一株产碳青霉烯酶IMP-26阴沟肠杆菌的分离鉴定

在过去的几年中,产碳青霉烯酶的肠杆菌科细菌不断被发现,这引起了临床工作者的强烈担忧.我院发现一株碳青霉烯类抗菌药物耐药的阴沟肠杆菌,此临床菌株分离自一位9个月的哮喘患儿,对亚胺培南和厄他培南表现为耐药,其最低抑菌浓度分别为4 μg/ml和16μg／ml.对青霉素类,头孢类及头孢与酶的复合制剂都表现为耐药.进一步的实验显示此菌株的Hodge试验阳性,确定该菌株产碳青霉烯酶.通过对亚胺培南与亚胺培南+EDTA的纸片的抑菌圈直径的比较,发现此种碳青霉烯酶能被EDTA抑制.通过PCR扩增,发现此阴沟肠杆菌含有blaInt11、blaIMP和blaTEM基因,通过测序和比对确定blaIMr的基因型为blaIMP-26,balTEM基因的基因型为blaTEM-104.通过等电聚焦电泳,也证实此阴沟肠杆菌产IMP和TEM酶.     

大肠埃希菌对碳青霉烯类药物的耐药机制研究

目的 研究我院普外科重症监护病房(ICU)出现的对碳青霉烯类抗菌药物耐药的大肠埃希菌分子流行病学特征和碳青霉烯耐药机制.方法 采用琼脂稀释法检测分离株的抗菌药物敏感性,采用脉冲场琼脂糖凝胶电泳(PFGE)研究碳青霉烯类耐药的大肠埃希菌分子流行病学机制,采用特异性PCR、DNA测序分析、接合试验、质粒提取和质粒转化试验、外膜蛋白分析等技术研究碳青霉烯耐药的分子机制.结果 我院分离的14株碳青霉烯耐药菌分别属于10个流行克隆型,均表现对包括亚胺培南和美罗培南在内多种抗菌药物耐药,碳青霉烯类耐药基因扩增显示均携带KPC-2型碳青霉烯酶基因,质粒提取与转化试验显示KPC-2定位于约56 kb大小的质粒上,SDS-PAGE分析发现耐药株多出现外膜蛋白的改变.结论 我院出现多个流行克隆型的碳青霉烯类耐药的大肠埃希菌,质粒型碳青霉烯酶KPC-2是我院泛耐型大肠埃希菌介导对碳青霉烯类耐药的主要原因,外膜孔蛋白改变参与介导大肠埃希菌对碳青霉烯类高度耐药.     

同时产ArmA型16S rRNA甲基化酶及KPC-2型β-内酰胺酶泛耐药阴沟肠杆菌的研究

目的 探讨上海交通大学医学院附属瑞金医院分离的5株泛耐药阴沟肠杆菌产碳青霉烯酶及16S rRNA甲基化酶的情况及两者之间的相关性.方法 用E test法检测5株泛耐药阴沟肠杆菌对10种抗菌药物的MIC值.PCR扩增16S rRNA甲基化酶基因armA、mtA、rmtB、rmtC、rmtD、npmA,β-内酰胺酶基因TEM、SHV、CTX-M-1、CTX-M-2、CTX-M-8、CTX-M-9、CTX-M-25、PER-1、VEB-1.鸟枪克隆法克隆碳青霉烯酶基因,并对克隆片段进行测序.质粒接合试验验证碳青霉烯酶基因及16S rRNA甲基化酶基因是否具有转移性.脉冲场凝胶电泳(PFGE)法对5株菌进行分子分型.等电聚焦电泳法检测β-内酰胺酶等电点.Southern杂交法对耐药决定因子进行定位.结果 5株阴沟肠杆菌对10种抗菌药物的MIC值均＞32 mg/L.克隆的碳青霉稀酶基因为blaKPC-2,酶编码基因上游为一转座酶基因,两侧为复制靶位,下游为ISKpn6的插入序列,该序列包括一个重复序列和tnpA转座子,酶编码基因位于54.2 kb的一个非接合性大质粒上.等电聚焦电泳显示5株菌均产4种β-内酰胺酶(TEM-1,pI5.4;KPC-2,pI6.7;SHV-12,pI8.2;CTX-M-14,pI8.4).16S rRNA甲基化酶基因位于接合性质粒上,而blaKPC-2基因则位于非接合性质粒上,5株菌PFGE型别一致.结论 5株泛耐药阴沟肠杆菌对碳青霉烯类耐药由产碳青霉烯酶KPC-2所介导.blaKPC-2与armA型16S rRNA甲基化酶基因由两条不同的质粒编码,不存在相关性.临床医院应加强监控,以防止交叉感染.     

儿科患者中对碳青霉烯类不敏感肠杆菌科细菌耐药性和耐药基因的研究

目的 研究儿科临床分离的对碳青霉烯类抗生素不敏感的肠杆菌科细菌耐药性和产生碳青霉烯酶的耐药基因特征.方法 收集2008年1月至2010年12月北京儿童医院住院患儿分离出的46株对碳青霉烯类抗生素不敏感的肠杆菌科细菌.使用琼脂稀释法进行药敏试验,测定抗菌药物的最低抑菌浓度(MIC)值,按照临床实验室标准化研究所(CLSI) 2011年推荐标准判断结果.使用改良Hodge试验和双纸片协同试验,进行产碳青霉烯酶的表型确证.使用PCR方法进行碳青霉烯酶相关耐药基因的检测.采用WHONET5.6软件进行数据分析.结果 46株对碳青霉烯类抗生素不敏感的肠杆菌科细菌,26株为肺炎克雷伯菌,占56.5％,13株为阴沟肠杆菌,占28.3％,7株为大肠埃希菌,占15.2％.对亚胺培南和美罗培南不敏感率分别为肺炎克雷伯菌69.2％和80.8％,阴沟肠杆菌76.9％和100％,大肠埃希菌85.7％和100％.46株肠杆菌科细菌,改良Hodge试验阳性40株(87.0％),双纸片协同试验阳性41株(89.1％).IMP基因阳性38株(82.6％),其余8株均未扩增出特异性条带检测均为阴性.结论 目前儿科临床分离对碳青霉烯类抗生素不敏感菌株中,肺炎克雷伯菌最多,占56.5％.肺炎克雷伯菌、阴沟肠杆菌和大肠埃希菌对碳青霉烯类抗生素亚胺培南不敏感程度低于美罗培南,对碳青霉烯不敏感肠杆菌科细菌主要产生B类金属酶,均为IMP基因型.     

肠杆菌科细菌中超广谱β-内酰胺酶(ESBL)的研究

目的 调查我院院内肠杆菌科细菌中产超广谱β-内酰胺酶（ESBL）菌株的发生率以及ESBL的表型和基因型。方法 对1999年2月-5月临床分离的162株肠杆菌科细菌，采用美国临床实验室标准化委员会规定的ESBL表型筛选和确证试验确定ESBL的发生率；等电聚集电泳，抑制试验，接合试验确定ESBL菌株中酶的表型；质粒的提取，脉冲场凝胶电泳（PFGE）、TEM-、SHV-、CTX-M-3特异引物的PCR和测序确定ESBL的基因型。结果 11.4%（5/44）的大肠杆菌、39.5%(17/43)的肺炎克雷伯菌、6.0%（3/50）的阴沟肠杆菌和8.0%(2/25)的弗劳地枸橼酸菌产ESBL。对于这27株菌，头孢噻肟的MIC明显高于头孢他啶，除1株阴沟肠杆菌外，其它26株菌对亚胺培南敏感，大多数菌株产2-6种酶，70.3%(19/27)的菌株产生CTX-M-3型的ESBL（等电点为8.6)。这种酶能被克拉维酸抑制，其编码的耐药性能够转移至敏感株，并且对头孢噻肟的耐药高于头孢他啶，对小部分菌株用PF-GE分型发现存在多个克隆。结论 产ESBL的肠杆菌科在院内较普遍；CTX-M-3型的ESBL最为常见。     

质粒介导KPC-2型碳青霉烯酶肺炎克雷伯菌儿童分离株耐药基因研究

目的 研究碳青霉烯类耐药肺炎克雷伯菌临床儿童分离株的耐药特点及分子流行病学特征.方法 收集温州医学院附属第二医院2010年7月-2011年6月从儿童标本中分离的耐碳青霉烯类肺炎克雷伯菌12株,所有菌株为非重复菌株,菌种鉴定采用全自动微生物分析仪.改良的Hodge试验筛选产碳青霉烯酶阳性菌株,采用PCR法检测KPC、IMP、bla(s)、VIM、SPM和整合酶基因,测序确定基因型.对菌株进行质粒结合试验、质粒消除试验检测质粒的转移性.脉冲场凝胶电泳(PFGE)分析耐药菌株的同源性.结果 12株耐碳青霉烯类肺炎克雷伯菌对庆大霉素、妥布霉素、阿米卡星、环丙沙星、左氧氟沙星、复方磺胺甲噁唑的敏感率分别为8.3％、41.7％、58.3％、8.3％、8.3％、33.3％;12株菌均携带有KPC-2基因,且同时携带有TEM-1和SHV型β-内酰胺酶基因,其中SHV-11-like和SHV-1 2-like各6株;11株携带CTX-M型基因,其中4株为CTX-M-14-like基因,6株CTX-M-15-like基因;2株携带有OXA-10型基因,1株携带有PER-1基因.未检出NDM-1、GIM、SPM、SIM、VIM型碳青霉烯酶基因.12株均为Ⅰ类整合酶基因(int1)阳性.2株通过接合试验把质粒传递给受体菌EC600.所有接合子blaTEM-1基因阳性、超广谱β-内酰胺酶(ESBL)基因阳性及对亚胺培南、庆大霉素、阿米卡星、妥布霉素和头孢噻肟耐药,接合子ESBL基因型与供菌一致.2株菌经质粒消除后对亚胺培南的MIC值均有较大程度降低,消除后KPC-2基因扩增为阴性.12株KPC-2基因阳性菌株经PFGE分成5个基因型,主要为B型和C型.结论 KPC-2型碳青霉烯酶基因已经在儿童肺炎克雷伯菌中播散,常伴随携带多种类型的ESBL基因和Ⅰ类整合酶基因,部分耐药基因可通过质粒播散.     

β-内酰胺酶合并OmpK36膜孔蛋白缺失引起肺炎克雷伯菌对碳青霉烯类抗生素敏感性的降低

目的 研究肺炎克雷伯菌(Klebsiella pneumoniae,Kp)对碳青霉烯类抗生素敏感性降低的分子机制.方法 临床分离到2株碳青霉烯敏感性降低的Kp Z2554和Z2110,经脉冲场凝胶电泳(PFGE)证实非同一克隆株.对2株细菌进行药物最低抑菌浓度测定(MIC)、接合试验、质粒图谱分析、等电聚焦电泳(IEF)、特异性PCR扩增和DNA序列分析、外膜蛋白分析以及羰酰氰氯苯腙(CCCP)抑制试验等.结果 Kp Z2554和Z2110对亚胺培南和美罗培南的MIC为1～8 μg/ml,前者对青霉素类、头孢菌素类和氨曲南等抗生素高度耐药,后者对上述抗生素的耐药程度稍弱于前者,但对头孢西丁的MIC大于512 μg/ml.IEF、PCR扩增和DNA序列分析证实Kp Z2554产TEM-1(等电点5.4)和CTX-M-14(等电点7.9)2种β-内酰胺酶,Kp Z2110产TEM-1、CTX-M-14和DHA-1(等电点7.8)3种酶.SDS-PAGE分析显示2株细菌均有相对分子质量(Mr)为39×103蛋白条带(OmpK36)的缺失.外膜蛋白基因(ompK35和ompK36)序列分析发现,Kp Z2554和Z2110的ompK35基因的个别位点存在点突变,但氨基酸序列不变.CCCP抑制试验显示CCCP不能提高Kp Z2554和Z2110对碳青霉烯类抗生素的敏感性.结论 β-内酰胺酶的产生合并OmpK36膜孔蛋白的缺失可引起Kp对碳青霉烯类抗生素敏感性的降低.     

临床分离产头孢菌素酶阴沟肠杆菌的耐药性研究

目的：研究我院产头孢菌素酶（AmpC酶）阴沟肠杆菌的检出率、耐药情况厦ampC基因型。方法：收集临床分离的耐药阴沟肠杆菌15株;三维试验检测AmpC酶;NCCLS方法检测超广谱β-内酰胺酶（ESBLs）;琼脂二倍稀释法测定MIC值;PCR扩增检测ampC基因及序列测定。结果：15株菌中8株菌（53,3％）产AmpC酶,3株菌（20．0％）产ESBLs。产AmpC酶的菌株除对亚胺培南全敏感外,对其它抗菌药不同程度耐药。5株菌的ampC基因与阴沟肠杆菌ECLC074的ampC基因100％同源,3株菌与之99％同源．2株菌的AmpC酶发生了1个氨基酸残基的改变。结论：产AmpC酶是阴沟肠杆菌对口．内酰胺类抗生素耐药的主要机制之一。阴沟肠杆菌ECLC074 ampC基因是我院主要的阴沟肠杆菌ampC基因型。产AmpC酶的阴沟肠杆菌常呈多重耐药,亚胺培南是治疗此类菌所致感染的最有效药物。     

摩根摩根菌中质粒介导KPC-2型碳青霉烯酶的检测

目的 研究碳青霉烯耐药摩根摩根菌的分子流行病学及其耐药机制.方法 2010年10月-2011年2月从杭州市中医院分离到7株碳青霉烯不敏感的摩根摩根菌.脉冲场凝胶电泳(PFGE)分析菌株之间的同源性;琼脂稀释法测定抗生素对细菌的最低抑菌浓度(MIC);接合试验、质粒图谱分析、特异性PCR扩增和序列分析等研究细菌对碳青霉烯耐药的分子机制.结果 分离自急诊监护病房的6株摩根摩根菌PFGE条带完全相同或相差1～3个条带;分离自重症监护病房的1株摩根摩根菌与其他6株PFGE条带差异明显.7株摩根摩根菌的耐药模式基本相同.亚胺培南、美罗培南和厄他培南的MIC值差异较大,分别为8μg/ml(耐药)、1μg/ml(敏感)和0.25～0.50μg/ml(敏感或中介耐药).7株摩根摩根菌对青霉素类、氨曲南和环丙沙星耐药,对头孢菌素类耐药或敏感,对阿米卡星敏感.接合试验使大肠埃希菌EC600对碳青霉烯类抗生素由敏感变为耐药,对其他β-内酰胺类抗生素也均耐药.摩根摩根菌及转移接合子均含有一个约为60kb的质粒.PCR扩增及测序表明摩根摩根菌及转移接合子均产KPC-2型碳青霉烯酶,且携带qnrS1基因.结论 首次在摩根摩根菌中检测到KPC-2型碳青霉烯酶,KPC-2是引起摩根摩根菌对碳青霉烯类不敏感的主要原因.     

亚胺培南耐药鲍曼不动杆菌16S rRNA甲基化酶基因检测及流行菌株分析

目的 研究我国多中心亚胺培南耐药的鲍曼不动杆菌的耐药性、16S rRNA甲基化酶的阳性率及分子流行病学特征.方法 收集2004年11月-2005年11月国内6省市19家医院临床分离的342株亚胺培南耐药的鲍曼不动杆菌.采用琼脂稀释法和E test法对18种抗菌药物测定分离株的最低抑菌浓度(MIC)值;脉冲场凝胶电泳(PFGE)分析菌株的同源性;PCR法筛选4种16S rRNA甲基化酶基因armA、rmtA、rmtB、rmtC,克隆测序明确基因型;接合试验、质粒抽提、电转化以及Southern blot确定甲基化酶耐药基因定位.结果 所有菌株均为多重耐药株,其中对阿米卡星、庆大霉素、妥布霉素、异帕米星、奈替米星耐药率分别为92.6%、98.6%、87.4%、90.9%和92.4%.PCR扩增、测序证实221株鲍曼不动杆菌检出甲基化酶armA基因;另3种甲基化酶基因rmtA、rmtB、rmtC均阴性.在上述342株鲍曼不动杆菌临床分离株中,298株可归类为6个流行克隆,44株为散发株.3个主要克隆(A、B、C)在全国广泛播散,分别在国内6家、3家、11家医院内流行.接合试验、质粒抽提、电转化及Southern blot表明armA编码基因存在于染色体上.结论 亚胺培南耐药鲍曼不动杆菌的表型均为多重耐药.介导氨基糖苷类抗生素高度耐药的16S rRNA甲基化酶基因armA在鲍曼不动杆菌中广泛存在,其主要传播方式为克隆播散,这必将引起临床的高度关注.     

Antibiotic susceptibility and beta-lactamase production in clinical isolates of Enterobacter spp

The in vitro susceptibility of 237 clinical isolates of Enterobacter spp. (E. aerogenes, E. agglomerans and E. cloacae; 41, 64 and 132 respectively) to 16 different antibiotics is described. Four quinolones (ciprofloxacin, lomefloxacin, norfloxacin and ofloxacin), two new cephalosporins (cefpirome and cefepime) and imipenem, all showed high activity against the three Enterobacter species tested (MIC50 less than or equal to 0.125 mg/l, MIC90 less than or equal to 0.5 mg/l). Also the aminoglycosides gentamicin and tobramycin were highly active antibiotics (MIC50 less than or equal to 0.5 mg/l, MIC90 less than or equal to 1.0 mg/l). The susceptibility of beta-lactam-antibiotics to beta-lactamase produced by Enterobacter spp. was evaluated, and imipenem and cefepime were found to be most stable. Different methods for detection of inducible beta-lactamases were used, the agar dilution method being more sensitive than the double-disc diffusion test. Elevated beta-lactamase production was detected, via induction, in 83% of E. aerogenes strains and 70% of E. cloacae strains, with cefamandole used as the substrate and cefoxitin as the inducer. Constitutive, high level enzyme production was detected in 7 and 13% respectively of the E. aerogenes and the E. cloacae strains. In all the strains of E. agglomerans, 10% of E. aerogenes and 13% of E. cloacae, no beta-lactamases could be detected with the methods studied.     

Emerging carbapenemases in Gram-negative aerobes

Carbapenemases may be defined as β -lactamases that significantly hydrolyze at least imipenem or/and meropenem. Carbapenemases involved in acquired resistance are of Ambler molecular classes A, B, and D. Class A, clavulanic acid-inhibited carbapenemases are rare. They are either chromosomally encoded (NMC-A, Sme-1 to Sme-3, IMI-1) in Enterobacter cloacae and Serratia marcescens , or plasmid encoded, such as KPC-1 in Klebsiella pneumoniae and GES-2 in Pseudomonas aeruginosa , the latter being a point-mutant of the clavulanic acid-inhibited extended-spectrum β -lactamase GES-1. The class B enzymes are the most clinically significant carbapenemases. They are metalloenzymes of the IMP or VIM series. They have been reported worldwide but mostly from South East Asia and Europe. Metalloenzymes, whose genes are plasmid and integron located, hydrolyze virtually all β -lactams except aztreonam. Finally, the class D carbapenemases are increasingly reported in Acinetobacter baumannii but compromise imipenem and meropenem susceptibility only marginally. The sources of the acquired carbapenemase genes remain unknown, as does the relative importance of the spread of epidemic strains as opposed to the spread of plasmid- or integron-borne genes. Because most of these carbapenemases confer only reduced susceptibility to carbapenems in Enterobacteriaceae, they may remain underestimated as a consequence of the lack of their detection.     

Use of imipenem-cilastatin in neonatal septicemias caused by gram-negative bacilli multiresistant to beta-lactam antibiotics

Seven neonates with septicemia due to Gram negative bacteria resistant to beta-lactam received imipenem-cilastatin therapy. Bacteria isolated were Enterobacter cloacae [3], Enterobacter aerogenes [1], Klebsiella pneumoniae [1], Serratia marcescens [1], Pseudomonas fluorescens [1]. The MICs of imipenem were lower 1 microgram/ml. In 3 children septicemia occurred during previous antimicrobial chemotherapy. 3 IV 60 mg/kg doses of imipenem with amikacin (15 mg kg/d) were administered every day. For five children blood cultures were negative after 48 hours of treatment. E. aerogenes septicemia required pefloxacin because blood cultures remained positive (d5) despite an increased dosage (90 mg/kg/d). All children were cured and imipenem-cilastatin was not responsible for any complication. Those results demonstrate the efficacy of imipenem in the treatment of septicemia in newborns due to multiresistant Gram negative bacteria.     

临床阴沟肠杆菌感染分布及耐药性分析

目的了解本院2006-2009年临床分离的阴沟肠杆菌的感染分布和耐药趋势特点,为临床合理应用抗生素提供依据。方法用MicroScanWalkAway40全自动细菌分析系统对临床分离的阴沟肠杆菌进行鉴定和药敏试验,并分析结果。结果 207株阴沟肠杆菌主要分布在呼吸科及ICU;标本的主要来源为痰及咽拭子（75.36%）;药敏试验结果显示,阴沟肠杆菌对亚胺培南、阿米卡星敏感性最高,对氨苄西林、阿莫西林/克拉维酸、头孢唑啉及头孢西丁的耐药性最严重,耐药率均〉80%。结论阴沟肠杆菌对β-内酰胺类抗生素耐药形势不容乐观,临床抗感染治疗应以分离菌株的体外抗菌药物敏感性为参考,同时注意分离菌株的流行趋势,合理选用抗生素,避免诱导性耐药的产生。     

环丙沙星耐药肠杆菌科细菌临床株qnr和aac(6')-Ⅰ b-cr基因的检测

目的 了解温州地区环丙沙星耐药肠杆菌科细菌临床株的qnr、aac(6')-Ⅰ b-cr基因的分布情况.方法 收集2005年8月-2008年4月间温州医学院附属第一医院环丙沙星耐药的肠杆菌科细菌共461株,其中大肠埃希菌370株,阴沟肠杆菌39株,克雷伯菌属细菌52株.应用PCR方法 检测qnr和aac(6')-Ⅰ b幕因,DNA测序检测qnrA、qnrB、qnrS和aac(6')-Ⅰ b-cr基因;接合传递试验方法 探讨细菌质粒介导的耐药性传递情况.结果 461株环丙沙星耐药的肠杆菌科细菌临床株中检出含qnr基因阳性菌株15株(3.25%),包括qnrA基因阳性株5株(4株阴沟肠杆菌和1株解鸟氨酸克雷伯菌)、qnrB基因阳性株4株(2株肺炎克雷伯菌和2株大肠埃希菌)、qnrS基因阳性株6株(2株肺炎克雷伯菌和4株大肠埃希菌);检出52株细菌(包括42株大肠埃希菌、4株阴沟肠杆菌和6株克雷伯菌属细菌)携带aac(6')-Ⅰ b-cr.15株qnr基因阳性的菌株同时携带aac(6')-Ⅰ b-cr,药敏结果 显示对业胺培南敏感但对多种抗生素耐药.15株qnr基因阳性的菌株中7株质粒接合传递试验成功,临床株对喹诺酮类和氨基糖苷类的耐药性部分传递给了受体株.结论 qnr基因在温州地区环丙沙星耐药的肠杆菌科细菌临床株中较少见,而aac(6')-Ⅰ b-cr基因存在较普遍.     

Occurrence and characterization of carbapenemase-producing Enterobacteriaceae: report from the SENTRY Antimicrobial Surveillance Program (2000-2004)

Emergence and dissemination of Enterobacteriaceae isolates harboring carbapenemases in various geographic regions represents a significant threat to the management of nosocomial infections. Enterobacteriaceae isolates from the SENTRY Antimicrobial Surveillance Program (2000-2004) demonstrating decreased susceptibility to imipenem and meropenem (minimum inhibitory concentration [MIC], > or =2 mg/L) were evaluated for the production of metallo-beta-lactamases and serine carbapenemases using disk approximation and polymerase chain reaction (PCR) tests. Carbapenemase-producing strains were epidemiologically typed by automated riboprinting and pulsed-field gel electrophoresis (PFGE) to establish clonality. Among 37,557 Enterobacteriaceae (5 genus groups) evaluated, 119 (0.32%) had increased carbapenem MIC values, and a carbapenemase was identified in 51 (42.9%) of these strains. KPC-2 and KPC-3 were the most frequently occurring carbapenemases (24 isolates, 20.2%) in the United States and were detected in Klebsiella spp, Citrobacter spp., Enterobacter spp., and Serratia marcescens strains isolated in New York, Arkansas, and Virginia. SME-2-producing S. marcescens were isolated in the New York City area, Texas, and Ohio, while NMC-A was found in one E. cloacae strain from New York. In contrast, metallo-beta-lactamases were prevalent in Europe. IMP-1-producing E. cloacae (11 isolates) were detected in Turkey, while VIM-1-producing strains were found in Italy (Enterobacter spp.) and Greece (Klebsiella pneumoniae). Clonal dissemination of carbapenemase-producing strains was observed in several medical centers on both continents. The occurrence of carbapenemases in various Enterobacteriaceae remains rare but appears to be spreading geographically (not in Latin America), mainly with metallo-beta-lactamases being found in Mediterranean Europe and KPC enzymes in the New York City area.     

Extended-spectrum-beta-lactamase-producing Enterobacteriaceae in Yaounde, Cameroon

Organisms producing extended-spectrum beta-lactamases (ESBLs) have been reported in many countries, but there is no information on the prevalence of ESBL-producing members of the family Enterobacteriaceae in Cameroon. A total of 259 Enterobacteriaceae strains were isolated between 1995 and 1998 from patients at the Yaounde Central Hospital in Cameroon. Enterobacterial isolates resistant to extended-spectrum cephalosporin and monobactam were screened for ESBL production by the double-disk (DD) synergy test. Thirty-one (12%) of these Enterobacteriaceae strains were shown to be positive by the DD synergy test, suggesting the presence of ESBLs. Resistance to oxyimino-cephalosporins and monobactams of 12 (38.7%) of the 31 strains-i.e., 6 Klebsiella pneumoniae, 4 Escherichia coli, 1 Citrobacter freundii, and 1 Enterobacter cloacae strain-was transferred to E. coli HK-225 by conjugation. Resistance to gentamicin, gentamicin plus trimethoprim-sulfamethoxazole, or trimethoprim-sulfamethoxazole was cotransferred into 6, 2, and 1 of these transconjugants, respectively. All 12 transconjugants were resistant to amoxicillin, piperacillin, all of the cephalosporins, and aztreonam but remained susceptible to cefoxitin and imipenem. Crude extracts of beta-lactamase-producing transconjugants were able to reduce the diameters of inhibition zones around disks containing penicillins, narrow- to expanded-spectrum cephalosporins or monobactams when tested against a fully susceptible E. coli strain but had no effect on such zones around cefoxitin, imipenem, and amoxicillin-clavulanate disks. The beta-lactamases produced by the 12 tranconjugants turned out to be SHV-12 by DNA sequencing. Therefore, the ESBL SHV-12 is described for the first time in Cameroon.     

Emergence of carbapenem-resistant Enterobacter cloacae carrying VIM-4 metallo-beta-lactamase and SHV-2a extended-spectrum beta-lactamase in a conjugative plasmid

Enterobacter cloacae strains are still infrequently resistant to carbapenems. A carbapenem-resistant clinical isolate of E. cloacae producing a plasmid-mediated metallo-beta-lactamase (MBL), VIM-4, was recovered from a Greek hospitalized patient. The bla(VIM-4) gene was located in the variable array of a class 1 integron structure repeatedly detected among bla(VIM-1)-bearing Gram-negative pathogens in Greece. The isolate possessed also on the same conjugative plasmid an extended-spectrum beta-lactamase (ESBL), SHV-2a, which contributed to the beta-lactam-resistant phenotype. This is the first report showing co-transfer of an ESBL with a VIM-type MBL. It suggests also that different VIM-type gene cassettes have been incorporated in a common integron structure, which seems indigenous of Gram-negative pathogens in our region.