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

目的：分析临床大肠埃希菌和肺炎克雷伯菌中超广谱β-内酰胺酶的基因型分布以及产ESBLs细菌中头孢菌素酶的存在情况。方法：质粒接合实验分析耐药性转移，根据超广谱β-内酰胺酶和头孢菌素酶各种已知基因序列设计引物，进行聚合酶链反应分析，确定超广谱β-内酰胺酶的基因型分布以及头孢菌素酶基因。结果：80.4％的菌株质粒接合实验成功，66％的菌株为TEM基因型，14％的菌株为SHV基因型，10％的菌株为CTX-M型。2株菌同时存在AmpC酶。TEM阳性的大肠埃希菌RAPD带型呈多样性。结论：临床分离的大肠埃希菌和肺炎克雷伯菌耐药性可以发生转移，ESBLs以TEM基因型为主，部分菌株为SHV、CTX-M型。同一菌株内可以产生2种不同基因型的超广谱β-内酰胺酶。产ESBL菌株不是以细菌克隆播散为主。     

临床分离弗劳地枸橼酸杆菌耐药性分析及质粒介导的AmpC酶基因检测

目的了解安徽地区临床分离弗劳地枸橼酸杆菌临床分布、耐药性及质粒介导Amp C酶(p Amp Cs)基因的流行情况。方法收集2012～2017年安徽省细菌耐药监控中心监测网内的34所医院报送的临床分离的104株弗劳地枸橼酸杆菌,采用琼脂稀释法进行抗生素敏感性实验。提取细菌质粒,聚合酶链式反应(PCR)检测质粒介导Amp C酶基因,对阳性扩增产物进行测序分析; Amp C酶阳性菌株做转移接合试验,PCR扩增并测序确定接合子的基因型;测定供体菌、受体菌和接合子对多种抗生素的最低抑菌浓度(MIC)。结果 104株弗劳地枸橼酸杆菌临床分布广泛,最多见于呼吸内科(30. 77%)及重症监护病房(24. 04%),标本来源以痰液、尿液最多。4株Amp C酶基因阳性的菌株中,有3株DHA-1基因阳性,1株ACT-1基因阳性。4株Amp C酶基因阳性的菌株有3株转移接合成功。这3株接合子与受体菌相比,对多种常用抗生素尤其是β-内酰胺类抗生素的MIC值提高了8～128倍。结论弗劳地枸橼酸杆菌临床分布广泛,耐药严重,携带有质粒介导的Amp C酶基因的菌株对多种β-内酰胺类抗生素耐药,并且可通过接合性质粒在不同菌属间播散。     

临床分离的肺炎克雷伯菌和大肠埃希菌中超广谱β-内酰胺酶的检测

目的：检测临床分离的临床分离的肺炎克雷伯菌和大肠埃希菌中产超广谱β-内酰胺酶（ESBL）菌株的发生率、耐药性、流行性以及酶的基因型别。方法：采用双纸片法、琼脂稀释法、PCR、脉冲场电泳对1999年复旦大学附属华山医院临床分离的559株肺克雷伯菌和427株大肠埃希菌进行检测。结果：肺炎克雷伯菌和大肠埃希菌中产ESBL菌株的发生率分别为51％和23.6％,产ESBL菌株主要分布于ICU和神经外科病房,对大多数的β-内酰胺酶和非β-内酰胺类抗菌药物耐药;PFGE显示,产ESBL菌株（尤其是产ESBL肺炎克雷伯菌株）在ICU病房中存在同一菌株的克隆传播;PCR检测显示TEM型是最为常见的β-内酰胺酶型别,CTX-M型ESBL亦较为普遍。结论：产ESBL菌株在临床分离的肺炎克雷伯菌和大肠埃希菌中较为普遍,而且为多重耐药菌株,在医院内中存在流行与传播。     

产ESBLs和AmpC肺炎克雷伯菌医院感染分子流行病学研究

目的:研究调查临床分离的120株非重复肺炎克雷伯菌中产超广谱β-内酰胺酶(Extendod spectrum β-laetamases,ESBLs)和头孢菌素酶(Amplerclass C β-lactamase,AmpC)菌株的发生率,以及ESBLs和AmpC:的表型及基因型.方法:采用美国国家临床实验室标准委员会规定的ESBLs表型,筛选和确证ESBLs的发生率.以分析性等电聚焦、接合实验确定ESBLs和AmpC菌株中酶的表型.以质粒为模板,用β-内酰胺酶通用引物、bhTEM、blaSHv及blaAmpC作PCR扩增,并以染色体为模板,用blaAmpC为引物作PCR扩增以确定酶的基因型.结果:120株非重复的肺炎克雷伯菌中,产β-内酰胺酶的有93株(77.5%),其中15株产EsBLs(12.5%),9株产AmpC(7.5%).医院感染ESBIs阳性率(25.5%)明显高于院外感染株(3.7%),所有产AmpC菌株均来自院内感染.所有菌株均对亚胺培南敏感.AmpC阳性菌株对β-内酰胺抗生素的耐药率高于AmpC阴性菌株.产ESBLs或AmpC的18株菌中有9株的编码基因可通过接合实验而转移.结论:肺炎克雷伯菌大多为产β-内酰胺酶菌株,其中产ESBLs和AmpC酶的频率较高,并常导致院内感染;其耐药质粒可通过接合转移给敏感菌,导致耐药性传播.     

产超广谱β-内酰胺酶大肠埃希菌质粒ampC基因检测和耐药性分析

目的 检测产超广谱β-内酰胺酶(ESBLs)大肠埃希菌的质粒ampC基因,并检测其对抗生素的耐药性,了解大肠埃希菌在院内的流行情况,指导临床合理用药.方法 在产ESBLs的大肠埃希菌中筛选出头孢西丁耐药菌株30株,通过热裂解法进行DNA抽提,对6组ampC特异引物进行PCR扩增,琼脂糖凝胶电泳区分PCR产物,进一步DNA测序确定其基因型.结果 5株同时产ampC酶和ESBLs大肠埃希菌的耐药性与195株只产ESBLs的相似,对β内酰胺类抗生素耐药强(P<0.01),符合ESBLs特点;同时符合ampC酶的特点,对头孢西丁和β-内酰胺酶抑制剂耐药(P<0.01),对四代头孢菌素和碳青霉烯类敏感.30株试验菌中有5株为阳性,基因型分别为CIT型2株,DHA型2株,EBC型1株.结论 大肠埃希菌可同时产ESBLs和Ampc酶菌株,由于其为质粒介导,具高传播性,易爆发院内感染,须高度重视.     

大肠埃希菌中共存两种携带ampC和/或超广谱β-内酰胺酶基因的质粒

目的：探讨从临床分离的大肠埃希菌（E．coli）中，由质粒编码的ampC和／或超广谱β-内酰胺酶（ESBLs）的基因质粒携带方式。方法：三维实验证实产AmpC酶的42株E．coli，用E．coliC600作为接合受体菌，采用头孢曲松和头孢他啶／头孢噻肟／棒酸两步淘筛法，进行对第三代头孢菌素耐药性的传递实验：用PCR法扩增质粒编码的ampC和ESBLs基因、三维酶实验和抗生素敏感实验对供体菌和接合子进行对比。结果：接合实验显示，有37侏供体菌对第三代头孢菌素的耐药性可传递，传递频率为10^-7～10^-5。其中23株菌仅传递了ESBLs耐药性，3株菌仅传递AmpC耐药性，11株菌传递了AmpC／ESBLs两种耐药性11株菌中，7株菌的AmpC／ESBLs耐药性携带在同一质粒上；另有4株菌各得到两种接合子，其中2株均得到携带ESBL耐药性和AmpC／ESBLs耐药性的2类接合子，2株均得到携带AmpC耐药性和ESBL耐药性的2类接合子结论：在同时表达AmpC和ESBLs两种酶的E．coli中，有些菌不能通过接合传递实验将两种耐药性分开传递，有些可分开传递。当AmpC和／或ESBLs耐药性基因在同一细菌中南2个质粒携带时，可在接合实验中得到两种不同的接合子。     

产超广谱β-内酰胺酶大肠埃希菌质粒ampC基因检测和耐药性分析

目的 检测产超广谱β-内酰胺酶(ESBLs)大肠埃希菌的质粒ampC基因,并检测其对抗生素的耐药性,了解大肠埃希菌在院内的流行情况,指导临床合理用药.方法 在产ESBLs的大肠埃希菌中筛选出头孢西丁耐药菌株30株,通过热裂解法进行DNA抽提,对6组ampC特异引物进行PCR扩增,琼脂糖凝胶电泳区分PCR产物,进一步DNA测序确定其基因型.结果 5株同时产ampC酶和ESBLs大肠埃希菌的耐药性与195株只产ESBLs的相似,对β内酰胺类抗生素耐药强(P<0.01),符合ESBLs特点;同时符合ampC酶的特点,对头孢西丁和β-内酰胺酶抑制剂耐药(P<0.01),对四代头孢菌素和碳青霉烯类敏感.30株试验菌中有5株为阳性,基因型分别为CIT型2株,DHA型2株,EBC型1株.结论 大肠埃希菌可同时产ESBLs和Ampc酶菌株,由于其为质粒介导,具高传播性,易爆发院内感染,须高度重视.     

产KPC酶超耐药阿斯肠杆菌EaH7的发现及其耐药伴生质粒的遗传特征

 目的  分析1株亚胺培南抗性阿斯肠杆菌(Enterobacter asburiae)的耐药机制及其遗传特征。方法  Vitek-2 Compact系统初步鉴定菌株并测定抗生素最小抑菌浓度,对16s rRNA基因测序以确定菌株;PCR法扩增β-内酰胺类、喹诺酮类和氨基糖苷类等17种耐药基因并测序确认;以CaCl₂诱导的化学法转化质粒;构建伴生质粒DNA文库并测序,以Glimmer 3.02 和BLASTP软件注释并预测伴生质粒序列的编码基因功能;采用接合试验验证伴生质粒对耐药性质粒转移的作用。结果  该菌株为阿斯肠杆菌,对亚胺培南等15种β-内酰胺及氨基糖苷类抗生素耐药,仅对左氧氟沙星和环丙沙星敏感;该菌株同时含有2种质粒,其中耐药性质粒pEa-1携带耐药基因blaKPC-2、blaCTX-M-15和blaTEM-1,而伴生质粒pEa-2则携带4种转移蛋白基因mobA、mobB、mobC和mobD;pEa-2可促进耐药质粒pEa-1接合转移。结论  在国内首次报道了产KPC-2酶的阿斯肠杆菌,该菌携带的质粒pEa-2具有促进耐药性质粒pEa-1接合转移的作用。     

肺炎克雷伯菌和大肠埃希菌产超广谱β-内酰胺酶的耐药表型和基因型研究

目的 检测临床分离的肺炎克雷伯菌和大肠埃希菌产超广谱β-内酰胺酶（ESBLs）的情况，了解产超广谱β-内酰胺酶临床分离菌的耐药特性，并探讨耐药性产生的基因背景。方法 通过对近两年（2004～2005）北京地区医院收集到的共295株大肠埃希菌和肺炎克雷伯杆菌，进行产ESBEs菌株表型鉴定、PCR检测基因型、等电聚焦电泳鉴定等电点，并对这些菌株耐药的特性和基因型进行了探讨。结果 ESBLs的检出率为18．6％，其中大肠埃希菌的检出率为27．7％，肺炎克雷伯杆菌的检出率为8．57％。引起耐药的基因型主要为TEN型81．8％（45／55），其次CTX—M型74．5％（41／55），然后是SHV型20％（11／55）。大肠埃希菌以TEN型为主（88．4％），肺炎克雷伯杆菌以CTX—M为主（91．7％）。经等电聚焦电泳验证，少数菌株的耐药由TEN、SHV、CTX型酶之外的ESBLs引起。结论 产ESBLs菌在临床上较为普遍，大肠埃希菌以TEN型为主，肺炎克雷伯菌以CTX—M型为主，CTX—M型酶对肺炎克雷伯杆菌的头孢噻肟耐药性起重要作用。     

CTX-M-3型超广谱β-内酰胺酶的序列分析与原核表达

目的对肺炎克雷伯菌所产CTX-M-3型超广谱β-内酰胺酶（EsBk）进行基因克隆和重组表达，探讨其特性。方法以产CTX-M-3型超广谱β-内酰胺酶肺炎克雷伯菌49号菌总基因组DNA为模板，PCR扩增CTX-M-3，将其克隆入pGEM-T载体后测定该核苷酸序列，再将CTX-M-3基因克隆到pBV220／DH5α系统进行重组，重组菌经42℃热诱导，SDS-PAGE电泳鉴定酶蛋白的表达。结果PCR扩增出大小为876bp的基因片段，与GenBank上同类酶的基因序列同源性为100％。大肠杆菌DH5α转化pBV220／CTX-M-3重组质粒后，ESBLs试验为阳性，药敏试验结果与原菌株相比耐药性下降。此基因能在大肠埃希菌中大量表达，SDS-PAGE电泳显示，蛋白分子质量大约为31KD。结论成功表达重组的CTX-M-3型酶，为进一步做酶动力学及酶的其他分子生物学特性研究奠定基础。     

肺炎克雷伯菌β-内酰胺酶基因型分布

目的阐明广州地区肺炎克雷伯菌β-内酰胺酶基因型分布。方法用药敏纸片法对67株耐三代头孢菌素类抗生素的肺炎克雷伯菌进行超广谱β-内酰胺酶(ESBLs)表型筛选,采用PCR检测β-内酰胺酶耐药基因,对部分ESBLs菌株进行脉冲场凝胶电泳(PFGE)。结果 67株菌中有58株菌ESBLs表型阳性,β-内酰胺酶基因检测显示,44株产CTX-M、43株产SHV(其中10株产超广谱的SHV)、14株产OXA-1群、38株产TEM,7株菌产AmpC。产CTX-M的菌株有20株产CTX-M-1群(包括15株产CTX-M-15)和24株产CTX-M-9群(包括14株产CTXM-14)。PFGE结果显示产ESBLs菌株同源性低。结论广州地区耐三代头孢肺炎克雷伯菌以产CTX-M-1群和CTX-M-9群ESBLs为主。     

Transconjugation and genotyping of the plasmid-mediated AmpC β-lactamase and extended-spectrum β-lactamase genes in Klebsiella pneumoniae

Bsckgroud AmpC β-lactamases and extended-spectrum β-lactamases （ESBLs） are becoming predominant causes of resistance to third and forth-generation cephalosporins in Klebsiella pneumoniae （K. pneumoniae）. It is very difficult to treat infectious diseases caused by multidrug-resistant K. pneumoniae. The purpose of the present study was to investigate transconjugation and characteristics of β-lactamase genes in K. pneumoniae producing AmpC β-lactamases and ESBLs.
Methods AmpC β-lactamases were detected by three-dimension test and ESBLs by disc confirmatory test. Minimum inhibitory concentrations （MICs） were determined by agar dilution. Transfer of resistance to EC600 （Rifr） was attempted by conjugation in broth and screened on agar containing cefotaxime （2 μg/ml） plus rifampin （1024 μg/ml）. The genes encoding AmpC or ESBLs and their transconjugants were detected by PCR and verified by DNA sequencing.
Results The resistant rates to ampicillin and piperacillin were 100% in 18 isolates of K. pneumoniae. However, imipenem was still of great bactericidal activity on K. pneumoniae, and its MIC50 was 0.5 μg/mL. Eleven β-lactamase genes, including TEM-1, TEM-11, SHV-13, SHV-28, CTX-M-9, CTX-M-22, CTX-M-55, OXA-1, LEN, OKP-6 and DHA-1, were found from 18 isolates. And at least one β-lactamase gene occurred in each isolate. To our surprise, there were six β-lactamase genes in the CZ04 strain. Among 18 isolates of K. pneumoniae, the partial resistant genes in 8 isolates were conjugated successfully, which had 100% homological sequence with donors by sequence analysis. Compared with donors, 8 transconjugants had attained resistance to most β-lactams, including ampicillin, piperacillin, cefoxitin, cefotaxime and aztreonam, or even amikacin and gentamicin.
Conclusions R plasmids can be easily transferred between the resistant and sensitive negative bacilli. It is very difficult to block and prevent the spread of antimicrobial resistance. So more attention should be paid to reducing the frequency, times and dosage of antimicrobials, especially third or fourth cephalosporins.     

临床大肠埃希氏菌耐药性检测及超广谱β-内酰胺酶流行病学调查

目的了解广州地区临床大肠埃希氏菌耐药及超广谱β-内酰胺酶(ESBLs)的流行情况。方法对临床分离鉴定的57株大肠埃希氏菌进行5大类15种抗菌药物的耐药性检测。通过表型确认试验筛选出产ESBLs菌株,采用PCR法检测15种临床重要且常见的耐药基因。结果 57株大肠埃希氏菌对15种抗菌药物产生了不同程度的耐药性。其中有23株产ESBLs,ESBLs产生率为40.4%;24株产TEM,13株产CTX-M(其中,8株产CTX-M-55、4株产CTX-M-14、1株产CTX-M-27),2株产OXA-1。结论广州地区大肠埃希氏菌耐药情况依然严峻。同时,ESBLs流行趋势发生了变化:以前以流行CTX-M-9型为主,现在以CTX-M-1型和CTX-M-9型同时流行为主要特征。     

Antimicrobial resistance, genotypic characterization and pulsed-field gel electrophoresis typing of extended spectrum β-lactamases-producing clinical Escherichia coli strains in Macao, China

Background  The rise of the production of CTX-M class extended spectrum β-lactamases (ESBLs) has been well documented in traveling countries but no data are found for Macao, an international travel city. The objectives of this study were to identify the antimicrobial resistance pattern, and determine the prevalence, genotype and clonal relationship of ESBLs in 209 clinical Escherichia coli strains from Macao, China.

Methods  Antimicrobial susceptibility test was performed to determine the resistance patterns of the isolates using the disk diffusion method with 17 antimicrobial agents. Phenotypic detection was screened and confirmed according to the Clinical and Laboratory Standards Institute. Genotypic characterization was detected by isoelectric focusing analysis, polymerase chain reaction and sequencing. The clonal relationship between the different ESBL isolates was studied by pulsed-field gel electrophoresis (PFGE).

Results  Imipenem and meropenem exhibited 100% susceptible among 209 strains. Overall, 82.3%, 67.3%, 52.9%, 51.2% and 51.0% of the isolates displayed resistance to ampicillin, tetracylcline, ciprofloxacin, sulfamethoxazole trimethoprin and gentamycin. The prevalence rate of ESBLs was 30.1%. Antibiotic resistances were found to be significantly higher among the ESBL producing group compared to non-ESBL producing group. We detected CTX-M-14 to be the major genotypic characterization of ESBLs (76.2%). Two strains showed indistinguishable patterns by PFGE.

Conclusions  The prevalence of antimicrobial resistance is alarming high in Macao. Antimicrobial resistance is significantly higher among the ESBL producing group. This study documented CTX-M-14 as the predominant ESBL type. Although indistinguishable pattern was found between two strains, it was too small to decide whether any of the investigated strains was epidemic. Our findings may be also pertinent for other geographic areas undergoing similar travel characteristics to understand the corresponding effects on bacterial populations.

     

云南某医院多耐药E. coli 和 K. pneumoni1e分子流行病学分析

目的分析云南某医院Eeoli和五pneumonia菌耐药基因分子流行病学特征。方法全自动微生物分析仪分类鉴定菌株及抗生素敏感试验。扩增并序列分析耐药基因。MIST及PFGE分析菌株遗传变异关系。结果共收集23株Ecoli菌和9株Kpneumonia菌,除了1株XDR彪pneumoniae,其余均为MDR菌。全部菌株对一代、二代头孢霉素,甚至三代头孢霉素CRO耐药。绝大多数菌株对氟化奎林酮类、氨基苷类及磺胺类抗生素耐药。39．1％和69．6％Eeo／i携带厶如嘶和厶‰基因,而44．4％和100．O％尼pneumoniae检测到讹。和Mam基因。胁。均为TEM-1基因型。CTX-M-55及CTX-M-15为该地区优势基因型。所有Kpneumonia菌株携带觚Hv基因,SHV-11为优势型。87．5％Ecoli和77．8％Kpneumoniae菌株携带ISEcpl。91．3％Eeoli和77．8％Kpneumoniae携带intl。44．4％尼pneumoniae携带ISCR1基因。PFGE及MIST研究显示菌株存在明显的遗传多态性。结论云南该医院多重耐药Eeoli和Kpneumonia菌呈高度流行,耐药基因在不同菌株及菌种间快速传播。     

国内ICU耐药黄杆菌携带β-内酰胺酶基因的研究

目的探讨ICU临床分离耐药黄杆菌携带β-内酰胺酶基因类型及基因介导方式.方法以市售试剂盒抽提质粒和染色体;纸片法定性检测细菌产β-内酰胺酶情况;PCR扩增法检测黄杆菌携带bla基因类型.结果纸片法显示3株菌均产β-内酰胺酶;PCR扩增显示3株黄杆菌的染色体和质粒分别携带3种和6种类型的酶基因.结论该组ICU耐药黄杆菌携带bla基因具有普遍性和多样性的特点,耐药基因可由质粒、染色体独立介导或两者共同介导.     

Prevalence and characterization of plasmid-mediated blaESBL with their genetic environment in Escherichia coli and Klebsiella pneumoniae in patients with pneumonia

Background  The extended spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are the major pathogens causing pneumonia and have a significant impact on the clinical course. Limited data exist on molecular characterization of ESBL-producing E. coli and K. pneumoniae that cause pneumonia. The aim of this study was to investigate the comprehensive multilevel characteristics of E. coli and K. pneumoniae causing pneumonia in China for the first time.

Methods  E. coli (17) and K. pneumoniae (21) isolates responsible for pneumonia were isolated from 1270 specimens collected in a prospective multi-center study in eight teaching hospitals in China from June to December in 2007. The susceptibilities, ESBL confirmation, sequence typing, bla_CTX-M and bla_SHV genes, their genetic environment and plasmid Inc/rep types were determined.

Results  Sixteen E. coli (94.1%) and eleven K. pneumoniae (52.4%) isolates were ESBL producers. About 77.8% and 66.7% of them were resistance to ciprofloxacin and levofloxacin, and 100% were susceptible to imipenem. The most prevalent ESBL gene was CTX-M-14, followed by SHV-2, CTX-M-15, CTX-M-3, CTX-M-65, SHV-12, SHV-26 and SHV-28. SHV-1 and SHV-11 were also detected and coexisted with bla_CTX-Ms in five strains, and three strains contained only SHV-1. All CTX-M-14 were detected ISEcp1 upstream and nine were found IS903 downstream and the majority of them (64.3%) were carried by IncF plasmids. All bla_SHV were flanked by recF and deoR, located on IncF, IncN, IncX and IncH plasmids. Two SHV-2, one SHV-1 and the only SHV-28 were further preceded by IS26. Genes lacY and lacZ were detected at further upstream of two bla_SHV-1. The K. pneumoniae carrying SHV-28 was susceptible to β-lactams, and no mutations or deletions in gene or promoter sequences were identified to account for susceptibility. Multilocus sequence typing experiments showed the ESBL-producing strains were genetically diverse.

Conclusions  The rate of occurrence of bla_ESBL in E. coli and K. pneumoniae causing pneumonia was high, and bla_CTX-M-14 was dominant and probably mobilized by ISEcp1 mainly on IncF plasmids. Importantly, unexpressed bla_ESBL genes may occur in susceptible isolates and hence may have clinical implications.

     

铜绿假单胞ESBLs基因分布及同源性分析

目的研究温州医学院附属第一医院临床分离铜绿假单胞菌超广谱β-内酰胺酶（EsBL8）产生情况、基因分布及流行特性,为临床合理使用抗菌药物、控制耐药性的传播提供理论依据。方法选取2005—2009年每年6-9月临床连续分离非重复铜绿假单胞菌共169株。PCR方法检测超广谱β-内酰胺酶基因（TEM、SHV、VEB、PER、GES、TLA、IBC、CTX—M-1、CTX—M-2、CTX—M-8、CTX—M-9、OXAⅠ、OXAⅡ、OXAⅢ、OXAⅣ和OXAⅤ）。采用脉冲场凝胶电泳技术（PFGE）分析产超广谱β-内酰胺酶的铜绿假单胞菌株间的同源性。结果169株铜绿假单胞菌,经PCR方法检测,检出产超广谱β-内酰胺酶菌株84株,阳性率为49．7％;169株PA中检出TEM、CTX—M-9、CTX—M-1、CTX—M-2、OXAⅠ、OXAⅣ、GES、OXAⅡ基因型各占32．0％,5．3％,4．7％,2．4％,1．8％,1．8％,1．2％,0．6％。在54株TEM阳性PA中,挑取同一病区不同患者不同标本分离的铜绿假单胞菌共21株,利用PFGE分析其同源性,多呈现散发流行,仅三株具有同源性。结论铜绿假单胞菌超广谱β-内酰胺酶基因型以TEM为主,GES、CTX—M-1、CTX—M-2、CTX—M-9、OXAⅠ、OXAⅡ、OXAⅣ等基因都有检出,未检出PER基因。脉冲场凝胶电泳结果显示,本院临床分离的铜绿假单胞菌大多无同源性,呈散发流行趋势,个别病区存在轻微的克隆播散现象。     

Distribution of TEM and SHV beta-lactamase genes among Klebsiella pneumoniae strains isolated from patients in Tehran.

BACKGROUND: There are numerous types of extended spectrum beta-lactamases (ESBLs), of which TEM and SHV are predominant among Klebsiella pneumoniae strains. Nosocomial infections with strains of K. pneumoniae are common in healthcare centers in Iran. However, no information is available on the prevalence of different phenotypes of ESBL strains in Tehran hospitals. MATERIAL/METHODS: To determine the resistance of K. pneumoniae to beta-lactam antibiotics in Tehran hospitals, 145 isolates were tested using the disk diffusion method. The MICs for ceftazidime were also determined using microbroth dilution assay. Isolates showing MIC >/=4 for ceftazidime were subjected to PCR to target the bla(SHV) and bla(TEM) genes and screened for ESBL production by the phenotypic confirmatory method. RESULTS: All strains were susceptible to imipenem. Resistance to ceftazidime and cefotaxime were 31% and 32%, respectively. Fifty-six isolates showed MIC >/=4 microg/ml for ceftazidime, of which 50 were positive for ESBL in the phenotypic confirmatory test. The prevalence of bla(SHV) and bla(TEM) among these isolates was 69.6% (n=39) and 32.1% (n=18), respectively. Resistance to ciprofloxacin was found among 32% of the ESBL strains. CONCLUSIONS: SHV is the dominant enzyme among the ESBL-producing strains of Klebsiella pneumoniae in Iran. The high rate of co-resistance to ceftazidime and ciprofloxacin is a matter of concern and treatment requires further attention to the results of susceptibility.     

应用DHPLC技术分析浙江沿海地区ESBLs基因类型的研究

目的应用变性高效液相色谱（denaturinghigh—performanceliquidchromatography,DHPLC）技术检测超广谱β-内酰胺酶（extendspectrumβ-lactamases,ESBLs）基因型,了解浙江沿海地区ESBLs基因型分布情况。方法收集171株多重耐药肠杆菌及标准菌株采用多重PCR进行头孢克肟-M型（CTX—M）扩增,扩增产物经DHPLC分析．确定临床菌株基因分型。结果171株多重耐药肠杆菌经表型试验有142株Il缶床菌株ESBLs表型阳性,经多重PCR扩增109株携带CTX—M基因,其中52株为CTX—M-1产物,57株为CTX—M-9产物．检出率达76．8％（109／142）。其中大肠埃希菌携带CTX—M基因比例最高（92．3％）,其次是肺炎克雷伯菌（72．3％）和阴沟肠杆菌（60．0％）。109株临床菌株PCR产物经DHPLC分析检测出4种CTX—M基因型,33株携带CTX—M-3、19株携带CTX—M-15、5株携带CTX—M-9、52株携带CTX—M-14。结论CTX—M型ESBLs菌株检测出4种基因型,CTX—M-3、CTX—M-15、CTX—M-9和CTX—M-14,其中CTX—M-14型是CTX—M型ESBLs主要型别。浙江沿海地区携带CTX—M型ESBLs的细菌以大肠埃希菌、肺炎克雷伯菌、阴沟肠杆菌为多见．