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.     

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.     

Discrimination of SHV β-Lactamase Genes by Restriction Site Insertion-PCR

Restriction site insertion-PCR (RSI-PCR) is a simple, rapid technique for detection of point mutations. This technique exploits primers with one to three base mismatches near the 3' end to modulate a restriction site. We have developed this technique to identify described mutations of the bla(SHV) genes for differentiation of SHV variants that cannot be distinguished easily by other techniques. To validate this method, eight standard strains were used, each producing a different SHV beta-lactamase: SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, SHV-6, SHV-8, and SHV-18. Mismatch primers were designed to detect mutations affecting amino acids at positions 8 (SspI), 179 (HinfI), 205 (PstI), 238 (Gly-->Ala) (BsrI), and 240 (NruI) of bla(SHV) genes. All amplimers of the bla(SHV) genes used in this study yielded the predicted restriction endonuclease digestion products. In addition, this study also makes theoretical identification of bla(SHV-6), bla(SHV-8), and 12 novel bla(SHV) variants using the PCR-restriction fragment length polymorphism (RFLP) technique possible. By using a combination of PCR-RFLP and RSI-PCR techniques, up to 27 SHV variants can now be distinguished rapidly and reliably. These simple techniques are readily applied to epidemiological studies of the SHV beta-lactamases and may be extended to the characterisation of other resistance determinants.     

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.     

Relationship between blaSHV-12 and blaSHV-2a in Korea

In contrast to the USA and Europe, where SHV-2, SHV-4 and SHV-5 are the prevalent extended-spectrum SHV enzymes, in Korea SHV-2a and SHV-12 are the most frequently identified extended-spectrum SHV enzymes. A 6.6 kb BamHI fragment containing the bla(SHV-12) gene of strain K7746 isolated from one university hospital in Korea was cloned into the pCRScriptCAM vector. Sequencing of the constructed recombinant plasmid pK7746-C1 revealed that the immediate upstream sequence of the bla(SHV-12) gene showed little similarity to the part of the prototype bla(SHV-1) gene due to the insertion of an IS26 element next to the -10 region. Instead, the upstream sequences of bla(SHV-12) retained 100% DNA identity with the part of plasmid pMPA2a from Klebsiella pneumoniae KPZU-3 carrying bla(SHV-2a). The restriction map of the inserted 6.6 kb DNA fragment of plasmid pK7746-C1 was also homologous to that of plasmid pMPA2a, suggesting a common lineage of bla(SHV-12) and bla(SHV-2a). We also studied, using PCR, the upstream non-coding region of several SHV beta-lactamase genes for the presence of IS26 sequence. The flanking IS26 sequence in the immediate upstream region of the bla(SHV) gene was not detected in five standard strains producing SHV-1, SHV-2, SHV-3, SHV-4 or SHV-5. However, IS26 was detected in all 69 clinical strains producing SHV-2a or SHV-12 isolated from three university hospitals in Korea during 1993-1999. The above findings suggest a direct evolution of SHV-12 from SHV-2a, not from SHV-2 to -5, and it is considered to be one of the reasons for the absolute predominance of SHV-2a and SHV-12 in Korea.     

Characterization of the extended-spectrum beta-lactamase reference strain, Klebsiella pneumoniae K6 (ATCC 700603), which produces the novel enzyme SHV-18

Klebsiella pneumoniae K6 (ATCC 700603), a clinical isolate, is resistant to ceftazidime and other oxyimino-beta-lactams. A consistent reduction in the MICs of oxyimino-beta-lactams by at least 3 twofold dilutions in the presence of clavulanic acid confirmed the utility of K. pneumoniae K6 as a quality control strain for extended-spectrum beta-lactamase (ESBL) detection. Isoelectric-focusing analysis of crude lysates of K6 demonstrated a single beta-lactamase with a pI of 7.8 and a substrate profile showing preferential hydrolysis of cefotaxime compared to ceftazidime. PCR analysis of total bacterial DNA from K6 identified the presence of a bla(SHV) gene. K6 contained two large plasmids with molecular sizes of approximately 160 and 80 kb. Hybridization of plasmid DNA with a bla(SHV)-specific probe indicated that a bla(SHV) gene was encoded on the 80-kb plasmid, which was shown to transfer resistance to ceftazidime in conjugal mating experiments with Escherichia coli HB101. DNA sequencing of this bla(SHV)-related gene revealed that it differs from bla(SHV-1) at nine nucleotides, five of which resulted in amino acid substitutions: Ile to Phe at position 8, Arg to Ser at position 43, Gly to Ala at position 238, and Glu to Lys at position 240. In addition to the production of this novel ESBL, designated SHV-18, analysis of the outer membrane proteins of K6 revealed the loss of the OmpK35 and OmpK37 porins.     

Carbapenem-resistant strain of Klebsiella oxytoca harboring carbapenem-hydrolyzing beta-lactamase KPC-2

We investigated a Klebsiella oxytoca isolate demonstrating resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The MICs of both imipenem and meropenem were 32 microg/ml. The beta-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. Isoelectric focusing studies demonstrated five beta-lactamases with pIs of 8.2 (SHV-46), 6.7 (KPC-2), 6.5 (unknown), 6.4 (probable OXY-2), and 5.4 (TEM-1). The presence of the bla(SHV) and bla(TEM) genes was confirmed by specific PCR assays and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the beta-lactamase with a pI of 6.7, Klebsiella pneumoniae carbapenemase-2 (KPC-2), was encoded on an approximately 70-kb conjugative plasmid that also carried SHV-46, TEM-1, and the beta-lactamase with a pI of 6.5. The bla(KPC-2) determinant was cloned in E. coli and conferred resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of KPC-2 showed a single amino acid difference, S174G, when compared with KPC-1, another carbapenem-hydrolyzing beta-lactamase from K. pneumoniae 1534. Hydrolysis studies showed that purified KPC-2 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and aztreonam. KPC-2 had the highest affinity for meropenem. The kinetic studies revealed that KPC-2 was inhibited by clavulanic acid and tazobactam. An examination of the outer membrane proteins of the parent K. oxytoca strain demonstrated that it expressed detectable levels of OmpK36 (the homolog of OmpC) and a higher-molecular-weight OmpK35 (the homolog of OmpF). Thus, carbapenem resistance in K. oxytoca 3127 is due to production of the Bush group 2f, class A, carbapenem-hydrolyzing beta-lactamase KPC-2. This beta-lactamase is likely located on a transposon that is part of a conjugative plasmid and thus has a very high potential for dissemination.     

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.     

Prevalence of Ambler class A and D beta-lactamases among clinical isolates of Pseudomonas aeruginosa in Korea

OBJECTIVES: Recently, resistance to extended-spectrum cephalosporins due to acquired beta-lactamases has been reported in Pseudomonas aeruginosa. The aim of this study was to investigate the prevalence of Ambler class A and D beta-lactamases and their extended-spectrum derivatives and antimicrobial susceptibilities of P. aeruginosa isolated from various parts of Korea. METHODS: A total of 252 consecutive, non-duplicate isolates of P. aeruginosa were studied for the presence of class A or D beta-lactamase. Antibiotic susceptibility tests and PCR amplification of genes encoding class A (bla(PSE-1), bla(PER-1), bla(VEB-1), bla(TEM), bla(SHV), bla(CTX-M) and bla(GES-1)) and class D beta-lactamases (bla(OXA-groupI), bla(OXA-groupII) and bla(OXA-groupIII)) were performed. For PCR-positive isolates, isoelectric focusing (IEF) analysis, sequencing and pulsed-field gel electrophoresis (PFGE) were performed. RESULTS: In 64 (25.4%) isolates, structural genes for PSE-1 (6.3%), OXA-10 (13.1%), OXA-4 (4.3%), OXA-30 (2.0%), OXA-2 (2.3%) and OXA-17 (0.4%) were found; their distribution varied between provinces. None harboured bla(PER-1), bla(VEB-1), bla(TEM), bla(SHV), bla(CTX-M) and bla(GES-1). The cross-class resistance rates to other antibiotics was significantly higher in class A and D beta-lactamase producers than in non-producers (P < 0.001 for aminoglycosides, ciprofloxacin and meropenem). CONCLUSIONS: OXA-type beta-lactamases are widespread, but their extended-spectrum derivatives are rare among P. aeruginosa in Korea. To our knowledge, this is the first report of OXA-17, an extended-spectrum derivative of OXA-10, outside the Middle East. In addition, combined resistance to ticarcillin and aminoglycosides was a useful indicator for P. aeruginosa producing PSE- or OXA-type beta-lactamases in this study.     

Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae

A Klebsiella pneumoniae isolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 microg/ml. The beta-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three beta-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence of bla(SHV) and bla(TEM) genes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the beta-lactamase with a pI of 6.7, KPC-1 (K. pneumoniae carbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene, bla(KPC-1), was cloned in E. coli and shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel carbapenem-hydrolyzing beta-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing beta-lactamase, Sme-1, from Serratia marcescens S6. Hydrolysis studies showed that purified KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest affinity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parent K. pneumoniae strain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present. We concluded that carbapenem resistance in K. pneumoniae strain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing beta-lactamase, KPC-1, although alterations in porin expression may also play a role.     

Comparative activities of clavulanic acid, sulbactam, and tazobactam against clinically important beta-lactamases.

Clavulanic acid, sulbactam, and tazobactam are inhibitors of a variety of plasmid-mediated beta-lactamases. However, inhibition data for these three inhibitors with a wide range of different plasmid-mediated beta-lactamases have not yet been compared under the same experimental conditions. A number of groups have inferred that clavulanic acid inhibits extended-spectrum TEM and SHV beta-lactamases, but inhibition data have rarely been published. In this study, the 50% inhibitory concentrations of these three beta-lactamase inhibitors for 35 plasmid-mediated beta-lactamases have been determined. Of these 35 beta-lactamases, 20 were extended-spectrum TEM- or SHV-derived beta-lactamases. The other 15 enzymes were conventional-spectrum beta-lactamases such as TEM-1 and SHV-1. Clavulanic acid was a more potent inhibitor than sulbactam for 32 of the 35 plasmid-mediated beta-lactamases tested. In particular, clavulanic acid was 60 and 580 times more potent than sulbactam against TEM-1 and SHV-1, respectively, currently the two most clinically prevalent gram-negative plasmid-mediated beta-lactamases. Statistical analysis of the data of the 50% inhibitory concentrations showed that clavulanic acid was 20 times more active overall than sulbactam against the conventional-spectrum enzymes. In addition, clavulanic acid was 14 times more potent than sulbactam at inhibiting the extended-spectrum enzymes. Tazobactam also showed significantly greater activity than sulbactam against the two groups of beta-lactamases. There were no significant differences between the overall activities of tazobactam and clavulanic acid against the extended-spectrum TEM and SHV enzymes and conventional-spectrum enzymes, although differences in their inhibition profiles were observed.     

Complexity and diversity of Klebsiella pneumoniae strains with extended-spectrum beta-lactamases isolated in 1994 and 1996 at a teaching hospital in Durban, South Africa

beta-Lactamase production was investigated in cultures of 25 Klebsiella pneumoniae isolates isolated at a hospital in Durban, South Africa, in 1994 and 1996. Twenty of these isolates gave ceftazidime MIC/ceftazidime plus clavulanate MIC ratios of >/=8, implying production of extended-spectrum beta-lactamases (ESBLs), and DNA sequencing identified an ESBL gene (bla(TEM-53)) in a further two isolates. Pulsed-field gel electrophoresis (PFGE) defined 4 distinct strains among the 12 isolates collected in 1994 and 9 distinct strains among the 13 isolates collected in 1996. In three cases, multiple isolates from single patients varied in their PFGE profiles and antibiograms, implying mixed colonization or infection. Isoelectric focusing and DNA hybridization found both TEM and SHV enzymes and their genes in all 25 isolates. Many isolates had multiple identical or different beta-lactamase gene variants, with at least 84 bla(SHV) and bla(TEM) gene copies among the 25 organisms. Sequencing identified the genes for the SHV-1, -2, and -5 enzymes and for four new SHV types (SHV-19, -20, -21, and -22). These new SHV variants had novel mutations remote from sites known to affect catalytic activity. Sequencing also found the genes for TEM-1, TEM-53, and one novel type, TEM-63. All the isolates had multiple and diverse plasmids. These complex and diverse patterns of ESBL production and strain epidemiology are far removed from the concept of an ESBL outbreak and suggest a situation in which ESBL production has become endemic and in which evolution is generating a wide range of enzyme combinations. This complexity and diversity complicates patient management and the design of antibiotic use policies.     

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.     

CTX-M-2 and a new CTX-M-39 enzyme are the major extended-spectrum beta-lactamases in multiple Escherichia coli clones isolated in Tel Aviv, Israel

The rate of occurrence of the extended-spectrum beta-lactamase (ESBL)-producing phenotype among Escherichia coli isolates in Tel Aviv is 12% (22). The aim of this study was to understand the molecular epidemiology of E. coli ESBL producers and to identify the ESBL genes carried by them. We studied 20 single-patient ESBL-producing E. coli clinical isolates. They comprised 11 distinct nonrelated pulsed-field gel electrophoresis (PFGE) genotypes: six isolates belonged to the same PFGE clone, four other clones included two isolates each, and six unrelated clones included only one isolate. All isolates produced various beta-lactamases with pIs ranging from 5.2 to 8.2, varying within similar PFGE clones. The most prevalent ESBL gene was bla(CTX-M); 16 isolates carried bla(CTX-M-2) and three carried a new ESBL gene designated bla(CTX-M-39). Three strains carried bla(SHV) (two bla(SHV-12) and one bla(SHV-5)), and two strains carried inhibitor-resistant ESBL genes, bla(TEM-33) and bla(TEM-30); 18 strains carried bla(TEM-1) and eight strains carried bla(OXA-2). Plasmid mapping and Southern blot analysis with a CTX-M-2 probe demonstrated that bla(CTX-M-2) is plasmid borne. The wide dissemination of ESBLs among E. coli isolates in our institution is partly related to clonal spread, but more notably to various plasmid-associated ESBL genes, occurring in multiple clones, wherein the CTX-M gene family appears almost uniformly. We report here a new CTX-M gene, designated bla(CTX-M-39), which revealed 99% homology with bla(CTX-M-26), with a substitution of arginine for glutamine at position 225.     

Predominance and genetic diversity of community- and hospital-acquired CTX-M extended-spectrum beta-lactamases in York, UK

OBJECTIVES: This study was conducted to detect the presence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae within the faecal flora of both community- and hospital-based patients in York and to characterize the bla(TEM), bla(SHV) and bla(CTX-M) genes present in these isolates. METHODS: One thousand faeces samples were collected and screened at York Hospital during October-December 2003. Ninety-five non-duplicate Enterobacteriaceae isolates resistant to third-generation cephalosporins were recovered; 22 isolates were selected for further study on the basis of a positive double disc diffusion test for ESBL production. Antibiotic susceptibility testing was performed to a range of antibiotics. The TEM, SHV and CTX-M genes were detected by PCR and the DNA sequenced. RESULTS: The distribution of ESBL-positive isolates from the hospital and community was 1.4:1. These included nine Escherichia coli, seven Enterobacter cloacae, four Citrobacter freundii and a single isolate each of Klebsiella spp. and Salmonella spp. A total of 17 isolates contained bla(CTX-M) (five bla(CTX-M-15), three bla(CTX-M-14) and nine bla(CTX-M-9)). ISEcp1 was present in isolates expressing CTX-M-14 and -15, but was absent upstream of In60-associated bla(CTX-M-9). E. coli isolates also contained either a bla(TEM-1) or bla(TEM-2), whereas six of the E. cloacae carried bla(SHV-12) and the Klebsiella spp. bla(SHV-36) in addition to bla(CTX-M-9). The single Salmonella spp. carried bla(SHV-12). CONCLUSIONS: The overall prevalence of ESBL in isolates of Enterobacteriaceae from York was 1.9%. ESBL-producing isolates were found in both the community and hospital, with the CTX-M type most common. This is also the first report of an ESBL-producing Salmonella in the UK.     

Detection of extended-spectrum beta-lactamases in members of the family Enterobacteriaceae: comparison of the double-disk and three-dimensional tests.

The three-dimensional and clavulanate double-disk potentiation tests were compared as procedures for the detection of extended-spectrum beta-lactamase production in 32 strains of Escherichia coli and Klebsiella pneumoniae, 31 of which produced TEM-1, TEM-2, TEM-3, TEM-4, TEM-5, TEM-7, TEM-8, TEM-9, TEM-10, TEM-12, TEM-101, SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, CAZ-2, MIR-1, or an unidentified extended-spectrum beta-lactamase with a pI of 5.95, with some strains producing multiple beta-lactamases. The three-dimensional test, which was performed in conjunction with a routine disk diffusion test, detected extended-spectrum beta-lactamase production in 26 of 28 (93%) of the strains that produced extended-spectrum beta-lactamases. The clavulanate double-disk potentiation test detected extended-spectrum beta-lactamases in only 22 of the 28 strains (79%) when it was performed as currently recommended. The three-dimensional test, when performed in conjunction with the disk diffusion test, offered the advantages of providing simultaneous information about both antibiotic susceptibility and extended-spectrum beta-lactamase production, coupled with a greater sensitivity and earlier detection of extended-spectrum beta-lactamases.     

SHV-12, SHV-5, SHV-2a and VEB-1 extended-spectrum beta-lactamases in Gram-negative bacteria isolated in a university hospital in Thailand.

Sixty-one extended-spectrum beta-lactamase (ESBL)-producing isolates were collected from Srinagarind Hospital, Thailand. These included 43 Enterobacteriaceae and 18 Pseudomonadaceae. The 43 Enterobacteriaceae were found to produce the following ESBLs: 26 (60.5%) SHV-12, 13 (30.2%) SHV-5, two (4.7%) SHV-2a, one (2.3%) VEB-1 and one (2.3%) unidentified. Twenty-four isolates (55.8%) also carried bla(TEM-1B), as well as bla(SHV) or bla(VEB-1). Plasmid DNA from transconjugants carrying the bla(SHV-12) gene showed various restriction patterns, indicating the distribution of the bla(SHV-12) gene among different antibiotic resistance plasmids. In contrast, bla(SHV-5) in 13 isolates was found on a single plasmid of c. 130 kb. Pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA from these isolates revealed that nine of 11 Klebsiella pneumoniae gave the same pattern, indicating clonal spread of the strain within the hospital, together with the occasional spread of the plasmid to other strains. Among the pseudomonad isolates, 16 Pseudomonas aeruginosa and one Pseudomonas putida had bla(VEB-like) and one P. aeruginosa had bla(SHV-12). Nine of the 16 isolates carrying bla(VEB-like) (56.3%) had identical PFGE patterns, suggesting the dissemination of this gene, also by clonal spread. At least six different bla(VEB-like-)containing integrons were found among the 18 isolates. This is the first report of bacteria producing SHV-12 and SHV-2a in Thailand and the first report of SHV-12 in P. aeruginosa, of VEB-1 in Citrobacter freundii and a VEB-1-like beta-lactamase in P. putida. These findings indicate that ESBL genes in the Far East are part of a gene pool capable of broad horizontal gene transfer, in that these genes can transfer between different families of Gram-negative bacilli.     

Unusual Salmonella enterica serotype Typhimurium isolate producing CMY-7, SHV-9 and OXA-30 beta-lactamases

Beta-lactam resistance in Salmonella isolates is increasing. This paper describes the combination of three different beta-lactamases, OXA-30, SHV-9 and CMY-7, expressed by an isolate of Salmonella enterica serotype Typhimurium. This is the first report of an isolate of Salmonella having both an extended-spectrum beta-lactamase and an AmpC beta-lactamase.     

SHV型β内酰胺酶新亚型SHV-71的发现

目的了解鲍曼不动杆菌中SHV型β内酰胺酶的基因型及其耐药性。方法采用纸片扩散法(K-B法)测定5株鲍曼不动杆菌对13种抗菌药物的敏感性;设计SHV型β内酰胺酶引物,采用PCR法获得SHV型酶编码基因的片段,并将PCR扩增产物克隆入pMD18-T载体,以双脱氧链终止法测定核苷酸序列,确定亚型。结果药敏试验结果显示:5株鲍曼不动杆菌对氨苄西林、哌拉西林、头孢西丁、头孢他啶和氨曲南均耐药。3株菌SHV基因型扩增结果呈阳性,其中2株与SHV-12的序列100%相同,另1株(No-03)经GenBank网上同源性比较,未发现完全相同的核苷酸和氨基酸序列,为一种新发现的SHV亚型β内酰胺酶耐药基因,其序列以SHV-71的名称在美国核酸数据库GenBank成功登录(GenBank注册号:DQ296194)。结论临床分离的鲍曼不动杆菌No-03株所产SHV型BLA为一种新SHV亚型β内酰胺酶。