产超广谱β-内酰胺酶的肺炎克雷伯菌相关耐药基因研究

目的了解常州地区肺炎克雷伯菌耐药状况及β-内酰胺类耐药基因。方法用琼脂稀释法检测氨苄西林、哌拉西林、哌拉西林-他唑巴坦、头孢噻肟、头孢他啶、头孢吡肟、氨曲南和亚胺培南对肺炎克雷伯菌的最低抑菌浓度(MIC)。采用聚合酶链反应(PCR)检测产超广谱β-内酰胺酶(ESBLs)肺炎克雷伯菌16种相关耐药基因,并用DNA测序仪测序。结果120株肺炎克雷伯菌ESBLs阳性率为44.2%(53株)。从53株肺炎克雷伯菌中检出TEM、SHV、CTX-M-1群、CTX-M-9群、OXA-1群、LEN、OKP和DHA 8种β-内酰胺酶基因,阳性率分别为75.5%、22.6%、18.9%、7.5%、11.3%、5.7%、3.8%和41.5%,其中2株菌同时检出6种β-内酰胺酶基因。基因测序证实为TEM-1、TEM-11、SHV-13、SHV-28、CTX-M-22、CTX-M-55、OXA-1和OKP-6等。结论本地区肺炎克雷伯菌已携带多种β-内酰胺酶耐药基因,是其对β-内酰胺类抗菌药物耐药的重要原因之一。     

肺炎克雷伯菌中超广谱β-内酰胺酶基因分布及转移研究

目的 研究肺炎克雷伯菌中超广谱β-内酰胺酶（ESBLs）基因类型及转移方式。方法 回顾性分析该院2011年1月至2013年1月收集的460例住院患者菌株的临床资料,并对其ESBLs基因进行分型和研究耐药性。结果 460株β-内酰胺酶的KPN检出6种β-内酰胺酶基因,其中bla TEM占总数的5％,bla SHV占总数的20％,bla CTX—M-1群占总数的5％,bla CTXM-9群占总数的25%,bla OXA-1群占总数的10％,bla DHA占总数的30％等;本研究共发现322种菌株耐药谱,菌株耐药在9种抗菌药物以上。阿莫西林／替卡西林／头孢噻吩等、阿莫西林／克拉维酸和头孢西丁、哌拉西林／克拉维酸、亚胺培南的耐药性分别为100％、60％、45％、35％和0。结论 肺炎克雷伯菌中ESBLs基因类型主要是bla TEM和bla CTX—M-1,并具有较为严重的耐药性。     

大肠埃希菌和肺炎克雷伯菌超广谱β-内酰胺酶的基因型与临床的关联

产超广谱和耐酶抑制剂β-内酰胺酶是革兰阴性菌对β-内酰胺类抗生素耐药的重要原因。近年来由于超广谱β-内酰胺抗生素在临床的大量应用，致使革兰阴性杆菌产生超广谱β-内酰胺酶ESBLs，ESBLs按编码基因同源性的不同主要分4型，即TEM型、SHV型、CTX—M型、和OXA型，而我国主要以CTX—M型为主，包括CTX—M-14-3922、27、24、28、29、13型等，其次为SHV型。在产ESBLs大肠埃希菌和肺炎克雷伯菌的ESBLs基因型中以CTX—M-14型和CTX—M-3型最常见，有的菌甚至产2种至3种酶型，造成临床多重耐药。因此，掌握它们的分型、分类、构效关系和耐药模式等方面的知识，对临床合理使用抗生素和防止耐药菌的产生具有重要的意义。     

医院感染铜绿假单胞菌产超广谱β-内酰胺酶基因型研究

目的研究医院感染铜绿假单胞菌产超广谱β-内酰胺酶（ESBLs）的基因型分布。方法用聚合酶链反应（PCR）对68株医院感染铜绿假单胞菌的SHV、TEM、CTX-M、OXA-10、VEB和PER等基因进行扩增和序列测定。结果68株医院感染铜绿假单胞菌中,检出26株产OXA-10群ESBLs,检出率为38．23％（26／68）;其中1株既产OXA-10群ESBLs又产TEM型广谱β-内酰胺酶;其余基因型未检出。经序列测定为OXA-10-like基因和TEM-1基因。结论医院感染铜绿假单胞菌所产的ESBLs主要为OXA-10群,且为多重耐药菌株。     

与ISEcp1—like插入序列相关的CTX—M型ESBLs基因传播机制研究

目的研究CTX—M—ESBLs在天津地区肺炎克雷伯菌中的传播与ISEcp1—like插入序列及1类整合子的关系。方法采用PCR-mapping、Southern印迹杂交及DNA序列分析等方法检测CTX-M-ESBLs基因，及其与ISEcp1—like插入序列和1类整合子的关系。结果46株产ESBLs肺炎克雷伯菌中44株（95．7％）携带CTX—M-1组和（或）CTX—M-9组基因，未发现CTX—M-2组基因。60％的CTX—M-1组β内酰胺酶基因和73．7％的CTX-M-9组β内酰胺酶基因上游存在ISEcp1—like插入序列。ISEcp1—like插入序列与CTX—M-3及CTX-M-14基因间隔区序列不同。46株中25株（54．3％）携带1类整合子基因，PCR—mapping及Southern印迹杂交未发现CTX—MB内酰胺酶基因在整合子上的证据。结论天津地区肺炎克雷伯菌临床株中存在CTX—M-ESBLs的流行，许多CTX—M—ESBLs基因上游存在ISEcp1—like插入序列。ISEcp1—like插入序列可能与CTX—M型超广谱酶基因在天津市肺炎克雷伯菌临床株中的播散有关。     

产超广谱β内酰胺酶大肠埃希菌与肺炎克雷伯菌的基因型和耐药性分析

目的研究我院住院患儿分离产ESBLs大肠埃希菌及肺炎克雷伯菌的基因型及其相关耐药性分析。方法收集2004年10月-2005年10月我院住院患儿呼吸道分离产ESBLs大肠埃希菌75株，肺炎克雷伯菌45株，PCR限制性片段长度多态性（RFLP）分析及PCR产物克隆测序等方法明确ESBLs基因型，并结合体外抗生素敏感试验研究基因分型与细菌对不同头孢菌素及氨曲南的敏感性的关系。结果上述120株细菌中112株（93．3％）产CTX—M型p内酰胺酶；未能扩增出SHV型、TEM型ESBL基因。CTX—M型基因亚型主要为CTX—M9簇中的CTX—M-14型（78、6％），其次为CTX—M-1簇中的CTX—M-3型（19．6％），仅有1．8％为CTX—M-8型；携带CTX-M-1簇的菌株对头孢他啶、头孢哌酮-舒巴坦、阿莫西林-克拉维酸、头孢吡肟及氨曲南的耐药率均明显高于CTX—M-9型。结论CTX—M基因型为武汉地区儿童分离大肠埃希菌以及肺炎克雷伯菌中最常见的ESBLs，CTX—M-14为最常见基因型，其次为CTX—M-3型；CTX—M-9簇及CTX-M-1簇对头孢他啶、头孢吡肟、头孢哌酮-舒巴坦和氨曲南的水解能力差异明显。     

产超广谱β-内酰胺酶肺炎克雷伯杆菌的基因型分型及耐药分析

目的：了解大连市中心医院产超广谱β-内酰胺酶（ESBLs）肺炎克雷伯杆菌耐药基因型及耐药情况。方法：采用，涵床实验室标准化委员会（CLSI）推荐的表型确认方法，筛选出该院2005年11月至2006年2月产ESBLs肺炎克雷伯杆菌51株。应用K-B纸片扩散法检测其对23种临床常用抗生素的耐药性；应用聚合酶链反应-毛细管电泳方法（PCR—CE）检测产ESBLs肺炎克雷伯杆菌的TEM、SHV、CTX．M基因。结果：51株产ESBLs肺炎克雷伯杆菌中．有50株呈阳性。仅携带一种基因型的分别为TEM型1株（1．96％），SHV型1株（1．96％），CTX—M．I型9株（17． 65％），CTX—M—II型8株（15．69％），CTX-M-Ⅲ型2株（3．92％），CTX—M—IV型6株（11．76％）。有23株（45． 10％）同时携带两种或两种以上基因型，且多为CTX-M型与TEM型或SHV型共同存在。药敏结果显示，碳青酶烯类抗生素对产ESBIs肺炎克雷伯杆菌表现出较高的抗菌活性，其耐药率为0．00％，四环素和复方新诺明耐药率分别为19．61％和27．45％一结论：本院产ESBLs肺炎克雷伯杆菌流行的主要耐药基因型为CTX—M型．碳青霉烯类抗生素是目前治疗产ESBLs肺炎克雷伯杆菌感染最有效的药物。[著者文摘]     

碳青霉烯类耐药肺炎克雷伯菌β-内酰胺酶检测和基因分型

摘要：目的：探讨碳青霉烯类耐药肺炎克雷伯菌产β-内酰胺酶情况和β-内酰胺酶基因分型研究。 方法：用Vitek-Ⅱ全自动微生物分析仪对本院临床标本中分离的对碳青霉烯类耐药的肺炎克雷伯菌进行菌种鉴定和药物敏感试验;用冻融法提取β-内酰胺酶,三维试验检测β-内酰胺酶［AmpC酶、超广谱β-内酰胺酶（ESBLs）、金属酶;改良Hodge试验检测KPC酶;用PCR扩增TEM、SHV、CTX-M、PER、VEB、DHA、MIR/ACT、KPC、IMP、VIM、SPM、GIM和NDM-1基因,并对阳性基因进行测序分析确定其基因型;REP-PCR检测分析其同源性。 结果: 4株对碳青霉烯类抗生素耐药肺炎克雷伯菌均表现为多重耐药,其中2株菌对所有测试的抗菌药物均耐药;1株菌产金属酶,由IMP-4型金属酶基因编码;4株菌均产生DHA型AmpC酶,2株菌产CTX-M-14型ESBLs;1株菌产TEM-71型ESBLs,均经测序证实;未检测出SHV、PER、VEB、MIR/ACT、KPC、VIM、SPM、GIM和NDM-1基因型。REP-PCR显示4株菌属于3个不同的克隆型。 结论：本院出现碳青霉烯类耐药的肺炎克雷伯菌,属于不同克隆型,产多种β-内酰胺酶（AmpC酶、ESBLs、金属酶）,基因型为DHA、IMP-4、CTX-M-14、TEM-71。     

大肠埃希菌和肺炎克雷伯菌产ESBLs株的耐药性与基因检测

目的研究深圳市人民医院2002年1月-2003年12月临床下呼吸道分离的产ESBLs大肠埃希菌和肺炎克雷伯菌的耐药性、ESBLs基因型以及基因型和耐药表型的相关性。方法共收集临床下呼吸道分离产ESBLs大肠埃希菌和肺炎克雷伯菌61株，采用酶抑制剂增强法作表型确证试验，琼脂稀释法作药敏试验，应用PCR、序列分析，确定ESBLs基因型。结果61株产ESBLs大肠埃希菌和肺炎克雷伯菌对亚胺培南的敏感率是100％，MIC50 0．5mg／L，两种细菌表现出不尽相同的耐药表型。共检出10种0内酰胺酶基因型，CTX-M型、SHV型和TEM型的检出率依次为83．6％、44．3％和37．7％，其中以CTXM14最多，共38株；SHV型仅见于肺炎克雷伯菌，SHV-12最常见，有20株；TEM型均为TEM-1。结论本院临床下呼吸道产ESBLs大肠埃希菌和肺炎克雷伯菌耐药情况严重，为多重耐药菌。ESBLs有7种基因型，CTX—M型是主要的基因型，其次是SHV型；两种菌中各基因型的分布不同，其耐药表型也不同。     

多重耐药肺炎克雷伯菌基因型和整合子的分析

目的了解多重耐药肺炎克雷伯菌的基因型和整合子存在情况。方法设计TEM、SHV和CTX-M型及Ⅰ、Ⅱ、Ⅲ类整合酶基因特异性引物,并将聚合酶链反应（PCR）扩增获得哥内酰胺酶编码基因,分别克隆入pMD18-T载体后测定其核苷酸序列,分析其基因亚型。结果该多重耐药株与TEM-1、SHV-12及CTX—M-3型编码基因序列的同源性为100％,Ⅰ类整合酶基因检测阳性,Ⅱ、Ⅲ类整合酶基因检测阴性。结论Ⅰ类整合子存在产ESBLs肺炎克雷伯菌中。     

Genes Encoding TEM-4, SHV-2, and CTX-M-10 Extended-Spectrum β-Lactamases Are Carried by Multiple Klebsiella pneumoniae Clones in a Single Hospital (Madrid, 1989 to 2000)

Over a 12-year period (1989 to 2000), 159 Klebsiella pneumoniae isolates harboring extended-spectrum beta-lactamases (ESBLs) (4.8% of the total number of K. pneumoniae isolates obtained) were recovered from 58 patients, who were mainly hospitalized in intensive care and surgery units. For 62 representative isolates from 58 patients, 31 clonal types harboring TEM-4 (n = 5), SHV-2 (n = 7), SHV2a (n = 4), SHV-5 (n = 1), CTX-M-10 (n = 13), or CTX-M-9 (n = 1) beta-lactamases were identified by pulsed-field gel electrophoresis. This is the first report to document the presence of the CTX-M-10 or the CTX-M-9 beta-lactamase in K. pneumoniae. These beta-lactamases were previously identified in Escherichia coli isolates from Spain. Only two of five K. pneumoniae TEM-4 clones caused more than a single case of infection, with one of them spreading for 9 months. A single plasmid was detected among these TEM-4 clones. Only two of seven K. pneumoniae clones containing SHV-2 and three of four strains harboring SHV-2a were detected in more than one case of infection. Plasmids encoding SHV-2 or SHV-2a were unrelated. Four of 13 K. pneumoniae CTX-M-10 clones were found in more than one patient, with two of them recovered 2 and 5 years apart. As in the case of the SHV-2 isolates, we were unable to document a common transmissible genetic element that could explain the polyclonal structure of our isolates. Nevertheless, the spread of a single gene may be suggested by the presence of a conserved set of noncoding polymorphisms in the sequences. Most ESBL-producing K. pneumoniae clones were ephemeral, being poorly selected and maintained in the hospital setting, but the genes encoding ESBL persisted successfully over the years that the strains were recovered, probably as a minority gene population in the hospital metagenome.     

Frequency and diversity of Class A extended-spectrum beta-lactamases in hospitals of the Auvergne, France: a 2 year prospective study

OBJECTIVES: To evaluate the frequency and diversity of extended-spectrum beta-lactamases (ESBLs) produced by Enterobacteriaceae and Pseudomonas aeruginosa in one French region. METHODS: During 2001-2002, all the non-duplicate isolates of P. aeruginosa resistant to ceftazidime and of Enterobacteriaceae intermediate or resistant to ceftazidime and/or cefotaxime and/or aminoglycosides with an AAC(6') I phenotype were collected in nine hospitals of the area. ESBL isoelectric points were determined, bla genes were amplified and sequenced and epidemic isolates were genotyped with ERIC2-PCR. RESULTS: ESBLs were observed in 297 Enterobacteriaceae (0.8%). The most frequent were TEM-3 like (n=152; 51.2%) and TEM-24 (n=115; 38.7%). Four new enzymes were observed, TEM-112 (pI 5.4), TEM-113 (pI 6.3), TEM-114 (pI 5.9) and TEM-126 (pI 5.4). Other TEMs were TEM-8, TEM-12, TEM-16, TEM-19, TEM-20, TEM-21, TEM-29 and TEM-71. The other ESBLs were SHV-4, SHV-5 and SHV-12, CTX-M-1, CTX-M-3, CTX-M-14 and CTX-M-15. In 37 P. aeruginosa (0.7%) only one ESBL was observed, PER-1. Five epidemic strains were detected, Serratia marcescens TEM-3 and four observed in several hospitals, Enterobacter aerogenes TEM-24, Citrobacter koseri TEM-3, Proteus mirabilis TEM-3 and P. aeruginosa PER-1. CONCLUSION: ESBL frequency was lower than in 1998, and CTX-M-type frequency higher (2.1% of ESBLs in 2001, 4.9% in 2002). This long-term survey detected new sporadic enzymes (TEM-112, TEM-113, TEM-114 and TEM-126) and interhospital epidemic strains while avoiding any overestimation of ESBL frequency that may otherwise have occurred because of acute epidemics.     

Extended-spectrum beta-lactamases in Klebsiella pneumoniae bloodstream isolates from seven countries: dominance and widespread prevalence of SHV- and CTX-M-type beta-lactamases

A huge variety of extended-spectrum beta-lactamases (ESBLs) have been detected during the last 20 years. The majority of these have been of the TEM or SHV lineage. We have assessed ESBLs occurring among a collection of 455 bloodstream isolates of Klebsiella pneumoniae, collected from 12 hospitals in seven countries. Multiple beta-lactamases were produced by isolates with phenotypic evidence of ESBL production (mean of 2.7 beta-lactamases per isolate; range, 1 to 5). SHV-type ESBLs were the most common ESBL, occurring in 67.1% (49 of 73) of isolates with phenotypic evidence of ESBL production. In contrast, TEM-type ESBLs (TEM-10 type, -12 type, -26 type, and -63 type) were found in just 16.4% (12 of 73) of isolates. The finding of TEM-10 type and TEM-12 type represents the first detection of a TEM-type ESBL in South America. PER (for Pseudomonas extended resistance)-type beta-lactamases were detected in five of the nine isolates from Turkey and were found with SHV-2-type and SHV-5-type ESBLs in two of the isolates. CTX-M-type ESBLs (bla(CTX-M-2) type and bla(CTX-M-3) type) were found in 23.3% (17 of 73) of isolates and were found in all study countries except for the United States. We also detected CTX-M-type ESBLs in four countries where they have previously not been described-Australia, Belgium, Turkey, and South Africa. The widespread emergence and proliferation of CTX-M-type ESBLs is particularly noteworthy and may have important implications for clinical microbiology laboratories and for physicians treating patients with serious K. pneumoniae infections.     

High diversity of extended-spectrum beta-lactamases among clinical isolates of Enterobacteriaceae from Portugal

OBJECTIVES: To investigate the occurrence and the diversity of Ambler class A ESBLs among Enterobacteriaceae from different Portuguese clinical settings over a 2 year period (2002-04). METHODS: One hundred and nine extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae isolates from five geographically distant health institutions in Portugal were studied. ESBLs were characterized by isoelectric focussing, PCR and further sequencing. Antibiotic susceptibility testing, transfer of resistance genes and clonal diversity were determined by standard procedures. Plasmid relatedness was established by comparison of random amplified polymorphic DNA (RAPD) patterns. RESULTS: ESBLs were identified as TEM (46%), SHV (30%), CTX-M (22%) and GES (2%) types; TEM-24, TEM-52, SHV-12 and CTX-M-15 enzymes being the most frequently found. Inter-hospital dissemination of epidemic strains harbouring the most prevalent ESBLs was detected, including the TEM-24-producing Enterobacter aerogenes European epidemic clone. Conjugative transfer of ESBLs was achieved for 67% of isolates and epidemic plasmids containing specific bla genes were detected (bla(CTX-M-15) and bla(TEM-24)). We describe two new ESBLs, SHV-90 (A187T, G238S and E240K) and SHV-91 (P20S and E240K), and a new TEM-type enzyme conferring a phenotype resembling that of a complex mutant TEM beta-lactamase, designated as TEM-154 (M69L and R164S). The broad-spectrum beta-lactamases SHV-26, SHV-36 and TEM-110 were first observed in our country. CONCLUSIONS: We describe a complex ESBL epidemiology in Portugal, including widespread dissemination of known strains and plasmids coding for TEM-24 and CTX-M-15 enzymes as observed in other European countries.     

Extended-spectrum beta-lactamase-producing Enterobacteriaceae in community and private health care centers

In 1999, 39 of 2,599 isolates of the family Enterobacteriaceae (1.5%) collected by eight private laboratories in the Aquitaine region in France produced an extended-spectrum beta-lactamase (ESBL). Among these were 19 Enterobacter aerogenes isolates; 8 Klebsiella pneumoniae isolates; 6 Escherichia coli isolates; 3 Proteus mirabilis isolates; and 1 isolate each of Serratia marcescens, Morganella morganii, and Providencia stuartii. ESBL producers were isolated from 38 patients, including 33 residents of 11 clinics or nursing homes and 5 ambulatory patients. Seven different ESBLs were characterized. These mainly consisted of TEM-24 (25 isolates) and TEM-21 (9 isolates), but TEM-15 (2 isolates) and TEM-3, TEM-19, SHV-4, and CTX-M-1 (1 isolate each) were also characterized. Seven strains showed the coexistence of different TEM- and/or SHV-encoding genes, including a new SHV-1 variant, SHV-44, defined by the substitution R205L previously reported for SHV-3 in association with S238G. The epidemiology of the ESBL producers was investigated by random amplification of polymorphic DNA, typing by enterobacterial repetitive intergenic consensus PCR, analysis of resistance cotransferred with the ESBL, and analysis of the restriction profiles of the ESBL-encoding plasmids. Of the TEM-24-expressing strains, 18 were E. aerogenes isolates, including 9 from the same clinic, that were representatives of the epidemic clone disseminating in France. Of the TEM-21-producing strains that belonged to different species of the family Enterobacteriaceae (E. coli, K. pneumoniae, and P. mirabilis), 8 were isolated in the same nursing home. Outbreaks due to strain and/or plasmid dissemination in these clinic and nursing home were demonstrated. The presence of ESBL producers in five ambulatory patients probably resulted from nosocomial acquisition. Our data highlight the serious need to monitor patients for ESBL-producing Enterobacteriaceae in general practice.     

Distribution of extended-spectrum beta-lactamases in clinical isolates of Enterobacteriaceae in Vietnam

Among 730 Escherichia coli, 438 Klebsiella pneumoniae, and 141 Proteus mirabilis isolates obtained between September 2000 and September 2001 in seven hospitals in Ho Chi Minh City, Vietnam, 26.6% were resistant to ceftazidime, 30% were resistant to cefotaxime, 31.5% were resistant to ceftriaxone, 15.9% were resistant to cefoperazone, and 6% were resistant to cefepime. Resistance to imipenem was found in 5.6% of the isolates. In 55 strains producing extended-spectrum beta-lactamases (32 E. coli isolates, 13 K. pneumoniae isolates, and 10 P. mirabilis isolates), structural genes for VEB-1 (25.5%), CTX-M (25.5%), SHV (38.1%), and TEM (76.3%) enzymes were detected alone or in combination. Sequencing of the PCR products obtained from the K. pneumoniae isolates revealed the presence of bla(VEB-1), bla(CTX-M-14), bla(CTX-M-17), bla(SHV-2), and bla(TEM-1). Molecular typing of the strains with a similar resistance phenotype to broad-spectrum cephalosporins indicated polyclonal spread. ISEcp1 was presumably responsible for dissemination of the bla(CTX-M-like) gene.     

浙江省台州地区不同来源的大肠埃希菌耐药性分析及产超广谱-内酰胺酶、头孢菌素酶的基因分型

目的 了解浙江省台州地区的分散式供水、食品从业人员粪便及临床标本分离的大肠埃希菌耐药状态、产超广谱-内酰胺酶(ESBLs) 和头孢菌素酶(AmpC)情况及相关耐药基因的类型。 方法 从分散式供水标本、食品从业人员肛拭及临床标本中分离培养的各100株大肠埃希菌进行药敏试验,用ESBLs确证试验、AmpC酶检测试验检测实验菌株的表型,以 PCR扩增和序列分析检测相关的耐药基因。 结果 从食品从业人员肛拭分离到的菌株耐药性高于分散式供水中分离的菌株,而临床标本分离到的菌株耐药性更高;从100株集中式供水分离的菌株中检测出产ESBLs菌6株,基因型为TEM-1合并CTX-M-9。100株食品从业相关人员肛拭分离的菌株中检测出产ESBLs 17株,基因型为TEM-1合并CTX-M-9,CTX-M-9;检出产AmpC酶3株,基因型为MOX-1、DHA-1。100株临床标本中分离出产ESBLs 33株,基因型为TEM-1合并CTX-M-9、CTX-M-9、CTX-M-25;检出产AmpC酶9株,基因型为MOX-1、DHA-1、FOX-1。 结论 在分散式供水及正常食品从业相关人员粪便中检出产ESBLs、AmpC酶的大肠埃希菌与在临床分离的标本中的产ESBLs、AmpC酶的大肠埃希菌携带的基因型有一定的相似性。