【作者】 张志坚；

【导师】 张钦宪；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 大肠埃希菌是目前社区获得性及院内感染的主要病原菌之一,在实验室检测过程中,有着较高的分离率。从临床上看,其临床感染类型多样,感染部位不一。对其所致感染治疗效果如何,直接影响到患者的预后及病程。基于此点,各地针对大肠埃希菌的研究逐步成为感染领域的一个热点。而针对其耐药机制的研究,则是焦点所在。基于广谱抗菌药物,尤其是三代头孢菌素的滥用及不合理应用,多重耐药大肠埃希菌不断涌现。临床分离菌株除了对β-内酰胺类药物耐药外,往往伴随有氨基糖甙类、喹诺酮类抗菌药物协同耐药。究其耐药机制,从表型上看,多涉及泵出机制、胞膜渗透性改变、产生抗菌药物钝化酶及修饰酶等；从分子水平上看,多为细菌携带并稳定遗传和或菌株间传递多种耐药基因。研究表明,野生菌株产超广谱β-内酰胺酶(Extended-spectrumβ-lactamases, ESBLs),携带单一或多种型别超广谱β-内酰胺酶耐药基因,是大肠埃希菌多重耐药的主要机制之一。超广谱β-内酰胺酶是β-内酰胺酶的一种,其编码基因多定位于质粒,少数存在于染色体上。此类酶可显著水解氧亚基β-内酰胺类抗菌药物,包括青霉素类、第一、二、三代头孢菌素及单环类药物。对于头霉烯、碳青霉烯类药物及酶抑制剂稳定敏感。自1983年首次报道以来,产酶菌株在世界范围内广泛流行。总的来看,其流行性有以下特点。(1)临床分离菌株所产酶型别增多,各酶型新的亚型不断出现。(2)除TEM、SHV型相对保守外,其他酶型亚型间核酸序列同源性较小。(3)各酶型多由TEM-1、2及SHV-1型酶突变而来。(4)菌株所携带酶型呈地域性分布。(5)各型及亚型酶抗药活性有所差异。河南地区自2000年刘新郑等人首次报道产超广谱β-内酰胺酶菌株以来,针对野生产酶大肠埃希菌耐药谱的报道不断增多,但对于其酶型及分布的研究涉之甚少。基于酶型地域性分布特征及各酶型问抗药活性差异,此种研究结果不能给临床抗感染治疗提供有力的实验室依据,势必造成医疗资源的浪费,加重患者的经济负担。作为人口众多,病源广泛,而经济相对落后的内陆省份,明确大肠埃希菌产超广谱β-内酰胺酶的型别分布及各亚型耐药谱,指导临床合理有效抗感染治疗,最大程度地降低医疗费用,势在必行。为了系统地了解河南地区大肠埃希菌产ESBLs的型别分布特征,初步明确ESBLs编码基因定位以及主要流行型别抗药活性,作者采用双纸片协同试验及酶抑制剂增强试验筛选产ESBLs大肠埃希菌；采用PCR扩增及DNA测序技术检测大肠埃希菌中ESBLs流行型别及菌株分布模式；采用E-test检测携带不同质粒谱及ESBLs基因型菌株抗药活性；采用X2检验分析质粒谱、基因型与抗药活性的相关性；采用基因重组技术构建表达载体并表达；采用液体稀释法检测转化子抗药活性。通过上述实验,最终可为临床抗感染治疗提供可靠的实验室资料。本课题包括以下三个部分。第一部分大肠埃希菌产超广谱β-内酰胺酶流行病学研究方法1.采用双纸片协同试验及酶抑制剂增强试验,对临床分离多重耐药大肠埃希菌进行产ESBLs筛选与确证。2.依据各型别ESBLs核苷酸同源性特征,采用DNAstar分析软件,完成特异性引物设计。3.采用PCR技术,对已确证138株产酶大肠埃希菌进行各型别ESBLs检测。4.采用DNA序列分析,明确河南地区产酶大肠埃希菌携带主要ESBLs型别。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ESBLs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ESBLs菌株确证试验阳性。3.琼脂糖凝胶电泳显示,PCR扩增阳性产物大小以及型间差异与设计结果相一致。4.DNA序列分析结果显示,河南地区产酶大肠埃希菌中主要携带ESBLs型别为TEM-1、SHV-12、CTX-M-1、CTX-M-14、CTX-M-25、CTX-M-38、OXA-1、OXA-20。5.138株大肠埃希菌中,TEM-1型广谱β-内酰胺酶最为流行(80.4%),然后依次为SHV-12(60.9%)、CTX-M-14(58.7%)、CTX-M-1(32.6%)、CTX-M-25 (6.5%)、OXA-1(6.5%)、CTX-M-38(4.3%)以及OXA-20(2.0%)。6.138株产酶大肠埃希菌中,单菌株可同时携带1-5种型别ESBLs,其中以携带两种型别为主(32.6%),然后依次为三种型别(26.1%)、单一型别(21.7%)、四种型别(17.4%)以及五种型别(2.2%)。7.单一型别ESBLs模式菌株,以携带TEM-1型最多见(13.0%),其次为CTX-M-14(4.3%)、SHV-12(2.2%)以及CTX-M-1(2.2%)。8.两种型别ESBLs模式菌株,以同时携带SHV-12+CTX-M-14为主(10.9%),然后依次为TEM-1+CTX-M-14(8.7%)、SHV-12+TEM-1(6.5%)、TEM-1+ CTX-M-1(2.2%)、TEM-1+CTX-M-38(2.2%)及OXA-1+CTX-M-1(2.2%).9.三种型别ESBLs模式菌株,以同时携带SHV-12+TEM-1+CTX-M-1为主(10.9%),其次为SHV-12+TEM-1+CTX-M-14(8.7%)、TEM-1+CTX-M-14+ CTX-M-38 (2.2%)、SHV-12+OXA-1+CTX-M-14 (2.2%)及SHV-12+OXA-20+CTX-M-14 (2.2%).10.四种型别ESBLs模式菌株,以同时携带SHV-12+TEM-1+CTX-M-1+ CTX-M-14为主(8.7%),其次为SHV-12+TEM-1+CTX-M-14+OXA-1(4.3%)、SHV-12+TEM-1+CTX-M-14+CTX-M-25 (2.2%)及SHV-12+TEM-1+CTX-M-1+CTX-M-25 (2.2%).11.五种型别ESBLs模式菌株,仅同时携带SHV-12+TEM-1+CTX-M-14+ CTX-M-25+CTX-M-1五种型别(2.2%)第二部分产超广谱β-内酰胺酶大肠埃希菌质粒谱、携带超广谱β-内酰胺酶型别及其耐药性相关性研究方法1.采用分子生物学技术,检测138株产ESBLs大肠埃希菌各菌株质粒谱。2.采用PCR及DNA序列分析,明确各菌株ESBLs基因型别分布模式。3.采用E-test法药敏试验,检测不同质粒分布模式菌株针对常用抗菌药物最小抑菌浓度(MIC)。4.采用E-test法药敏试验,检测ESBLs基因型别分布模式菌株MIC。5.统计学处理：所有数据均通过SPSS10.0统计学软件分析处理。质粒谱与抗药活性、质粒谱与基因型、基因型与抗药活性之间均采用X2检验分析,显著性水准α=0.05.结果1.产ESBLs大肠埃希菌可携带1～5种质粒。各质粒分子量依次为21kb、9.0kb、7.0kb、3.Okb以及2.5kb。2.138株产ESBLs大肠埃希菌有5种质粒谱模式。其中21kb质粒为各菌株均携带质粒。携带两种质粒(21kb+2.5kb)最多见(50株),其次为单一质粒(21kb,27株)、五种质粒(22株)、四种质粒(无9.0kb质粒,21株)以及三种质粒(21kb+3.0kb+2.5kb,18株)。3.不同质粒条带的各菌株具有以下特点：均对青霉素类、头孢一、二代及三代中头孢噻肟、头孢曲松耐药。对头孢他啶表现为体外试验敏感。对亚胺培南以及阿米卡星稳定敏感。各菌株对头孢毗肟的耐药性不一4.不同质粒条带菌株间MIC结果统计学分析后,X2值为0.00/0.21,p值均>0.5/0.9,组间耐药性无显著性差异,表明各菌株耐药性与质粒条带数相关性不强。5.质粒条带数与相应菌株携带ESBLs型别之间X2值为29.39,p>0.05,表明组间无显著性差异,提示两者无显著相关性。6.不同型别ESBLs菌株及其对不同抗菌药物MIC值之间X2为0.00/0.21,p值均>0.5/0.9,表明组间无显著性差异,提示各菌株耐药性与所携带ESBLs型别分布相关性不强。第三部分大肠埃希菌产超广谱β-内酰胺酶主要酶型抗药活性研究方法1.采用分子生物学技术,分别构建pET-28a-TEM-1、pET-28a-SHV-12以及pET-28a-CTX-M-14表达载体。2.采用氯化钙转化技术,制备转化子BL21-TEM-1、BL21-CTX-M-14以及JM109-SHV-12。3.采用液体稀释法体外药物敏感试验,分别检测宿主菌BL21、JM109及转化子BL21-TEM-1、BL21-CTX-M-14、JM109-SHV-12抗药活性。结果1.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。2.BL21、JM109大肠杆菌对于各种类型抗菌药物均表现敏感。3.产TEM-1转化子对于青霉素类药物显著耐药,氨苄西林、派拉西林MIC均大于32μg/ml。对部分头孢一代抗菌药物有水解作用,头孢唑啉MIC大于16μg/ml。对于头孢三代、四代抗菌药物显著敏感,头孢噻肟、头孢他啶MIC均小于1μg/ml。β-内酰胺酶酶抑制剂克拉维酸、舒巴坦及他唑巴坦可明显抑制TEM-1水解活性,从而降低菌株耐药性,阿莫西林/克拉维酸、氨苄西林/舒巴坦及派拉西林/他唑巴坦MIC均小于8/4μg/ml。碳青霉烯类及单环类抗菌药物稳定敏感,亚胺培南MIC小于4μg/ml,氨曲南MIC小于16μg/ml。4.产SHV-12转化子对于青霉素类具有广泛水解活性,氨苄西林及哌拉西林MIC均大于16μg/ml。对头孢一代、三代抗菌药物表现为耐药,头孢唑林与头孢噻肟MIC均大于16μg/ml,而头孢他啶MIC大于32μg/ml。β-内酰胺酶抑制剂可降低该酶的抗药活性,含酶抑制剂药物均表现为敏感。氨曲南MIC等于16μg/ml,表现为中度敏感,考虑转化子表达酶量或产物活性改变所致。碳青霉烯类抗菌药物稳定敏感,亚胺培南MIC小于4μg/ml。5.产CTX-M-14转化子对氨苄西林、派拉西林显著耐药(MIC>32μg/ml及64μg/ml),对头孢唑啉、头孢呋辛、头孢吡肟、头孢克洛MIC值均>16μg/ml,而头孢曲松则>32μg/ml,均表现为显著耐药。同时,该型酶对头孢塞肟及头孢他啶的的水解能力相差32倍以上,MIC分别为>32μg/ml及≤1μg/ml,前者耐药,后者表现为敏感。另,CTX-M-14对于氨曲南稳定耐药(MIC)16μg/ml),而对于亚胺培南稳定敏感(MIC≤4μg/ml)。对于加酶抑制剂的抗菌药物稳定敏感(阿莫西林／棒酸、氨苄西林／舒巴坦、派拉西林／他唑巴坦及头孢哌酮／舒巴坦)。此外,该型ESBL对于喹诺酮、四环素类药物多敏感。结论1.河南地区大肠埃希菌产ESBLs有TEM-1、SHV-12、CTX-M-1、CTX-M-14、CTX-M-25、CTX-M-38、OXA-1、OXA-20等型别存在,其中主要流行型别为TEM-1、SHV-12以及CTX-M-14;同一菌株可具有1-5种ESBLs基因型别模式,其中以两种型别最为多见。2.菌株携带质粒谱与其耐药活性无显著相关性；菌株携带质粒谱与其携带ESBL型别无显著相关性；菌株携带ESBL型别与其耐药活性无显著相关性。3. BL21-TEM-1表达产物属于广谱β-内酰胺酶,转化子主要对青霉素类耐药,β-内酰胺酶抑制剂可抑制酶活性。BL21-CTX-M-14、JM109-SHV-12表达产物属于超广谱β-内酰胺酶,转化子对青霉素类、头孢菌素类抗菌药物均显著耐药,β-内酰胺酶抑制剂可抑制酶活性。更多还原

【Abstract】 Escherichia coli (E.coli) is the most important pathogen in community-acquired and nosocomial infections. The isolated rate of E. coli keeps at a high level in the laboratory of clinical microbiology and it is frequently reported all over the world. The therapeutic efficacy to E. coli has been regarded as a common factor affected the patients’ prognosis and pathogenesis. In order to solve this problem and obtain better anti-infection efficacy, the research on the E. coli has become a hot topic in the anti-infective territory. The study focusing on the mechanism of resistance is the key point.With the overuse or abuse of broad spectrum antibiotics, especially the third generation cephalosporins application, multiple antibiotic resistant E.coli possess a high rate of occurrence. The strains separated from clinical laboratory not only are capable of hydrolyzing mostβ-lactams, but also are highly resistant to fluoroquinolones and aminoglycosides. In phenotype, the mechanism of resistance includes producing inactive and modification enzyme、changing membrane permeability and forming efflux pump. On the other hand, vertical transmission and plasmid-mediated transmission are the main mechanism of resistant gene spreading among strains in molecular level. According to the results of latest research, producing extended-spectrumβ-lactamases (ESBLs) play an important role in the multiple antibiotic resistant of wild-type E.coli. More than one ESBLs type lie in the same strain provides the resisrant ability obviously.ESBLs belongs toβ-lactamases. Due to the DNA sequence diversities, ESBLs can be divided into different types, and most types are plasmid-mediated. This kind ofβ-lactamases can hydrolyze oximino-cephalosporins obviously.that is to say, the ESBL-produing strains is highly resistant to the first、second and third generation cephalosporins, penicillins and monobactam. On the other hand, it is susceptible to cephamycin、carbapenem, andβ-lactamase inhibitors can inhibit its hydrolyzing antivity. ESBL-producing strain was first reported in 1983, and nowdays, it has become an increasing problem in daily clinical life worldwide. Prevalence characterization of extended-spectrum beta-lactamases among Escherichia coli Isolates divided into five parts. (1) The ESBLs from wild-type strains turns on diverse types, and new subtypes are frequently reported. (2) the whole genome sequence nucleotide homology of TEM or SHV subtypes is very higher, but the homology of other ESBLs subtypes is very lower. (3) most ESBLs derived from TEM-1、TEM-2 or SHV-1 enzymes. (4) the prevalence of ESBL-types differs from country to country and from laboratory to laboratory. (5) the antimicrobial-resistant phenotype among all of ESBL types and subtypes is different.In HeNan province, the first ESBL-producing strain was confirmed by liu xinzheng in 2000. And researchs most focus on The antibiotics resistant profile of wild-type ESBL-producing E.coli nowdays. Only a few studies regarded the topic that reveals the prevalence and the diversity of ESBLs among Escherichia coli isolates have been reported. Without effective information of the prevalence characterization and difference among all kinds of ESBLs types, the clinic is difficult to treat infections. As the result, patients have to afford much more. Henan has a large number population, and its ecnomics is at low level. To determine the prevalence and the diversity of ESBL among Escherichia coli isolates, and confirm the antibiotics resistant profile of different subtypes is very important for the clinic to treat infection reasonably and reduce the cost of medicine.In order to explore the prevalent characteration and diversity of ESBLs among Escherichia coli isolates, to find the role of ESBLs encoded-gene localization, and to explore the antibiotics resistant profile of ESBL enzymes dominated in ESBL-positive E.coli isolates in henan,138 nonduplicate ESBL-positive isolates were collected from the first affiliated hospital, the third affiliated hospital of zhengzhou university and the people’s hospital of henan province. ESBL-Producing isolates were confirmed by double-disc synergy test and enzymes inhibitor enhancing test. ESBL epidemiology was described by PCR and DNA sequencing. Detecting the antibiotics sensitivity of all strains containing different plasmid profiles and ESBL-types by E-test. The statistics were analyzed by chi-square criterion. The ESBLs expressing vectors was constructed by gene recombination. The antibiotics sensitivities of transformant were determined by microdilution.Through above tests, we try to describe the a complex ESBLs epidemiology in henan, and to reveal the relationship in plasmid profile and ESBL-type, plasmid profile and antibiotics sensitivity, ESBL-type and antibiotics sensitivity, and to identify the antibiotics resistant of ESBL dominated. At last, we provide a new theory for the treatment of patients infected with E.coli.The experiment is divided into three parts. Part I:The study on the epidemiology of E.coli producing ESBLsMehtods:1. Multiple antibiotic resistant E.coli isolates were detected corresponding to the phenotype of producing ESBLs detected by double-disc synergy screening test, and confirmation for producing ESBLs was carried out by enzymes inhibitor enhancing test.2. Acorrding to nucleotide homology diversity of all kinds of ESBLs types, universal primers and specific primers were designed by DNAstar analysing software.3. ESBL-producing isolates confirmated by enzymes inhibitor enhangcing test were analysed by PCR to detect ESBLs type.4. The ESBLs types among E.coli dominated in henan were identified by DNA sequeemng.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 products of PCR and difference among all the ESBL types corresponded to the design.4. The DNA sequeening result revealed thay these were TEM-1、SHV-12、CTX-M-1、CTX-M-14、CTX-M-38、OXA-1 and OXA-20 ESBLs types among E.coli in henan province.5. In 138 E.coli isolates, TEM-1 typeβ-lactamases was encoded in 80.4%and donimated in ESBL-positive E.coli isolates in henan. Respectively, SHV-12、CTX-M-1、CTX-M-14、CTX-M-38、OXA-1 and OXA-20 ESBL type were encoded in 60.9%,32.6%,58.7%,6.5%,4.3%and 2.0%.6. In 138 ESBL-producing strains,1-5 ESBLs types could exist in single isolate. There were 32.6%,26.1%,21.7%,17.4%and 2.2%strains which contained two types、three types, one type, four types and five types respectively.7. TEM-1 typeβ-lactamases donimated in ESBL-positive isolates which only contained one ESBL type. And then, CTX-M-14. SHV-12 and CTX-M-1 were encoded in 4.3%.2.2%and 2.2%respectively.8. SHV-12+CTX-M-14 donimated in ESBL-positive isolates which contained two ESBL types (10.9%). And then, TEM-1+CTX-M-14. SHV-12+TEM-1. TEM-1+CTX-M-1. TEM-1+CTX-M-38 and OXA-1+CTX-M-1 were encoded in 8.7%.6.5%.2.2%.2.2%and 2.2%respectively.9. SHV-12+TEM-1+CTX-M-1 donimated in ESBL-positive isolates which contained three ESBL types (10.9%). And then, SHV-12+TEM-1+CTX-M-14. TEM-1+CTX-M-14+CTX-M-38. SHV-12+OXA-1+CTX-M-14 and SHV-12+ OXA-20+CTX-M-14 were encoded in 8.7%.2.2%.2.2%and 2.5%respectively.10. SHV-12+TEM-1+CTX-M-1+CTX-M-14 donimated in ESBL-positive isolates which contained four ESBL types (8.7%).and then, SHV-12+TEM-l+CTX-M-14+OXA-1、SHV-12+TEM-1+CTX-M-14+CTX-M-25 and SHV-12+TEM-1+ CTX-M-1+CTX-M-25 were encoded in 4.3%.2.2%and 2.2%respectively.11. SHV-12+TEM-1+CTX-M-1+CTX-M-25+CTX-M-14 was the only one pattern in the isolates which contained five ESBL types. PartⅡ:the relationships among the plasmid maps、ESBLs type patterns and antibiotics resistance of ESBL-producing E.coliMethods:1. Using molecular biological technique to reveal the plasmid map of 138 ESBL-producing strains of E.coli.2. The ESBL-type pattern among strains was confirmed by PCR and DNA sequencing.3. The common antibacterials minimum inhibition concentrations of ESBL-producers which contained different plasmid map was detected by E-test.4. The common antibacterials minimum inhibition concentration of ESBL-producers which contained different ESBL-type patterns was detected by E-test.5. All of the experimental data were analyzed by SPSS 10.0 statistical package program.the relationships among the plasmid maps、ESBLs type patterns and antibiotics resistance of ESBL-producing strains of E.coli were analyzed by chi-square criterion,and the level of significant difference wasα=0.05.Results:1. The ESBL-producing strains of E.coli had 1-5 plasmids, which molecule was about 21kb、9.0kb、7.0kb、3.0kb and 2.5kb.2. There were five kinds of plasmid maps in the 138 strains. The plasmid of 21kb existed in every isolate. The strain which contained two kinds of plasmid (21kb+2.5kb) was encoded in 50, single plasmid (21kb)、five kinds、four kinds of plasmid (without 9.0kb) and three kinds of plasmid(21kb、3.0kb、 2.5kb) were encoded in 27、22、21 and 18 respectively.3. The strains which contained different plasmid map were resistant to penicillins, the first and second generation cephalosporins, cefotaxime and Ceftriaxone (MIC≥256μg/ml). All of them were sensitive to imipenem and amikacin (MIC≦12μg/ml and 0.25μg/ml).and every strain was sensitive to Ceftazidime in vitro (MIC≦4μg/ml). Different strain had a different result to cefepime (MICs was32、48、48、16、64μg/ml)4. There had no significant difference between the drug resistence and plasmid map of ESBL-producing strains of E.coli (p>0.5/0.9)5. There had no significant difference between the ESBL-type patterns and plasmid map of ESBL-producing strains of E.coli (p>0.05)6. There had no significant difference between the drug resistence and the ESBL-type patterns of ESBL-producing strains of E.coli (p>0.5/0.9)PartⅢ:Study on the antibiotics resistance of dominated ESBLs among E.coli isolatesMethods:1. Using gene recombination technical to construct the expressing vector of pET-28a-TEM-1、pET-28a-SHV-12 and pET-28a-CTX-M-14.and the vector was identified by PCR.2. Using CaCl2 transforming method to introduce the expressing vector of pET-28a-TEM-1 and pET-28a-CTX-M-14 into E.coli BL21, and introduce the expressing vector of pET-28a-SHV-12 into E.coli JM109. Selecting the positive strains by kanamycin resistant and PCR.3. Using microdilution to detected the antibiotics resistence of the E.coli BL21、 E.coli JM109 and the transformants of BL21-TEM-1、BL21-CTX-M-14 and JM109-SHV-12. the results were judged according to the standard of NCCLs.Results:1. Using PCR to detect the plasmids derived from transformants, the products located at 900bp in the agarose gel electrophoresis. All of these results indicated that the expressing vectors were constructed successfully.2. The E.coli BL21 and E.coli JM109 were sensitive to all kinds of antibiotics in vitro.3. TEM-1 transformant was obviously resisitant to penicillins. The MICs of ampicillin and piperacillin were more than 32μg/ml respectively. Parts of the first generation cephalosporins could be hydrolyzed by TEM-1 transformant. The MIC of cephazoline was more than 16μg/ml. The third、quaternary generation cephalosporins tested in the study could efficiently inhibit the TEM-1 transformant. The MICs of cefotaxime and Ceftazidime were less than 1μg/ml. Theβ-lactamase inhibitors including clavulanic acid、sulbactam and tazobactam could inhibit the activity of the enzyme and reduce the resistance of TEM-1 producing strains. The MICs of the Amoxicillin/clavulanic、ampicillin/sulbactam and piperacillin/tazobactam were all less than 8/4μg/ml. the transformant was stably sensitive to carbapenems and monobactam tested in the study. The MICs of imipenem and aztreonam was less than 4μg/ml and 16μg/ml respectively.4. The SHV-12 transformant could effectively hydrolyze all the penicillins tested in the study. The MICs of ampicillin and piperacillin were more than 16μg/ml respectively. And the trasnformant was resistant to the first、third generation cephalosporins, the MICs of cephazoline and cefotaxime were more than 16μg/ml respectively,and the MIC of Ceftazidime was 32μg/ml.β-lactamase inhibitors including clavulanic acid、sulbactam and tazobactam could inhibit the activity of the enzyme and reduce the resistance of SHV-12 producing strains, the antibiotics containing the inhibitor could inhibit the strains producing enzyme. the transformant was stably sensitive to carbapenems tested in the study, the MIC of imipenemwas less than 4μg/ml. The SHV-12 transformant was intermediate to the aztreonam and its MIC was 16μg/ml, indicated that the transformant expressed a few enzyme or the activity of the enzyme had changed.5. The CTX-M-14 transformant was obviously resisitant to ampicillin and piperacillin, and their MICs were more than 32μg/ml、64μg/ml respectively. 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 32 times as much as it to the ceftazidime, the former was resistant and its MIC was more than 32μg/ml, and the latter was sensitive and its MIC was 1μg/ml. On the other hand, The CTX-M-14 transformant was stably resitant to aztreonam and its MIC was more than 16μg/ml. the MIC of imipenemwas less than 4μg/ml indicated than the transformant was stably sensitive to it. The transformant was also sensitive to Amoxicillin/clavulanic、ampicillin/sulbactam and piperacillin/tazobactam, and their MICs were all less than 8/4μg/ml. Quinolones and tetracyclines antibiotics tested in the study could inhibit the transformant effectively, the MIC of ciprofloxacin and tetracycline was less than 0.06μg/ml、less than 4μg/ml respectively.Conclusion:1. There are TEM-1、SHV-12、CTX-M-1、CTX-M-14、CTX-M-38、OXA-1 and OXA-20 ESBL types among E.coli in henan province, and TEM-1、SHV-12 and CTX-M-14 are the main ESBLs type which donimate in the ESBL-positive E.coli strains.1-5 ESBLs types can exist in single isolate. The strains which contain two ESBL types at the same time are the main prevalent isolates in henan.2. There are no significant difference between the plasmid map and ESBL types、ESBL types and drug resistance、drug resistance and plasmid map.3. The expressing products of BL21-TEM-1 belonged toβ-lactamase, and the transformant is obviously resistant to penicillins. The inhibitor can inhibit the activity of TEM-1 type enzyme. The expressing products of BL21-CTX-M-14 and JM109-SHV-12 belong to ESBLs, and the transformants are obviously resistant to penicillins and cephalosporins, The inhibitor can inhibit the activity of. CTX-M-14 and SHV-12 type ESBL.更多还原