella pneumoniae-produced extended-spectrum beta-lactamase (ESBL) in a hospital with high prevalence of this infection[J].Int J Infect Dis,2006,10(1):56～60.
[7]James H, Jorgensen M L, Me Elmeel. Detection of CTX-M-Type Extended-Spectrum Beta-Lactamase (ESBLs) by Testing with MicroScan Overnight and ESBL Confirmation Panels[J]. Clinical Microbiology,2010,48(1):120～123
[8]Mena A, Plasencia V, Garcia L, et al. Characterization of a large outbreak by CTX-M-1-producing Klebsiella pneumoniae and mechanisms leading to in vivo carbapenem resistance development[J]. Clin Microbiol,2006,44 (8):2831～2837
[9]Paterson DL, Bonnamo RA. Extend-spectum beta-lactmases:a clinical update[J].Clin Microbiol Rev,2005,18(4):657～686
[10]李菁,林彤,宋帅,等.基因工程抗体研究进展[J].生物技术通报,2009,20(10)：40～44
[11]王秀红,周素芳.基因工程抗体研究进展[J].生物技术通讯,2007,18(2)：304～306
[12]De Bernardez Clark E, Schwarz E, Rudolph R.Inhibition of aggregation side reactions during in vitro protein folding[J].Methods Enzymol,1999,309:217～236
[13]Ejima D, Watanabe M, Sato Y, et al.High yield refolding and punification process for recombinant human interleukin-6 expressed in Escherichia coli[J].Biotechnol bioeng, 1999,62 (3):301～310
[14]唐玉钗,刘丽霞.应用辛酸法提取血清和腹水中的免疫球蛋白[J].上海免疫学杂志,1989,9(6)：370～371
[15]吴爱琴,元刚,吴昭怡,等CTX-M型超广谱β-内酰胺酶的研究进展[J].检验医学与临床,2009,6(6)：450～452
[16]朱戌冬,吴伟元,陈民钧.超广谱β-内酰胺酶CTX-M型新进展[J].中国抗感染化疗杂志,2001,1(2)：109～110
[1]Shu JC, Chia JH, Kuo AJ, et al. A 7-year surveillance for ESBL-producing Escherichia coli and Klebsiella pneumoniae at a university hospital in Taiwan:the increase of CTX-M-15 in the ICU[J]. Epidemiol Infect,2010,138 (2):253～263
[2]Jones CH, Tuckman M, Keeney D, et al. Characterization and sequence analysis of extended-spectrum-{beta}-lactamase-encoding genes from Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates collected during tigecycline phase 3 clinical trials[J]. Antimicrob Agents Chemother,2009,53 (2):465～475
[3]沈关心,周汝麟.现代免疫学实验技术.第二版[M].武汉：湖北科学技术出版社,2002,102～106
[4]Burgess DS, Hall RG 2nd, Lewis JS 2nd, et al. Clinical and microbiologic analysis of a hospital's extended-spectrum beta-lactamase-producing isolates over a 2-year period[J]. Pharmacotherapy,2003,23 (10):1232～1237
[5]徐敏超,王晶,严群,等.宽噬噬菌体治疗实验性小鼠产超广谱β-内酰胺酶大肠埃希菌感染的研究[J].中华医院感染学杂志,2006,16(3)：252～255
[6]陈民钧.美国临床实验室标准化研究所2005年版有关药敏试验标准化更新要点[J]．中华检验医学杂志,2005,28(4)：449～451
[7]孙长贵,陈汉美.超广谱β-内酰胺酶及其实验室筛检[J].临床检验杂志,2000,18(1)：52～55
[8]李巧菊,韩明,张再伟.医院感染ESBLs菌株的分子生物学测定[J].现代预防医学,2006,33(12)：2425～2427
[9]吕世静.临床免疫学检验.第一版[M].北京：中国医药科技出版社,2004,173～174
[10]吕世静.临床免疫学检验.第一版[M].北京：中国医药科技出版社,2004,207～209
[11]Rowe Magnus DA, Mazel D. The role of integrons in antibiotic resistance gene capture[J]. Int Med Microbiol,2002,292 (2):115～125
[12]Lavigne JP, Gaillard JB, Bourg G, et al.Extended-spectrum beta-lactamases-producing Stenotrophomonas maltophilia strains:CTX-M enzymes detection and virulence study[J]. Pathol Biol (Paris),2008,56 (7-8):447～453
[13]Yu WL, Chuang YC. Wahher-Rasmussen J. Extended-spectrum beta-lactamases in Taiwan: epidemiology, detection, treatment and infection control[J].Microbiol Immunol Infect, 2006,39(4):264～277
[14]郭小兵,张志坚,阎志勇,等.CTX-M-14型β-内酰胺酶原核表达载体的构建及其抗药活性分析[J].山东医药,2009,49(11)：21～23
[15]张志坚,郭小兵,阎志勇,等.郑州地区大肠埃希菌中CTX-M型超广谱β-内酰胺酶的基因型分布[J].中华医院感染学杂志,2009,19(18)：2382～2384
[16]杜艳霞.酶联免疫吸附试验测定操作中的体会[J].中国实用医药,2009,4(27)：139～140
[17]孙桂梅,韩金玲,赵莉,等.免疫诊断(主要是ELISA)试验操作的几点体会[J].中国现代药物应用,2009,3(3)：161～162
[1]Jacoby GA, Munoz-Price LS. The new beta-lactamases[J]. N Engl J Med,2005,352(4): 380～391
[2]Bradford PA. Extended-spectrum β-lactamases in the 21st century:characterization, epidemiology, and detection of this important resistance threat[J]. Clin Microbiol Rev 2001,14 (4):933～951
[3]Aranzazu Valverde, Teresa M. Coquel, et al.Complex molecular epidemiology of extended-spectrumβ-lactamases in Klebsiella pneumoniae:a long-term perspective from a single institution in Madrid[J]. Antimicrobial Chemotherapy,2008,61 (1):64～72
[4]Livermorel DM, Canton R, Gniadkowski M, et al. CTX-M:changing the face of ESBLs in Europe[J]. Antimicrobial Chemotherapy,2007,59 (2):165～174
[5]Ben-Hamouda T, Foulon T, Ben-Mahrez K. Involvement of SHV-12 and SHV-2a encoding plasmids in outbreaks of extended-spectrum β-lactamase-producing Klebsiella pneumoniae in a Tunisian neonatal ward[J]. Microb Drug Resist,2004,10(2):132～138.
[6]Gangoue-Pieboji J, Bedenic B, Koulla-Shiro S, et al. Extended spectrum-β-lactamase-producing Enterobacteriaceae in YaoundeCameroon[J]. Clin Microbiol,2005,43 (7): 3273～3277
[7]Gangoue-Pieboji J, Miriagou V, Vourli S, et al. Emergence of CTX-M-15-producing enterobacteria in Cameroon and characterization of a blaCTX-M-15-carrying element[J]. Antimicrob Agents Chemother,2005,49 (1):441～443
[8]Blomberg B, Jureen R, Manji KP, et al. High rate of fatal cases of pediatric septicemia caused by gram-negative bacteria with extendedspectrum β-lactamases in Dar es Salaam, Tanzania[J]. Clin Microbiol,2005,43 (2):745～749
[9]Gray KJ, Wilson LK, Phiri A, et al.Identification and characterization of ceftriaxone resistance and extended-spectrum β-lactamases in Malawian bacteraemic Enterobacteriaceae[J].Antimicrob Agents Chemother,2006,57 (4),661～667
[10]Perilli M, Dell'Amico E, Segarore B, et al. Molecular characterization of extended-spectrum beta-lactamases produced by nosocomial isolates of Enterobacteriaceae from an Italian national wide survey [J].Microbiol,2002,40(2):611～614
[11]Coque TM, Oliver A, PerezDiaz JC, et al.Genes encoding TEM-4, SHV-2 and CTX-M-10 extended-spectrum beta-lactamases are carried by multiple Klebsiella pneumoniae clones in a single hospital(Madrid,1989 to 2000) [J].Antimicrob Agents Chemother,2002,46(2): 500～510
[12]Yu WL, Chuang YC Wahher-Rasmussen J. Extended-spectrum beta-lactamases in Taiwan: epidemiology, detection, treatment and infection control [J].Microbiol Immunol Infect, 2006,39(4):264～277
[13]Ho PL, shek RH, chow KH, et al. Detection and characterization of extended-spectrum (3-lactamases among bloodstream isolates of Enterobacter spp. in Hong Kong, 2000-2002[J]. Antimicrobial Chemotherapy,2005,55(3):326～332
[14]俞云松.超广谱β-内酰胺酶研究进展[J].中华医学杂志,2006,86(9)：641～644
[15]Severin JA, Mertaniasih NM, Kuntaman K,et al.Molecular characterization of extended-spectrum beta-lactamases in clinical Escherichia coli and Klebsiella pneumoniae isolates from Surabaya, Indonesia[J]. J Antimicrob Chemother,2010,65(3):465-469
[16]Blanco M, Alonso MP, Nicolas-Chanoine MH,et al. Molecular epidemiology of Escherichia coli producing extended-spectrum{beta}-lactamases in Lugo (Spain): dissemination of clone O25b:H4-ST131 producing CTX-M-15[J]. J Antimicrob Chemother, 2009,63(6):1135～1141
[17]Yu WL, Chuang YC, Walther-Rasmussen J. Extended-spectrum beta-lactamases in Taiwan: epidemiology, detection, treatment and infection control[J]. J Microbiol Immunol Infect, 2006,39 (4):264～277
[18]Empel J, Baraniak A, Literacka E, et al. Molecular survey of beta-lactamases conferring resistance to newer beta-lactams in Enterobacteriaceae isolates from Polish hospitals[J]. Antimicrob Agents Chemother,2008,52 (7):2449～2454
[19]Oliveira CF, Dal Forno NL, Alves IA, et al. Prevalence of the TEM, SHV and CTX-M families of extended-spectrum beta-lactamases in Escherichia coli and Klebsiella spp at the University Hospital of Santa Maria, State of Rio Grande do Sul[J].Rev Soc Bras Med Trop,2009,42 (5):556～560
[20]Doublet B, Granier SA, Robin F, et al.Novel plasmid-encoded ceftazidime-hydrolyzing CTX-M-53 extended-spectrum beta-lactamase from Salmonella enterica serotypes Westhampton and Senftenberg[J]. Antimicrob Agents Chemother,2009,53(5):1944～1951
[21]陈民钧.美国临床实验室标准化研究所2005年版有关药敏试验标准化更新要点[J].中华检验医学杂志,2005,28(4)：449～451
[22]陈民钧,俞云松,王辉,等.改进药敏折点标准指导临床合理选择抗菌药物[J]．中华检验医学杂志,2005,28(6)：565～568
[23]Steward CD, Rasheed JK, Hubert SK, et al. Characterization of clinical isolates of Klebsiella pneumoniae from 19 laboratories using the National Committee for Clinical Laboratory Standards extended-spectrum beta-lactamase detection methods[J].J Clin Microbiol,2001,39(8):2864～2872
[24]Queenan AM, Foleno B, Gownley C, et al. Effects of inoculum and be-ta-laetamase activity in AmpC-and extended-spectrum beta-lactamlase(ESBL)-producing Escherichia coli and Klebsiella pneumoniae clinical isolates tested by using National Committee for Clinical Laboratory Standards ESBL methodology[J].J Clin Microbiol,2004,42(1): 269～275
[25]Cagatay AA, Kocagez T, Emksoy H.Dio-Sensimedia:a novel culturemediam for rapid detection of extended spectrum beta-lactamases. BMC Infect Dis,2003,25(3):22
[26]Menon T, Bindu D, Kumar CP, et al. Comparison of double disc and three dimensional methods to screen for ESBL producers in a tertiary care hospital[J].Indian J Med Microbiol, 2006,24(2):117～120
[27]Marra AR, Pemira CA, Castelo A, et al. Health and economic out-comes of the detection of Klebsiella pneumoniae-produced extended-spectrum beta-lactamase (ESBL) in a hospital with high prevalence of this infection[J].Int J Infect Dis,2006,10(1):56～60.
[28]Leverstein-vail Hall MA, Fluit AC, Paauw A, et al. Evaluation of the E-test ESBL and the BD Phoenix, VITEK 1, and VITEK 2 Automated Instruments for detection of extended-spectrum beta-lactamase-in multi-resistant Eschefichia coli and Klebaiella spp[J].J Clin Microbiol,2002,40(10):3703～3711
[29]Dashti AA, west P, Paton R, etal. Characterization of extended-spectrum beta-lactamase (ESBL)-producing Kuwait and UK strains identified by the Vitek system, and subsequent comparison of the Vitek system with other commercial ESBL-testing systems using these strains[J].J Med Microbiol,2006,55(Pt4):417～421
[30]Skippen I, Shemko M, Palmer C, etal Laboratory diagnosis of bloodstream infections caused by extended-spectrum beta-lactamase-producing E coli and Klebaiella species[J]. Br J Biomed Sci,2006,63(1):1-4
[31]Spanu T, Sanguinetti M, Tumbarello M, et al. Evaluation of the new VITEK 2 extended-spectrum beta-lactamase (ESBL)test for rapid detection of ESBL production in Enterobacteriaceae isolates[J].J Clin Microbiol,2006,44(9):3257～3262
[32]Schwaber MJ, Navon-Venezia S, Chmelnitsky I, et al Utility of the VITEK 2 Advanced Expert System for identification of extended-spectrum beta-lactamase production in Enterobacter spp[J].J Clin Microbiol,2006,44(1):241～243
[33]Limcott AJ, Brown WJ. Evaluation of four commercially available extended-spectrum beta-lactamase phenotypie confirmation tests[J].J Chn Microbiol,2005,43(3):1081～1085
[34]James H, Jorgensen M L, Mc Elmeel. Detection of CTX-M-Type Extended-Spectrum Beta-Lactamase (ESBLs) by Testing with MicroScan Overnight and ESBL Confirmation Panels[J]. Clinical Microbiology,2010,48(1):120～123
[35]Bagattini M, Crivaro V, Di Popolo A, et al. Molecular epidemiology of extended-spectrum β-lactamase-producing Klebsiella pneumoniae in a neonatal intensive care unit[J]. Antimicrob Chemother,2006,57(5):979～982
[36]Romero L, Lopez L, Rodn'guez-Bano J, et al. Long-term study of the frequency of Escherichia coli and Klebsiella pneumoniae isolates producing extended-spectrum β-lactamases[J]. Clin Microbiol Infect,2005,11(8):625～631
[37]Garci'a San Miguel L, Cobo J, Valverde A, et al. Clinical variables associated with the isolation of Klebsiella pneumoniae expressing different extended-spectrum β-lactamases[J]. Clin Microbiol Infect,2007,13(5):532～538
[38]Mena A, Plasencia V, Garcia L, et al. Characterization of a large outbreak by CTX-M-1-producing Klebsiella pneumoniae and mechanisms leading to in vivo carbapenem resistance development[J]. Clin Microbiol,2006,44(8):2831～2837
[39]Yan JJ, Ko WC, Wu HM, et al. Complexity of Klebsiella pneumoniae isolates resistant to both cephamycins and extended-spectrum cephalosporins at a teaching hospital in Taiwan[J]. Clin Microbiol,2004,42(11):5337-5340
[40]Lehner S, Grabein B, Pfaller P, et al. Relevance of ESBL-producing pathogens for clinical surgery:diagnostics, therapy, and prevention[J]. Chirurg,2009,80(6):527-536.
[41]J. Farber, KA. Moder, F Layer, et al. Extended-Spectrum Beta-Lactamase Detection with Different Panels for Automated Susceptibility Testing and with a Chromogenic Medium[J]. Clin Microbiol,2008,46(11):3721-3727
[42]Paterson DL, Bonnamo RA. Extend-spectum beta-lactmases:a clinical update[J].Clin Microbiol Rev,2005,18(4):657-686
[43]Tenover FC, Raney PM, Williams PP, et al. Evaluation of the NCCLS extended-spectrum beta-lactamase confirmation methods for Escherichia coli with isolates collected during Project ICARE[J].J Clin Microbiol Rev,2003,41(7):3142-3146
[44]Jemima SA, Verghese Susan. Multiplex PCR for blaCTX-M & blaSHV in the extended spectrum beta lactamase (ESBL) producing Gram-negative isolates[J]. Indian Med Res, 2008,128(3):313～317
[45]Cynthia O, Ayla E, Thierry N, et al. Rapid detection of CTX-M-producing Enterobacteriaceae in urine samples[J]. Antimicrob Chemother.2009,64(5):986～989
[46]Barlaan EA, Sugimofi M, Furukawa S, et al. Profiling and monitoring of microbial populations by denaturing high-performance liquid chromatography[J].J Microbiol Methods,2005,61(3):399～412.
[47]刘朝晖,王汉平,陈劲龙,等.运用变性高效液相色谱对肺炎克雷伯菌产ESBL进行基因分型[J].中华微生物学和免疫学杂志,2005,25(9)：764～767
[48]张盛斌,刘朝晖,杨银梅,等.运用变性高效液相色谱技术快速检测肺炎克雷伯菌耐药性[J]中华医院感染学杂志,2006,16(9)：1075～1077
[49]刘朝晖,张盛斌,王汉平,等.运用变性高效液相色谱快速检测CTX-M型超广谱β内酰胺酶[J].中国感染与化疗杂志,2006,6(6)：384～388
[50]Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for β-lactamases and its correlation with molecular structure[J]. Antimicrob Chemother,1995,19(6): 1211～1233
[51]Paterson DL, Ko WC, Von Gottberg A, et al. Antibiotic therapy for Klebsiella pneumoniae bacteremia:implications of production of extended-spectrum β-lactamases[J].Clin Infect Dis,2004,39 (1):31～37
[52]Sohei Harada MD, Yoshikazu Ishii M.D, Keizo Yamaguchi MD.Extended-spectrum β-Lactamases:Implications for the Clinical Laboratory and Therapy[J]. Korean Lab Med, 2008,28 (6):401～412
[53]Vora S, Auckenthaler R. What is the significance of extended spectrum betalactamases in clinical practice. Rev Med Suisse[J].2009,5(220):1991～1994
[54]Isturiz R.Global resistance trends and the potential impact on empirical therapy[J]. Antimicrob Agents.,2008,32, Suppl(4):201～206
[55]Zahar JR, Lortholary O, Martin C, et al. Addressing the challenge of extended-spectrum beta-lactamases[J].Curr Opin Investing Drugs,2009,10(2):172～180
[56]Shahid M, Sobia F, Singh A, et al. Beta-lactams and beta-lactamase-inhibitors in current-or potential-clinical practice:a comprehensive update[J]. Crit Rev Microbiol,2009,35(2): 81～108
[57]Bassetti M, Righi E, Viscoli C. Novel beta-lactam antibiotics and inhibitor combinations[J]. Expert Opin Investig Drugs,2008,17(3):285～296
[58]徐敏超,王晶,严群,等.宽噬噬菌体治疗实验性小鼠产超广谱β-内酰胺酶大肠埃希菌感染的研究[J].中华医院感染学杂志,2006,16(3)：252～255
[59]Ren N, Wang F, Niu JQ. A study of 10-23 DNAzyme inhibiting the expression of hepatitis B virus genes[J].Zhonghua Gan Zang Bing Za Zhi.2005,13(10):745～748
[60]洪洁华,刘晓波,吕小迅.“10-23”型脱氧核酶的研究进展[J].国外医学药学分册,2007,34(2)：99～102
[61]Zaborowska Z, Furste JP, Erdmann VA, et al.Sequence requirements in the catalytic core of the "10-23" DNA enzyme[J].J Biol Chem,2002,277 (43):40617-60622
[62]侯征,孟静茹,扈本荃,等.脱氧核酶抑制耐药基因mecRl的表达逆转MRSA耐药性[J].中国抗生素杂志,2006,31(3)：144～148
[63]谢勇恩,凌保东,余娴,等.10-23脱氧核酶抑制细菌β-内酰胺酶基因表达的实验研究[J].中国病理生理学杂志,2006,22(4)：652～655
[64]刘刚,凌保东,谢勇恩,等.脱氧核酶抑制细菌TEM-1β-内酰胺酶基因表达的实验研究[J].中国抗生素杂志,2007,32(11)：693～696
[65]Lai R, Liu H, Hui Lee, et al. An anionic antimicrobial peptide from toad Bombina maxima [J]. Biochem Biophys Res Commun,2002,295 (4):796～799
[66]Brogden, K A, De Lucca AJ, Bland J, et al.Isolation of an ovine pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica[J]. Proc Natl Acad Sci U S A,1996,93 (1):412-416
[67]Schittek B, Hipfel R, Sauer B, et al. Dermcidin:a novel human antibiotic peptide secreted by sweat glands[J]. Nat Immunol,2001,2 (12):1133-1137
[68]Andersson M, Boman A, Boman H G Ascaris nematodes from pig and human make three antibacterial peptides:isolation of cecropin PI and two ASABF peptides[J]. Cell Mol Life Sci,2003,60 (3):599～606
[69]Otvos LJr. The short proline-rich antibacterial peptide family[J].Cell Mol Life Sci,2002, 59 (7):1138～1150
[70]Shamova O, Brogden K A, Zhao C, et al. Purification and properties of proline-rich antimicrobial peptides from sheep and goat leukocytes [J]. Infect Immun,1999,67 (8): 4106～4111
[71]Park Y, Lec DG, Jang SH, et al. A leu lys rich antimicrobial peptide:activity and mechanism[J].Biochim Biophys Acta,2003,1645 (2):172～182
[72]De Zoysa M, Whang I, Lee S, et al. Defensin from disk abalone Haliotis discus discus: molecular cloning, sequence characterization and immune response against bacterial infection[J]. Fish Shellfish Immunol,2010,28 (2):261～266
[73]Vaara M.New approaches in peptide antibiotics[J].Curr Opin Pharmacol,2009,9(5):571～ 576
[74]Juretic D, Vukicevic D, Ilic N, et al. Computational design of highly selective antimicrobial peptides[J]. J Chem Inf Model,2009,49 (12):2873～2882
[75]Chou HT, Kuo TY, Chiang JC, et al. Design and synthesis of cationic antimicrobial peptides with improved activity and selectivity against Vibrio spp[J]. Int J Antimicrob Agents,2008,32 (2):130～138