Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum beta-lactamase encoded by an Escherichia coli integron gene

A clinical isolate, Escherichia coli MG-1, isolated from a 4-month-old Vietnamese orphan child, produced a beta-lactamase conferring resistance to extended-spectrum cephalosporins and aztreonam. In a disk diffusion test, a typical synergistic effect between ceftazidime or aztreonam and clavulanic acid was observed along with an unusual synergy between cefoxitin and cefuroxime. The gene for VEB-1 (Vietnamese extended-spectrum beta-lactamase) was cloned and expressed in E. coli JM109. The recombinant plasmid pRLT1 produced a beta-lactamase with a pI of 5.35 and conferred high-level resistance to extended-spectrum (or oxyimino) cephalosporins and to aztreonam. Vmax values for extended-spectrum cephalosporins were uncommonly high, while the affinity of the enzyme for ceftazidime and aztreonam was relatively low. blaVEB-1 showed significant homology at the DNA level with only blaPER-1 and blaPER-2. Analysis of the deduced protein sequence showed that VEB-1 is a class A penicillinase having very low levels of homology with any other known beta-lactamases. The highest percentage of amino acid identity was 38% with PER-1 or PER-2, two uncommon class A extended-spectrum enzymes. Exploration of the genetic environment of blaVEB-1 revealed the presence of gene cassette features, i.e., (i) a 59-base element associated with blaVEB-1; (ii) a second 59-base element just upstream of blaVEB-1, likely belonging to the aacA1-orfG gene cassette; (iii) two core sites (GTTRRRY) on both sides of blaVEB-1; and (iv) a second antibiotic resistance gene 3' of blaVEB-1, aadB. blaVEB-1 may therefore be the first class A extended-spectrum beta-lactamase that is part of a gene cassette, which itself is likely to be located on a class 1 integron, as sulfamide resistance may indicate. Furthermore, blaVEB-1 is encoded on a large (> 100-kb) transferable plasmid found in a Klebsiella pneumoniae MG-2 isolated at the same time from the same patient, indicating a horizontal gene transfer.     

Genetic-biochemical analysis and distribution of the Ambler class A beta-lactamase CME-2, responsible for extended-spectrum cephalosporin resistance in Chryseobacterium (Flavobacterium) meningosepticum

In vitro synergy between extended-spectrum cephalosporins and either clavulanic acid or cefoxitin was found for Chryseobacterium meningosepticum isolates during a double-disk assay on an agar plate. An extended-spectrum beta-lactamase (ESBL) gene from a C. meningosepticum clinical isolate was cloned and expressed in Escherichia coli DH10B. Its protein conferred resistance to most beta-lactams including extended-spectrum cephalosporins but not to cephamycins or to imipenem. Its activity was strongly inhibited by clavulanic acid, sulbactam, and tazobactam, as well as by cephamycins and imipenem. Sequence analysis of the cloned DNA fragment revealed an open reading frame (ORF) of 891 bp with a G+C content of 33.9%, which lies close to the expected range of G+C contents of members of the Chryseobacterium genus. The ORF encoded a precursor protein of 297 amino acids, giving a mature protein with a molecular mass of 31 kDa and a pI value of 9.2 in E. coli. This gene was very likely chromosomally located. Amino acid sequence comparison showed that this beta-lactamase, named CME-2 (C. meningosepticum ESBL), is a novel ESBL of the Ambler class A group (Bush functional group 2be), being weakly related to other class A beta-lactamases. It shares only 39 and 35% identities with the ESBLs VEB-1 from E. coli MG-1 and CBL-A from Bacteroides uniformis, respectively. The distribution of bla(CME-2) among unrelated C. meningosepticum species isolates showed that bla(CME-2)-like genes were found in the C. meningosepticum strains studied but were absent from strains of other C. meningosepticum-related species. Each C. meningosepticum strain produced at least two beta-lactamases, with one of them being a noninducible serine ESBL with variable pIs ranging from 7.0 to 8.5.     

Control of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in a children's hospital by changing antimicrobial agent usage policy

OBJECTIVES: This ambidirectional intervention study was performed to examine the impact of a change in antibiotic policy on extended-spectrum beta-lactamase (ESBL) prevalence in a children's hospital with a high prevalence of ESBL production among Escherichia coli and Klebsiella pneumoniae. METHODS: The use of extended-spectrum cephalosporins was restricted and use of beta-lactam/ beta-lactamase inhibitor combinations was encouraged from 2002. All strains of E. coli and K. pneumoniae isolated from sterile body fluids from 1999 to 2005 were analysed for beta-lactamase production and the prevalences of ESBL production were compared at three periods; pre-intervention (1999-2001), transitional period (2002-03) and post-intervention (2004-05). RESULTS: Comparing the pre- and post-intervention periods, overall piperacillin/tazobactam use increased from 2.2 to 108.0 days on antibiotics/1000 patient admission days/year (AD) (P for trend < 0.001), whereas extended-spectrum cephalosporin use decreased from 175.0 to 96.9 AD (P for trend < 0.001). Among 252 strains of E. coli (n = 128) and K. pneumoniae (n = 124), the overall prevalence of ESBL producers decreased from 39.8% (41/103) to 22.8% (18/79) (P for trend = 0.018). This decreasing trend of ESBL production was more evident for K. pneumoniae (64.1% to 25.6%; P for trend < 0.001) than E. coli (25.0% to 19.4%; P for trend = 0.514). The mortality rates of invasive disease caused by E. coli or K. pneumoniae remained unchanged. CONCLUSIONS: The substitution of piperacillin/tazobactam for extended-spectrum cephalosporins successfully decreased the prevalence of ESBL production of K. pneumoniae and E. coli in an institute for children where ESBLs were endemic. The impact of change in antibiotic policy was more evident in K. pneumoniae than E. coli.     

AmpC beta-lactamase in an Escherichia coli clinical isolate confers resistance to expanded-spectrum cephalosporins

Cloning, sequencing, and biochemical analysis identified a novel AmpC-type beta-lactamase conferring resistance to extended-spectrum cephalosporins in an Escherichia coli clinical isolate. This enzyme, exhibiting 14 amino acid substitutions compared to a reference AmpC cephalosporinase of E. coli, hydrolyzed ceftazidime and cefepime significantly.     

Haemophilus influenzae bla(ROB-1) mutations in hypermutagenic deltaampC Escherichia coli conferring resistance to cefotaxime and beta-lactamase inhibitors and increased susceptibility to cefaclor

The clinical use of cefaclor has been shown to enrich Haemophilus influenzae populations harboring cefaclor-hydrolyzing ROB-1 beta-lactamase. Such a selective process may lead to the increased use of extended-spectrum cephalosporins or beta-lactams plus beta-lactamase inhibitors and, eventually, resistance to these agents, which has not previously been observed in H. influenzae. In order to establish which bla(ROB-1) mutations, if any, could confer resistance to extended-spectrum cephalosporins and/or to beta-lactamase inhibitors, a plasmid harboring bla(ROB-1) was transformed into hypermutagenic strain Escherichia coli GB20 (DeltaampC mutS::Tn10), and this construct was used in place of H. influenzae bla(ROB-1). Strain GB20 with the cloned gene was submitted to serial passages in tubes containing broth with increasing concentrations of selected beta-lactams (cefotaxime or amoxicillin-clavulanate). Different mutations in the bla(ROB-1) gene were obtained during the passages in the presence of the different concentrations of the selective agents. Mutants resistant to extended-spectrum cephalosporins harbored either the Leu169-->Ser169 or the Arg164-->Trp164 substitution or the double amino acid change Arg164-->Trp164 and Ala237-->Thr237. ROB-1 mutants that were resistant to beta-lactams plus beta-lactamase inhibitors and that harbored the Arg244-->Cys244 or the Ser130-->Gly130 replacement were also obtained. The cefaclor-hydrolyzing efficiencies of the ROB-1 variants were strongly decreased in all mutants, suggesting that if bla(ROB-1) mutants were selected by cefaclor, this drug would prevent the further evolution of this beta-lactamase toward molecular forms able to resist extended-spectrum cephalosporins or beta-lactamase inhibitors.     

Epidemiology of conjugative plasmid-mediated AmpC beta-lactamases in the United States

A sample of 752 resistant Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli strains from 70 sites in 25 U.S. states and the District of Columbia was examined for transmissibility of resistance to ceftazidime and the nature of the plasmid-mediated beta-lactamase involved. Fifty-nine percent of the K. pneumoniae, 24% of the K. oxytoca, and 44% of the E. coli isolates transferred resistance to ceftazidime. Plasmids encoding AmpC-type beta-lactamase were found in 8.5% of the K. pneumoniae samples, 6.9% of the K. oxytoca samples, and 4% of the E. coli samples, at 20 of the 70 sites and in 10 of the 25 states. ACT-1 beta-lactamase was found at eight sites, four of which were near New York City, where the ACT-1 enzyme was first discovered; ACT-1 beta-lactamase was also found in Massachusetts, Pennsylvania, and Virginia. FOX-5 beta-lactamase was also found at eight sites, mainly in southeastern states but also in New York. Two E. coli strains produced CMY-2, and one K. pneumoniae strain produced DHA-1 beta-lactamase. Pulsed-field gel electrophoresis and plasmid analysis suggested that AmpC-mediated resistance spread both by strain and plasmid dissemination. All AmpC beta-lactamase-containing isolates were resistant to cefoxitin, but so were 11% of strains containing transmissible SHV- and TEM-type extended-spectrum beta-lactamases. A beta-lactamase inhibitor test was helpful in distinguishing the two types of resistance but was not definitive since 24% of clinical isolates producing AmpC beta-lactamase had a positive response to clavulanic acid. Coexistence of AmpC and extended-spectrum beta-lactamases was the main reason for these discrepancies. Plasmid-mediated AmpC-type enzymes are thus responsible for an appreciable fraction of resistance in clinical isolates of Klebsiella spp. and E. coli, are disseminated around the United States, and are not so easily distinguished from other enzymes that mediate resistance to oxyimino-beta-lactams.     

Plasmid-mediated beta-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli resistant to ceftazidime.

Low-level transferable resistance to ceftazidime was detected in seven strains of Klebsiella pneumoniae and one strain of Escherichia coli. Six of the Klebsiella strains and the E. coli strain were shown to produce a novel beta-lactamase (CAZ-lo) with a pI of 5.6 that hydrolyzed broad-spectrum cephalosporins at low but comparable levels. One strain of K. pneumoniae was of a serotype different from that of the other strains and produced a plasmid-encoded cefuroximase (FUR) with a pI of 7.5 that mediated moderate levels of resistance to different broad-spectrum cephalosporins. High-level resistance to ceftazidime was detected in one other strain of K. pneumoniae, which produced a beta-lactamase with a pI of 6.5 (CAZ-hi). Apart from its pI, this enzyme differed from CAZ-lo by a specific and high hydrolytic activity against ceftazidime. The epidemiological context suggested that CAZ-hi may be a mutant of CAZ-lo, and this hypothesis was supported by the isolation of laboratory mutants of CAZ-lo showing properties identical to those of the clinical CAZ-hi enzyme.     

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.     

Biochemical properties of a carbapenem-hydrolyzing beta-lactamase from Enterobacter cloacae and cloning of the gene into Escherichia coli.

A clinical isolate of Enterobacter cloacae, strain NOR-1, exhibited resistance to imipenem and remained susceptible to extended-spectrum cephalosporins. Clavulanic acid partially restored the susceptibility of the strain to imipenem. Two beta-lactamases with isoelectric points (pI) of 6.9 and > 9.2 were detected in strain E. cloacae NOR-1; the higher pI corresponded to AmpC cephalosporinase. Plasmid DNA was not detected in E. cloacae NOR-1 and imipenem resistance could not be transferred into Escherichia coli JM109. The carbapenem-hydrolyzing beta-lactamase gene was cloned into plasmid pACYC184. One recombinant plasmid, pPTN1, harbored a 5.3-kb Sau3A fragment from E. cloacae NOR-1 expressing the carbapenem-hydrolyzing beta-lactamase. This enzyme (pI 6.9) hydrolyzed ampicillin, cephalothin, and imipenem more rapidly than it did meropenem and aztreonam, but it hydrolyzed extended-spectrum cephalosporins only weakly and did not hydrolyze cefoxitin. Hydrolytic activity was partially inhibited by clavulanic acid, sulbactam, and tazobactam, was nonsusceptible to chelating agents such as EDTA and 1,10-o-phenanthroline, and was independent of the presence of ZnCl2. Its relative molecular mass was 30,000 Da. Induction experiments concluded that the carbapenem-hydrolyzing beta-lactamase biosynthesis was inducible by cefoxitin and imipenem. Subcloning experiments with HindIII partial digests of pPTN1 resulted in a recombinant plasmid, designated pPTN2, which contained a 1.3-kb insert from pPTN1 and which conferred resistance to beta-lactam antibiotics. Hybridization studies performed with a 1.2-kb HindIII fragment from pPtN2 failed to determine any homology with ampC of E. cloacae, with other known beta-lactamase genes commonly found in members of the family Enterobacteriaceae (bla(TEM-1)) and bla(SHV-3) derivatives), and with previously described carbapenemase genes such as those from Xanthomonas maltophilia, Bacillus cereus, Bacteroides fragilis (cfiA), and Aeromonas hydrophila (cphA). This work describing the biochemical properties of a novel chromosome-encoded beta-lactamase from E. cloacae indicates that this enzyme differs from all the previously described carbapenemases. This is the first reported cloning of a carbapenem-hydrolyzing gene from a member of the family Enterobacteriaceae.     

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.     

TEM-4, a new plasmid-mediated beta-lactamase that hydrolyzes broad-spectrum cephalosporins in a clinical isolate of Escherichia coli.

A clinical isolate of Escherichia coli, strain CB-134, recovered in 1986 from an abdominal abscess, exhibited resistance to penams, oxyimino-beta-lactams including broad-spectrum cephalosporins (cefotaxime, ceftriaxone, ceftazidime), and aztreonam but remained susceptible to cephamycins (cefoxitin, cefotetan) and to moxalactam and imipenem. Clavulanate (2 micrograms/ml) restored the susceptibility of the strain to broad-spectrum cephalosporins and aztreonam. A beta-lactamase with an isoelectric point (pI) of 5.9 was detected in strain CB-134, and the corresponding gene was transferred by conjugation to E. coli together with the associated aminoglycoside resistance determinant [AAC(3)-II] and tetracycline, trimethoprim, and sulfonamide resistance. The beta-lactamase efficiently hydrolyzed cefotaxime and ceftriaxone but only moderately hydrolyzed ceftazidime and was inhibited by clavulanate and sulbactam (1 microM) and by anti-TEM-1 and anti-TEM-2 sera. This extended-spectrum beta-lactamase, conferring resistance to cefotaxime, ceftriaxone, ceftazidime, and aztreonam, was comparable to CTX-1 (TEM-3) but differed from it by pI. Agarose gel electrophoresis of the plasmid DNA indicated that this new enzyme was coded by pUD16, a plasmid of 220 kilobases which belongs to the Inc6 incompatibility group. Hybridization with an intragenic probe for TEM-1 revealed that this beta-lactamase derives from TEM-type beta-lactamases and hence it was named TEM-4.     

In vitro activities of the potent, broad-spectrum carbapenem MK-0826 (L-749,345) against broad-spectrum beta-lactamase-and extended-spectrum beta-lactamase-producing Klebsiella pneumoniae and Escherichia coli clinical isolates

An important mechanism of bacterial resistance to beta-lactam antibiotics is inactivation by beta-lactam-hydrolyzing enzymes (beta-lactamases). The evolution of the extended-spectrum beta-lactamases (ESBLs) is associated with extensive use of beta-lactam antibiotics, particularly cephalosporins, and is a serious threat to therapeutic efficacy. ESBLs and broad-spectrum beta-lactamases (BDSBLs) are plasmid-mediated class A enzymes produced by gram-negative pathogens, principally Escherichia coli and Klebsiella pneumoniae. MK-0826 was highly potent against all ESBL- and BDSBL-producing K. pneumoniae and E. coli clinical isolates tested (MIC range, 0.008 to 0.12 microgram/ml). In E. coli, this activity was associated with high-affinity binding to penicillin-binding proteins 2 and 3. When the inoculum level was increased 10-fold, increasing the amount of beta-lactamase present, the MK-0826 MIC range increased to 0.008 to 1 microgram/ml. By comparison, similar observations were made with meropenem while imipenem MICs were usually less affected. Not surprisingly, MIC increases with noncarbapenem beta-lactams were generally substantially greater, resulting in resistance in many cases. E. coli strains that produce chromosomal (Bush group 1) beta-lactamase served as controls. All three carbapenems were subject to an inoculum effect with the majority of the BDSBL- and ESBL-producers but not the Bush group 1 strains, implying some effect of the plasmid-borne enzymes on potency. Importantly, MK-0826 MICs remained at or below 1 microgram/ml under all test conditions.     

Characterization of a novel extended-spectrum beta-lactamase from Pseudomonas aeruginosa.

A clinical isolate of Pseudomonas aeruginosa RNL-1 showed resistance to extended-spectrum cephalosporins which was inhibited by clavulanic acid. Although this strain contained three plasmids ca. 80, 20, and 4 kb long, the resistance could not be transferred by mating-out assays with P. aeruginosa or Escherichia coli. Cloning of a 2.1-kb Sau3A fragment from P. aeruginosa RNL-1 into plasmid pACYC184 produced pPZ1, a recombinant plasmid that encodes a beta-lactamase. This beta-lactamase (PER-1) had a relative molecular mass of 29 kDa and a pI of 5.4 and was biosynthesized by P. aeruginosa RNL-1 along with a likely cephalosporinase with a pI of 8.7. PER-1 showed a broad substrate profile by hydrolyzing benzylpenicillin, amoxicillin, ticarcillin cephalothin, cefoperazone, cefuroxime, HR 221, ceftriaxone, ceftazidime, and (moderately) aztreonam but not oxacillin, imipenem, or cephamycins. Vmax values for extended-spectrum cephalosporins were uncommonly high, and the affinity of the enzyme for most compounds was relatively low (i.e., high Km). PER-1 activity was inhibited by clavulanic acid, sulbactam, imipenem, and cephamycins but not by EDTA. A 1.1-kb SnaBI fragment from pPZ1 failed to hybridize with plasmids that encode TEM-, SHV-, OXA-, or CARB/PSE-type beta-lactamase or with the ampC gene of P. aeruginosa. However, the same probe appeared to hybridize with chromosomal but not plasmid DNA from P. aeruginosa RNL-1. This study reports the properties of a novel extended-spectrum beta-lactamase in P. aeruginosa which may not be derived by point mutations from previously known enzymes of this species.     

A novel extended-spectrum beta-lactamase CTX-M-23 with a P167T substitution in the active-site omega loop associated with ceftazidime resistance

OBJECTIVE: In recent years, cefotaximases of the CTX-M type have become a predominant cause of resistance to extended-spectrum cephalosporins in Gram-negative bacteria. Although most enzymes provide higher levels of resistance to cefotaxime than to ceftazidime, mutants with enhanced catalytic efficiency against ceftazidime have recently been described. This report identifies another ceftazidime-resistant mutant of the CTX-M class of enzymes. METHODS: Two ceftazidime-resistant strains, Escherichia coli IFI-1 and Klebsiella pneumoniae IFI-2, were isolated from a 46-year-old man during treatment of postoperative peritonitis with ceftazidime. Susceptibility testing, mating-out assays, isoelectric focusing as well as PCR and sequencing techniques were carried out to investigate the underlying mechanism of resistance. RESULTS: E. coli IFI-1 and K. pneumoniae IFI-2 exhibited a clavulanic acid-inhibited substrate profile that included extended-spectrum cephalosporins. Notably, both strains had up to a 32-fold higher level of resistance to ceftazidime than to cefotaxime. Further characterization revealed that a novel bla(CTX-M) gene encoding a beta-lactamase with a pI of 8.9 was implicated in this resistance: CTX-M-23. Along with the substitutions D114N and S140A, CTX-M-23 differed from CTX-M-1, the most closely related enzyme, by a P167T replacement in the active-site omega loop, which has not previously been observed in other CTX-M enzymes. By analogy with what was observed with certain TEM/PSE/BPS-type beta-lactamases, the amino acid substitution in the omega loop may explain ceftazidime resistance, which has only rarely been reported for other CTX-M enzymes. CONCLUSION: The emergence of a new ceftazidime-resistant CTX-M-type mutant provides evidence that these enzymes are able to broaden their substrate spectrum towards ceftazidime, probably due to substitutions in the active-site omega loop.     

Transposition of the gene encoding a TEM-12 extended-spectrum beta-lactamase.

An isolate of Klebsiella oxytoca from the blood culture of a child with leukemia was found to produce two beta-lactamases, at least one of which conferred resistance to ceftazidime. Genes encoding both enzymes were located on a single self-transmissible 100-kb plasmid, pOZ201. This plasmid was introduced into Escherichia coli UB5201 (pACYC184), and the gene encoding one beta-lactamase was transposed onto plasmid pACYC184 by exploiting a gene dosage effect. The transposable gene was found to encode a TEM-12 enzyme as determined by nucleotide sequencing. This gene was subsequently transposed onto plasmid pUB307. The transposable element encoding the TEM-12 enzyme has been designated Tn841. Both plasmids pACYC184::Tn841 and pUB307::Tn841 were shown to encode a beta-lactamase with the same isoelectric point and substrate profile as the TEM-12 beta-lactamase. Transposon Tn841, at approximately 7 kb, is larger than TnA (4.8 kb) and transposes at a lower frequency. Although it produced a resolvase which can complement the resolvase of Tn3, its transposase function was not able to complement the transposition of a TnA element which lacked transposase. The occurrence of a gene encoding an extended-spectrum beta-lactamase on a transposable element in a clinically significant bacterium is potentially a cause for concern for the spread of resistance to the extended-spectrum cephalosporins.     

TLA-1: a new plasmid-mediated extended-spectrum beta-lactamase from Escherichia coli

Escherichia coli R170, isolated from the urine of an infected patient, was resistant to expanded-spectrum cephalosporins, aztreonam, ciprofloxacin, and ofloxacin but was susceptible to amikacin, cefotetan, and imipenem. This particular strain contained three different plasmids that encoded two beta-lactamases with pIs of 7.0 and 9.0. Resistance to cefotaxime, ceftazidime, aztreonam, trimethoprim, and sulfamethoxazole was transferred by conjugation from E. coli R170 to E. coli J53-2. The transferred plasmid, RZA92, which encoded a single beta-lactamase, was 150 kb in length. The cefotaxime resistance gene that encodes the TLA-1 beta-lactamase (pI 9.0) was cloned from the transconjugant by transformation to E. coli DH5alpha. Sequencing of the bla(TLA-1) gene revealed an open reading frame of 906 bp, which corresponded to 301 amino acid residues, including motifs common to class A beta-lactamases: (70)SXXK, (130)SDN, and (234)KTG. The amino acid sequence of TLA-1 shared 50% identity with the CME-1 chromosomal class A beta-lactamase from Chryseobacterium (Flavobacterium) meningosepticum; 48.8% identity with the VEB-1 class A beta-lactamase from E. coli; 40 to 42% identity with CblA of Bacteroides uniformis, PER-1 of Pseudomonas aeruginosa, and PER-2 of Salmonella typhimurium; and 39% identity with CepA of Bacteroides fragilis. The partially purified TLA-1 beta-lactamase had a molecular mass of 31.4 kDa and a pI of 9.0 and preferentially hydrolyzed cephaloridine, cefotaxime, cephalothin, benzylpenicillin, and ceftazidime. The enzyme was markedly inhibited by sulbactam, tazobactam, and clavulanic acid. TLA-1 is a new extended-spectrum beta-lactamase of Ambler class A.     

Antibiotic prescribing in general practice: striking differences between Italy (Ravenna) and Denmark (Funen)

The aim of this study was to evaluate the incidence of decreased susceptibility to broad-spectrum cephalosporins in Enterobacteriaceae that lack inducible chromosomal bla genes, and to determine the enzymes responsible for resistance. From all clinically relevant Enterobacteriaceae strains isolated between 1994 and 1996, 88 of 7054 Escherichia coli, seven of 581 Klebsiella pneumoniae and 23 of 166 Klebsiella oxytoca strains were studied because of their decreased susceptibilities to broad-spectrum cephalosporins (as reflected in intermediate susceptibilities and/or positive synergy tests and/or irregular crenellated inhibition zones). The most frequent mechanism implicated in decreased susceptibility to broad-spectrum cephalosporins displayed by E. coli and K. oxytoca was hyperproduction of chromosomal beta-lactamase, followed by plasmid-mediated SHV-1 hyperproduction in E. coli. In our hospital, the incidence of plasmid-mediated extended-spectrum beta-lactamases (ESBLs) between 1994 and 1996 was low. ESBLs were found in only 10 (0.14%) E. coli strains (six CTX-M-9, two TEM-12 and two SHV-2), in one (0.17%) K. pneumoniae strain (SHV-2) and in no K. oxytoca strains. The relatively wide variety of beta-lactamases that were detected among these common bacteria isolated from a single medical centre, including non-TEM- and non-SHV-derived ESBLs, appears epidemiologically remarkable.     

Evaluation of the MicroScan ESBL plus confirmation panel for detection of extended-spectrum beta-lactamases in clinical isolates of oxyimino-cephalosporin-resistant Gram-negative bacteria

OBJECTIVE: We aimed to assess the performance of the MicroScan ESBL plus confirmation panel using a series of 87 oxyimino-cephalosporin-resistant Gram-negative bacilli of various species. METHODS: Organisms tested included 57 extended-spectrum beta-lactamase (ESBL) strains comprising Enterobacter aerogenes (3), Enterobacter cloacae (10), Escherichia coli (11), Klebsiella pneumoniae (26), Klebsiella oxytoca (3) and Proteus mirabilis (4). Also included were 30 strains resistant to oxyimino cephalosporins but lacking ESBLs, which were characterized with other resistance mechanisms, such as inherent clavulanate susceptibility in Acinetobacter spp. (4), hyperproduction of AmpC enzyme in Citrobacter freundii (2), E. aerogenes (3), E. cloacae (3), E. coli (4), Hafnia alvei (1) and Morganella morganii (1), production of plasmid-mediated AmpC beta-lactamase in K. pneumoniae (3) and E. coli (3) or hyperproduction of K1 enzyme in K. oxytoca (6). RESULTS: The MicroScan MIC-based clavulanate synergy correctly classified 50 of 57 ESBL strains as ESBL-positive and 23 of 30 non-ESBL strains as ESBL-negative (yielding a sensitivity of 88% and a specificity of 76.7%, respectively). False negatives among ESBL producers were highest with Enterobacter spp. due to masking interactions between ESBL and AmpC beta-lactamases. False-positive classifications occurred in two Acinetobacter spp., one E. coli producing plasmid-mediated AmpC beta-lactamase and two K. oxytoca hyperproducing their chromosomal K1 beta-lactamase. CONCLUSION: The MicroScan clavulanate synergy test proved to be a valuable tool for ESBL confirmation. However, this test has limitations in detecting ESBLs in Enterobacter spp. and in discriminating ESBL-related resistance from the K1 enzyme and from inherent clavulanate susceptibility in Acinetobacter spp.     

CFE-1, a novel plasmid-encoded AmpC beta-lactamase with an ampR gene originating from Citrobacter freundii

A clinical isolate of Escherichia coli from a patient in Japan, isolate KU6400, was found to produce a plasmid-encoded beta-lactamase that conferred resistance to extended-spectrum cephalosporins and cephamycins. Resistance arising from production of a beta-lactamase could be transferred by either conjugation or transformation with plasmid pKU601 into E. coli ML4947. The substrate and inhibition profiles of this enzyme resembled those of the AmpC beta-lactamase. The resistance gene of pKU601, which was cloned and expressed in E. coli, proved to contain an open reading frame showing 99.8% DNA sequence identity with the ampC gene of Citrobacter freundii GC3. DNA sequence analysis also identified a gene upstream of ampC whose sequence was 99.0% identical to the ampR gene from C. freundii GC3. In addition, a fumarate operon (frdABCD) and an outer membrane lipoprotein (blc) surrounding the ampR-ampC genes in C. freundii were identified, and insertion sequence (IS26) elements were observed on both sides of the sequences identified (forming an IS26 composite transposon); these results confirm the evidence of the translocation of a beta-lactamase-associated gene region from the chromosome to a plasmid. Finally, we describe a novel plasmid-encoded AmpC beta-lactamase, CFE-1, with an ampR gene derived from C. freundii.     

Risk factors and outcomes of extended-spectrum beta-lactamase-producing E. coli peritonitis in CAPD patients.

OBJECTIVE: To determine the risk factors and outcomes of peritonitis caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in continuous ambulatory peritoneal dialysis (CAPD). PATIENTS AND METHODS: Episodes of E. coli CAPD peritonitis in our unit from October 1994 to August 2003 were reviewed. Demographic data, underlying medical conditions, recent use of gastric acid inhibitors (including H2 antagonist and proton pump inhibitor), recent antibiotic therapy, antibiotic regimen for peritonitis episodes, sensitivity test results of the E. coli isolated, and clinical outcomes were examined. RESULTS: Over a 10-year study period, 88 episodes of E. coli peritonitis were recorded; 11 of the 88 cases were caused by ESBL-producing E. coli. Recent use of cephalosporins and gastric acid inhibitor were associated with the development of ESBL-producing E. coil peritonitis. Compared with non-ESBL-producing E. coli peritonitis, more cases in the ESBL-producing E. coli group developed treatment failure (45.5% vs 13.0%, p = 0.02) and died of sepsis (27.3% vs 3.9%, p = 0.02). Peritoneal failure rate was higher in the ESBL-producing E. coli group, although the difference was not statistically significant (18.2% vs 3.9%, p = 0.12). CONCLUSION: Peritonitis caused by ESBL-producing E. coli is associated with worse clinical outcomes. The use of cephalosporins and gastric acid inhibitors may contribute to its development. Further studies are warranted to investigate and determine the predisposing factors for ESBL-producing E. coli peritonitis.