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.     

Variety of TEM-, SHV-, and CTX-M-type beta-lactamases present in recent clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae from Taiwan

A total of 171 hospitals' isolates of E. coli, K. pneumoniae, and E. cloacae with a minimum inhibitory concentration (MIC) of > or =2 microg/ml for ceftazidime or cefotaxime were evaluated for the production of beta-lactamases. PCR amplification with specific primers for the bla (SHV), bla (TEM), and bla (CTX) genes revealed that a total of 53, 81, and 43 of these genes were amplified, respectively. Sequencing results confirmed that TEM-1, CTX-M-3 and -14, SHV-1, -5, -11, -12, and -33, OXY-1a, and LEN-1 were presented among these isolates. No specific large cluster of isolates carried the same beta-lactamases, indicating the wide diversity of the collected strains. Plasmid spread between E. coli and K. pneumoniae was identified in few isolates. Combinations of TEM, SHV, and CTX beta-lactamase genes, including extended-spectrum beta-lactamase genes, were observed in all three species.     

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.     

Beta-lactamase characterization in Escherichia coli isolates with diminished susceptibility or resistance to extended-spectrum cephalosporins recovered from sick animals in Spain

A total of 1439 Escherichia coli isolates from sick animals were received from the Spanish Network of Veterinary Antimicrobial Resistance Surveillance (VAV) from 1997 to 2001. Antimicrobial susceptibility tests were performed and diminished susceptibility to cefotaxime and ceftazidime was identified in 2.5% and 2.8% of the isolates, respectively. Beta-lactamase characterization was carried out in the group of 20 E. coli isolates with both characteristics. The MIC ranges of different beta-lactams showed by these 20 isolates were as follows (in microg/ml): ampicillin (64-->256), amoxicillin-clavulanic acid (4-64), ticarcillin (8-->128), cefazolin (32-->256), cefoxitin (4-->128), cefotaxime (1-64), ceftazidime (2-->64), ceftriaxone (0.5-64), imipenem (< or = 0.06-0.25), and aztreonam (2-->32). TEM, SHV, CMY, and FOX beta-lactamase genes were analyzed by PCR and sequencing. The beta-lactamase genes detected were the following ones (number of isolates): bla(TEM-1b) (3), bla(TEM-1a) (1), bla(TEM-30f) (2), bla(TEM-1b) + bla(CMY-2) (2), and bla(SHV-12) (1). Sequences of the promoter and/or attenuator region of the chromosomal ampC gene were studied in all the 20 isolates. Mutations at position -42 or -32 were detected in 16 isolates and these mutations were associated with the presence of a TEM type beta-lactamase in 6 isolates. Besides, a high variety of plasmidic beta-lactamases was detected including TEM-30 and CMY-2. To our knowledge, this is the first time that TEM-30 beta-lactamase has been detected in E. coli isolates of animal origin.     

Molecular characterisation of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp. isolates at a tertiary-care centre in Lebanon.

The prevalence of bla CTX-M, bla TEM and bla SHV genes among extended-spectrum beta-lactamase (ESBL)-producing clinical isolates of Escherichia coli (n = 50) and Klebsiella spp. (n = 50) from Lebanon was 96%, 57% and 67%, and 40%, 82% and 84%, respectively. Genotyping revealed that the clonal diversity was unrelated to the presence of bla genes. Sequence analysis of 16 selected isolates identified the bla CTX-M-15, bla TEM-1, bla OXA-1 and six bla SHV genes, as well as the gene encoding the quinolone-modifying enzyme AAC(6')-Ib-cr. The genes encoding CTX-M-15 and AAC(6')-Ib-cr were carried on a 90-kb plasmid of the pC15-1a or pCTX-15 type, which transferred both ESBL production and quinolone resistance from donors to transconjugants.     

Using nucleic acid microarrays to perform molecular epidemiology and detect novel β-lactamases: a snapshot of extended-spectrum β-lactamases throughout the world

The worldwide dissemination of extended-spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae is a major concern in both hospital and community settings. Rapid identification of these resistant pathogens and the genetic determinants they possess is needed to assist in clinical practice and epidemiological studies. A collection of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis isolates, including phenotypically ESBL-positive (n = 1,093) and ESBL-negative isolates (n = 59), obtained in 2008-2009 from a longitudinal surveillance study (SMART) was examined using an in vitro nucleic acid-based microarray. This approach was used to detect and identify bla(ESBL) (bla(SHV), bla(TEM), and bla(CTX-M) genes of groups 1, 2, 9, and 8/25) and bla(KPC) genes and was combined with selective PCR amplification and DNA sequencing for complete characterization of the bla(ESBL) and bla(KPC) genes. Of the 1,093 phenotypically ESBL-positive isolates, 1,041 were identified as possessing at least one bla(ESBL) gene (95.2% concordance), and 59 phenotypically ESBL-negative isolates, used as negative controls, were negative. Several ESBL variants of bla(TEM) (n = 5), bla(SHV) (n = 11), bla(CTX-M) (n = 19), and bla(KPC) (n = 3) were detected. A new bla(SHV) variant, bla(SHV-129), and a new bla(KPC) variant, bla(KPC-11), were also identified. The most common bla genes found in this study were bla(CTX-M-15), bla(CTX-M-14), and bla(SHV-12). Using nucleic acid microarrays, we obtained a "molecular snapshot" of bla(ESBL) genes in a current global population; we report that CTX-M-15 is still the dominant ESBL and provide the first report of the new β-lactamase variants bla(SHV-129) and bla(KPC-11).     

Development of a sensitive and specific enzyme-linked immunosorbent assay for detecting and quantifying CMY-2 and SHV beta-lactamases

Polyclonal rabbit antibodies against SHV-1 and CMY-2 beta-lactamases were produced and characterized, and enzyme-linked immunosorbent assays (ELISAs) were developed. Immunoblots revealed that the anti-SHV-1 antibody recognized SHV-1 but did not recognize TEM-1, K-1, OXA-1, or any AmpC beta-lactamase tested. The anti-CMY-2 antibody detected Escherichia coli CMY-2, Enterobacter cloacae P99, Klebsiella pneumoniae ACT-1, and the AmpC beta-lactamases of Enterobacter aerogenes, Morganella morganii, and Citrobacter freundii. No cross-reactivity of the anti-CMY-2 antibody was seen against laboratory strains of E. coli possessing TEM-1, SHV-1, K-1, or OXA-1 beta-lactamases. Operating conditions for performing ELISAs were optimized. Both anti-CMY-2 and anti-SHV-1 antibodies detected picogram quantities of purified protein in ELISAs. The reactivity of the anti-CMY-2 antibody was tested against a number of AmpC beta-lactamases by assaying known quantities of purified enzymes in ELISAs (AmpC beta-lactamases of M. morganii, C. freundii, E. coli, and E. cloacae). As the homology to CMY-2 beta-lactamase decreased, the minimum level needed for detection increased (e.g., 94% homology recognized at 1 ng/ml and 71% homology recognized at 10 ng/ml). The ELISAs were used to assay unknown clinical isolates for AmpC and SHV beta-lactamases, and the results were confirmed with PCR amplification of bla(AmpC) and bla(SHV) genes. Overall, we found that our ELISAs were at least 95% sensitive and specific for detecting SHV and AmpC beta-lactamases. The ELISA format can facilitate the identification of AmpC and SHV beta-lactamases and can be used to quantify relative amounts of beta-lactamase enzymes in clinical and laboratory isolates.     

Detection of SHV-1 beta-lactamase in Pseudomonas aeruginosa strains by genetic methods

Twelve multidrug-resistant Pseudomonas aeruginosa (MDRPA) isolates were recovered over a period of two years in the National Bone Marrow Transplant Centre of Tunisia. MDRPA isolates were isolated from seven patients and from three environmental samples. Isoelectric focusing revealed pIs of 8.2, 5.5 and 7.6 in all MDRPA isolates. These strains produced the OXA-18 extended spectrum beta-lactamase and an SHV type beta-lactamase as shown by screening PCR analysis. DNA hybridization confirmed this inference, detecting bla(SHV) gene in these isolates. Pulsed-field gel electrophoresis (PFGE) defined one predominant genomic group; group A (seven isolates) and four different genotypes containing one to two isolates. Clonally related isolates were recovered from three patients and from two washbasins. Sequencing DNA of cluster representative strains identified the classical bla(SHV-1) gene. For these strains, the nucleotide sequence of the structural bla(SHV-1) gene was nearly identical to those previously described. Such enzyme has not been reported from P. aeruginosa. This is the first report of the SHV-1 penicillinase in epidemic P. aeruginosa strain.     

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.     

Application of minimal sequence quality values prevents misidentification of the blaSHV type in single bacterial isolates carrying different SHV extended-spectrum beta-lactamase genes

Nucleotide sequencing is the standard molecular method for determination of the beta-lactamase gene present in an isolate. Using minimal sequence quality values prevents misidentification of bla(SHV) genes, as illustrated by three strains of three different species that each contained two different bla(SHV) alleles, SHV-2 and SHV-12.     

Prevalence of beta-lactamases among ampicillin-resistant Escherichia coli and Salmonella isolated from food animals in Denmark

The genetic background for beta-lactamase-mediated resistance to beta-lactam antibiotics was examined by PCR and sequencing in 160 ampicillin-resistant isolates (109 Escherichia coli and 51 Salmonella) obtained from healthy and diseased food animals in Denmark. Sequencing revealed three different variants of bla (TEM-1), of which bla (TEM-1b) was the most frequently detected (80 E. coli and 47 Salmonella), followed by bla (TEM-1a) (eight E. coli, one Salmonella) and bla (TEM-1c) (seven E. coli). A few isolates were found to express OXA, TEM-30, or PSE beta-lactamases. Mutations in the ampC promoter leading to increased production of the AmpC beta-lactamase were demonstrated in 11 cefoxitin-resistant or intermediate E. coli isolates. Nine of these isolates did not contain any bla (TEM) genes, whereas the remaining two did. No genes encoding SHV or extended-spectrum beta-lactamases were detected. Two new variants of bla (TEM) were detected, which have been designated bla (TEM-127) and bla (TEM-128). In TEM-127, amino acid 158 is substituted from His to Asn, whereas a substitution from Asp to Glu is seen at amino acid 157 in TEM-128. According to MIC determinations, these novel enzymes do not possess activity against extended-spectrum beta-lactams.     

Occurrence of extended spectrum beta-lactamases among isolates of Enterobacteriaceae from urinary tract infections in southern Italy

The present investigation was undertaken to assess the prevalence of extended-spectrum beta-lactamases (ESbetaLs) among urinary tract infection (UTI) isolates. During 4 months in 2004, a total of 650 Enterobacteriaceae strains from UTIs was collected by five clinical microbiology laboratories located in southern Italy and the beta-lactamase production was investigated. A total of 50 of the 650 isolates were double-disk positive and suspected of producing an ESbetaL; Escherichia coli (36.0%) and Klebsiella pneumoniae (32.0%) were the most common species among all ESbetaL producers. Characterization of ESbetaL determinants was carried out by the colony blot hybridization method, and polymerase chain reaction (PCR) and DNA sequencing in order to identify the presence of bla (TEM), bla (SHV), bla (PER), and bla (CTX-M) determinants. The ESbetaL variants found in this study were the following: TEM-15, TEM-24, TEM-52, TEM-134, SHV-12, CTX-M-1, CTX-M-3, CTX-M-15, and PER-1. As expected, the majority of the isolates were found to be susceptible to imipenem (94%), cefepime (54%) and piperacillin-tazobactam (54%). The results of this survey show the prevalence of ESbetaL enzymes among enterobacterial pathogens causing UTIs in southern Italy.     

Occurrence and diversity of integrons and beta-lactamase genes among ampicillin-resistant isolates from estuarine waters

The aim of the present study was to assess the occurrence and molecular diversity of beta-lactamase genes and integrons among Gram-negative ampicillin-resistant bacteria from Ria de Aveiro. Ampicillin-resistant isolates were selected and subjected to genotyping using REP-PCR. Representatives from each REP pattern were affiliated with the following taxa by sequencing the 16S rRNA gene: Aeromonas caviae, A. hydrophila, A. media, A. molluscorum, A. veronii, A. salmonicida, Aeromonas sp., Pseudomonas putida, Pseudomonas sp., Escherichia coli, Escherichia sp., Shigella sonnei, Shigella sp., Klebsiella pneumoniae, K. oxytoca, Raoultella ornithinolytica, R. terrigena, R. planticola, Citrobacter freundii, Morganella morganii and Enterobacter sp. Isolates affiliated with genera Escherichia or Shigella were identified as Escherichia coli using phenotypic-based tests. PCR was used to assess beta-lactamase encoding sequences (bla(TEM), bla(SHV), bla(CARB), bla(CTX-M), bla(IMP), bla(VIM), bla(CphA/IMIS), bla(OXA-A), bla(OXA-B), bla(OXA-C)), class 1 and class 2 integrases, and integron variable regions. Sequence analysis of PCR products was performed. beta-Lactamase genes were detected in 77.8% of the Enterobacteriaceae and in 10.5% of the Aeromonas. The most frequently detected gene was bla(TEM), followed by bla(SHV,)bla(OXA-B), bla(CphA/IMIS) and bla(CARB). Retrieved sequences shared high homology with previously described beta-lactamases. The intI1 gene was present in 29.6% of the Enterobacteriaceae and in 21% of the Aeromonas isolates. The intI2 gene was present in 4 isolates. A total of 13 cassettes included in 12 different cassette arrays were identified. The most frequently found resistance gene cassettes were aadA variants. Previous investigations based on cultivation-independent approaches revealed higher molecular diversity among beta-lactamase-encoding sequences in this estuary. This fact reinforces the hypothesis that cultivation-dependent approaches may underestimate the prevalence of antibiotic resistance genes in environmental samples and may introduce bias in the recovery of their molecular variants.     

Molecular characterization and occurrence of extended-spectrum beta-lactamase resistance genes among Salmonella enterica serovar Corvallis from Thailand, Bulgaria, and Denmark

Fifty nine Salmonella Corvallis isolates from humans and food products in Bulgaria, Denmark, and Thailand were examined for antimicrobial susceptibility and characterized by pulsed-field gel electrophoresis (PFGE). Cephalosporin-resistant isolates were examined for the presence of genes encoding beta-lactamases by PCR and sequencing. Ten different PFGE types were observed. One type (30 isolates) was recovered in all three countries; three types were found only in Bulgaria, two only in Denmark, two only in Thailand, and two both in Denmark and Thailand. Ten isolates were susceptible to all antimicrobial agents tested, whereas 41 were resistant to three or more antimicrobials. Most resistance was observed among the isolates from Bulgaria. Of the 25 isolates from Bulgaria, 20 displayed resistance to ampicillin and the cephalosporins ceftiofur and cephalothin. All 20 isolates tested negative for bla (CMY-1), bla (CMY-2), and bla (ACC), but positive for bla (SHV), of which five were sequenced to bla (SHV-2). Plasmid profiling and hybridization revealed that the bla (SHV) gene was located on plasmids of approximately 70 kb. Five plasmid profiles were found among these 20 isolates. The plasmid profiling confirmed the PFGE-type and was able to further subdivide the strains. Seventeen of these 20 isolates contained also bla (TEM), of which nine representatives were sequenced to bla (TEM-1B), or bla (TEM-1H). One isolate contained bla (CTX-M-15), bla (SHV-2), and bla (TEM-1H), with the bla (CTX-M-15), and bla (TEM-1H) genes located on a 63-kb transferable plasmid. This study showed a high frequency of resistance among S. Corvallis isolated from humans and food products in Bulgaria, with a lower frequency in Thailand and Denmark. The clonal relatedness among the isolates from three countries could indicate a recent spread of this serovar.     

Effects of phenotype and genotype on methods for detection of extended-spectrum-beta-lactamase-producing clinical isolates of Escherichia coli and Klebsiella pneumoniae in Norway

Consecutive clinical isolates of Escherichia coli (n = 87) and Klebsiella pneumoniae (n = 25) with reduced susceptibilities to oxyimino-cephalosporins (MICs > 1 mg/liter) from 18 Norwegian laboratories during March through October 2003 were examined for bla(TEM/SHV/CTX-M) extended-spectrum-beta-lactamase (ESBL) genes, oxyimino-cephalosporin MIC profiles, ESBL phenotypes (determined by the ESBL Etest and the combined disk and double-disk synergy [DDS] methods), and susceptibility to non-beta-lactam antibiotics. Multidrug-resistant CTX-M-15-like (n = 23) and CTX-M-9-like (n = 15) ESBLs dominated among the 50 ESBL-positive E. coli isolates. SHV-5-like (n = 9) and SHV-2-like (n = 4) ESBLs were the most prevalent in 19 ESBL-positive K. pneumoniae isolates. Discrepant ESBL phenotype test results were observed for one major (CTX-M-9) and several minor (TEM-128 and SHV-2/-28) ESBL groups and in SHV-1/-11-hyperproducing isolates. Negative or borderline ESBL results were observed when low-MIC oxyimino-cephalosporin substrates were used to detect clavulanic acid (CLA) synergy. CLA synergy was detected by the ESBL Etest and the DDS method but not by the combined disk method in SHV-1/-11-hyperproducing strains. The DDS method revealed unexplained CLA synergy in combination with aztreonam and cefpirome in three E. coli strains. The relatively high proportion of ESBL-producing E. coli organisms with a low ceftazidime MIC in Norway emphasizes that cefpodoxime alone or both cefotaxime and ceftazidime should be used as substrates for ESBL detection.     

First outbreak of multidrug-resistant Klebsiella pneumoniae producing both SHV-12-type extended-spectrum beta-lactamase and DHA-1-type AmpC beta-lactamase at a Korean hospital

PURPOSE: Coexistence of different classes of beta-lactamases in a single bacterial isolate may pose diagnostic and therapeutic challenges. We investigated a spread of Klebsiella pneumoniae isolates co-producing an AmpC beta-lactamase and an extended-spectrum beta-lactamase (ESBL) in a university hospital. MATERIALS AND METHODS: Over a three-month period, a total of 11 K. pneumoniae isolates, which exhibited resistance to cefotaxime, aztreonam, and cefoxitin, were isolated. These isolates showed positive to ESBLs by double disk tests. Minimal inhibitory concentrations (MICs) were determined by broth microdilution testing. All isolates were examined by isoelectric focusing, PCR and sequence analysis to identify bla(SHV) and bla(DHA), and molecular typing by pulsed-field gel electrophoresis (PFGE). RESULTS: All 11 isolates were highly resistant (MIC, > or = 128microg/ml) to ceftazidime, aztreonam, and cefoxitin, while they were susceptible (MIC, < or = 2microg/ml) to imipenem. The bla(SHV-12) and bla(DHA-1) genes were detected by PCR and sequence analysis. PFGE revealed a similar pattern in 10 of the 11 strains tested. CONCLUSION: This is the first outbreak report of K. pneumoniae in Korea which co-produced SHV-12 and DHA-1 beta-lactamase, and we suggest a clonal spread of multidrug-resistant K. pneumoniae at a hospital.     

Dissemination of SHV-12 and characterization of new AmpC-type beta-lactamase genes among clinical isolates of enterobacter species in Korea

To determine the prevalence and genotype of an extended-spectrum beta-lactamase and new chromosomal AmpC beta-lactamases among clinical isolates of Enterobacter species, we performed antibiotic susceptibility testing, pI determination, induction tests, transconjugation, enterobacterial repetitive consensus (ERIC) PCR, sequencing, and phylogenetic analysis. Among the 51 clinical isolates collected from a university hospital in Korea, 6 isolates have been shown to produce SHV-12 and inducible AmpC beta-lactamases. These also included three isolates producing TEM-1b and one strain carrying TEM-1b and CMY-type beta-lactamases with a pI of 8.0. The results from ERIC PCR revealed that six isolates were genetically unrelated, suggesting that dissemination of SHV-12 was responsible for the spread of resistance to extended-spectrum beta-lactams in Korea. Six genes of inducible AmpC beta-lactamases that are responsible for the resistance to cephamycins (cefoxitin and cefotetan), amoxicillin, cephalothin, and amoxicillin-clavulanic acid were cloned and characterized. A 1,165-bp DNA fragment containing the ampC genes was sequenced and found to have an open reading frame coding for a 381-amino-acid beta-lactamase. The nucleotide sequence of four ampC genes (bla(EcloK992004.1), bla(EcloK995120.1), bla(EcloK99230), and bla(EareK9911729)) shared considerable homology with that of AmpC-type class C beta-lactamase genes of gram-negative bacteria, especially that of the chromosomal ampC gene (bla(EcloMHN1)) of Enterobacter cloacae MHN1 (99.9, 99.7, 99.6, and 99.6% identity, respectively). The sequences of two ampC genes (bla(EcloK9973) and bla(EcloK9914325)) showed close similarity to the chromosomal ampC gene (bla(EcloQ908R)) of E. cloacae Q908R (99.7% identity). The results from phylogenetic analysis suggested that six ampC genes could originate from bla(EcloMHN1) or bla(EcloQ908R).