大肠埃希菌和肺炎克雷伯菌质粒中AmpC酶基因型的检测

目的探讨台州地区大肠埃希菌和肺炎克雷伯菌临床分离株质粒介导的AmpC酶基因型流行状况。方法应用VITEK-60型全自动细菌鉴定仪鉴定细菌,按NCCLS推荐的确证试验测定β-内酰胺酶(ESBLs);采用头孢西丁纸片扩散法筛选疑产AmpC酶阳性菌株,并通过酶粗提物头孢西丁三维试验、接合试验和PCR测序等实验分析该菌株的基因型及基因表型。结果299株大肠埃希菌和肺炎克雷伯菌ESBLs检出率为19.73%(59/299),AmpC酶纸片扩散法筛选阳性率为12.04%(36/299),且36株AmpC酶筛选阳性菌株均为ESBLs阳性株;该菌株中有15株经三维试验证实为AmpC酶阳性,阳性率5.02%(15/299);15株产AmpC菌经接合试验得到15株接合子,经PCR及测序证实与原菌株表型基本一致。质粒型AmpC基因中13株为CIT型,2株为DHA型。结论台州地区大肠埃希菌和肺炎克雷伯菌临床分离株中已出现质粒携带的AmpC基因,基因型以CIT型为主,其次是DHA型。AmpC基因可能与编码ESBLs的基因存在于同一质粒上,编码的AmpC酶的质粒可在细菌间传递。     

老年患者大肠埃希菌产质粒AmpC酶基因型特点及耐药性分析

目的了解临床分离老年患者大肠埃希菌产质粒AmpC酶的基因型及其耐药(DR)性特点,为临床合理选用抗生素提供依据。方法收集2009年度老年患者临床不重复分离大肠埃希菌233株,用三维试验和确证试验分别进行羟氨苄青霉素(AmpC)酶和超广谱β内酰胺酶(ES-BLs)检测,用接合试验证实酶基因型的转移,通过PCR检测及序列分析进行质粒AmpC酶基因型确定,采用药敏纸片扩散法(K-B法)进行药敏试验。结果 233株大肠埃希菌中,头孢西丁抑菌环直径≤17mm的有53株,占22.7%,酶提取物三维试验检测7株阳性,4株(1.7%)PCR扩增出CMY-2型质粒AmpC酶,有1株接合试验阳性;ESBLs阳性占68.7%(160/233)。药敏结果显示产质粒AmpC酶大肠埃希菌对二、三代头孢菌素、氨曲南、头孢西丁和含酶抑制剂的DR率明显高于非产酶株(P0.05),所有大肠埃希菌对亚胺培南和美罗培南均100%敏感。结论从老年患者中成功分离出产CMY-2型质粒AmpC酶大肠埃希菌,产质粒AmpC酶菌株对β-内酰胺类抗生素的耐药性明显高于非产酶株。     

耐多药大肠埃希菌AmpC酶基因型及耐药性

目的探讨临床分离耐多药大肠埃希菌(E.coli)产AmpCβ-内酰胺酶(AmpC酶)现状。方法初筛试验及三维试验:检测产AmpC酶菌株;纸片扩散法(K-B法):测定临床分离71株大肠埃希菌对头孢噻肟(CTX)等10种抗菌药物的抗菌活性;PCR技术检测三维实验阳性菌株的AmpC酶基因。阳性扩增片段送上海生工生物技术公司进行基因测序。结果在71株大肠埃希菌中粗筛阳性产AmpC酶菌株共11株,通过头孢西丁三维试验分离出产AmpC酶的菌株8株,占总菌株数11.27%。药敏结果显示对亚胺培南全敏感,对阿米卡星等9种抗菌药物均有不同程度耐药;耐药模式中以耐多药菌株24株(33.80%);产AmpC酶菌株对多种常用抗生素的耐药率明显高于非产AmpC酶菌株(P<0.05);检测发现2株质粒AmpC酶基因阳性。结论大肠埃希菌的多重耐药的现象突出;产AmpC酶菌株的耐药率明显高于非产AmpC酶菌株;质粒介导的AmpC酶的基因型表现为DHA-1。     

产超广谱β内酰胺酶菌株中质粒头孢菌素酶的研究

目的 调查产超广谱β内酰胺酶(ESBLs)的大肠埃希菌和肺炎克雷伯菌中质粒头孢菌素酶(AmpC酶)的发生率及其基因型。方法 收集北京朝阳医院2001年1～12月头孢西丁耐药的产ESBLs的24株大肠埃希菌、8株肺炎克雷伯菌,采用等电聚焦电泳测定β内酰胺酶的等电点;接合试验证实酶基因有无可转移性;脉冲场凝胶电泳(PFGE)确定耐药株的亲缘关系;对AmpC酶基因进行多重PCR及序列分析确定其基因型。结果 2001年北京朝阳医院ESBLs的发生率,大肠埃希菌为16.8％(49／292),肺炎克雷伯菌为160.5％(35／212);ESBLs株中质粒AmpC酶的发生率,大肠埃希菌为2．0％(1／49),肺炎克雷伯菌为17．1％(6／35)。这7株菌均产生DHA-1型AmpC酶;1株肺炎克雷伯菌可将头孢西丁耐药性传给受菌体。该7株菌都产TEM-1酶、5株产CTX-M-3型ESBL、2株产SHV-12型ESBL;该7株菌携带质粒2～5个,且都有约33～36kb的大质粒。PFGE发现这7株菌来自多个不同的克隆株。结论 北京朝阳医院ESBL阳性的大肠埃希菌和肺炎克雷伯菌中,有7株既产DHA-1型质粒AmpC酶,又产CTX-M-3或SHV-12 ESBL。这7株菌来自多个不同的克隆株。     

96株大肠埃希菌耐药性及其产ESBLs、AmpC酶菌株基因型检测

目的 了解大肠埃希菌耐药现状及其及产超广谱β-内酰胺酶（ESBLs）和AmpCβ-内酰胺酶（AmpC酶）菌株基因型,以指导临床医师合理使用抗菌药物。方法自本院尿路感染患者标本中分离大肠埃希菌96株,采用确证试验和改良三维试验检测其ESBLs、AmpC酶表达情况,用K-B法测定其对亚胺培南等16种抗菌药物的敏感性,用PCR法检测TEM、SHV、PER、CTX-M-1群、CTX-M-2群、CTX—M-3群、DHA和ACT-1基因型分布情况,用接合转移试验了解耐药基因转移方式。结果96株大肠埃希菌单产ESBLs30株,同时产ESBLs和AmpC酶4株,单产AmpC酶2株;产ESBLs大肠埃希菌对氨苄西林、头孢唑啉、头孢呋辛、头孢噻肟四种抗生素耐药率达到100％,而对左氧氟沙星、复方新诺明和呋喃妥因的耐药率亦〉80％,对亚胺培南和美罗培南均敏感;ESBLs基因型以CTX—M和TEM型为主,AmpC酶基因为DHA型,两酶能通过接合转移方式将质粒携带的耐药性传递至受体菌。结论大肠埃希菌所致感染对碳青霉烯类药物较为敏感;临床应及时了解本地区产ESBLs、AmpC酶大肠埃希菌的耐药表型和基因型,以指导临床合理使用抗生素、延缓细菌耐药性产生、控制耐药菌株播散和流行。     

大肠埃希菌和肺炎克雷伯菌超广谱β-内酰胺酶检测及药敏结果分析

目的 了解广东东莞地区分离的大肠埃希菌和肺炎克雷伯菌产超广谱β-内酰胺酶(ESBLs)的检测和耐药情况。方法 用确证试验对从临床分离的219株大肠埃希菌和73株肺炎克雷伯菌做ESBLs检测，ATB自动细菌分析仪做药敏试验。结果 219株大肠埃希菌检出产ESBLs94株，检出率为42．9％。73株肺炎克雷伯菌检出产ESBLs30株，检出率为41％。产ESBLs与非产ESBLs菌株的耐药率差异有显著性。结论 本院分出的大肠埃希菌和肺炎克雷伯菌产ESBLs严重，产酶菌株耐药率很高，应引起临床高度重视。     

产质粒介导Ⅰ型头孢菌素酶细菌的耐药性及基因型研究

目的 了解华南地区产β内酰胺酶中质粒介导的Ⅰ型头孢菌素(AmpC)酶细菌的耐药性及其基因型特征。方法 收集革兰阴性菌临床分离无重复菌株共1187株，采用头孢西丁三维试验检测AmpC酶，Kirby-Bauer琼脂扩散法检测耐药性；质粒结合试验，聚合酶链反应(PCR)通用引物扩增各组基因及测序以确定AmpC酶基因型。结果 革兰阴性菌头孢西丁三维试验阳性率为5．9％(70／1187)，其中质粒介导的AmpC β内酰胺酶的检出率为大肠杆菌4．2％(19／451)，克雷伯菌属4．7％(16／339)，肠杆菌属2．1％(4／190)，产碱杆菌属5．3％(1／19)，不动杆菌属2．2％(1／45)，总检出率为3．5％(41／1187)。药敏显示结合子对头霉素和氨苄西林耐药，对头孢吡肟和亚胺培南敏感。PCR扩增和测序结果证实为blaDHA-1基因和blaACT-1。基因，主要分布于克雷伯菌属和大肠杆菌属。结论 华南地区质粒介导AmpC酶的基因型以DHA-1和ACT-1为主。第四代头孢菌素和碳青霉烯类药物可作为治疗产质粒介导的AmpC酶细菌感染的有效药物。     

重症监护病房产AmpC酶大肠埃希菌的检测及耐药特性研究

目的探讨我院重症监护病房中大肠埃希菌产AmpC酶的情况及耐药特性。方法采用头孢西丁三相试验检测大肠埃希菌的产AmpC酶情况,采用纸片扩散法做药敏试验。结果重症监护病房分离的大肠埃希菌产AmpC酶情况比较严重,达到30.8%,耐药情况比较严峻。结论对于重症监护病房分离的大肠埃希菌多数为多重耐药菌株,对产AmpC酶株的大肠埃希菌临床经验用药应首选碳青酶烯类抗生素。     

产超广谱β-内酰胺酶大肠埃希菌和肺炎克雷伯菌的分布及耐药性分析

目的了解医院产超广谱β-内酰胺酶（ESBLs）大肠埃希菌和肺炎克雷伯菌的分布及耐药性特点。方法对我院2004年7月～2006年7月间各类临床标本分离出的大肠埃希菌307株和肺炎克雷伯菌202株，用CLSI／NCCLS推荐的表型确证法检测其ESBLs，采用K-B纸片琼脂扩散法进行药敏试验，分析产ESBLs菌株的分布及耐药性。结果大肠埃希菌和肺炎克雷伯菌产ESBLS菌株的检出率分别为38．4％（118／307）和31．7％（64／202），主要分布于尿和痰、咽拭子中，病区主要集中于肺科、神经外科、感染科、泌尿外科、ICU室。产ESBLs菌株对亚胺培南和美罗培南呈高度敏感，对哌托西林／三唑巴坦、头孢哌酮／舒巴坦、头孢西丁耐药率较低，对其他抗菌药物均出现较高耐药。结论产ESBLS的大肠埃希菌和肺炎克雷伯菌分布在不同病区的不同标本中，碳青霉烯类抗生素亚胺培南和美罗培南是治疗产ESBLS菌株感染的较佳药物。     

耐头孢西丁革兰阴性杆菌高产AmpC酶发生率及其基因型和耐药性

目的了解耐头孢西丁革兰阴性杆菌高产AmpC酶发生率及其基因型分布和耐药性状况。方法收集2004年9月-2005年3月分离自南京军区福州总院、福建省立医院、解放军476医院、福州?第二医院住院患者155株耐头孢西丁无重复革兰阴性杆菌,采用酶提取物三维试验检测AmpC酶;API药敏试验板和K-B法测定高产AmpC酶菌株对抗菌药物的敏感性;质粒转化试验定位耐药基因;PCR通用引物扩增AmpC酶与ESBLs基因及其序列测定,确定其基因亚型。结果在155株菌中有36株高产AmpC酶,高产AmpC酶发生率23.2%,分布在13种菌种中,其中大肠埃希菌、肺炎克雷伯菌、鲍氏不动杆菌和阴沟肠杆菌的发生率,分别为29.3%、13.9%、6/10、2/6。在36株高产AmpC酶菌株中检测出DHA-1型2株(肺炎克雷伯菌)、CMY-2型4株(大肠埃希菌),新发现基因CMY-22型1株(大肠埃希菌,GenBank登录号:DQ256079),5株携带CMY基因的大肠埃希菌分别同时带有TEM-1型广谱酶,TEM-144(新发现基因,GenBank登录号:DQ256080)、CTX-M-27、和CTX-M-14超广谱β-内酰胺酶。高产AmpC酶菌株耐药性严重,且呈多重耐药,但对亚胺培南和美罗培南的敏感率>87%。结论耐头孢西丁革兰阴性杆菌高产AmpC酶发生率较高,菌种分布较宽,耐药性强,治疗相关菌造成的感染应以亚胺培南和美罗培南为首选药物。福州地区临床分离株发现产DHA-1型AmpC酶肺炎克雷伯菌、产CMY-2型和CMY-22型AmpC酶大肠埃希菌。CMY-2型和CTX-M-27型为中国大陆首次报告,CMY-22型和TEM-144型为国内外首次发现。     

同时产DHA-1型头孢菌素酶和SHV-12型超广谱β内酰胺酶的肺炎克雷伯菌

目的研究头孢菌素酶(AmpC)和超广谱β内酰胺酶(ESBLs)在临床分离肺炎克雷伯菌中的流行、表型及其基因特性。方法先后用标准纸片扩散法、三维试验、等电聚焦、酶抑制试验以及微量稀释法等进行表型检测。然后用接合试验、多重聚合酶链反应(PCR)以及基因测序等方法进行分子生物学研究。结果受试的86株细菌中有4株三维试验阳性，等电聚焦以及酶抑制试验表明这些菌株都产一种等电点(PI)为7．8并具有AmpC性质的β内酰胺酶，基因测序表明和DHA01型AmpC酶一致；它们同时伴随产生一种PI为8．2的ESBLs，基因测序表明其来源为SHV-12。微量稀释法检测表明上述菌株不但对多种三代头孢菌素耐药，而且出现了对头孢吡肟耐药，但对碳青霉烯类抗生素仍敏感。结论本研究发现同时产DHA-1型高产AmpC酶和SHV-12型ESBLs的肺炎克雷伯菌及其耐药表型。此类菌株的出现，将为临床抗感染治疗带来新的困难。     

Prevalent phenotypes and antibiotic resistance in Escherichia coli and Klebsiella pneumoniae at an Indian tertiary care hospital: plasmid-mediated cefoxitin resistance.

BACKGROUND: The beta-lactam antibiotics, in combination with aminoglycosides, are among the most widely prescribed antibiotics. However, because of extensive and unnecessary use, resistance to these drugs continues to increase. In recent years, resistance in the Indian bacterial population has increased markedly, the majority showing complex mechanisms. Due to increased transcontinental movement of the human population, it would be wise to know the prevalence and resistance complexity of these strains, well in advance, in order to formulate a policy for empirical therapy. METHODS: One hundred and eighty-one isolates of Escherichia coli and 61 isolates of Klebsiella pneumoniae obtained from 2655 non-repeat samples of pus (912) and urine (1743) were studied, and their resistance rates and patterns were noted. The isolates were analyzed for prevalent aminoglycoside and cephalosporin resistance phenotypes and for the presence of extended spectrum beta-lactamase (ESBL) and AmpC enzymes by spot-inoculation and modified three-dimensional tests developed in our laboratory. Fourteen isolates of E. coli and six of K. pneumoniae, resistant to all of the antibiotics tested, were selected for plasmid screening, curing, and transconjugation experiments, and for comparative evaluation of the double disk synergy test (DDST) and modified three-dimensional test (TDT) for detection of beta-lactamases. RESULTS: Urinary E. coli isolates showed maximum susceptibility to amikacin (57.1%), followed by tobramycin (38.5%) and gentamicin (31.9%). Eighteen (19.8%) isolates were susceptible to cefotaxime, whereas 11 (12.1%) were susceptible to ceftriaxone. The K. pneumoniae isolates from urine samples showed maximum susceptibility to tobramycin (63.6%) followed by amikacin (54.5%). Of the K. pneumoniae isolates, 31.8% were susceptible to cefotaxime and 13.6% were susceptible to ceftriaxone. A more or less similar trend of antibiotic susceptibility was noted in E. coli and K. pneumoniae isolates from pus samples. Twenty-six (14.4%) E. coli and 15 (24.6%) K. pneumoniae isolates were found to be ESBL-producers by NCCLS-ESBL phenotypic confirmatory test. Eighteen (9.9%) E. coli and 19 (31.1%) K. pneumoniae isolates were found to be AmpC enzyme-producers by our modified TDT. The simultaneous occurrence of ESBL and AmpC enzymes was noted in 7.7% and 9.8% isolates of E. coli and K. pneumoniae, respectively. CONCLUSIONS: The prevalence of multidrug-resistant bacterial isolates is quite high in our bacterial population. On comparative evaluation of DDST and TDT in resistant isolates, TDT was found to be the better method, detecting ESBLs in 80% of isolates compared to 15% with DDST. A 19.9-kb plasmid was consistently present in all the screened isolates of E. coli and K. pneumoniae, and was inferred to encode cefoxitin and tetracycline resistance based on curing and transconjugation experiments.     

Surveillance of nalidixic acid-resistant and extended-spectrum beta-lactamase-producing Salmonella spp. isolated from human feces

Nalidixic acid (NA)-resistant and extended-spectrum beta-lactamase (ESBL)-producing Salmonella sp. isolates from human specimens are associated with clinical failure or delayed response in subjects treated with fluoroquinolone or third-generation cephalosporins. We studied drug susceptibility in 604 Salmonella enterica isolates from human feces in 2007. Of these, 39 (6.5%) were resistat to NA. Of these, 46% were resistant to two or more drugs and 2% susceptible to NA were resistant to multiple drugs (p < 0.001). Three ESBL-producing Salmonella sp. isolated were of the CTX-M family gene type. One strain of plasmid-mediated AmpC beta-lactamase belonged to the CMY-2 family gene type. Our results thus showed that NA-resistant isolates were resistant to antimicrobial agents and confirmed the presence of a small number of isolates producing ESBL and AmpC beta-lactamase.     

产金属β-内酰胺酶铜绿假单胞菌的基因检测及耐药性研究

目的　研究本地区铜绿假单胞菌临床分离株中产金属 β -内酰胺酶流行株的主要基因型和耐药性。 方法　用纸片扩散法进行抗生素协同敏感试验 ,检测产金属 β -内酰胺酶的铜绿假单胞菌 ,用琼脂稀释法检测其最低抑菌浓度 (MIC) ,分别用金属酶IMP - 1,IMP - 2 ,VIM - 1和VIM - 2引物进行聚合酶链反应 (PCR)并对扩增产物进行序列分析。结果　在 82株耐亚胺培南铜绿假单胞菌中 ,抗生素协同敏感试验阳性 7株 ,PCR检测金属 β -内酰胺酶阳性株也为这 7株 ,且基因型均为VIM - 2型。产酶株对亚胺培南、头孢噻肟、头孢他啶、哌拉西林 /他唑巴坦等常用抗铜绿假单胞菌 β -内酰胺类抗生素不敏感 ,对氨曲南较为敏感。结论　本地区铜绿假单胞菌所产金属 β-内酰胺酶为VIM - 2型 ,且产酶株耐药性强 ,应注意对其进行临床检测和监控。     

Simultaneous presence of blaTEM and blaSHV genes on a large conjugative plasmid carried by extended-spectrum beta-lactamase-producing Klebsiella pneumoniae

BACKGROUND: Among members of the family Enterobacteriaceae, the production of plasmid-mediated extended-spectrum beta-lactamases (ESbetaLs) has emerged as an important mechanism of the resistance to beta-lactams. METHODS: The molecular basis of extended-spectrum beta-lactamase in 48 strains of Klebsiella pneumoniae, recovered from neonatal patients with nosocomial septicemia during an outbreak that occurred in November 2000 at a Neonatal Intensive Care Unit of The Andes University Hospital in Venezuela, were investigated. RESULTS: The isolates were resistant to expanded-spectrum cephalosporins, aztreonam, gentamicin, kanamycin, tetracycline, and chloramphenicol, but remained susceptible to cefoxitin, imipenem, amikacin, and tobramycin. Production of ESbetaL activity was confirmed by restoring susceptibility to ceftazidime in the presence of clavulanic acid. All isolates harbored an 87-kilobase plasmid. Analysis of the outer membrane protein patterns did not reveal relevant changes of the porins profile. All resistance markers were transferable to Escherichia coli by conjugation and were lost en bloc after treatment with acridine orange. Isoelectric focusing for beta-lactamases was performed on transconjugants, obtaining 2 bands with isoelectric points of 5.4 and 8.2. Genes encoding both enzymes are located on the single, self-transferable 87-kilobase plasmid pKAM542. Analysis of the plasmid by hybridization revealed the presence of both blaTEM and blaSHV determinants. Cloning and sequencing identified them as blaTEM-1 and blaSHV-5, respectively; the latter was responsible for the ESbetaL activity among nosocomial isolates of K pneumoniae. CONCLUSIONS: Microbiologists, epidemiologists, and clinicians must be aware of potential ESbetaL-encoding organisms to assess proper antimicrobial managements and effective infection controls.     

Klebsiella pneumoniae and other Enterobacteriaceae producing novel plasmid-mediated beta-lactamases markedly active against third-generation cephalosporins: epidemiologic studies

Analysis of the enzymes produced by clinical isolates of multiresistant Klebsiella pneumoniae from hospitals in France revealed two novel broad-spectrum beta-lactamases. The first, characterized by an isoelectric point of 6.3 and a high hydrolytic activity on cefotaxime, is designated CTX-1. This beta-lactamase was encoded by a 95-kilobase plasmid (incompatibility group 7M) and cotransferred with resistance to tetracyclines, sulfonamides, and aminoglycosides (AAC [6']-IV). From 1984 to June 1987, 490 CTX-1-producing strains of Enterobacteriaceae were isolated. The second plasmid-mediated beta-lactamase (CAZ-1) was isolated in 1987 from three K. pneumoniae strains more resistant to ceftazidime than to other third-generation cephalosporins. This broad-spectrum beta-lactamase differed from CTX-1 by its isoelectric point--close to 5.6--and its high hydrolytic activity on ceftazidime and was encoded by a 150-kilobase plasmid. It was demonstrated that these expanded-spectrum beta-lactamases are TEM derivatives.     

Conjugative resistance to tazobactam plus piperacillin among extended-spectrum beta-lactamase-producing nosocomial Klebsiella pneumoniae

We studied the genetic origins of piperacillin-tazobactam resistance among nosocomial Klebsiella pneumoniae strains. A total of 30 nosocomial isolates resistant to piperacillin-tazobactam were obtained from various regions of Turkey. Isoelectric focusing demonstrated at least 2 enzymes common to all strains: I at a pI of 8.0 and the other at 5.4. Piperacillin-tazobactam resistance was successfully transferred from all of the strains to Escherichia coli. Of the piperacillin-tazobactam-resistant transconjugates, 23 were also resistant to ceftazidime. However, 7 transconjugates were susceptible to ceftazidime but resistant to piperacillin-tazobactam, producing a single enzyme focusing at pI 5.4. Piperacillin resistance caused by this enzyme was reversed by clavulanate and by increased amounts of tazobactam, which indicates that this enzyme confers resistance due to its high amount. Sequence analysis revealed this enzyme to be TEM-1. This study demonstrates that transferable hyper-produced TEM-1 causes piperacillin-tazobactam resistance in Klebsiella strains in Turkish hospitals.