肠杆菌科临床株产超广谱β-内酰胺酶的基因多样性研究

目的 研究本地肠杆菌科临床株产超广谱β-内酰胺酶（ESBLs)的基因型。方法 采用K-B法，接合传递试验，质粒谱分析，PCR，PCR-限制性片段长度多态性分析（RFLP）及DNA序列分析等方法对33株产ESBLs肠杆菌科临床株的表型及基因型进行分析。结果 33株临床株对头孢噻肟的耐药率为85%，明显高于头孢他啶，PCR检测结果为28株临床株携带blaCTX-M-ESBLs，24株携带blaTEMDNA,9株携带blaSHVDNA，PCR-RFLP检测结果为临床株EC98A7的TEM酶含有E104K突变，未发现发生G238S突变的SHV酶，DNA序列分析及同源性比较表明4株临床株（EC98A7，EB56，CFR78及KP9941）的blaCTX-MDNA片段属于CTX-M亚组1，分别与blaCTX-M-3和blaCTX-M-12的同一性最高，结论 本地肠杆菌科临床株85%携带CTX-M型，多数菌株同时携带2种以上β-内酰胺酶，大肠杆菌EC98A7同时携带CTX-M型和TEM型两种ESBLs。     

78株阴沟肠杆菌产酶分布与耐药分析

阴沟肠杆菌是医院内感染的常见病原菌。快速检测持续高产AmpC酶或ESBLs或同时表达两种酶的阴沟肠杆菌的分布与耐药特征,对指导临床合理应用抗生素、控制院内感染具有重要的临床意义。     

多药耐药阴沟肠杆菌临床株ampC基因的克隆和表达

目的克隆一株临床分离的多药耐药阴沟肠杆菌的ampC基因,并研究其编码的AmpCβ-内酰胺酶的特性。方法聚合酶链反应(PCR)扩增阴沟肠杆菌ECLC074的ampC基因,将扩增的目标片段连接入pMD18-T进行双链测序后,进一步克隆入pACYC184质粒,用获得的pACYC184/ampC重组质粒转化大肠杆菌MC4100。采用琼脂稀释法测定阴沟肠杆菌ECLC074,大肠杆菌MC4100及大肠杆菌MC4100重组菌的抗生素敏感性,采用三维试验及等电聚焦电泳研究β-内酰胺酶的特性。结果DNA测序及限制性酶切分析结果表明,阴沟肠杆菌ECLC074的ampC基因已被克隆入pACYC184质粒,大肠杆菌MC4100重组菌和阴沟肠杆菌ECLC074均产生一种等电点为7.8的β-内酰胺酶,该酶具有AmpCβ-内酰胺酶的耐药表型特征。结论阴沟肠杆菌的ampC基因可以被克隆入pACYC184质粒,并能在大肠杆菌MC4100中表达,这为深入研究染色体AmpCβ-内酰胺酶创造了条件。     

医院检出阴沟肠杆菌耐药性及耐药基因

目的了解临床分离阴沟肠杆菌耐药性与耐药基因情况,为临床合理选用抗菌药物提供理论依据。方法收集赣南医学院第一附属医院临床分离的阴沟肠杆菌40株,琼脂稀释法与纸片扩散法检测常用抗菌药物的敏感性,采用PCR方法对12种耐药基因进行检测。将该院检出的40株阴沟肠杆菌作为研究样本,采用琼脂稀释法与纸片扩散法行药敏试验,而后采用序列分析法与PCR(聚合酶反应)对12种耐药相关基因进行分析。结果经分析后的药物敏感结果显示,40株阴沟肠杆菌标本对头孢吡肟耐药率以15.0%为最低,而对美罗培南与亚胺培南敏感率均为100%,其他抗菌耐药率范围为42.0%~92.5%;耐药基因共8类,分别为sull、Int I 1、aac(3,')-I、Amp C、CTX-M-9、CTX-M-3、SHV-2a、TEM-1,sull+Int I1为大部分菌株所携带;耐药谱分为9型(A-I),其中以A、D占比较高,且基因分型与其分型存在一定的相关性。结论阴沟肠杆菌有高度与多重耐药性特征,其耐药机制较为复杂且为多种耐药机制共同作用。     

综合性医院2009~2014年多重耐药肠杆菌分布及耐药性分析

目的：通过对南京医科大学第一附属医院2009~2014年间多重耐药肠杆菌(包括大肠杆菌、肺炎克雷伯杆菌、肠杆菌属)分布状况及药敏结果的研究,旨为临床治疗及控制多重耐药菌感染提供依据。方法所有菌株分离自我院2009年1月至2014年12月的临床标本,且均经法国梅里埃公司 Vitek2compact 全自动微生物分析系统或 API 细菌鉴定系统鉴定。药敏判断标准及超广谱β-内酰胺酶(ESBLs)检测按照当年的美国实验室标准化协会(CLSI)文件。数据采用 Whonet5.4软件统计分析。结果2009~2014年,大肠杆菌及肺炎克雷伯杆菌的 ESBLs 阳性率呈先升后降趋势,而肠杆菌属细菌的 ESBLs 阳性率呈逐年下降趋势。多重耐药肠杆菌主要分布在普外科、ICU、老年科及肾内科。产 ESBLs 的肠杆菌主要分离自呼吸道及泌尿道。2014年我院多重耐药的肠杆菌对碳青霉烯类抗生素及哌拉西林/他唑巴敏感率仍高。结论多重耐药菌院内感染控制初见成效,但多重耐药肠杆菌分离率仍高,应加强对临床标本特别是呼吸道及泌尿道感染标本的微生物学检测;碳青霉烯类抗生素及β-内酰胺类/β-内酰胺酶抑制剂仍是治疗多重耐药肠杆菌感染的有效抗生素。     

多重耐药阴沟肠杆菌致感染性腹泻一例

患者男,33岁,因“乏力、纳差、尿黄7周余,加重伴腹泻6d”于2 0 0 2年9月30日入院。7周前诊断为“急性肝炎”,经保肝治疗1个月余,症状无缓解,巩膜、皮肤黄染加深,9月2 4日出现腹泻,呈稀水样便,每天十余次,无发热、腹痛,无黏液脓血便及里急后重感,给予“氟哌酸、阿米卡星”等治疗效果差。入院体检:神志清楚,轻度脱水貌,全身皮肤、巩膜重度黄染,心肺未见异常,肝脾肋下未及,肝区轻叩痛,腹水征阳性,肠鸣音亢进,5～7次/min ,双下肢无水肿。抗HBs和抗 HBc阳性,甲、丙、丁、戊型标志物均阴性;白蛋白2 2 g/L,总胆红素4 4 2 .2 μmol/L,直接胆红素3…     

革兰阴性杆菌产AmpC酶及超广谱β-内酰胺酶的研究

为探讨临床分离的对第三代头孢菌素耐药的革兰阴性杆菌AmpCβ－内酰胺酶（AmpC)和超广谱β－内酰胺酶（ESBLs)的分布情况，采用头孢西丁三维试验检测AmpC酶，纸片确证试验和头孢曲松三维试验检测ESBLs。结果显示，227株革兰阴性杆菌中产AmpC酶者97株（占42．6％），产ESBLs者38株（占16．7％）。提示AmpC酶和ESBLs是导致革兰阴性杆菌耐药的两类主要酶。AmpC酶和ESBLs的单克隆抗体有望为这类细菌感染的免疫生物治疗提供新方法。     

阴沟肠杆菌感染与耐药机制的研究进展

阴沟肠杆菌为兼性厌氧的革兰阴性菌,主要引起肺部感染,是近几年常见的机会致病菌之一,治疗和耐药情况受到越来越多的关注,针对该菌的药敏情况和耐药机制的研究也更加深入,本文就阴沟肠杆菌的感染和耐药机制进行了简要综述。     

上海部分地区肠杆菌科细菌产超广谱β内酰胺酶情况及 …

目的 了解上海地区肠杆菌科细菌产超广谱β－内酰胺酶（ＥＳＢＬｓ）的情况;比较产ＥＳＢＬｓ菌与非产ＥＳＢＬｓ菌对１１种抗生素的耐药率。方法 收集１９９９年３月￣１９９９年１０月上海地区４家医院分离的肠杆菌细菌１０２６株,用双纸片协同扩散法检测ＥＳＢＬｓ,用Ｋｉｒｂｙ－Ｂａｕｅｒ琼脂扩散法作药敏。结果 １０２６株肠杆菌科细菌中,共检出产ＥＳＢＬｓ菌３５２株,检出率为３４．３１％,其中肺炎克雷伯菌为３７     

产新型β-内酰胺酶阴沟肠杆菌的耐药性及RAPD分型分析

目的了解医院感染阴沟肠杆菌的现状、耐药与流行状况。探讨产新型β-内酰胺酶阴沟肠杆菌的耐药机制与基因分型。方法细菌的药敏试验采用K-B法;产超广谱β-内酰胺酶、AmpC酶检测分别采用双纸片确证试验和三维试验;基因分型采用随机扩增多态性DNA（RAPD）法。结果60株阴沟肠杆菌中产超广谱β-内酰胺酶、AmpC酶分别为23株和32株,产新型β-内酰胺酶株14株。RAPD将60株阴沟肠杆菌分为7种株型。其中Ⅰ、Ⅱ型、Ⅲ型占68.3%,是主要的流行株。产新型β-内酰胺酶阴沟肠杆菌对11种常用抗生素的耐药率为21.3%～100%。结论医院感染阴沟肠杆菌对不同抗生素具有较高的耐药性,不同产酶类型对抗生素的耐药性不同。产新型β-内酰胺酶阴沟肠杆菌的出现与流行是阴沟肠杆菌多重耐药与高耐药的重要原因。     

Heterogeneity in ampR-ampC gene interaction in Enterobacter cloacae

The ampR gene and its regulation of AmpC beta-lactamase synthesis were investigated for Enterobacter cloacae 1194E, a wild-type strain producing a group A (pI 8.7) enzyme. Expression of the cloned E. cloacae 1194E ampR-ampC region was examined initially in Escherichia coli HB101. However, transformants showed only constitutive beta-lactamase expression. For study of enzyme expression in a more closely related host, the cloned E. cloacae 1194E ampR-ampC region was transformed into E. cloacae 55, a wild-type strain producing a group B (pI 7.8) enzyme. Results indicated a functional E. cloacae 1194E ampR gene that could not be transcomplemented by E. cloacae 55. A comparative analysis of ampR nucleotide and amino acid-sequence data from E. cloacae 1194E and E. cloacae MHN1 revealed related but divergent genes. Thermal induction studies of AmpC beta-lactamase also indicated a difference between E. cloacae 1194E and E. cloacae 55 in ampR-ampC interaction. Thus, it appears that, in at least some strains of Enterobacter, significant intraspecies divergence of ampR has occurred. This heterogeneity in ampR would not have been detected with beta-lactamase expression studies conducted exclusively in E. coli.     

阴沟肠杆菌医院感染分布及耐药性分析

目的探讨阴沟肠杆菌(ECL)在临床标本中的分布及耐药性,为临床医师合理用药提供科学依据。方法采用微量稀释法对临床送检标本分离的ECL菌株进行药敏试验,分析其耐药性。结果 136株ECL菌株主要分布在重症监护病房(ICU)(37株)、呼吸内科(36株)、肾内科(18株)、血液内科(14株)和神经内科(14株)等科室;主要来源于痰液(75株),占55.1%。ECL对IPM的敏感率为97%;对头孢吡肟、环丙沙星、庆大霉素、左旋氧氟沙星、哌拉西林/他唑巴坦的敏感率在70%～81%,对哌拉西林、妥布霉素的敏感率在60%～70%之间,对头孢他啶、头孢曲松、氨曲南的敏感率在50%～60%,且均呈下降趋势;对氨苄西林、头孢唑啉、氨苄西林/舒巴坦敏感率仅为5.9%、2.2%和27.9%。结论阴沟肠杆菌耐药性已十分严重,临床应根据药敏结果合理使用抗生素。     

Emergence of resistance to ceftazidime during therapy for Enterobacter cloacae infections

The mechanism of resistance to ceftazidime in two clinical isolates of Enterobacter cloacae that emerged during therapy with broad-spectrum beta-lactam antibiotics was studied. Both isolates acquired broad resistance to advanced-spectrum beta-lactam drugs other than imipenem. Biotyping confirmed strain identity in both cases, and no new plasmids were detected in the resistant isolates. Both resistant isolates produced beta-lactamase constitutively. Slow but definite hydrolysis of ceftazidime was demonstrated by using purified beta-lactamase in a spectrophotometric assay. Further evidence that beta-lactamase is responsible for resistance in these organisms was provided by the demonstration that cefoxitin, a potent inducer of beta-lactamase, antagonized the activity of ceftazidime against these isolates. This antagonism could be prevented by inhibition of derepression of beta-lactamase with clindamycin. Clindamycin also prevented regrowth of ceftazidime-treated cells in time-kill studies and markedly reduced production of beta-lactamase in induced cultures at concentrations as low as 2 micrograms/ml.     

Development of beta-lactam-resistant Enterobacter cloacae in mice

We compared the ability of four newer beta-lactam compounds to produce resistance in an experimental model of Enterobacter cloacae infection. Mice infected intraperitoneally developed resistance depending on antibiotic treatment and the dose given. Percentages of mice in which resistance was observed were as follows: 100% after ceftriaxone (50 mg/kg, two doses); 87% after ceftriaxone (50 mg/kg, one dose); 35% after ceftriaxone (500 mg/kg, one dose); and 21% after carumonam (25 mg/kg, two doses). No resistance occurred after therapy with either BMY 28142 (25 mg/kg, two doses) or Sch 34343 (50 mg/kg, two doses). Heterogeneous resistance to beta-lactams among the cells within a given Enterobacter population accounted for these differences. The minimal concentration inhibiting the growth of the preexisting resistant variants, together with the antibiotic concentrations obtained in the peritoneal fluid, were associated with further emergence of resistance in the mouse treated with this antibiotic.     

院内阴沟肠杆菌的感染状况及耐药性的4年分析

目的了解阴沟肠杆菌的医院感染现状及其耐药性变迁。方法对我院2004-2007年分离出阴沟肠杆菌的药敏试验结果进行回顾分析。结果132株阴沟肠杆菌标本主要源于痰(66株,50.00)和血液(31株,23.49);菌株分布于住院部各个病区;阴沟肠杆菌对碳青酶烯类、头孢吡肟、头孢哌酮/舒巴坦的敏感性较高,耐药率均<20.00。结论阴沟肠杆菌是临床感染性疾病的常见病原菌之一,危重患者是阴沟肠杆菌感染的易感人群,其耐药率较高,合理选择抗菌药物要以药敏实验为依据。     

Impact of the ampD gene and its product on beta-lactamase production in Enterobacter cloacae

In an investigation of the influence of the ampD gene on beta-lactamase production and induction in Enterobacter cloacae, the ampR-ampC gene region cloned into a plasmid and the ampD gene cloned into another vector were transferred to a strain of Escherichia coli. The genetically manipulated E. coli strains served as a model for study of the inducibility of beta-lactamases in E. cloacae. In addition, beta-lactamase induction in E. cloacae bearing the previously mentioned plasmids was studied. After induction of the beta-lactamase with cefoxitin, the specific hydrolytic activity, the viable cell count, and the degradation of cefoxitin were determined. beta-Lactamase expression decreased with an increasing amount of the ampD gene product. The cefoxitin concentration decreased in proportion to the amount of enzyme, but the induction of beta-lactamase seemed not to be an important factor influencing the viable cell count of E. cloacae as long as cefoxitin concentrations exceeded the MIC. Despite different beta-lactamase concentrations, the decrease in the viable cell count was nearly identical in all experiments.     

Genetic control of beta-lactamase production in Enterobacter cloacae

In Enterobacter cloacae, mutations in favor of overproduction of beta-lactamase--leading to resistance to third-generation cephalosporins--occur at frequencies of 10(-4)-10(-7). Cloning experiments reveal that at least three genes are involved in the regulation of chromosomal beta-lactamase expression. The structural gene, ampC, is located adjacent to the regulatory gene, ampR, coding for a protein that can serve as an activator in the presence of an inducer. An example of an ampR mutant that is independent of an inducer has been studied. More important for the development of cefotaxime resistance in E. cloacae are mutations in the ampD gene and other proposed regulatory genes. Inactivation of the ampD gene leads to elevated beta-lactamase production. Thus, ampD negatively controls ampC expression. Evidence for the existence of a third regulatory gene, ampE, has been found.     

Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase.

The structure of the class C ampC beta-lactamase (cephalosporinase) from Enterobacter cloacae strain P99 has been established by x-ray crystallography to 2-A resolution and compared to a class A beta-lactamase (penicillinase) structure. The binding site for beta-lactam (penicillinase) structure. The binding site for beta-lactam antibiotics is generally more open than that in penicillinases, in agreement with the ability of the class C beta-lactamases to better bind third-generation cephalosporins. Four corresponding catalytic residues (Ser-64/70, Lys-67/73, Lys-315/234, and Tyr-150/Ser-130 in class C/A) lie in equivalent positions within 0.4 A. Significant differences in positions and accessibilities of Arg-349/244 may explain the inability of clavulanate-type inhibitors to effectively inactivate the class C beta-lactamases. Glu-166, required for deacylation of the beta-lactamoyl intermediate in class A penicillinases, has no counterpart in this cephalosporinase; the nearest candidate, Asp-217, is 10 A from the reactive Ser-64. A comparison of overall tertiary folding shows that the cephalosporinase, more than the penicillinase, is broadly similar to the ancestral beta-lactam-inhibited enzymes of bacterial cell wall synthesis. On this basis, it is proposed that the cephalosporinase is the older of the two beta-lactamases, and, therefore, that a local refolding in the active site, rather than a simple point mutation, was required for the primordial class C beta-lactamase to evolve to the class A beta-lactamase having an improved ability to catalyze the deacylation step of beta-lactam hydrolysis.