运用变性高效液相色谱对肺炎克雷伯菌产ESBL进行基因分型

目的 通过运用变性高效液相色谱（DHPLC）技术对前期研究已确认产超广谱β-内酰胺酶（ESBL）的肺炎克雷伯菌临床分离株TEM型质粒进行基因分型,试图建立一种方便快捷的用于ESBL分子诊断及其流行病学监测的新方法.方法 利用PCR技术从肺炎克雷伯菌临床分离株中扩增出TEM型质粒的编码序列,扩增产物运用DHPLC技术进行分析,分析提示,异常的样本通过测序确定其基因突变的类型,最后通过比对确定其基因型.结果 共分析了101例肺炎克雷伯菌临床分离株,全部样本均扩增出TEM型质粒的编码序列,经过DHPLC分析,52例（51.4%）样本表现为单一的洗脱峰,其形态与TEM-1标准菌株的峰型相一致,测序确定它们的碱基序列亦相一致,不存在变异,为TEM-1型;49例（48.6%）样本表现为异常的洗脱峰,它们均为双峰,形态一致,但异源双链峰的高度有差异,测序结果表明它们均存在四种相同的基因突变,在NCBI网站比对后确定为TEM-116;测序结果还提示,部分样本中TEM-1和TEM-116混合存在,其比例的不同表现为DHPLC时异源双链峰高度的差异;文献检索表明,本次确定的TEM-116为一新的基因亚型,为国内首次报道.结论 DHPLC具有简便快捷、高通量和自动化的特点,重复性好,不仅可对已知突变作出即时诊断,还可发现新的基因亚型,不失为一种较好的ESBL分子诊断方法及其流行病学监测手段.     

吉林地区大肠埃希菌和肺炎克雷伯菌ESBLs的基因分型

目的对产超广谱β-内酰胺酶(ESBLs)的大肠埃希菌和肺炎克雷伯菌携带的ESBLs耐药基因进行分型,为临床合理应用抗生素提供理论依据。方法收集住院患者标本中分离出的51株大肠埃希菌和32株肺炎克雷伯菌,经PCR对上述菌株所携带的ESBLs耐药基因进行分型。结果产ESBLs大肠埃希菌中耐药质粒编码TEM型、SHV型和非TEM非SHV型超广谱β-内酰胺酶基因的百分率依次为80.4%,7.8%和11.8%;而在肺炎克雷伯菌中的百分率依次为78.1%,71.9%和25.0%。多数产ESBLs肺炎克雷伯菌同时产生一种以上的β-内酰胺酶。结论获得了吉林地区大肠埃希菌和肺炎克雷伯菌ESBLs的不同基因型,ESBLs基因型具有地区性差异。     

北京发现同时产DHA-1质粒AmpC型及CTX-M型超广谱β-内酰胺酶的肺炎克雷伯菌

目的研究大肠埃希菌和肺炎克雷伯菌同时产超广谱β内酰胺酶(ESBL)及质粒型Ampc酶菌株的比率及其基因型。方法收集北京两家教学医院2001--2002年产ESBL且对头孢西叮耐药的59株大肠埃希菌和21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，并用碱裂解法提取质粒；采用多重聚合酶链式反应(multiplex polymerase chain reaction，MPCR)及序列分析确定质粒AmpC酶的基因型。脉冲场凝胶电泳(pulsed field gelel ectrophoresis，PFGE)确定耐药菌株的亲缘关系。结果北京两家医院ESBL中质粒型AmCZ酶的发生率，大肠埃希菌分别为0和2％，肺炎克雷伯菌则分别为9．7％和17．1％。1株大肠埃希菌及9株肺炎克雷伯菌产生DHA-1型质粒AmpC酶，同时也产CTX-3型(6株)或CTX—M-14(1株)或SHV-12(3株)型ESBL。10株中，3株肺炎克雷伯菌可将头孢西叮耐药性传给受体菌。这10株菌均至少携带1个约33—36kb的大质粒，未发现质粒的传播。PFGE发现这10株菌来自不同的克隆株。结论北京地区发现同时产DHA-1质粒AmpC型及CTX—M型ESBL的肺炎克雷伯菌，它们来自不同的克隆。     

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.     

产ESBL肺炎克雷伯菌中16S rRNA甲基化酶的分布与相关耐药性的研究

目的 调查我院产ESBL肺炎克雷伯菌临床分离株16S rRNA甲基化酶基因的分布以及与耐药谱的关系,并初步探讨其在分子流行病学分析中的作用.方法 收集我院临床2010年3月至9月分离出69株非重复产ESBL肺炎克雷伯菌,采用PCR法检测16S rRNA 甲基化酶基因,并对阳性菌株进行ESBL基因及整合子基因分析,通过DNA直接测序确定.质粒接合试验和质粒消除试验确定16S rRNA甲基化酶基因的传播途径,利用ERIC-PCR技术进行基因分型.结果 69株产ESBL肺炎克雷伯菌中有rmtB阳性菌株20株(28.9％),其中2株同时携带有rmtB和armA.在20株产16SrRNA甲基化酶菌株中,均携带有CTX-M基因,测序显示14株CTX-M-14基因,6株CTX-M-15基因;14株携带有TEM-1基因;8株携带有SHV基因,测序显示5株SHV-12基因,3株SHV-11基因;3株携带有OXA-10基因;3株携带有VBE-1基因.另有12株携带有int1阳性,含有5种不同的耐药基因盒,分别携带drfA25、drfA1、drfA12、aadA1、aadA2、sat和blaVEB-1基因.ERIC-PCR法显示20株16SrRNA甲基化酶基因阳性的肺炎克雷伯菌主要分为5型,A型为优势流行克隆株.质粒接合和消除试验发现A型克隆株KP5和KP16 rmtB均位于一质粒上并通过接合传播.结论 本院产ESBL肺炎克雷伯菌临床分离株中存在16S rRNA甲基化酶基因rmtB的普遍流行,导致对多种氨基糖苷类抗生素高水平耐药.rmtB可通过水平基因传播和克隆传播的两种方式进行播散,并且存在同时产ESBLs、16S rRNA甲基化酶和Ⅰ类整合子的肺炎克雷伯菌的传播.     

产超广谱β-内酰胺酶肺炎克雷伯菌药物敏感性和基因型研究

【摘要】 目的 研究产超广谱β-内酰胺酶（ESBLs）肺炎克雷伯菌基因型和药敏特征。方法 对2003年12月～2004年12月哈尔滨地区临床分离的74株产ESBLs肺炎克雷伯菌，测定菌株对常用β-内酰胺类和非β-内酰胺类抗菌药物的敏感性，并采用SHV型和TEM型特异性引物检测ESBLs的基因型别。结果 产ESBLs细菌对头孢吡肟、氨曲南、阿米卡星和环丙沙星的敏感率依次71.6%、20.3%、54.1%、31.1%，对头孢哌酮/舒巴坦的敏感率为54.1%，69株（93.2%）对亚氨培南敏感。74株产ESBLs细菌中携带blaSHV和blaTEM基因的阳性例数分别是45（60.8%）和23（31.1%）。结论 本地区产ESBLs肺炎克雷伯菌对常用β-内酰胺类和非β-内酰胺类抗菌药物的敏感性降低，临床上需慎用酶抑制剂复合物及第四代头孢菌素，亚氨培南仍是治疗产ESBLs菌感染的首选药物。产ESBLs肺炎克雷伯菌blaSHV的流行频率高于blaTEM。     

产ESBLs革兰阴性菌的TEM、SHV、CTX-M基因分型研究

目的:检测本院临床产超广谱β-内酰胺酶(ESBLs)革兰阴性菌的TEM、SHV、CTX-M基因型特征。方法:双纸片法确定产ESBLs的临床分离菌,提取质粒DNA,PCR法扩增ESBLs的TEM、SHV、CTX-M基因片段,对临床分离的12株肺炎克雷伯菌,17株大肠埃希氏菌和16株铜绿假单胞菌,共45株革兰阴性菌进行TEM、SHV、CTX-M基因分型研究。结果:本院临床分离出的45株产ESBLs临床分离菌的质粒中,有36株扩增出TEM基因片段,33株扩增出SHV基因片段,31株扩增出CTX-M基因片段,TEM、SHV、CTX-M基因片段的检出率分别为80%、74%和68%。结论:TEM、SHV、CTX-M已成为本院临床分离产ESBLs菌的主要耐药基因型,有必要进一步加强临床耐药基因的监测,为临床用药和医院感染监控提供参考。     

武汉儿童医院住院患者质粒AmpC酶和超超广谱β-内酰胺酶分子流行病学研究

随着同时产ESBL和质粒型AmpC酶（超超广谱β-内酰胺酶，SSBL）的耐药菌株日益增多，导致菌株产生更强的耐药性，给感染控制带来很大困难。为了了解本院住院患儿大肠埃希菌（E.coli）及肺炎克雷伯菌（Kpn）高产质粒AmpC酶和SSBL的分子流行病学特征。     

Solexa测序法研究肺炎克雷伯菌质粒基因组的耐药基因

目的 通过Solexa高通量测序法研究肺炎克雷伯菌的质粒与耐药性的关系.方法 菌株为7年临床分离的206株肺炎克雷伯菌.提取所有菌的全部质粒DNA,Solexa高通量测序获得大规模的短序列.SOAP软件对质粒基因进行分析拼接,分析结果与相关数据库进行比对.MAQ软件分析质粒基因组包含的超广谱β-内酰胺酶(ESBL)多样性及单核苷酸多态性(SNP)情况.结果 肺炎克雷伯菌质粒基因组中已知的直接与耐药相关的基因就有13种.质粒基因组中存在多个ABC主动外排转运系统.发现4种编码β-内酰胺酶的ORF,其中SHV型ESBLs分布最广.系统分析了206株肺炎克雷伯菌质粒基因组中SHV型ESBLs的SNPs位点,发现存在着大量的非同义替换SNPs位点.结论 发现质粒中SHV型ESBLs基因可能受到选择压力,存在着大量的非同义替换SNPs位点.肺炎克雷伯菌质粒存在外排药物耐药方式,从而形成低水平的非特异多重耐药.     

产生超广谱β-内酰胺酶的肺炎克雷伯菌的药物敏感性及其基因型研究

目的研究产生超广谱β-内酰胺酶（ESBLs）的肺炎克雷伯菌基因型及其药敏特征。方法对2003年12月～2004年12月哈尔滨地区临床分离的74株产ESBLs肺炎克雷伯菌,测定菌株对常用β-内酰胺类和非β-内酰胺类抗菌药物的敏感性,并采用SHV型和TEM型特异性引物检测ESBLs的基因型。结果产ESBLs细菌对头孢吡肟、氨曲南、阿米卡星和环丙沙星的敏感率依次为71．6％、20．3％、54．1％、31．1％,对头孢哌酮／舒巴坦的敏感率为54．1％,69株（93．2％）对亚胺培南敏感。74株产ESBLs细菌中携带blaSHV和6kTEM基因的阳性例数分别是45例（60．8％）和23例（31．1％）。结论哈尔滨地区产ESBLs肺炎克雷伯菌对常用β-内酰胺类和非β-内酰胺类抗菌药物的敏感性降低,临床上需慎用酶抑制剂复合物及第4代头孢菌素,亚胺培南仍是治疗产ESBLs菌感染的首选药物。产ESBLs肺炎克雷伯菌blaSHV的流行频率高于blaTEM.     

Molecular characterization of extended-spectrum beta-lactamases produced by nosocomial isolates of Enterobacteriaceae from an Italian nationwide survey

Extended-spectrum beta-lactamases (ESBLs) are widespread in hospital settings worldwide. The present investigation was undertaken to assess the distribution and prevalence of ESBLs belonging to the TEM and SHV families in 448 ESBL-producing clinical isolates of Enterobacteriaceae collected from 10 different Italian hospitals. The natures of TEM and SHV determinants were identified by direct sequencing of PCR-amplified genes. TEM-52 and SHV-12 were the most common variants, and they were found in most hospitals and in several different species. Other less frequent variants included TEM-5, TEM-12, TEM-15, TEM-19, TEM-20, TEM-24, TEM-26, TEM-43, TEM-60, TEM-72, TEM-87, SHV-2a, SHV-5, and SHV-11. Proteus mirabilis was the most common producer of TEM-type ESBLs, while Klebsiella pneumoniae was the most common producer of SHV-type ESBLs. The distribution of TEM- and SHV-type ESBL variants in Enterobacteriaceae from Italian hospitals exhibited notable differences from those from other geographical settings.     

Involvement of SHV-12 and SHV-2a encoding plasmids in outbreaks of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a Tunisian neonatal ward

Previous genotypic investigations of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae recovered in a Tunisian neonatal ward revealed the spread of two epidemic strains and a high number of genetically unrelated isolates. The aim of the present study was to determine the role of the dissemination of self-transferrable plasmids harboring bla genes in the outbreaks experienced by the ward. The 49 previously identified clinical isolates of ESBL-producing K. pneumoniae were examined for relationships between their enzymes and plasmids. Analysis of crude extracts by isoelectric focusing showed four beta-lactamase-activities at pI 8.2, 7.6, 6, and 5.4. Clinical isolates contained large plasmids that could be transferred by conjugation and transformation conferring resistance to expanded-spectrum cephalosporins. DNA amplification and sequencing were performed to confirm the identities of transferred beta-lactamases. Nucleotide sequence analysis of SHV-specific PCR products from six isolates identified two bla(SHV) genes corresponding to SHV derived ESBLs, SHV-12 and SHV-2a. PstI digestion of plasmid DNA from transformants revealed six restriction patterns. The occurrence of the prevalent plasmid pattern in both epidemic strains and unrelated isolates indicated that diffusion and endemic persistence of the bla(SHV-ESBL) genes in the ward were due to concomitant spread of epidemic strains and plasmid dissemination among unrelated strains.     

Development of a multiplex PCR and SHV melting-curve mutation detection system for detection of some SHV and CTX-M beta-lactamases of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae in Taiwan

Infection by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae has been increasing in Taiwan. Accurate identification of the ESBL genes is necessary for surveillance and for epidemiological studies of the mode of transmission in the hospital setting. We describe herein the development of a novel system, which consists of a multiplex PCR to identify bla(SHV), bla(CTX-M-3)-like, and bla(CTX-M-14)-like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalent bla(SHV) genes (bla(SHV-1), bla(SHV-2), bla(SHV-2a), bla(SHV-5), bla(SHV-11), and bla(SHV-12)) in Taiwan. Sixty-five clinical isolates, which had been characterized by nucleotide sequencing of the bla(SHV) and bla(CTX-M) genes, were identified by the system. The system was then used to genotype the ESBLs from 199 clinical isolates, including 40 Enterobacter cloacae, 68 Escherichia coli, and 91 Klebsiella pneumoniae, collected between August 2002 and March 2003. SHV-12 (80 isolates) was the most prevalent type of ESBL identified, followed in order of frequency by CTX-M-3 (65 isolates) and CTX-M-14 (36 isolates). Seventeen (9%) of the 199 clinical isolates harbored both SHV- and CTX-M-type ESBLs. In contrast to Enterobacter cloacae, the majority of which produced SHV-type ESBLs, E. coli and K. pneumoniae were more likely to possess CTX-M-type ESBLs. Three rare CTX-M types were identified through sequencing of the bla(CTX-M-3)-like (CTX-M-15) and bla(CTX-M-14)-like (CTX-M-9 and CTX-M-13) genes. The system appears to provide an efficient differentiation of ESBLs among E. coli, K. pneumoniae, and Enterobacter cloacae in Taiwan. Moreover, the design of the system can be easily adapted for similar purposes in areas where different ESBLs are prevalent.     

SHV-5 extended-spectrum beta-lactamases in clinical isolates of Escherichia coli in Malaysia

Escherichia coli isolates resistant to ceftazidime isolated in the University Malaya Medical Center (UMMC) Kuala Lumpur, Malaysia, between the years 1998 and 2000 were studied for extended-spectrum beta-lactamase (ESBL) production. All strains were analysed phenotypically and genotypically and found to be ESBL-producing organisms harbouring SHV-5 beta-lactamase. This was confirmed by PCR-SSCP and nucleotide sequencing of the blaSHV amplified gene. As there was no evidence of ESBL activity in E. coli prior to this, coupled with the fact that there was a predominance of SHV-5 beta-lactamases in Klebsiella pneumoniae isolates in UMMC, we postulate that the E. coli obtained the SHV-5 beta-lactamase genes by plasmid transfer from the ESBL-producing K. pneumoniae.     

Molecular epidemiology of Klebsiella pneumoniae producing SHV-5 beta- lactamase: parallel outbreaks due to multiple plasmid transfer.

Over a period of 22 months, 32 patients treated in three independent intensive care units of the Innsbruck University Hospital were infected with extended-spectrum beta-lactamase-producing members of the family Enterobacteriaceae (30 Klebsiella pneumoniae isolates, 1 Klebsiella oxytoca isolate, and 1 Escherichia coli isolate). As confirmed by sequencing of a bla gene PCR fragment, all isolates expressed the SHV-5-type beta-lactamase. Genomic fingerprinting of epidemic strains with XbaI and pulsed-field gel electrophoresis grouped 20 of 21 isolates from ward A into two consecutive clusters which included 1 of 3 ward B isolates. All six K. pneumoniae isolates from ward C formed a third cluster. Stool isolates of asymptomatic patients and environmental isolates belonged to these clusters as well. Additionally, 2,600 routine K. pneumoniae isolates from the surrounding provinces (population, 900,000) were screened for SHV-5 production. Only one of six nonepidemic isolates producing SHV-5 beta-lactamase was matched with the outbreak strains by genomic fingerprinting. Plasmid fingerprinting, however, revealed the epidemic spread of a predominant R-plasmid, with a size of approximately 80 kb, associated with 29 of the 30 K. pneumoniae isolates. This plasmid was also present in the single K. oxytoca and E. coli isolates from ward C and in three nonepidemic isolates producing SHV-5. Our results underline that strain typing exclusively on the genomic level can be misleading in the epidemiological investigation of plasmid-encoded extended-spectrum beta-lactamases. Our evidence for multiple events of R-plasmid transfer between species of the family Enterobacteriaceae in this nosocomial outbreak stresses the need for plasmid typing, especially because SHV-5 beta-lactamase seems to be regionally spread predominantly via plasmid transfer.     

Molecular characterization of extended-spectrum beta-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli from a Korean nationwide survey

To determine the prevalence and genotypes of extended-spectrum beta-lactamases (ESBLs) among clinical isolates of Klebsiella pneumoniae and Escherichia coli, we performed antibiotic susceptibility testing, pI determination, induction testing, transconjugation, and DNA sequencing analysis. Among the 509 isolates collected from 13 university hospitals in Korea, 39.2% produced ESBLs. ESBL-producing isolates were detected in every region in Korea. A total of 44.6% of the isolates produced both TEM- and SHV-type ESBLs, and 52% of ESBL-producing isolates transferred resistance to ceftazidime by transconjugation. The ESBLs were TEM-19, TEM-20, TEM-52, SHV-2a, SHV-12, and one new variant identified for the first time in Korea, namely, TEM-116. TEM-1 and SHV-12 were by far the most common variants. TEM-1, TEM-116, and SHV-12 showed a high prevalence in K. pneumoniae. Two isolates (E. coli SH16 and K. pneumoniae SV3) produced CMY-1-like beta-lactamases, which play a decisive role in resistance to cefoxitin and cefotetan, as well as TEM-type enzymes (TEM-20 and TEM-52, respectively). Using MIC patterns and DNA sequencing analysis, we postulated a possible evolution scheme among TEM-type beta-lactamases in Korea: from TEM-1 to TEM-19, from TEM-19 to TEM-20, and from TEM-20 to TEM-52.     

Identification of SHV-type extended spectrum beta-lactamase genes in Pseudomonas aeruginosa by PCR-restriction fragment length polymorphism and insertion site restriction-PCR

We propose a simple and rapid method to discriminate SHV-type extended spectrum beta-lactamase (ESBL) genes in P. aeruginosa based on PCR techniques (PCR-RFLP and RSI-PCR). We studied 22 producing ESBL P. aeruginosa strains isolated from seven immunocompromised patients (19 isolates) and from environmental swabs (three isolates) at the Bone Marrow Transplantation Center of Tunis. Screening PCR with primer pairs designed to detect gene encoding TEM, SHV, OXA group I, OXA group II, OXA-18 and PER-1 ESBL was positive for bla(OXA18) and bla(SHV) genes in all isolates. Pulsed field gel electrophoresis using SpeI endonuclease defined five genotypic groups. For at least one isolate corresponding to each genotype observed, restriction of PCR products by DdeI and BsrI revealed the same restriction pattern that the bla(SHV-1) negative control; in the same way, RSI-PCR products digestion by NruI, thus excluding 35, 238 and 240 mutations characterizing reported ESBL in P. aeruginosa (SHV-2a, SHV5 et SHV12), and suggesting that studied bla(SHV) genes were not ESBL ones. Genomic DNA hybridization by southern blot with probe consisting in bla(SHV-1) gene was positive in these isolates. Sequencing the full-length open reading frame revealed nucleotide sequence of the bla(SHV-1). PCR-RFLP and RSI-PCR results were then confirmed. This approach is effective for screening P. aeruginosa for ESBL genes carriage in epidemiological studies and for detecting new variants.     

Outbreak of infection with extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a Mexican hospital

Thirty-one strains of Klebsiella pneumoniae (including 10 duplicates) from 21 septicemic pediatric patients (age, <2 months) were studied during a 4-month period (June to October 1996) in which the fatality rate was 62% (13 of 21). These isolates identified by the API 20E system yielded the same biotype. Pulsed-field gel electrophoresis experiments revealed the same clone in 31 strains. The isolates were multidrug-resistant but were still susceptible to ciprofloxacin, imipenem, and cefoxitin. A 135-kb plasmid was harbored in all of the isolates. No transconjugants were obtained that were resistant to ampicillin, cefotaxime, tetracycline, or gentamicin. Isoelectric focusing for beta-lactamases was performed on all strains, and three bands with pIs of 5.4, 7.6, and 8.2 were obtained. Of these, the pI 8.2 beta-lactamase had an extended-spectrum beta-lactamase phenotype. PCR amplification of both TEM- and SHV-type genes was obtained. The sequence analysis of the SHV PCR product indicated a mutation corresponding to the SHV-5 beta-lactamase.