鲍曼不动杆菌耐药性及β内酰胺酶基因型研究

目的 明确浙江湖州地区临床分离的鲍曼不动杆菌耐药性及β内酰胺酶基因型。方法 采用ATB药敏试验条板微量肉汤法测定临床分离的60株鲍曼不动杆菌对18种抗菌药物的敏感性，采用聚合酶链反应(PCR)及序列分析的方法分析β内酰胺酶基因型。结果 鲍曼不动杆菌对亚胺堵南、美洛培南的耐药率为1．7％，哌拉西林／他唑巴坦的耐药率为48．3％，对其他抗菌药物的耐药率均在50％以上。有18株菌株扩增到SHV型β内酰胺酶，17株为ESBLs SHV—12，另1株(HZ01株)扩增序列全长含826个核苷酸，与SHV—1相比，第35、191、238、240位氨基酸有不同，被命名为SHV-48(GenBank登录号：AY259164)。结论 浙江湖州地区临床分离的鲍曼不动杆菌耐药严重，至少存在3种不同类型的β内酰胺酶，基因型分别为SHV-12、SHV-48、TEM-1。     

一种新SHV基因型β-内酰胺酶的发现及其临床意义

[目的]查明中国浙江省湖州地区临床分离的鲍曼不动杆菌的耐药情况及其β-内酰胺酶(BLA)耐药基因类型。[方法]采用微量稀释法测定临床分离的鲍曼不动杆菌HZ01株对21种抗菌药物的敏感性、三维试验法检测超广谱β-内酰胺酶(ES—BLs)，采用聚合酶链反应(PCR)技术检测TEM、SHV、OXA、CTX—M-1、CTX—M-2、CTX—M-9、PER及VEB型BLA耐药基因、并进行序列分析。[结果]临床分离的鲍曼不动杆菌HZ01株产生SHV型BLA，其扩增片段含826个核苷酸，核苷酸及相应的氨基酸序列经GenBank查询无相同序列，为新发现的一种BLASHV-48型(GenBank注册号：AY259164)。[结论]临床分离的鲍曼不动杆菌HZ01株所产BLA为SHV-48亚型，为一种新发现的新型BLA。     

下呼吸道感染鲍曼不动杆菌ESBLs表型及基因型检测

目的分析引起下呼吸道感染产超广谱β-内酰胺酶(ESBk)鲍曼不动杆菌的耐药情况及其β-内酰胺酶(BLA)耐药基因类型.方法采用微量稀释法测定临床分离的35株鲍曼不动杆菌对21种抗菌药物的敏感性、三维试验法检测ESBLs采用聚合酶链反应(PCR)及序列分析的方法分析BLA基因型.结果 29株(82.9%)ESBLs阳性,其中14株(40.0%)合并产AmpC酶.所有菌株均对亚胺培南、美洛培南、头孢哌酮/舒巴坦和多粘菌素E敏感,其余抗生素的耐药率在40.0～100.0%之间.35株TEM型扩增全部阳性,任选2株测序结果均为TEM-1型.有15株菌株扩增到SHV型BLA基因,经测序13株为SHV-12型ESBLs,另2株(HZ01株和HZ29株)为2种新发现的SHV型BLA,分别被命名为SHV-48和SHV-56.结论临床分离的引起下呼吸道感染鲍曼不动杆产ESBLs比例很高,多重耐药状况极其严重,至少存在4种不同类型的β内酰胺酶基因,基因型分别为TEM-1、SHV-12、SHV-48和SHV-56.     

下呼吸道感染鲍曼不动杆菌ESBLs表型及基因型检测

目的分析引起下呼吸道感染产超广谱β-内酰胺酶(ESBLs)鲍曼不动杆菌的耐药情况及其β-内酰胺酶(BLA)耐药基因类型。方法采用微量稀释法测定临床分离的35株鲍曼不动杆菌对21种抗菌药物的敏感性、三维试验法检测ESBLs采用聚合酶链反应(PCR)及序列分析的方法分析BLA基因型。结果29株(82.9%)ESBLs阳性,其中14株(40.0%)合并产AmpC酶。所有菌株均对亚胺培南、美洛培南、头孢哌酮/舒巴坦和多粘菌素E敏感,其余抗生素的耐药率在40.0～100.0%之间。35株TEM型扩增全部阳性,任选2株测序结果均为TEM-1型。有15株菌株扩增到SHV型BLA基因,经测序13株为SHV-12型ESBLs,另2株(HZ01株和HZ29株)为2种新发现的SHV型BLA,分别被命名为SHV-48和SHV-56。结论临床分离的引起下呼吸道感染鲍曼不动杆产ESBLs比例很高,多重耐药状况极其严重,至少存在4种不同类型的β内酰胺酶基因,基因型分别为TEM-1、SHV-12、SHV-48和SHV-56。     

一种新基因型TEM-128β-内酰胺酶的发现及其临床意义

目的 分析产超广谱β-内酰胺酶(ESBLs)鲍曼不动杆菌TEM型β-内酰胺酶(BLA)的编码基因并确定其亚型。方法 采用聚合酶链反应(PCR)扩增BLA编码基因TEM、SHV、OXA、CTX—M-1、CTX—M-2、CTX—M-9、PER和VEB片段，双脱氧链终止法测定核苷酸序列、确定亚型。结果临床分离的鲍曼不动杆菌HZ38株产生TEM型BLA，其扩增片段含1049个核苷酸，核苷酸及相应的氨基酸序列经Gen—Bank查询无相同序列，为新发现的一种BLA TEM-128型(GenBank注册号：AY359287)。结论 临床分离的鲍曼不动杆菌HZ38株所产BLA为TEM-128亚型，为一种新发现的新型BLA。     

超广谱β-内酰胺酶在多重耐药鲍曼不动杆菌中的作用

目的探讨超广谱β-内酰胺酶(ESBLs)在多重耐药鲍曼不动杆菌中的作用。方法收集该院2014年临床分离的158株鲍曼不动杆菌,采用纸片扩散法(K-B)测定其对临床常用20种抗菌药物的敏感性。采用PCR对其中100株多重耐药进行ESBLs基因扩增,通过序列分析明确基因型。结果鲍曼不动杆菌对多黏菌素B均敏感,对米诺环素、亚胺培南、头孢哌酮-舒巴坦和丁胺卡那霉素的耐药率分别为3.9%、45.8%、48.1%和39.1%,其余抗菌药物耐药率均大于50%。多重耐药菌株中,32株SHV-12型基因阳性,54株PER-1型基因阳性,16株TEM-1型基因阳性,有2株同时携带这3种基因,6株同时携带SHV-12和PER-1型基因。结论鲍曼不动杆菌耐药情况较严重,多重耐药与携带SHV-12、PER-1和TEM-1型基因相关。     

广州地区鲍曼不动杆菌产β内酰胺酶基因型调查

目的调查广州地区临床分离的鲍曼不动杆菌耐药性及其主要β内酰胺酶分布。方法用K-B纸片扩散法测定临床分离的211株鲍曼不动杆菌对13种抗菌药物的敏感性,采用PCR对85株耐药株进行β内酰胺酶基因扩增,通过序列分析明确基因型。结果鲍曼不动杆菌对美罗培南、亚胺培南、头孢哌酮-舒巴坦和替卡西林-克拉维酸的耐药率分别为0、0.9%、31.4%和31.1%,其余抗菌药物耐药率在39.3%~80.1%;85株耐药菌株中,57株扩增到ampC基因、12株SHV-12型基因阳性,14株PER-1型基因阳性,7株CTX-M-14型基因阳性,8株TEM-1型基因阳性;同时携带2种基因有11株,携带3种基因有10株。结论鲍曼不动杆菌耐药与携带ampC基因、SHV-12、PER-1、CTX-M-14和TEM-1型等基因密切相关。     

多重耐药肺炎克雷伯菌β-内酰胺酶编码基因分子生物学研究

目的确定浙江省嘉兴地区临床分离的多重耐药肺炎克雷伯菌E3株所产β-内酰胺酶编码基因序列及亚型.方法肺炎克雷伯菌E3株经酶抑制剂增强的肉汤稀释法鉴定为产超广谱β-内酰胺酶(ESBLs)细菌,聚合酶链反应(PCR)扩增其β-内酰胺酶编码基因片段,克隆入pGEM-Teasy载体,双脱氧链终止法测定核苷酸序列并确定亚型,重组细菌表型鉴定.结果E3株同时产SHV和TEM型2种β-内酰胺酶,其中SHV基因片段含812个核苷酸,编码产物有266个氨基酸,应为SHV-11型β-内酰胺酶;TEM基因片段含973个核苷酸,编码产物有288个氨基酸,应为TEM-1型β-内酰胺酶.含TEM-1和SHV-11编码基因的重组细菌经酶抑制剂增强的肉汤稀释法鉴定为阴性.结论E3株肺炎克雷伯菌同时产生SHV-11和TEM-1 2种ESBLs,其可影响ESBLs的表型检测结果.     

关于ICU病房鲍曼不动杆菌耐药性及基因型研究

目的 调查分析聊城地区ICU病房109株鲍曼不动杆菌耐药性及β-内酰胺酶基因型.方法 用K-B法测定临床分离的109株鲍曼不动杆菌对13种抗菌药物的耐药性,采用PCR法检测β-内酰胺酶耐药基因型.结果 除IMP外,109株鲍曼不动杆菌对其余13种抗菌药物的耐药率均超过50%,PCR扩增对IMP中介或耐药的53株鲍曼不动杆菌有11株扩增到blaOXA-23,产ESBLs组29株中有5株扩增出blasHv、blaCTX-M-14、blaCTX-M-3,2株出现blaSHV、blaCTX-M-1.结论 聊城地区ICU病房鲍曼不动杆菌多重耐药常见,β-内酰胺酶产生率高,碳青霉烯酶以blaOXA-23型为主,ESBLs、AmpC酶两种β-内酰胺酶基因的多重PCR阳性率均低.     

一种新基因型SHV-28 β-内酰胺酶的发现及其特性的初步研究

目的 鉴定产超广谱β－内酰胺酶（ESBLs)肺炎克雷伯菌SHV型β－内酰胺酶（BLSA）的编码基因、BLA亚型并初步研究其特性。方法 聚合酶链反应（PCR）扩增BLA编码基因片段,克隆入pGEM-Teasy载体,双脱氧链终止法测定核苷酸序列、确定亚型,并做接合试验。结果 临床分离的8株肺炎克雷伯菌,均产生SHV型BLA,其扩增片段含812个核苷酸,核苷酸及相应的氨基酸序列经GenBank查询无相同序列,为新发现的一种BLA SHV－28型（GenBank注册号：AF299299）。接合试验的结果证明耐药基因位于质粒上。结论 8株临床分离的产ESBLs肺炎克雷伯菌的产BLA为SHV－28亚型,为新发现的一种新型BLA。     

Diversity of SHV and TEM beta-lactamases in Klebsiella pneumoniae: gene evolution in Northern Taiwan and two novel beta-lactamases, SHV-25 and SHV-26

A total of 113 blood culture isolates of Klebsiella pneumoniae from 10 hospitals in northern Taiwan were studied for SHV and TEM beta-lactamase production. bla(SHV) was amplified from all isolates by PCR. TEM-type resistance, was found in 32 of the isolates and was of the TEM-1 type in all isolates. SHV-1, -2, -5, -11, and -12 and two novel enzymes were identified. These novel enzymes were designated SHV-25 and SHV-26 and had pIs of 7.5 and 7.6, respectively. Amino acid differences in comparison to the amino acid sequence of bla(SHV-1) were found at positions T18A (ThrACC-->AlaGCC), L35Q (LeuCTA-->GluCAA), and M129V (MetATG-->ValGTG) for SHV-25 and at position A187T (AlaGCC-->ThrACC) for SHV-26. The results of substrate profiles and MIC determinations showed that the novel enzymes did not hydrolyze extended-spectrum cephalosporins, rendering the isolates susceptible to these agents. Inhibition profiles revealed that the 50% inhibitory concentration for SHV-26 was higher than those for SHV-1 and SHV-25, resulting in an intermediate resistance to amoxicillin-clavulanic acid. Forty-nine ribotypes were identified, suggesting that major clonal spread had not occurred in any of the hospitals. According to the amino acid sequence, SHV beta-lactamases in Taiwan may basically be derived through stepwise mutation from SHV-1 or SHV-11 and further subdivided by four routes. The stepwise mutations initiated from SHV-1 or SHV-11 to SHV-2, SHV-5, and SHV-12 comprise the evolutionary change responsible for extended-spectrum beta-lactamase (ESBL) production in Taiwan. The stepwise mutations that lead to a non-ESBL (SHV-25) and the beta-lactamase (SHV-26) with reduced susceptibility to clavulanic acid are possibly derived from SHV-11 and SHV-1, respectively. The results suggest a stepwise evolution of SHV beta-lactamases in Taiwan.     

Molecular analysis of the simultaneous production of two SHV-type extended-spectrum beta-lactamases in a clinical isolate of Enterobacter cloacae by using single-nucleotide polymorphism genotyping

Bacteria that simultaneously produce multiple extended-spectrum beta-lactamases are frequently isolated. We report an Enterobacter cloacae isolate, ES24, producing four different beta-lactamases (AmpC type beta-lactamase, TEM-1, SHV-7, and a novel extended-spectrum beta-lactamase, SHV-30). Direct sequencing of bla(SHV) gene products gave a "double peak" at position 703, suggesting the presence of more than one allele. Using fluorescence resonance energy transfer real-time PCR to detect single-nucleotide polymorphisms, we were able to distinguish two different bla(SHV) genes in a single isolate. This may prove to be a useful technique in surveys of beta-lactamase production in contemporary clinical isolates.     

Evolution and spread of SHV extended-spectrum beta-lactamases in gram-negative bacteria.

Resistance to beta-lactam antibiotics has been a problem for as long as these drugs have been used in clinical practice. In clinically significant bacteria the most important mechanism of resistance is the production of one or more beta-lactamases, enzymes that hydrolyse the beta-lactam bond characteristic of this family of antibiotics. Prominent among the beta-lactamases produced by the Enterobacteriaceae is the SHV family. The first reported SHV beta-lactamase had a narrow spectrum of activity. By the accumulation of point mutations at sites that affect the active site of the enzyme, a family of derivatives of SHV-1 has evolved. Derivatives of SHV-1 either have an extended spectrum of activity, capable of inactivating third-generation cephalosporins, or are resistant to beta-lactamase inhibitors. This review describes the evolution and spread of the SHV family of beta-lactamases, introducing the structure-function analysis made possible by DNA sequence analysis. It also reviews the methods used to characterize members of this family of beta-lactamases, indicating some of the difficulties involved.     

Rapid discriminatory detection of genes coding for SHV beta-lactamases by ligase chain reaction

Ligase chain reaction (LCR) is a recently developed technique that employs a thermostable ligase and allows for the discrimination of DNA sequences differing in only a single base pair. The method has been adapted and applied to differentiation of bla(SHV) genes. We have developed an LCR typing method to characterize point mutations in genes for SHV-derived extended-spectrum beta-lactamases with four different sets of biotinylated LCR primers. To evaluate the applicability of the current technique, we tested seven Escherichia coli strains producing SHV-1, SHV-2, SHV-2a, SHV-3, SHV-4, SHV-5, and SHV-12. With the LCR typing, seven SHV genes can be distinguished according to their incorporating point mutations. In an attempt to characterize SHV beta-lactamases by LCR typing in clinical isolates, 46 strains carrying bla(SHV) genes (32 Klebsiella pneumoniae, 10 Enterobacter cloacae, and 4 E. coli) were subjected to antibiotic susceptibility testing, isoelectric focusing, and LCR typing. LCR typing allowed the characterization of beta-lactamases, and genotypes obtained by LCR typing were in accordance with phenotypes such as antibiotic resistance profile and pI value of beta-lactamase. Therefore, we concluded that LCR typing may permit defining the SHV families with simplicity and reliability and can be applied to the detailed characterization and molecular epidemiology of SHV-type beta-lactamases.     

Patterns and mechanisms of resistance to beta-lactams and beta-lactamase inhibitors in uropathogenic Escherichia coli isolated from dogs in Portugal.

Little is known about beta-lactam and beta-lactamase inhibitor susceptibilities of uropathogenic Escherichia coli isolates from animals. Seventy-two isolates collected from canine urinary tract infections were studied by disc diffusion and microdilution methods. The mechanisms responsible for amoxicillin resistance were associated with the production of beta-lactamases in 26 (36%) isolates. These beta-lactamases were further characterized by isoelectric focusing (IEF) and PCR, and bla(TEM), bla(OXA-1), bla(SHV) and ampC genes were detected. The isolates were highly resistant to amoxicillin and ticarcillin, with MIC(90)s of 2048 mg/L. The MIC(90) of cefalothin was 128 mg/L, but the MIC(90)s of ceftazidime, ceftriaxone, cefotaxime and aztreonam were lower (0.5, 0.06, 0.25 and 0.5 mg/L, respectively). Fourteen isolates were not inhibited by clavulanate. The mechanisms of resistance to beta-lactams and beta-lactamase inhibitors involved the presence of TEM-1 beta-lactamase in 20 isolates, which had an isoelectric point (pI) of 5.4 and were positive for the presence of the bla(TEM) gene. Fourteen of these isolates produced TEM-1 beta-lactamase alone, and the other six showed an additional band at pI 9.0-9.2 on IEF and the ampC gene by PCR, indicating the simultaneous production of AmpC enzyme. IEF showed that one isolate produced AmpC alone and PCR detected the presence of the ampC gene. Three of the 26 beta-lactamases with a pI of 7.6 belonged to the SHV family, which was confirmed by the presence of the bla(SHV) gene. The remaining two beta-lactamases were OXA-1 focusing at 7.4, and were encoded by the bla(OXA-1) gene. Resistance to beta-lactamase inhibitors was mediated mainly by TEM-1 alone (six of 26) or together with AmpC (four of 26), AmpC alone (one of 26), SHV (one of 26) and OXA-1 (two of 26) enzymes. Clear resistance to extended-spectrum cephalosporins, ceftazidime and ceftriaxone (64 mg/L), was found in one isolate. Isolates producing either AmpC or OXA-1 enzymes or producing high levels of TEM-1 beta-lactamases had susceptibility patterns that were difficult to distinguish without IEF and/or amplification of the corresponding specific genes. This work supports the need for antimicrobial resistance surveillance in veterinary medicine.     

Extended-spectrum beta-lactamases among Enterobacter isolates obtained in Tel Aviv, Israel

The extended-spectrum beta-lactamase (ESBL)-producing phenotype is frequent among Enterobacter isolates at the Tel Aviv Sourasky Medical Center, Tel Aviv, Israel. We examined the clonal relatedness and characterized the ESBLs of a collection of these strains. Clonal relatedness was determined by pulsed-field gel electrophoresis. Isoelectric focusing (IEF) and transconjugation experiments were performed. ESBL gene families were screened by colony hybridization and PCR for bla(TEM), bla(SHV), bla(CTX-M), bla(IBC), bla(PER), bla(OXA), bla(VEB), and bla(SFO); and the PCR products were sequenced. The 17 Enterobacter isolates studied comprised 15 distinct genotypes. All isolates showed at least one IEF band (range, one to five bands) whose appearance was suppressed by addition of clavulanate; pIs ranged from 5.4 to > or = 8.2. Colony hybridization identified at least one family of beta-lactamase genes in 11 isolates: 10 harbored bla(TEM) and 9 harbored bla(SHV). PCR screening and sequence analysis of the PCR products for bla(TEM), bla(SHV), and bla(CTX-M) identified TEM-1 in 11 isolates, SHV-12 in 7 isolates, SHV-1 in 1 isolate, a CTX-M-2-like gene in 2 isolates, and CTX-M-26 in 1 isolate. In transconjugation experiments with four isolates harboring bla(TEM-1) and bla(SHV-12), both genes were simultaneously transferred to the recipient strain Escherichia coli HB101. Plasmid mapping, PCR, and Southern analysis with TEM- and SHV-specific probes demonstrated that a single transferred plasmid carried both the TEM-1 and the SHV-12 genes. The widespread presence of ESBLs among Enterobacter isolates in Tel Aviv is likely due not to clonal spread but, rather, to plasmid-mediated transfer, at times simultaneously, of genes encoding several types of enzymes. The dominant ESBL identified was SHV-12.     

Prevalence of SHV-12 among clinical isolates of Klebsiella pneumoniae producing extended-spectrum beta-lactamases and identification of a novel AmpC enzyme (CMY-8) in Southern Taiwan

Twenty (8.5%) of 234 nonrepetitive clinical isolates of Klebsiella pneumoniae from southern Taiwan were found to produce extended-spectrum beta-lactamases (ESBLs): 10 strains produced SHV-12, 4 produced SHV-5, 2 produced a non-TEM non-SHV ESBL with a pI of 8.3, 3 produced a novel AmpC beta-lactamase designated CMY-8 with a pI of 8.25, and 1 produced SHV-12 and an unidentified AmpC enzyme with a pI of 8.2. The CMY-8 enzyme confers a resistance phenotype similar to CMY-1 and MOX-1, and sequence comparisons showed high homologies (>95%) of nucleotide and amino acid sequences among these three enzymes. Plasmid and pulse-field gel electrophoresis analyses revealed that all isolates harboring an SHV-derived ESBL were genetically unrelated, indicating that dissemination of resistance plasmids is responsible for the spread of SHV ESBLs among K. pneumoniae in this area. All three isolates carrying CMY-8 had identical genotypic patterns, suggesting the presence of an epidemic strain.     

Extended-spectrum beta-lactamases in clinical isolates of Enterobacter gergoviae and Escherichia coli in China.

Resistance to ceftazidime, detected in isolates of Escherichia coli 5518 and Enterobacter gergoviae 3773 from our hospital, was transferred, together with resistance to aminoglycosides, trimethoprim, sulfonamide, and other beta-lactam antibiotics, by conjugation to E. coli JP559. Both E. coli transconjugants were resistant to ampicillin, all cephalosporins, and aztreonam but remained susceptible to cefoxitin and imipenem. The enzymes of the two transconjugant strains readily hydrolyzed cephalosporins in a spectrophotometric assay. Hybridization results suggested that the extended-spectrum beta-lactamase produced by E. coli 5518 was a non-TEM, non-SHV enzyme, the origin of which is currently unknown. The beta-lactamase produced by E. gergoviae 3773 was of the SHV type and was further proved to be SHV-2 by DNA sequencing. Thus, extended-spectrum beta-lactamases are occurring in China as well as in other parts of the world.     

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.     

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.