河南地区大肠埃希菌产超广谱β-内酰胺酶的研究
|
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。
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 ESBLs
Mehtods:
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.coli
Methods:
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 isolates
Methods:
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.
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。
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 ESBLs
Mehtods:
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.coli
Methods:
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 isolates
Methods:
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.
引文
[1]Jacoby GA.Epidemiology of extended-spectrum β-lactamases [J].Clin Infect Dis,1998, 27(1):81～83
[2]Gniadkowski M.Evolution and epidemiology of extended-spectrum beta-lactamases (ESBLSs) and ESBLs-producing microorganisms [J].Clin Microbiol Infect,2001,7(11): 597～608
[3]张玲.革兰阴性杆菌产超广谱β-内酰胺酶研究进展.中华医院感染学杂志,2008,18(6)：897～900
[4]Bonnet R, Dutour C, Sampaio JLM, et al. Novel cefotaximase (CTX-M-16) with increased catalytic efficiency due to substitution Asp-240→Gly.2001, Antimicrob Agents Chemother,45:2269～2275
[5]Cao V, Lambert T, Nhu DQ, et al. Distribution of extended-spectrum B-lactamases in clinical isolates of Enterobacteriaceae in Vietnam.2002, Antimicrob Agents Chemother,46: 3739～3743
[6]Casin I, Hanau-Bercot I, Podglajen H, et al. Salmonella enterica serovar Typhimurium blaPER-1-carrying plasmid pSTI1 encodes an extended-spectrum aminoglycoside 6'-N-acetyltransferase of type Ib.2003, Antimicrob Agents Chemother,47:697～703
[7]De Champs C, Poirel L, Bonnet R, et al. Prospective survey of B-lactamases produced by ceftazidime-resistant Pseudomonas aeruginosa isolated in a French hospital in 2000.2002, Antimicrob Agents Chemother,46:3031～3034
[8]De Champs C, Sirot D, Chanal C, et al. A 1998 survey of extended-spectrum β-lactamases in Enterobacteriaceae in France.2000, Antimicrob Agents Chemother 44:3177～3179
[9]Gniadkowski M, Schneider I, Palucha A, et al. Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland:identification of a new CTX-M-3 cefotaxime-hydrolyzing β-lactamase that is closely related to the CTX-M-1/MEN-1 enzyme.1998, Antimicrob Agents Chemother,42:827～832
[10]Vahaboglu H, Ozturk R, Aygiin G, et al. Widespread detection of PER-1-type extended-spectrum B-lactamases among nosocomial Acinetobacter and Pseudomonas aeruginosa isolates in Turkey:a nationwide multicentre study.1997, Antimicrob Agents Chemother,41:2265～2269
[11]Girlich D, Poirel L, Leelaporn A, et al. Molecular epidemiology of the integron-located VEB-1 extended-spectrum β-lactamase in nosocomial enterobacterial isolates in Bangkok, Thailand.2001, J Clin Microbio,139:175～182
[12]季淑娟,顾怡明,谭文涛,等.中国部分地区大肠埃希菌和肺炎克雷伯菌超广谱β内酰胺酶基因型研究.中华医学检验杂志,2004,27(9)：590～593
[13]王燕,李苏利,李杨.细菌产超广谱β-内酰胺酶的研究状况.现代中西医结合杂志,2006,15(23)：3312～3314
[14]Goossens H. MYSTIC program:summary of European data from 1997 to 2000. Diagn Microbiol Infect Dis,2001,41:183～189
[15]管希周,刘又宁,王睿,等.临床产ESBLs细菌耐药特性及其基因分型的研究.中国抗生素杂志,2001,26(6)：468～472
[16]Yu YS, ZhouWL, Chen YG, et al. Ep idemiological and antibiotic resistant study on extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Zhejiang Province.ChinMed J,2002,115:1479～1482
[17]朱德妹,熊自忠,汪复,等.超广谱β-内酰胺酶和细菌耐药性.中华传染病杂志,2000,18(3)：151～154
[18]陈民钧,王辉.中国重症监护病房革兰阴性菌耐药性连续7年监测研究.中华医学杂志,2003.83(5)：375～381
[19]陆坚,唐英春,吴本权,等.华南地区质粒介导超广谱β-内酰胺酶基因分型研究.中华微生物学和免疫学杂志,2002,22(6)：638～643
[20]郑巧敏,张秀霞,朱涛,等.大肠埃希菌耐药性水平传播试验研究.中国微生态杂志,2008,20(1)：44～48
[21]付英梅,张凤民,张文莉,等.产超广谱β-内酰胺酶大肠埃希菌和肺炎克雷伯菌Ⅰ类整合子及其与ESBLSs基因关系的研究.中华医院感染学杂志,2007,17(3)：241～245
[22]叶惠芬,陈惠玲,刘平,等.产ESBLs大肠埃希菌和肺炎克雷伯菌耐药性和基因型分布.中国感染与化疗杂志,2007,7(2)：127～129
[23]张洁,包其郁,张洪勤,等.多重耐药性大肠埃希菌质粒谱与ESBLs基因型分析.中国抗生素杂志,2006,31(4)：198～201
[24]Bart helemy M, Peduzzi J, Labia R. Distinction between the primary structures of TEM-1 and TEM-2 beta-lactamases [J].Ann Inst Pasteur Microbiol,1985,136:311～321
[25]Gniadkowski M. Evolution and epidemiology of extended-spectrum beta-lactamases (ESBLs) and ESBLSs-producing microorganisms [J]. Clin Microbiol Infect,2001,7 (11): 597～608
[1]肖庆忠,苏丹虹,江洁华,等.广州地区3500株革兰阴性细菌的分布特征及耐药性特点[J].第一军医大学学报,2005,25(2)：132～138
[2]陈文光,蒋景华,王宗欣,等.大肠埃希菌的科室分布和耐药性分析[J].中华医院感染学杂志,2008,18(1)：119～120
[3]许桂芳,金春光.产超广谱β-内酰胺酶大肠埃希菌耐药性及基因同源性研究[J].中国卫生检验杂志,2009,19(8)：1843～1844
[4]魏殿军,宋师铎.医院内、外感染大肠埃希菌、肺炎克雷伯菌药物敏感性分析[J].中华医院感染学杂志,2003,13(7)：677～680
[5]Aminzadeh Z, Sadat Kashi M, Sha'bani M.Bacteriuria by extended-spectrum Beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae:isolates in a governmental hospital in South of Tehran, Iran[J].Iran J Kidney Dis.2008,2(4):197～200
[6]Song S, Lee EY, Koh EM, etc. Antibiotic resistance mechanisms of Escherichia coli Isolates from urinary specimens [J]. Korean J Lab Med,2009,29(1):17～24
[7]Johnson JR, Johnston B, Kuskowski MA, etc. Molecular epidemiology and phylogenetic distribution of the Escherichia coli pks genomic island[J]. J Clin Microbiol,2008, 46(12):3906～11
[8]Manges AR, Tabor H, Tellis P, etc. Endemic and epidemic lineages of Escherichia coli that cause urinary tract infections[J]. Emerg Infect Dis,2008,14(10):1575～83
[9]萨姆布鲁克,弗里奇,曼尼阿蒂斯,等.分子克隆实验指南[M].第2版.北京：科学出版社,2002
[10]苏内尔,菲利普斯.质粒生物学[M].第1版.北京：化学工业出版社,2009
[11]苏健裕,石磊,杨连生.临床大肠埃希菌第Ⅰ类整合子检测及耐药基因盒分析[J].检验医学与临床,2008,5(7)：385～387
[12]刘朝霞,刘又宁.质粒介导的喹喏酮类药物耐药性研究[J].国际呼吸杂志,2006,26(4)：304～306
[13]徐艳,郭丽双,付英梅,等.细菌对氨基糖苷类抗生素的耐药机制[J].中国微生态杂志,2008,20(2)：191～193
[14]石福艳,白亚娜,樊景春,等.医院感染中大肠埃希菌耐药质粒的同源性分析[J].中国公共卫生,2006,22(9)：1091～1092
[15]张洁,包有郁,张洪勤,等.多重耐药性大肠埃希菌质粒谱与ESBLs基因型分析.中国抗生素杂志,2006,31(4)：198～201
[16]年华,褚云卓,王倩.医院感染大肠埃希菌耐药性变化分析[J].中华医院感染学杂志,2007,17(4)：452～454
[17]耿燕,张王刚,王香玲,等.泌尿系感染产超广谱β内酰胺酶(ESBLs)大肠埃希菌的耐药表型和基因分型[J].第四军医大学学报,2005,26(7)：622～624
[18]张志坚,郭小兵,张钦宪.产ESBLs大肠埃希菌质粒谱与耐药性分析[J].第四军医大学 学报,2008,29(21)：1970～1972
[19]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
[20]张之文,王红宁.整合子结构及在细菌耐药性扩散中的作用[J].国外医药抗生素分册,2007,24(4)：150～153
[21]廖伟娇,江洁华,易建云,等.多重耐药革兰阴性杆菌Ⅰ类整合子的检测[J].热带医学杂志,2006,6(6)：667～669
[22]ROWE-MANGUS DA, MAZEL D.The role of integrons in antibiotic resistance gene capture[J]. Int J Med Microbiol,2002,292(2):115～125.
[23]FLUIT AG, SCHMITZ FJ. Resistance integrons and superintegrons[J]. Clin Microbiol Infect[J],2004,10(4):272～288.
[24]杨维青,刘晓峰,黄震.大肠埃希菌中Ⅰ类整合子耐药基因的分析[J].中国抗生素杂志,2009,34(7)：434～436
[25]李婷,马平,赵冰,等.健康人群中大肠埃希菌耐药整合子的检测[J].华西药学杂志,2009,24(4)：372～374
[26]苏健裕,石磊,杨连生.临床大肠埃希菌第Ⅰ类整合子检测及耐药基因盒分析[J].检验医学与临床,2008,5(7)：385～387
[1]娄茜,黄长武,吴倩.泌尿系感染大肠埃希菌的耐药性分析[J].检验医学与临床,2007,4(10)：940～941
[2]张凤珍,张蕴莉,许德顺.产超广谱β-内酰胺酶大肠埃希菌感染的监测[J].中国实验诊断学,2005,9(1)：83～84
[3]Wada K, Kariyama R, Mitsuhata R, et al. Experimental and Clinical Studies on Fluoroquinolone-insusceptible Escherichia coli Isolated from Patients with Urinary Tract Infections from 1994 to 2007[J]. Acta Med Okayama,2009,63(5):263～72
[4]王燕,李苏利,李扬.细菌产超广谱β-内酰胺酶的研究概况[J].现代中西医结合杂志,2006,15(23)：3312～3314
[5]Philippon A,Labia R,Jacoby G. Extended-spectrum β-lactamases[J]. Antimicrob Agents Chemother,1989,33(1):13
[6]章清,魏丽,魏光美,等.大肠埃希菌、肺炎克雷伯菌的耐药性变迁[J].中华医院感染血杂志,2004,14(2)：220～222
[7]俞云松.超广谱β-内酰胺酶研究概况[J].中国抗生素杂志,2003,12(28)：712-718
[8]Bradford PA. Extended Spectrum β-Lactamses in the 21st century:charcterization, epidemiology, and detection of this important resistance threat [J]. Clin MicrobiolRev, 2001,14(9):993～951
[9]程训民,李敏,张琪.超广谱β-内酰胺酶研究进展[J].淮海医药,2005,23(4)：343-345
[10]马越,李景云,张新妹.2002年临床常见细菌耐药性监测[J].中华检验医学杂志,2004,27(1)： 38～45
[11]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
[12]Bradford P A.Extended-spectrum beta-lactamases in the 21st century:characterization, epidemiology, and detection of this important resistance threat [J]:Clin MicrobiolRev, 2001,14(4):933-951
[13]Livermore DM. Beta-lactamases in laboratory and clinical resistance[J].Clin Microbiol Rev,1995,8(4):557～584.
[14]Bart helemy M, Peduzzi J, Labia R.Distinction between the primary structures of TEM-1 and TEM-2 beta-lactamases[J].Ann Inst Pasteur Microbiol,1985,136:311～321
[15]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
[16]李虹玲,刘文恩.SHV型超广谱β-内酰胺酶的研究进展[J].实用预防医学,2007,14(1)：253～256
[17]Heritage J, M'Zali F, Gascoyne-Binzi D, et al. Evolution and spread of SHV extended-spectrum β-lactamases in Gram-negative baceria [J].J Antimicrob Chemother,1999, 44:309～318
[18]Szabo D, Melan MA, Hujer AM, et al. Molecular analysis of the simultaneous production of two SHV-type extended-spectrum beta-lactamases in a clinical isolate of Enterobacter cloacae by using single-nucleotide polymorphism genotyping [J].Antimicrob Agents Chemother,2005,49(11):4716-4720
[19]Poirel L, Heritier C, Podglajen I, et al.Emergence in Klebsiella pneumoniae of a Chromosome-encoded SHV β-lactamase that compromise the efficacy of Imipenem[J]. Antimicrobial Agents and Chemotherapy,2003,47(2):755-758
[20]Huletskt A, Knox JR, Levesque RC.Role of ser-238 and Lys-240 in the hydrolysis of third-generation cephhalosporins by SHV-type beta-lactamases probed by site-directed mutagenesis and three-dimensional modeling[J].J Biol Chem,1933,268:3690～3697
[21]张哲,蒋晓飞,李敏,等.SHV-12型超广谱β-内酰胺酶特性的研究[J].检验医学,2006,21(1)：31～34
[22]季淑娟,顾怡明,谭文涛,等.中国部分地区大肠埃希菌和肺炎克雷伯菌超广谱β内酰胺酶基因型研究[J].中华医学检验杂志,2004,27(9)：590～593
[23]张盛斌,刘朝辉CTX-M型ESBLs的研究进展[J].中华感染与化疗杂志,2006,6(3)：212～215
[24]熊自忠,朱德妹,汪复,等.CTX-M-14和CTX-M-24编码基因的检测及其功能表达[J].中华医学杂志,2004,84(17)：1454～1459
[25]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
[26]Cartelle M, del-Mar Tomas M, Molina F, et al.High level resistance to ceftazidime conferred by a novel enzyme, CTX-M-32, derived from CTX-M-1 through a single Asp240-Gly substitution[J].Antimicrob Agents Chemother,2004,48 (6):2308～2313
[27]李金钟CTX-M型ESBLs的研究进展[J].国际检验医学杂志,2007,28(6)：543～545
[1]陆一平,李晖,陈艳华,等.大肠埃希菌感染的临床分布与耐药性[J].山西医药杂志,2006,35(4)：371～372
[2]张传栋,刘存津.产ESBLs大肠埃希菌临床分布及耐药性分析[J].中华医院感染学杂志,2009,19(13)：1728～1729
[3]金文君,黄永禄,徐志江,等.临床分离大肠埃希菌的耐药性分析[J].中国卫生检验杂志,2009,19(10)：2332～2333
[4]金文君,黄永禄,徐志江,等ESBLs阳性大肠埃希菌和肺炎克雷伯菌产AmpC酶的检测与体外抗菌活性分析[J].中华医院感染学杂志,2008,18(6)：855～858
[5]BALL P.Quinolone generations:natural history or natural selection? [J] J Antimicrob Chemother,2000,46(supplT1):17～24
[6]ANDERSSON M I, MACGOWAN A P. Development of the quinolones [J] J Antimicrob Chemother,2003,51(suppl 1):1～11
[7]林东昉,王明贵.喹诺酮类抗菌药物的研究进展[J].新医学,2007,38(1)：49～50
[8]Jcoby GA. Mechanisms of resistance to quinolones[J]. Clin infect dis,2005,41 (2): 120～126
[9]Nikaido H, Thanassi DG.Penetration of lipophilic agents with multiple protonation sites into bacterial cells:tetracylines and fluoroquinones as examples Anticrob[J]. Agents chemother,1993,37(7):1393
[10]方治平,徐炜,刘小康,等.大肠杆菌体内氟喹诺酮类药物耐药机制的研究[J].四川大学学报(医学版),2005,36(1)：86～89
[11]贾蓓.膜孔蛋白及其再细菌耐药中的作用[J].国外医药抗生素分册,1997,18(6)：444～447
[12]钟利,范昕建.大肠杆菌膜孔膜蛋白研究进展[J].国外医药抗生素分册,1998,19(3)：208～210
[13]Van bambeke F, Balzi E, Tulkens PM. Antibiotic efflux pimps [J]. Biochem Pharmacol, 2000,60 (4):457～470
[14]Touze T, Eswaran J, Bokma E, et al. Interactions underlying assembly of the Escherichia coli AcrAB-TolC multidrug efflux system [J]. Mol microbiol,2004,53 (2):697～706
[15]Sennhauser G, Amstutz P, Briand C, et al. Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors[J]. PLoS Biol,2007,5(1):e7
[16]Fujihira E, Tamura N, Yamaguchi A. Membrane topology of a multidrug efflux transporter, AcrB, in Escherichia coli[J]. J Biochem,2002,131(1):145～151
[17]Tikhonova EB, Zgurskaya HI. AcrA, AcrB, and TolC of Escherichia coli Form a Stable Intermembrane Multidrug Efflux Complex[J]. J Biol chem,2004,279 (31):32116～24
[18]Seeger MA, Diederichs K, Eicher T, et al. The AcrB efflux pump:conformational cycling and peristalsis lead to multidrug resistance[J]. Curr Drug Targets,2008,9(9):729～49
[19]Eicher T, Brandstatter L, Pos KM. Structural and functional aspects of the multidrug efflux pump AcrB[J].Biol Chem,2009,390(8):693～9
[20]HOOPER DC.Mechanism of action and resistance of older and newer fluoroquinolones [J]. Clin infect Dis,2000,31(suppl2):S24～S28
[21]HOOPER DC.Mechanism of action of antimicrobials:focus on fluoroquinolones [J]. Clin infect Dis,2001,32(suppl 1):S9-S15
[22]Bebear CM, Charron A, Bove JM, et al. Cloning and nucleotide sequence of the topoisomerase IV parC and pare genes of Mycoplasma hominis[J].Antimicrob Agents Chemother.1998,42(8):2024～2029
[23]Fournier B and Hooper DC. Effects of mutations in Gr1A of topoisomerase Ⅳ from Staphylococcus aureu Effects of mutations in Gr1A of topoisomerase Ⅳ on quinoline and coumarin activity[J].Antimicrob Agents Chemother.1998,4 (8):2019～2024
[24]Luzzaro F. Fluoroquinolones and Gram-negative bacteria:antimicrobial activity and mechanisms of resistance[J]. Infez Med.2008,160 (Suppl2):5～11
[25]Truong QC, Van N, Shlaes D, et al. A novel, double mutation in DNA gyrase A of Escherichia coli conferring resistance to quinolone antibiotics[J]. Antimicrob Agents Chemother.1997,41(1):85～93
[26]Cond S, Oethinger M, Kaifel K, et al. gyrA mutations in high-level fluoroquinolone-resistant clinical isolates of Escherichia coli[J]. Antimicrob Agents Chemother.1996, 38(4):443～451
[27]Kumagai Y, Kato JI, Hoshino K,et al. Quinolone-resisitance mutants of Escherichia coli DNA topoisomerase Ⅳ parCgene[J]. Antimicrob Agents Chemother.1996,40(3):710～ 716
[28]Bachoual R, Analysis of the mutations involved in fluoroquinolone-resisitant of in vivo and in vitro mutants of Escherichia coli [J]. Microb Drug Rsist,1998 4 (4):271～279
[29]Chen JY, Siu LK, Lu PL, et al. Molecular epidemioliogy and mutations at gyrA and parC genes of ciprofloxacin-resistant Escherichia coli isolated from a Taiwan medical center[J]. Microb Drug Rsist,2001,7(1):47～53
[30]Cattoir V, Lesprit P, Lascols C,et al. In vivo selection during ofloxacin therapy of Escherichia coli with combined topoisomerase mutations that confer high resistance to ofloxacin but susceptibility to nalidixic acid[J]. J Antimicrob Chemother,2006,58(5): 1054-1057
[31]Petersen A, Christensen JP, Kuhnert P. Vertical transmission of a fluoroquinolone-resisitant Escherichia coli within an integrated broiler operation[J].Vet Microbiol,2006,116(1-3): 120～128
[32]蒋萍,陈恒屹,张坚磊,等.耐喹诺酮类大肠埃希菌耐药机制的研究[J].中华医院感染学杂志,2007,17(6)：635～638
[33]Niwa H, Hobo S, Anzai T. A nucleotide mutation associated with fluoroquinolone resistance observed in gyrA of in vitro obtained Rhodococcus equi mutants[J].Vet Microbiol,2006,115(1):264～268
[34]Mart nez LM, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid[J]. Lancet,1998,351:797～799
[35]Li XZ. Quinolone resistance in bacetrica:emphasis on plasmid-mediated mechanisms[J]. Int J Antimicrob Agent,2005,25 (6):453～463
[36]Suzuki MHM, Matsumoto M, Takahashi M, et al. Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella flexneri 2b[J].Antimicrob Agents Chemother,2005,49(2):801～803
[37]Tran JH, Jacoby GA, Hooper DC. Interaction of the plasmid-encoded quinolone resistance protein qnrA with Escherichia coli topoisomerase Ⅳ[J]. Antimicrob Agents Chemother, 2005,49(7):3050～3052
[38]Jacoby GA, Kelley E, Walsh, et al.qnrB, Another plasmid-mediated gene for quinolone resistance protein qnrA with Escherichia coli topoisomerase Ⅳ[J]. Antimicrob Agents Chemother,2006,50 (4):1178～1182
[39]Rbicsek A, Strahilevita J, Jacoby GA, et al. Fluoroquinolone-modifying enzyme:a new adaptation of a common aminoglycoside acetyltransferase[J].Nat med,2006,12(1):83～88
[40]Perichon BP, Galimand CM. Transferable resistance to aminoglycosides by methylation of G1405 in 16s rRNA and to hydrophilic fluoroquinolones by QepA-mediated efflux in Escherichia coli[J]. Antimicrob Agents Chemother,2007,51(7):2464～2469
[41]Yamane KJ, Wachino S, Suzuki K, et al. New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate[J]. Antimicrob Agents Chemother,2007,51 (9):3354～3360
[42]张致平.β-内酰胺类抗生素研究的进展[J].中国新药杂志,2002,11(1)：61～67
[43]韩刚,蒋立建,韩冲,等.新型β-内酰胺类抗生素及其特点[J].东南大学学报(医学版),2008,27(4)：316～318
[44]肖时超,吕火祥,沈蓓琼,等.2003年至2007年大肠埃希菌的临床分离及耐药趋势分析[J].中国微生态学杂志,2008,20(4)：407～409
[45]沈继录.超广谱p-内酰胺酶研究进展[J].国外医学临床生物化学与检验学分册.2004,25(3)：234～237
[46]周炎腾,贾杰,苏林光,等.410株大肠埃希菌耐药性分析[J].中国热带医学,2003,3(2)： 207
[47]Knothe H, Shah P, Kremery V, et al.Transferable resistance to ce-fotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of klebsiella pneumoniae and serratia marcescens[J].Infection,1983,11 (6):315～317
[48]Anna Baraniak, Janusz Fiett, Agnieszka Mro'wka, et al.Evolution of TEM-type Extended spectrum lactamases in Clinical Enterobacteriaceae Strains in Poland[J].Antimicrobial Agents and Chemotherpy,2005,49(5):1872～80
[49]Chaibi EB, Sirot D, Paul G, et al.Inhibitor-resistant TEM-β-lactamas:phenotypic, genetic and biochemical characteristics[J].Antimicrob Chemother,1999,43 (4):447～458
[50]Robin F, Delmas J, Chanal C, et al. TEM-109(CMT-5), a Natural complex mutant of TEM-lbeta-Lactamase combining the Amino Acid Substitutions of TEM-6 and TEM-33 (IRF-5)[J]. Antimicrob Agents Chemother,2005,49 (11):4443～4447
[51]李虹玲,刘文恩,陈腊梅,等.湖南地区SHV型超广谱β-内酰胺的研究[J].中华医院感染学杂志,2009,19(11)：1328～1331
[52]Bradford PA, Urban C, Jaiswal A, et al.SHV-7, a novel cefotaxime-hydrolyzing beta-lactamase, identified in Escherichia coil isolates from hospitalized nursing home patients[J]. Antimicrob Agents Chemother,1995,39 (7):1646
[53]Hujer AM, Hujer KM, Helfand MS, et al.Amino acid substitutions at Ambler position Gly238 in the SHV-1 beta-lactamase:exploring sequence requirements for resistance to penicillins and cephalosporins[J]. Antimicrob Agents Chemother,2002,46(12):3971～7
[54]Giakkoup IP, Miriagou V, Gazoul IM, et al.Properties of mutant SHV-5beta-lactamases constructed by substitution of isoleucine or valine for methionine at position 69[J]. Antimicrob Agents Chemother,1998,42 (5):1281～3
[55]Helfand MS, Hujer AM, Sonnichsen FD, et al.Unexpected advanced generation cephalosporinase activity of the M69F variant of SHV beta-lactamase[J].J-Biol-Chem, 2002,277 (49):47719～23
[56]Prinarakis EE, Miriagou V, Tzelepi E, et al. Emergence of an inhibitor-resistant beta-lactamase (SHV-10) derived from an SHV-5 variant[J]. Antimicrob Agents Chemother, 1997,41(4):838～40
[57]Poirel L, Heritier C, Podglajen I, et al. Emergence in Klebsiella pneumoniae of a chromosome-encoded SHV beta-lactamase that compromises the efficacy of imipenem[J]. Antimicrob Agents Chemother,2003,47(2):755～8
[58]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
[59]刘文恩,陈腊梅,邹明祥,等.湖南地区CTX-M型超广谱β-内酰胺酶的研究[J].中华检验医学杂志,2006,29(10)：278～881
[60]Cartelle M,del-MarTomas M,Molina F,et al.High-level resistance to ceftazidime conferred by a novel enzyme,CTX2M232,derived f rom CTX-M-1 through a single Asp240-Gly substitution [J].Antimicrob Agents Chemot her,2004,48(6):2308～2313.
[61]Bonnet R,Sampaio JL,Labia R, et al.A novel CTX-M beta-lactamase (CTX-M-8) in cefotaxime-resistant Enterobacteriaceae isolated in Brazil[J]. Antimicrob Agents Chemother,2000,44(7):1936～1942
[62]杨玉荣.超广谱β-内酰胺酶研究进展[J].中国兽药杂志,2004,38(12)：31～34
[63]Joumana N, Samaha-K foury, George F Araj.Recent development in β-lactamases and extended spectrum β-lactamases[J]. English Clinical Journal 2004,7(4):220～224
[64]Scoulica EV, Neonakis IK, Gikas LI, et al.Spread of bla VIM-1 producing E.coli in a university hospital in Greece.Genetic analysis of the integron carrying the bla VIM-1 metaallo-β-lactamase gene[J].Diagn Microbiol infect Dis,2004,48 (3):167～172
[65]张永标,张扣兴,唐英春,等.产质粒介导AmpC酶和ESBLs细菌的耐药性及β-内酰 胺酶基因型研究[J].中华微生物学和免疫学杂志,2004,24(7)：577～582
[66]Lee SH, Jeong SH,Park YM.Characterization of blaCMY-10, a novel plasmid-encoded AmpC type β-lactamases gene in a clinical isolate of Enterobacter aerogens[J].J Appl Microbiol,2003,44 (10):744～752
[67]Mulvey MR,Bryce E, Boyd DA, et al.Molecular characterization of cefoxitin-resistant Escherichia coli from Canadian hospitals[J].Antimicrob Agents Chemother,2005,49 (1): 358～365
[68]Siu LK, Lu PL, Chen JY, et al.High-level expression of ampC beta-lactamase due to insertion of nucleotides between-10 and-35 promoter sequences in Escherichia coli clinical isolates:cases not responsive to extended-spectrum-cephalosporin treatment[J]. Antimicrob Agents Chemother,2003,47 (7):2138～2144
[69]多丽波,刘晶,张联博,等.高产AmpC酶大肠埃希菌的分子生物学研究[J].中华医院感染学杂志,2007,17(7)：786～789
[70]蒋燕群,曹娜,陈泰尧,等.革兰阴性杆菌中质粒ampC基因的检测与分析[J].上海医学检验杂志,2003,18(6)：328～330
[71]Galimand M, Courvalin P, Lambert T.Plasmid-mediated high-level resistance to aminoglycosides in Enterbacteriaceae due to 16s rRNA methylation[J]. Antimicrob Agents Chemother,2003,47 (8):2565～2571
[72]张馨琢,黄永茂.大肠埃希菌产氨基糖苷类修饰酶的研究进展[J].西南军医,2008,10(6)：105～107
[73]YOKOYAMA K, DOIY, YAMANE K, et al.Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa[J].Lancet,2003,362 (9399):1888～1893
[74]Yam ane K, Wachino J, Suzuki S, et al.16S rRNA methylase-producing, Gram-negative pathogens, Japan[J].EID,2007,14 (4):642～646
[75]林宁,孙海平,糜祖煌.多重耐药大肠埃希菌16S rRNA甲基化酶、氨基糖苷类修饰酶基因研究[J].中国抗生素杂志,2008,33(9)：536～539
[76]韩玲,聂大平.革兰阴性菌中16S rRNA甲基化酶基因的检测[J].中国感染与化疗杂志,2009,9(4)：267～271
[77]Shaw KJ, Rather PN, Hare RS, et al.Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes[J].Microbiol Rev, 1993,57 (1):138～163
[78]Chow JW, Kak V, You I, et al. aminoglycoside resistance genes aph(2")-Ib and aac(6')-Im detected together in strains of both Escherichia coli and Enterococcus faccium[J]. Antimicrob Agents Chemother,2001,45 (10):2691
[79]陈接根,吕建新,娄永良,等.大肠埃希菌氨基糖苷类耐药株AAC(3)-基因保守区分析[J].遗传,2004,26(2)：202～204
[80]Jakobsen L, Sandvang D, Jensen VF, et al.Gentamicin susceptibility in Escherichia coli related to the genetic background:problems with breakpoints[J].Clin Microbiol infect, 2007,13 (8):830
[81]Fihman V, Lartique MF, Jacqier H, et al.Appearance of aac(6')-Ib-Cr gene among extended-spectrum beta-lactamase-producing Enterobacteriaceae in a French hospital [J]. infect,2008,56 (6):454
[82]Kehrenberg C, Schwarz S.Nucleotide sequence and organization of plasmid pMVSCS1 from Mannheimia varigena:identification of amultiresistance gene cluster[J].J Antimicrob Chemother,2002,49 (2):383～386
[83]姚蕾,陈琼,胡昌勤,等.64株大肠埃希菌对氨基糖苷类抗生素的耐药表型与钝化酶基因型的分析[J].中华微生物与免疫学杂志,2005,25(9)：727～732
[84]李智山,周乐翔,赵建忠,等.大肠埃希菌新的氨基糖苷类修饰酶基因研究[J].中华医院感染学杂志,2007,17(8)：914～916
[2]Gniadkowski M.Evolution and epidemiology of extended-spectrum beta-lactamases (ESBLSs) and ESBLs-producing microorganisms [J].Clin Microbiol Infect,2001,7(11): 597～608
[3]张玲.革兰阴性杆菌产超广谱β-内酰胺酶研究进展.中华医院感染学杂志,2008,18(6)：897～900
[4]Bonnet R, Dutour C, Sampaio JLM, et al. Novel cefotaximase (CTX-M-16) with increased catalytic efficiency due to substitution Asp-240→Gly.2001, Antimicrob Agents Chemother,45:2269～2275
[5]Cao V, Lambert T, Nhu DQ, et al. Distribution of extended-spectrum B-lactamases in clinical isolates of Enterobacteriaceae in Vietnam.2002, Antimicrob Agents Chemother,46: 3739～3743
[6]Casin I, Hanau-Bercot I, Podglajen H, et al. Salmonella enterica serovar Typhimurium blaPER-1-carrying plasmid pSTI1 encodes an extended-spectrum aminoglycoside 6'-N-acetyltransferase of type Ib.2003, Antimicrob Agents Chemother,47:697～703
[7]De Champs C, Poirel L, Bonnet R, et al. Prospective survey of B-lactamases produced by ceftazidime-resistant Pseudomonas aeruginosa isolated in a French hospital in 2000.2002, Antimicrob Agents Chemother,46:3031～3034
[8]De Champs C, Sirot D, Chanal C, et al. A 1998 survey of extended-spectrum β-lactamases in Enterobacteriaceae in France.2000, Antimicrob Agents Chemother 44:3177～3179
[9]Gniadkowski M, Schneider I, Palucha A, et al. Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland:identification of a new CTX-M-3 cefotaxime-hydrolyzing β-lactamase that is closely related to the CTX-M-1/MEN-1 enzyme.1998, Antimicrob Agents Chemother,42:827～832
[10]Vahaboglu H, Ozturk R, Aygiin G, et al. Widespread detection of PER-1-type extended-spectrum B-lactamases among nosocomial Acinetobacter and Pseudomonas aeruginosa isolates in Turkey:a nationwide multicentre study.1997, Antimicrob Agents Chemother,41:2265～2269
[11]Girlich D, Poirel L, Leelaporn A, et al. Molecular epidemiology of the integron-located VEB-1 extended-spectrum β-lactamase in nosocomial enterobacterial isolates in Bangkok, Thailand.2001, J Clin Microbio,139:175～182
[12]季淑娟,顾怡明,谭文涛,等.中国部分地区大肠埃希菌和肺炎克雷伯菌超广谱β内酰胺酶基因型研究.中华医学检验杂志,2004,27(9)：590～593
[13]王燕,李苏利,李杨.细菌产超广谱β-内酰胺酶的研究状况.现代中西医结合杂志,2006,15(23)：3312～3314
[14]Goossens H. MYSTIC program:summary of European data from 1997 to 2000. Diagn Microbiol Infect Dis,2001,41:183～189
[15]管希周,刘又宁,王睿,等.临床产ESBLs细菌耐药特性及其基因分型的研究.中国抗生素杂志,2001,26(6)：468～472
[16]Yu YS, ZhouWL, Chen YG, et al. Ep idemiological and antibiotic resistant study on extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Zhejiang Province.ChinMed J,2002,115:1479～1482
[17]朱德妹,熊自忠,汪复,等.超广谱β-内酰胺酶和细菌耐药性.中华传染病杂志,2000,18(3)：151～154
[18]陈民钧,王辉.中国重症监护病房革兰阴性菌耐药性连续7年监测研究.中华医学杂志,2003.83(5)：375～381
[19]陆坚,唐英春,吴本权,等.华南地区质粒介导超广谱β-内酰胺酶基因分型研究.中华微生物学和免疫学杂志,2002,22(6)：638～643
[20]郑巧敏,张秀霞,朱涛,等.大肠埃希菌耐药性水平传播试验研究.中国微生态杂志,2008,20(1)：44～48
[21]付英梅,张凤民,张文莉,等.产超广谱β-内酰胺酶大肠埃希菌和肺炎克雷伯菌Ⅰ类整合子及其与ESBLSs基因关系的研究.中华医院感染学杂志,2007,17(3)：241～245
[22]叶惠芬,陈惠玲,刘平,等.产ESBLs大肠埃希菌和肺炎克雷伯菌耐药性和基因型分布.中国感染与化疗杂志,2007,7(2)：127～129
[23]张洁,包其郁,张洪勤,等.多重耐药性大肠埃希菌质粒谱与ESBLs基因型分析.中国抗生素杂志,2006,31(4)：198～201
[24]Bart helemy M, Peduzzi J, Labia R. Distinction between the primary structures of TEM-1 and TEM-2 beta-lactamases [J].Ann Inst Pasteur Microbiol,1985,136:311～321
[25]Gniadkowski M. Evolution and epidemiology of extended-spectrum beta-lactamases (ESBLs) and ESBLSs-producing microorganisms [J]. Clin Microbiol Infect,2001,7 (11): 597～608
[1]肖庆忠,苏丹虹,江洁华,等.广州地区3500株革兰阴性细菌的分布特征及耐药性特点[J].第一军医大学学报,2005,25(2)：132～138
[2]陈文光,蒋景华,王宗欣,等.大肠埃希菌的科室分布和耐药性分析[J].中华医院感染学杂志,2008,18(1)：119～120
[3]许桂芳,金春光.产超广谱β-内酰胺酶大肠埃希菌耐药性及基因同源性研究[J].中国卫生检验杂志,2009,19(8)：1843～1844
[4]魏殿军,宋师铎.医院内、外感染大肠埃希菌、肺炎克雷伯菌药物敏感性分析[J].中华医院感染学杂志,2003,13(7)：677～680
[5]Aminzadeh Z, Sadat Kashi M, Sha'bani M.Bacteriuria by extended-spectrum Beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae:isolates in a governmental hospital in South of Tehran, Iran[J].Iran J Kidney Dis.2008,2(4):197～200
[6]Song S, Lee EY, Koh EM, etc. Antibiotic resistance mechanisms of Escherichia coli Isolates from urinary specimens [J]. Korean J Lab Med,2009,29(1):17～24
[7]Johnson JR, Johnston B, Kuskowski MA, etc. Molecular epidemiology and phylogenetic distribution of the Escherichia coli pks genomic island[J]. J Clin Microbiol,2008, 46(12):3906～11
[8]Manges AR, Tabor H, Tellis P, etc. Endemic and epidemic lineages of Escherichia coli that cause urinary tract infections[J]. Emerg Infect Dis,2008,14(10):1575～83
[9]萨姆布鲁克,弗里奇,曼尼阿蒂斯,等.分子克隆实验指南[M].第2版.北京：科学出版社,2002
[10]苏内尔,菲利普斯.质粒生物学[M].第1版.北京：化学工业出版社,2009
[11]苏健裕,石磊,杨连生.临床大肠埃希菌第Ⅰ类整合子检测及耐药基因盒分析[J].检验医学与临床,2008,5(7)：385～387
[12]刘朝霞,刘又宁.质粒介导的喹喏酮类药物耐药性研究[J].国际呼吸杂志,2006,26(4)：304～306
[13]徐艳,郭丽双,付英梅,等.细菌对氨基糖苷类抗生素的耐药机制[J].中国微生态杂志,2008,20(2)：191～193
[14]石福艳,白亚娜,樊景春,等.医院感染中大肠埃希菌耐药质粒的同源性分析[J].中国公共卫生,2006,22(9)：1091～1092
[15]张洁,包有郁,张洪勤,等.多重耐药性大肠埃希菌质粒谱与ESBLs基因型分析.中国抗生素杂志,2006,31(4)：198～201
[16]年华,褚云卓,王倩.医院感染大肠埃希菌耐药性变化分析[J].中华医院感染学杂志,2007,17(4)：452～454
[17]耿燕,张王刚,王香玲,等.泌尿系感染产超广谱β内酰胺酶(ESBLs)大肠埃希菌的耐药表型和基因分型[J].第四军医大学学报,2005,26(7)：622～624
[18]张志坚,郭小兵,张钦宪.产ESBLs大肠埃希菌质粒谱与耐药性分析[J].第四军医大学 学报,2008,29(21)：1970～1972
[19]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
[20]张之文,王红宁.整合子结构及在细菌耐药性扩散中的作用[J].国外医药抗生素分册,2007,24(4)：150～153
[21]廖伟娇,江洁华,易建云,等.多重耐药革兰阴性杆菌Ⅰ类整合子的检测[J].热带医学杂志,2006,6(6)：667～669
[22]ROWE-MANGUS DA, MAZEL D.The role of integrons in antibiotic resistance gene capture[J]. Int J Med Microbiol,2002,292(2):115～125.
[23]FLUIT AG, SCHMITZ FJ. Resistance integrons and superintegrons[J]. Clin Microbiol Infect[J],2004,10(4):272～288.
[24]杨维青,刘晓峰,黄震.大肠埃希菌中Ⅰ类整合子耐药基因的分析[J].中国抗生素杂志,2009,34(7)：434～436
[25]李婷,马平,赵冰,等.健康人群中大肠埃希菌耐药整合子的检测[J].华西药学杂志,2009,24(4)：372～374
[26]苏健裕,石磊,杨连生.临床大肠埃希菌第Ⅰ类整合子检测及耐药基因盒分析[J].检验医学与临床,2008,5(7)：385～387
[1]娄茜,黄长武,吴倩.泌尿系感染大肠埃希菌的耐药性分析[J].检验医学与临床,2007,4(10)：940～941
[2]张凤珍,张蕴莉,许德顺.产超广谱β-内酰胺酶大肠埃希菌感染的监测[J].中国实验诊断学,2005,9(1)：83～84
[3]Wada K, Kariyama R, Mitsuhata R, et al. Experimental and Clinical Studies on Fluoroquinolone-insusceptible Escherichia coli Isolated from Patients with Urinary Tract Infections from 1994 to 2007[J]. Acta Med Okayama,2009,63(5):263～72
[4]王燕,李苏利,李扬.细菌产超广谱β-内酰胺酶的研究概况[J].现代中西医结合杂志,2006,15(23)：3312～3314
[5]Philippon A,Labia R,Jacoby G. Extended-spectrum β-lactamases[J]. Antimicrob Agents Chemother,1989,33(1):13
[6]章清,魏丽,魏光美,等.大肠埃希菌、肺炎克雷伯菌的耐药性变迁[J].中华医院感染血杂志,2004,14(2)：220～222
[7]俞云松.超广谱β-内酰胺酶研究概况[J].中国抗生素杂志,2003,12(28)：712-718
[8]Bradford PA. Extended Spectrum β-Lactamses in the 21st century:charcterization, epidemiology, and detection of this important resistance threat [J]. Clin MicrobiolRev, 2001,14(9):993～951
[9]程训民,李敏,张琪.超广谱β-内酰胺酶研究进展[J].淮海医药,2005,23(4)：343-345
[10]马越,李景云,张新妹.2002年临床常见细菌耐药性监测[J].中华检验医学杂志,2004,27(1)： 38～45
[11]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
[12]Bradford P A.Extended-spectrum beta-lactamases in the 21st century:characterization, epidemiology, and detection of this important resistance threat [J]:Clin MicrobiolRev, 2001,14(4):933-951
[13]Livermore DM. Beta-lactamases in laboratory and clinical resistance[J].Clin Microbiol Rev,1995,8(4):557～584.
[14]Bart helemy M, Peduzzi J, Labia R.Distinction between the primary structures of TEM-1 and TEM-2 beta-lactamases[J].Ann Inst Pasteur Microbiol,1985,136:311～321
[15]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
[16]李虹玲,刘文恩.SHV型超广谱β-内酰胺酶的研究进展[J].实用预防医学,2007,14(1)：253～256
[17]Heritage J, M'Zali F, Gascoyne-Binzi D, et al. Evolution and spread of SHV extended-spectrum β-lactamases in Gram-negative baceria [J].J Antimicrob Chemother,1999, 44:309～318
[18]Szabo D, Melan MA, Hujer AM, et al. Molecular analysis of the simultaneous production of two SHV-type extended-spectrum beta-lactamases in a clinical isolate of Enterobacter cloacae by using single-nucleotide polymorphism genotyping [J].Antimicrob Agents Chemother,2005,49(11):4716-4720
[19]Poirel L, Heritier C, Podglajen I, et al.Emergence in Klebsiella pneumoniae of a Chromosome-encoded SHV β-lactamase that compromise the efficacy of Imipenem[J]. Antimicrobial Agents and Chemotherapy,2003,47(2):755-758
[20]Huletskt A, Knox JR, Levesque RC.Role of ser-238 and Lys-240 in the hydrolysis of third-generation cephhalosporins by SHV-type beta-lactamases probed by site-directed mutagenesis and three-dimensional modeling[J].J Biol Chem,1933,268:3690～3697
[21]张哲,蒋晓飞,李敏,等.SHV-12型超广谱β-内酰胺酶特性的研究[J].检验医学,2006,21(1)：31～34
[22]季淑娟,顾怡明,谭文涛,等.中国部分地区大肠埃希菌和肺炎克雷伯菌超广谱β内酰胺酶基因型研究[J].中华医学检验杂志,2004,27(9)：590～593
[23]张盛斌,刘朝辉CTX-M型ESBLs的研究进展[J].中华感染与化疗杂志,2006,6(3)：212～215
[24]熊自忠,朱德妹,汪复,等.CTX-M-14和CTX-M-24编码基因的检测及其功能表达[J].中华医学杂志,2004,84(17)：1454～1459
[25]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
[26]Cartelle M, del-Mar Tomas M, Molina F, et al.High level resistance to ceftazidime conferred by a novel enzyme, CTX-M-32, derived from CTX-M-1 through a single Asp240-Gly substitution[J].Antimicrob Agents Chemother,2004,48 (6):2308～2313
[27]李金钟CTX-M型ESBLs的研究进展[J].国际检验医学杂志,2007,28(6)：543～545
[1]陆一平,李晖,陈艳华,等.大肠埃希菌感染的临床分布与耐药性[J].山西医药杂志,2006,35(4)：371～372
[2]张传栋,刘存津.产ESBLs大肠埃希菌临床分布及耐药性分析[J].中华医院感染学杂志,2009,19(13)：1728～1729
[3]金文君,黄永禄,徐志江,等.临床分离大肠埃希菌的耐药性分析[J].中国卫生检验杂志,2009,19(10)：2332～2333
[4]金文君,黄永禄,徐志江,等ESBLs阳性大肠埃希菌和肺炎克雷伯菌产AmpC酶的检测与体外抗菌活性分析[J].中华医院感染学杂志,2008,18(6)：855～858
[5]BALL P.Quinolone generations:natural history or natural selection? [J] J Antimicrob Chemother,2000,46(supplT1):17～24
[6]ANDERSSON M I, MACGOWAN A P. Development of the quinolones [J] J Antimicrob Chemother,2003,51(suppl 1):1～11
[7]林东昉,王明贵.喹诺酮类抗菌药物的研究进展[J].新医学,2007,38(1)：49～50
[8]Jcoby GA. Mechanisms of resistance to quinolones[J]. Clin infect dis,2005,41 (2): 120～126
[9]Nikaido H, Thanassi DG.Penetration of lipophilic agents with multiple protonation sites into bacterial cells:tetracylines and fluoroquinones as examples Anticrob[J]. Agents chemother,1993,37(7):1393
[10]方治平,徐炜,刘小康,等.大肠杆菌体内氟喹诺酮类药物耐药机制的研究[J].四川大学学报(医学版),2005,36(1)：86～89
[11]贾蓓.膜孔蛋白及其再细菌耐药中的作用[J].国外医药抗生素分册,1997,18(6)：444～447
[12]钟利,范昕建.大肠杆菌膜孔膜蛋白研究进展[J].国外医药抗生素分册,1998,19(3)：208～210
[13]Van bambeke F, Balzi E, Tulkens PM. Antibiotic efflux pimps [J]. Biochem Pharmacol, 2000,60 (4):457～470
[14]Touze T, Eswaran J, Bokma E, et al. Interactions underlying assembly of the Escherichia coli AcrAB-TolC multidrug efflux system [J]. Mol microbiol,2004,53 (2):697～706
[15]Sennhauser G, Amstutz P, Briand C, et al. Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors[J]. PLoS Biol,2007,5(1):e7
[16]Fujihira E, Tamura N, Yamaguchi A. Membrane topology of a multidrug efflux transporter, AcrB, in Escherichia coli[J]. J Biochem,2002,131(1):145～151
[17]Tikhonova EB, Zgurskaya HI. AcrA, AcrB, and TolC of Escherichia coli Form a Stable Intermembrane Multidrug Efflux Complex[J]. J Biol chem,2004,279 (31):32116～24
[18]Seeger MA, Diederichs K, Eicher T, et al. The AcrB efflux pump:conformational cycling and peristalsis lead to multidrug resistance[J]. Curr Drug Targets,2008,9(9):729～49
[19]Eicher T, Brandstatter L, Pos KM. Structural and functional aspects of the multidrug efflux pump AcrB[J].Biol Chem,2009,390(8):693～9
[20]HOOPER DC.Mechanism of action and resistance of older and newer fluoroquinolones [J]. Clin infect Dis,2000,31(suppl2):S24～S28
[21]HOOPER DC.Mechanism of action of antimicrobials:focus on fluoroquinolones [J]. Clin infect Dis,2001,32(suppl 1):S9-S15
[22]Bebear CM, Charron A, Bove JM, et al. Cloning and nucleotide sequence of the topoisomerase IV parC and pare genes of Mycoplasma hominis[J].Antimicrob Agents Chemother.1998,42(8):2024～2029
[23]Fournier B and Hooper DC. Effects of mutations in Gr1A of topoisomerase Ⅳ from Staphylococcus aureu Effects of mutations in Gr1A of topoisomerase Ⅳ on quinoline and coumarin activity[J].Antimicrob Agents Chemother.1998,4 (8):2019～2024
[24]Luzzaro F. Fluoroquinolones and Gram-negative bacteria:antimicrobial activity and mechanisms of resistance[J]. Infez Med.2008,160 (Suppl2):5～11
[25]Truong QC, Van N, Shlaes D, et al. A novel, double mutation in DNA gyrase A of Escherichia coli conferring resistance to quinolone antibiotics[J]. Antimicrob Agents Chemother.1997,41(1):85～93
[26]Cond S, Oethinger M, Kaifel K, et al. gyrA mutations in high-level fluoroquinolone-resistant clinical isolates of Escherichia coli[J]. Antimicrob Agents Chemother.1996, 38(4):443～451
[27]Kumagai Y, Kato JI, Hoshino K,et al. Quinolone-resisitance mutants of Escherichia coli DNA topoisomerase Ⅳ parCgene[J]. Antimicrob Agents Chemother.1996,40(3):710～ 716
[28]Bachoual R, Analysis of the mutations involved in fluoroquinolone-resisitant of in vivo and in vitro mutants of Escherichia coli [J]. Microb Drug Rsist,1998 4 (4):271～279
[29]Chen JY, Siu LK, Lu PL, et al. Molecular epidemioliogy and mutations at gyrA and parC genes of ciprofloxacin-resistant Escherichia coli isolated from a Taiwan medical center[J]. Microb Drug Rsist,2001,7(1):47～53
[30]Cattoir V, Lesprit P, Lascols C,et al. In vivo selection during ofloxacin therapy of Escherichia coli with combined topoisomerase mutations that confer high resistance to ofloxacin but susceptibility to nalidixic acid[J]. J Antimicrob Chemother,2006,58(5): 1054-1057
[31]Petersen A, Christensen JP, Kuhnert P. Vertical transmission of a fluoroquinolone-resisitant Escherichia coli within an integrated broiler operation[J].Vet Microbiol,2006,116(1-3): 120～128
[32]蒋萍,陈恒屹,张坚磊,等.耐喹诺酮类大肠埃希菌耐药机制的研究[J].中华医院感染学杂志,2007,17(6)：635～638
[33]Niwa H, Hobo S, Anzai T. A nucleotide mutation associated with fluoroquinolone resistance observed in gyrA of in vitro obtained Rhodococcus equi mutants[J].Vet Microbiol,2006,115(1):264～268
[34]Mart nez LM, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid[J]. Lancet,1998,351:797～799
[35]Li XZ. Quinolone resistance in bacetrica:emphasis on plasmid-mediated mechanisms[J]. Int J Antimicrob Agent,2005,25 (6):453～463
[36]Suzuki MHM, Matsumoto M, Takahashi M, et al. Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella flexneri 2b[J].Antimicrob Agents Chemother,2005,49(2):801～803
[37]Tran JH, Jacoby GA, Hooper DC. Interaction of the plasmid-encoded quinolone resistance protein qnrA with Escherichia coli topoisomerase Ⅳ[J]. Antimicrob Agents Chemother, 2005,49(7):3050～3052
[38]Jacoby GA, Kelley E, Walsh, et al.qnrB, Another plasmid-mediated gene for quinolone resistance protein qnrA with Escherichia coli topoisomerase Ⅳ[J]. Antimicrob Agents Chemother,2006,50 (4):1178～1182
[39]Rbicsek A, Strahilevita J, Jacoby GA, et al. Fluoroquinolone-modifying enzyme:a new adaptation of a common aminoglycoside acetyltransferase[J].Nat med,2006,12(1):83～88
[40]Perichon BP, Galimand CM. Transferable resistance to aminoglycosides by methylation of G1405 in 16s rRNA and to hydrophilic fluoroquinolones by QepA-mediated efflux in Escherichia coli[J]. Antimicrob Agents Chemother,2007,51(7):2464～2469
[41]Yamane KJ, Wachino S, Suzuki K, et al. New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate[J]. Antimicrob Agents Chemother,2007,51 (9):3354～3360
[42]张致平.β-内酰胺类抗生素研究的进展[J].中国新药杂志,2002,11(1)：61～67
[43]韩刚,蒋立建,韩冲,等.新型β-内酰胺类抗生素及其特点[J].东南大学学报(医学版),2008,27(4)：316～318
[44]肖时超,吕火祥,沈蓓琼,等.2003年至2007年大肠埃希菌的临床分离及耐药趋势分析[J].中国微生态学杂志,2008,20(4)：407～409
[45]沈继录.超广谱p-内酰胺酶研究进展[J].国外医学临床生物化学与检验学分册.2004,25(3)：234～237
[46]周炎腾,贾杰,苏林光,等.410株大肠埃希菌耐药性分析[J].中国热带医学,2003,3(2)： 207
[47]Knothe H, Shah P, Kremery V, et al.Transferable resistance to ce-fotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of klebsiella pneumoniae and serratia marcescens[J].Infection,1983,11 (6):315～317
[48]Anna Baraniak, Janusz Fiett, Agnieszka Mro'wka, et al.Evolution of TEM-type Extended spectrum lactamases in Clinical Enterobacteriaceae Strains in Poland[J].Antimicrobial Agents and Chemotherpy,2005,49(5):1872～80
[49]Chaibi EB, Sirot D, Paul G, et al.Inhibitor-resistant TEM-β-lactamas:phenotypic, genetic and biochemical characteristics[J].Antimicrob Chemother,1999,43 (4):447～458
[50]Robin F, Delmas J, Chanal C, et al. TEM-109(CMT-5), a Natural complex mutant of TEM-lbeta-Lactamase combining the Amino Acid Substitutions of TEM-6 and TEM-33 (IRF-5)[J]. Antimicrob Agents Chemother,2005,49 (11):4443～4447
[51]李虹玲,刘文恩,陈腊梅,等.湖南地区SHV型超广谱β-内酰胺的研究[J].中华医院感染学杂志,2009,19(11)：1328～1331
[52]Bradford PA, Urban C, Jaiswal A, et al.SHV-7, a novel cefotaxime-hydrolyzing beta-lactamase, identified in Escherichia coil isolates from hospitalized nursing home patients[J]. Antimicrob Agents Chemother,1995,39 (7):1646
[53]Hujer AM, Hujer KM, Helfand MS, et al.Amino acid substitutions at Ambler position Gly238 in the SHV-1 beta-lactamase:exploring sequence requirements for resistance to penicillins and cephalosporins[J]. Antimicrob Agents Chemother,2002,46(12):3971～7
[54]Giakkoup IP, Miriagou V, Gazoul IM, et al.Properties of mutant SHV-5beta-lactamases constructed by substitution of isoleucine or valine for methionine at position 69[J]. Antimicrob Agents Chemother,1998,42 (5):1281～3
[55]Helfand MS, Hujer AM, Sonnichsen FD, et al.Unexpected advanced generation cephalosporinase activity of the M69F variant of SHV beta-lactamase[J].J-Biol-Chem, 2002,277 (49):47719～23
[56]Prinarakis EE, Miriagou V, Tzelepi E, et al. Emergence of an inhibitor-resistant beta-lactamase (SHV-10) derived from an SHV-5 variant[J]. Antimicrob Agents Chemother, 1997,41(4):838～40
[57]Poirel L, Heritier C, Podglajen I, et al. Emergence in Klebsiella pneumoniae of a chromosome-encoded SHV beta-lactamase that compromises the efficacy of imipenem[J]. Antimicrob Agents Chemother,2003,47(2):755～8
[58]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
[59]刘文恩,陈腊梅,邹明祥,等.湖南地区CTX-M型超广谱β-内酰胺酶的研究[J].中华检验医学杂志,2006,29(10)：278～881
[60]Cartelle M,del-MarTomas M,Molina F,et al.High-level resistance to ceftazidime conferred by a novel enzyme,CTX2M232,derived f rom CTX-M-1 through a single Asp240-Gly substitution [J].Antimicrob Agents Chemot her,2004,48(6):2308～2313.
[61]Bonnet R,Sampaio JL,Labia R, et al.A novel CTX-M beta-lactamase (CTX-M-8) in cefotaxime-resistant Enterobacteriaceae isolated in Brazil[J]. Antimicrob Agents Chemother,2000,44(7):1936～1942
[62]杨玉荣.超广谱β-内酰胺酶研究进展[J].中国兽药杂志,2004,38(12)：31～34
[63]Joumana N, Samaha-K foury, George F Araj.Recent development in β-lactamases and extended spectrum β-lactamases[J]. English Clinical Journal 2004,7(4):220～224
[64]Scoulica EV, Neonakis IK, Gikas LI, et al.Spread of bla VIM-1 producing E.coli in a university hospital in Greece.Genetic analysis of the integron carrying the bla VIM-1 metaallo-β-lactamase gene[J].Diagn Microbiol infect Dis,2004,48 (3):167～172
[65]张永标,张扣兴,唐英春,等.产质粒介导AmpC酶和ESBLs细菌的耐药性及β-内酰 胺酶基因型研究[J].中华微生物学和免疫学杂志,2004,24(7)：577～582
[66]Lee SH, Jeong SH,Park YM.Characterization of blaCMY-10, a novel plasmid-encoded AmpC type β-lactamases gene in a clinical isolate of Enterobacter aerogens[J].J Appl Microbiol,2003,44 (10):744～752
[67]Mulvey MR,Bryce E, Boyd DA, et al.Molecular characterization of cefoxitin-resistant Escherichia coli from Canadian hospitals[J].Antimicrob Agents Chemother,2005,49 (1): 358～365
[68]Siu LK, Lu PL, Chen JY, et al.High-level expression of ampC beta-lactamase due to insertion of nucleotides between-10 and-35 promoter sequences in Escherichia coli clinical isolates:cases not responsive to extended-spectrum-cephalosporin treatment[J]. Antimicrob Agents Chemother,2003,47 (7):2138～2144
[69]多丽波,刘晶,张联博,等.高产AmpC酶大肠埃希菌的分子生物学研究[J].中华医院感染学杂志,2007,17(7)：786～789
[70]蒋燕群,曹娜,陈泰尧,等.革兰阴性杆菌中质粒ampC基因的检测与分析[J].上海医学检验杂志,2003,18(6)：328～330
[71]Galimand M, Courvalin P, Lambert T.Plasmid-mediated high-level resistance to aminoglycosides in Enterbacteriaceae due to 16s rRNA methylation[J]. Antimicrob Agents Chemother,2003,47 (8):2565～2571
[72]张馨琢,黄永茂.大肠埃希菌产氨基糖苷类修饰酶的研究进展[J].西南军医,2008,10(6)：105～107
[73]YOKOYAMA K, DOIY, YAMANE K, et al.Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa[J].Lancet,2003,362 (9399):1888～1893
[74]Yam ane K, Wachino J, Suzuki S, et al.16S rRNA methylase-producing, Gram-negative pathogens, Japan[J].EID,2007,14 (4):642～646
[75]林宁,孙海平,糜祖煌.多重耐药大肠埃希菌16S rRNA甲基化酶、氨基糖苷类修饰酶基因研究[J].中国抗生素杂志,2008,33(9)：536～539
[76]韩玲,聂大平.革兰阴性菌中16S rRNA甲基化酶基因的检测[J].中国感染与化疗杂志,2009,9(4)：267～271
[77]Shaw KJ, Rather PN, Hare RS, et al.Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes[J].Microbiol Rev, 1993,57 (1):138～163
[78]Chow JW, Kak V, You I, et al. aminoglycoside resistance genes aph(2")-Ib and aac(6')-Im detected together in strains of both Escherichia coli and Enterococcus faccium[J]. Antimicrob Agents Chemother,2001,45 (10):2691
[79]陈接根,吕建新,娄永良,等.大肠埃希菌氨基糖苷类耐药株AAC(3)-基因保守区分析[J].遗传,2004,26(2)：202～204
[80]Jakobsen L, Sandvang D, Jensen VF, et al.Gentamicin susceptibility in Escherichia coli related to the genetic background:problems with breakpoints[J].Clin Microbiol infect, 2007,13 (8):830
[81]Fihman V, Lartique MF, Jacqier H, et al.Appearance of aac(6')-Ib-Cr gene among extended-spectrum beta-lactamase-producing Enterobacteriaceae in a French hospital [J]. infect,2008,56 (6):454
[82]Kehrenberg C, Schwarz S.Nucleotide sequence and organization of plasmid pMVSCS1 from Mannheimia varigena:identification of amultiresistance gene cluster[J].J Antimicrob Chemother,2002,49 (2):383～386
[83]姚蕾,陈琼,胡昌勤,等.64株大肠埃希菌对氨基糖苷类抗生素的耐药表型与钝化酶基因型的分析[J].中华微生物与免疫学杂志,2005,25(9)：727～732
[84]李智山,周乐翔,赵建忠,等.大肠埃希菌新的氨基糖苷类修饰酶基因研究[J].中华医院感染学杂志,2007,17(8)：914～916