This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Poirel, L.
Right arrow Articles by Nordmann, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poirel, L.
Right arrow Articles by Nordmann, P.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, December 2001, p. 3355-3361, Vol. 45, No. 12
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.12.3355-3361.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

CTX-M-Type Extended-Spectrum beta -Lactamase That Hydrolyzes Ceftazidime through a Single Amino Acid Substitution in the Omega Loop

Laurent Poirel,1 Thierry Naas,1 Isabelle Le Thomas,2 Amal Karim,1 Edouard Bingen,2 and Patrice Nordmann1,*

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre Cedex,1 and Service de Microbiologie, Hôpital Robert Debré, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Xavier-Bichat, 75018 Paris,2 France

Received 18 December 2000/Returned for modification 11 April 2001/Accepted 21 August 2001


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Escherichia coli ILT-1, Klebsiella pneumoniae ILT-2, and K. pneumoniae ILT-3 were isolated in May 1999 in Paris, France, from a rectal swab of a hospitalized 5-month-old girl. These isolates had a clavulanic acid-inhibited substrate profile that included expanded-spectrum cephalosporins. The MICs of cefotaxime were higher for E. coli ILT-1 and K. pneumoniae ILT-2 than for K. pneumoniae ILT-3, while the opposite was found for the MICs of ceftazidime. Genetic and biochemical analyses revealed that E. coli ILT-1 and K. pneumoniae ILT-2 produced the CTX-M-18 beta -lactamase, while K. pneumoniae ILT-3 produced the CTX-M-19 beta -lactamase. The amino acid sequence of the CTX-M-18 beta -lactamase differed from that of the CTX-M-9 beta -lactamase by an Ala-to-Val change at position 231, while CTX-M-19 possessed an additional Pro-to-Ser change at position 167 in the omega loop of Ambler class A enzymes. The latter amino acid substitution may explain the CTX-M-19-mediated hydrolysis of ceftazidime, which has not been reported for other CTX-M-type enzymes. The blaCTX-M-18 and blaCTX-M-19 genes were located on transferable plasmids that varied in size (ca. 60 and 50 kb, respectively) but that showed similar restriction patterns.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Several clavulanic acid-inhibited Ambler class A expanded-spectrum beta -lactamases (ESBLs) have been reported, mostly in members of the family Enterobacteriaceae, in addition to the TEM- and SHV-type ESBLs (21, 22). Among them, the CTX-M-type beta -lactamases are typical ESBLs. The designation "CTX" refers to their powerful spectrum of hydrolysis of cefotaxime (32). The CTX-M beta -lactamases hydrolyze ceftazidime with a very low catalytic efficiency. Thus, CTX-M beta -lactamases confer to both wild-type and laboratory-obtained enterobacterial hosts high levels resistance to cefotaxime, ceftriaxone, and aztreonam but have only marginal effects on the MIC of ceftazidime.

The first CTX-M beta -lactamase (CTX-M-1/MEN-1) was characterized in Escherichia coli strains isolated from German and Italian patients (4-6). This family of enzymes now comprises 14 members: CTX-M-1 to CTX-M-12, Toho-1, and Toho-2 (7, 23, 28, 32). These enzymes share 71 to 98% amino acid sequence identities. Escherichia coli and Salmonella enterica serotype Typhimurium are the enterobacterial species most frequently reported to produce the CTX-M beta -lactamases (28, 32). The CTX-M-type beta -lactamase-producing strains are endemic in Latin America, Japan, and some areas in Eastern Europe (6, 9, 12, 14, 24, 32). Rare reports signal the presence of CTX-M enzymes in enterobacterial isolates from other countries such as France, Kenya, Spain, and Greece (18, 28, 31).

The CTX-M beta -lactamase genes are mostly located on plasmids, and Toho-1-like beta -lactamase genes have also been found to be located on chromosomes (35). The progenitors of some of these plasmid-mediated enzymes could be the chromosomally encoded beta -lactamases of the enterobacterial species Kluyvera ascorbata (GenBank accession number no. AJ251722).

This report identifies two isogenic CTX-M-type beta -lactamases from enterobacterial strains isolated from the same patient. One of them was peculiar since it hydrolyzed ceftazidime much more than cefotaxime, a property not reported previously for a CTX-M-type enzyme.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Bacterial strains and plasmids. The bacterial strains and plasmids used in the present work are listed in Table 1. E. coli ILT-1, Klebsiella pneumoniae ILT-2, and K. pneumoniae ILT-3 clinical isolates were identified by the API 20E system (bioMérieux, Marcy l'Etoile, France).

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   Strains and plasmids used in this study

PFGE. Plug preparation was done according to the instructions of Bio-Rad (Ivry/Seine, France). Whole-cell DNAs from K. pneumoniae ILT-2 and ILT-3 were digested with XbaI at 37°C overnight. Electrophoresis in a 1% agarose gel in 0.5× Tris-borate-EDTA buffer was performed with a CHEF DRII apparatus (Bio-Rad). Migration conditions, staining, and chromosomal fingerprints compared and assigned to pulsed-field gel electrophoresis (PFGE) types and subtypes were as described previously (13).

Susceptibility testing. Antibiotic-containing disks were used for antibiotic susceptibility testing by the disk diffusion assay (Sanofi-Diagnostics Pasteur, Marnes-la-Coquette, France), as described previously (26). The double-disk synergy test was performed with ceftazidime-, cefotaxime-, aztreonam-, and amoxicillin-clavulanic acid-containing disks on Mueller-Hinton (MH) agar plates; and the results were interpreted as described previously (16). MICs were determined by an agar dilution technique on MH agar (Sanofi-Diagnostics Pasteur) with an inoculum of 104 CFU per spot, as described previously (27). All plates were incubated at 37°C for 18 h. The MICs of the beta -lactams were determined alone or in combination with a fixed concentration of clavulanic acid (2 µg/ml) or tazobactam (4 µg/ml). MIC results were interpreted according to the guidelines of the National Committee for Clinical Laboratory Standards after incubation at 37°C for 18 h (21).

PCR, cloning experiments, recombinant plasmid analysis, and DNA sequencing. Whole-cell DNAs from E. coli ILT-1 and K. pneumoniae ILT-2 and ILT-3 were extracted as described previously(27). They were used as templates in standard PCR experiments (29) with sets of laboratory-designed primers for detection of class A beta -lactamase genes and their extended-spectrum derivatives, blaTEM, blaSHV, blaPER-1, blaPER-2, blaVEB-1, blaToho-1, blaToho-2, blaSFO-1, blaGES-1, blaCTX-M-2 (26), and blaCTX-M-3 (primer CTXM-A, 5'-CGCTTTGCGATGTGCAG-3'; primer CTXM-B, 5'-ACCGCGATATCGTTGGT-3').

By using external primers for blaToho-1 (primer PreCTX-M-A, 5'-TGATGTAACACGGATTGACC-3'; primer PreCTX-M-B, 5'-TTACAGCCCTTCGGCGATGA-3'), the fragments obtained by PCR were cloned into the SrfI site of plasmid pPCRScript-Cam SK(+) (Stratagene, Amsterdam, The Netherlands) and expressed in E. coli JM109. Recombinant plasmids were selected on amoxicillin (100 µg/ml)- and chloramphenicol (30 µg/ml)-containing Trypticase soy (TS) agar plates. Recombinant plasmids pMA-1, pMA-2, and pMA-3 were obtained using E. coli ILT-1 and K. pneumoniae ILT-2 and ILT-3 as templates, respectively. Both strands of the cloned DNA fragments, inserted into recombinant plasmids, were sequenced with an Applied Biosystems sequencer (model ABI 377). The nucleotide and deduced amino acid sequences were analyzed and compared to sequences available over the Internet at the National Center for Biotechnology Information website (http: //www.ncbi.nlm.nih.gov).

Plasmid content, conjugation, and hybridization. Plasmid DNAs from E. coli ILT-1, K. pneumoniae ILT-2 and ILT-3, and recombinant clones were extracted with Maxi columns (Qiagen, Courtaboeuf, France). Plasmid DNAs were analyzed by electrophoresis on a 0.8% agarose gel (Gibco BRL-Life Technologies, Cergy-Pontoise, France) containing 0.15 µg of ethidium bromide per ml for 18 h at 90 V. A 1-kb DNA ladder (Gibco BRL-Life Technologies) was used as a reference DNA size standard. Conjugation experiments were performed between E. coli ILT-1, K. pneumoniae ILT-2 and ILT-3, and nalidixic acid- and rifampin-resistant E. coli JM109 in solid and liquid media at 37°C, as described previously (27). Transconjugants were selected on TS agar plates containing 150 µg of rifampin per ml or 100 µg of nalidixic acid and 100 µg of amoxicillin per ml. In order to locate the beta -lactamase genes, plasmid DNAs of E. coli ILT-1, K. pneumoniae ILT-2 and ILT-3, and their E. coli transconjugants were extracted with a Qiagen Maxi column and restricted with HindIII and BamHI. The DNA fragments were run on a 0.8% agarose gel, transferred onto a nylon membrane (Hybond N+; Amersham Pharmacia Biotech, Orsay, France) by the Southern technique (29), and hybridized with a PCR-generated probe of 550 bp whose sequence was specific for a region internal to the blaCTX-M-3 sequence (primers CTX-M-A and CTX-M-B) and a PCR-generated probe of 850 bp whose sequence was specific for a region internal to the blaTEM sequence (27). The nonradioactive enhanced chemiluminescence random prime system (Amersham Pharmacia Biotech) was used to label the DNA probes and for detection.

beta -Lactamase extracts and purification. Cultures of E. coli JM109 harboring recombinant plasmids pMA-1 and pMA-3 were grown overnight at 37°C in 4 liters of TS broth containing amoxicillin (100 µg/ml) and chloramphenicol (30 µg/ml). The beta -lactamase extracts were obtained as described previously (26) and were resuspended in 30 ml of 20 mM Tris-HCl (pH 9.2). Similarly, beta -lactamase extracts were obtained from 10-ml cultures of E. coli ILT-1, K. pneumoniae ILT-2 and ILT-3, and their E. coli transconjugants and were subsequently resuspended in 0.1 ml of sodium phosphate buffer (pH 7).

The beta -lactamase extracts of cultures of E. coli JM109(pMA-1) or E. coli JM109(pMA-3) were dialyzed overnight against 20 mM Tris-HCl (pH 9.2) at 4°C in order to eliminate spermine and to adjust the pH to 9.2. The enzyme extracts were loaded onto a preequilibrated Q-Sepharose column (1.6 by 5 cm; Amersham Pharmacia Biotech) with the same buffer. The resulting enzyme extract was recovered in the flowthrough and was dialyzed against 50 mM phosphate buffer (pH 6) overnight at 4°C. This extract was then loaded onto a preequilibrated S-Sepharose column, and the proteins were eluted with a linear NaCl gradient (0 to 0.5 M). The beta -lactamase activity was eluted with NaCl at a concentration of 50 mM in phosphate buffer. The fractions with the highest beta -lactamase activities were pooled and dialyzed against 50 mM phosphate buffer (pH 7), prior to concentration 10-fold with Centrisart-C30 microcentrifuge filters (Sartorius, Göttingen, Germany). The purified beta -lactamase extracts were used for determination of enzymatic activity. Their purity was estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (29).

IEF analysis. beta -Lactamase extracts from cultures of E. coli ILT-1, K. pneumoniae ILT-2 and ILT-3, and their E. coli transconjugants and purified enzymes from cultures of E. coli JM109 harboring recombinant plasmid pMA-1 or pMA-3 were subjected to analytical isoelectric focusing (IEF) analysis on a polyacrylamide gel with ampholine (pH 3.5 to 9.5), as described previously (27). The focused beta -lactamases were detected by overlaying the gel with 1 mM nitrocefin (Oxoid, Dardilly, France) in 100 mM phosphate buffer (pH 7.0). The pI values were determined and compared to those of known beta -lactamases.

Kinetic parameters. Purified beta -lactamase extracts were used for kinetic measurements performed at 30°C in 100 mM sodium phosphate (pH 7.0). The initial rates of hydrolysis were determined with an ULTROSPEC 2000 UV spectrophotometer (Amersham Pharmacia Biotech) and were analyzed by computer with Swift II software (Amersham Pharmacia Biotech). The kcat and Km values were determined by analyzing beta -lactam hydrolysis under initial rate conditions by using the Eadie-Hoffstee linearization of the Michaelis-Menten equation, as described previously (27).

The 50% inhibitory concentrations (IC50s) were determined for clavulanic acid, tazobactam, and sulbactam. Various concentrations of these inhibitors were preincubated with the purified enzyme for 3 min at 30°C to determine the concentrations that reduced the rate of hydrolysis of 1 µM cephaloridine by 50%. Results are expressed in units of micromolar. The specific activities of the purified beta -lactamase from E. coli JM109 harboring pMA-1 and E. coli JM109 harboring pMA-3 were obtained as described previously (27). One unit of enzyme activity was defined as the activity which hydrolyzed 1 µM benzylpenicillin per min per mg of protein. Specific activities were determined with 100 µM benzylpenicillin as the substrate. Additionally, the specific activity of nonpurified beta -lactamase extract from cultures of E. coli JM109 harboring pMA-1 or pMA-3 was determined with 100 µM piperacillin, a substrate that was hydrolyzed similarly by beta -lactamases from cultures of E. coli JM109(pMA-1) or E. coli JM109(pMA-3). The protein content was measured by the Bio-Rad DC Protein assay.

Nucleotide sequence accession numbers. The nucleotide sequence data for the CTX-M-18 and CTX-M-19 beta -lactamases reported in this paper have been assigned GenBank nucleotide sequence database accession nos. AF325133 and AF325134, respectively.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Characterization of clinical isolates and preliminary antibiotic susceptibility testing. E. coli ILT-1 and K. pneumoniae ILT-2 and ILT-3 were isolated from a 5-month-old girl in May 1999 at the Robert Debré Hospital (Paris, France) as a result of systematic rectal screening of patients admitted to the pediatric intensive care unit (ICU) for multidrug-resistant bacteria. The child was born in Vietnam and was transferred to the cardiovascular unit of Insitute Jacques Cartier (Paris, France), where she was hospitalized for 15 days for surgical cure of intraventricular communication. During the follow-up after surgery, she developed cardiovascular failure, for which she received treatment with cefotaxime, vancomycin, and netilmicin for 1 week. Rectal screening for multidrug-resistant bacteria performed 2 weeks after her admission to the ICU was positive for ESBL-producing isolates, but the rectal screening performed on the day of her admission was negative. No other enterobacterial isolate with a similar ESBL resistance profile was identified in the same ICU concomitantly or during the following 4-month period (data not shown).

Antibiotic susceptibility testing by disk diffusion suggested that the extended-spectrum cephalosporin resistance profile was due to the presence of an ESBL. Synergies were observed between clavulanate and ceftazidime, cefotaxime, and aztreonam (data not shown). E. coli ILT-1 and K. pneumoniae ILT-2 were more resistant to cefotaxime than to ceftazidime (Table 2). On the other hand, K. pneumoniae ILT-3 was more resistant to ceftazidime than to cefotaxime (Table 2). E. coli ILT-1 and K. pneumoniae ILT-2 and ILT-3 were also resistant to amikacin, gentamicin, kanamycin, netilmicin, tobramycin, and chloramphenicol and were susceptible to sulfonamides and fluoroquinolones. E. coli ILT-1 and K. pneumoniae ILT-2 were susceptible to rifampin, while K. pneumoniae ILT-3 was resistant to rifampin (data not shown).

                              
View this table:
[in this window]
[in a new window]
  		TABLE 2.   MICs of beta -lactams for clinical isolates, their E. coli JM109 transconjugants, E. coli JM109 harboring recombinants plasmids, and reference strain E. coli JM109

PFGE analysis of XbaI-restricted DNAs of K. pneumoniae ILT-2 and ILT-3 showed that they have distinguishable patterns and thus were not epidemiologically related (data not shown).

PCR experiments and cloning of beta -lactamase genes. Preliminary PCR amplification experiments with primers designed to amplify several internal fragments of ESBL genes gave positive results for blaTEM and blaCTX-M-3 for each clinical strain and positive results for blaSHV for the two K. pneumoniae clinical isolates. External primers for blaCTX-M-3 were used to PCR amplify a 900-bp fragment of a blaCTX-M-3-like gene by using whole-cell DNAs of E. coli ILT-1 and K. pneumoniae ILT-2 and ILT-3 as templates. The corresponding PCR amplimers were cloned into the SrfI site of pPCRScript-Cam (SK+), giving rise to recombinant plasmids pMA-1, pMA-2, and pMA-3, respectively.

DNA sequencing. Analysis of the inserted nucleotide sequences from recombinant plasmids pMA-1, pMA-2, and pMA-3 showed in each case an 876-bp open reading frame encoding a 291-amino-acid protein. On the basis of the protein alignments, two CTX-M-type enzymes were identified: CTX-M-18 encoded by pMA-1 and pMA-2 and CTX-M-19 encoded by pMA-3. CTX-M-18 differed from the previously characterized CTX-M-9 enzyme by an alanine-to-valine substitution at position 231, according to the designation of class A enzymes of Ambler et al. (1), and CTX-M-19 had an additional proline-to-serine substitution at position 167 (Fig. 1). The latter substitution lay close to conserved residue Glu166 in the omega loop of class A beta -lactamases. CTX-M-18 and CTX-M-19, like CTX-M-9, share 88% amino acid identity with the most closely related plasmid-mediated CTX-M-type enzyme, Toho-2, and 80% amino acid identity with the chromosome-encoded penicillinase of K. ascorbata (GenBank accession number no. AJ251722).


View larger version (39K):
[in this window]
[in a new window]
  		FIG. 1.   Comparison of the amino acid sequences of CTX-M-18 and CTX-M-19 to that of CTX-M-9. Dashes indicate identical amino acids. The numbering is according to the designation of Ambler et al. (1). Highlighted amino acids are those strictly conserved in class A beta -lactamases (17), and the amino acids of the omega loop are underlined.

Since PCR experiments also gave positive results for blaTEM with clinical strains, the PCR amplicons were sequenced. In all cases, the same blaTEM-1 gene was identified (data not shown).

Kinetic parameters. The specific activities of the purified CTX-M-18 and CTX-M-19 beta -lactamases from cultures of E. coli JM109(pMA-1) and E. coli JM109(pMA-3) were 30 and 10 µmol · min-1 · mg of protein-1, respectively, when 100 µM benzylpenicillin was used as the substrate. Their overall rate of recovery was 70% with 60-fold purification. Their purities were estimated to be 90%. The kinetic parameters for the purified CTX-M-18 beta -lactamase showed that it had strong activity against most beta -lactams including cefotaxime, ceftriaxone, cefepime, and cefpirome (Table 3). The catalytic activity of CTX-M-19 was lower than that of CTX-M-18 for all substrates except piperacillin and ceftazidime. The activity of CTX-M-18 against ceftazidime was not detectable (Table 3). In contrast, kinetic parameters could be calculated for ceftazidime hydrolysis for CTX-M-19. However, the specific activity of CTX-M-19 for ceftazidime remained low (0.5 mU/mg). The catalytic activity of CTX-M-19 against aztreonam, cefepime, and cefpirome was not detectable.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 3.   Steady-state kinetic parameters of CTX-M-18 and CTX-M-19 beta -lactamases

Inhibition studies as measured by determination of the IC50s with cephaloridine as the substrate showed that CTX-M-18 and CTX-M-19 were inhibited by clavulanic acid (60 and 90 nM, respectively) and sulbactam (500 and 150 nM, respectively). However, tazobactam inhibited more CTX-M-18 activity (IC50, 5 nM) than CTX-M-19 activity (IC50, 40 nM).

Transfer of antibiotic resistance, plasmid analysis, and hybridizations. E. coli transconjugants with E. coli ILT-1 and K. pneumoniae ILT-2 and ILT-3 as donors were obtained at high frequencies. Each transconjugant showed an ESBL phenotype. Transconjugants of E. coli ILT-1 and K. pneumoniae ILT-2 harbored plasmids pILT-1 and pILT-2 of ca. 60 kb, respectively, with indistinguishable restriction patterns (Fig. 2A). Transconjugants of K. pneumoniae ILT-3 had plasmid pILT-3 of ca. 50 kb. Plasmid pILT-3 was related to plasmids pILT-1 and pILT-2 but differed by at least deletions of 1.6- and 3-kb HindIII-restricted fragments, deletion of an additional 3-kb HindIII-restricted fragment, and deletion of ca.10 kb of a BamHI-restricted fragment (Fig. 2A). Plasmids pILT-1, pILT-2, and pILT-3 conferred the same pattern of resistance to non-beta -lactam antibiotics, including resistance to gentamicin, kanamycin, netilmicin, tobramycin, and chloramphenicol. HindIII- and BamHI-restricted plasmids pILT-1, pILT-2, and pILT-3 hybridized with an internal probe for blaCTX-M on DNA fragments of different sizes. However, for a given restriction digest, the hybridizing DNA fragments were identical for plasmids pILT-1 to pILT-3 (Fig. 2B). Similarly, hybridization of HindIII- and BamHI-restricted plasmids pILT-1 to pILT-3 with an internal probe for blaTEM-1 gave different hybridizing DNA fragments that were identical for all plasmids for a given restriction digest (Fig. 2C). These results showed that restricted DNA fragments of pILT-1, pILT-2, and pILT-3 that contained either the blaTEM or the blaCTX-M gene were different and that these natural plasmids were structurally related. Additionally, probes for blaTEM or blaCTX-M hybridized at the migration position of chromosomal DNA (Fig. 2B and C).


View larger version (40K):
[in this window]
[in a new window]
  		FIG. 2.   Plasmid analysis of E. coli JM109 transconjugants harboring natural plasmids pILT-1, pILT-3, and pILT-2 from E. coli ILT-1, K. pneumoniae ILT-3, and K. pneumoniae ILT-2, respectively. (A) Agarose gel electrophoresis of crude DNA lysates. Lane 1, pILT-1; lane 2, pILT-3; lane 3, pILT-2; lane 4, HindIII-restricted pILT-1; lane 5, HindIII-restricted pILT-3; lane 6, HindIII-restricted pILT-2; lane 7, BamHI-restricted pILT-1; lane 8, BamHI-restricted pILT-3; lane 9, BamHI-restricted pILT-2; lane M, molecular size marker. (B) Corresponding autoradiograph after Southern transfer and hybridization with the blaCTX-M probe. (C) Corresponding autoradiograph after Southern transfer and hybridization with the blaTEM probe. Hybridizing DNA fragments and their sizes are indicated by horizontal arrows. Numbers on the right of each gel are in kilobases.

IEF analysis. IEF analysis showed that E. coli ILT-1, K. pneumoniae ILT-2 and ILT-3, and their transconjugants expressed two beta -lactamases with pIs of 5.4 and 8. The beta -lactamase with a pI of 5.4 corresponded to TEM-1. The beta -lactamase with a pI of 8 corresponded to CTX-M-18 for E. coli ILT-1, K. pneumoniae ILT-2, their transconjugants, E. coli JM109 harboring recombinant plasmid pMA-1, and E. coli JM109 harboring recombinant plasmid pMA-2 and to CTX-M-19 for K. pneumoniae ILT-3, its transconjugant, and E. coli JM109(pMA-3). An additional beta -lactamase with a pI of 7.6 likely corresponded to the chromosomally encoded SHV-type beta -lactamase of K. pneumoniae for isolates ILT-2 and ILT-3.

Detailed analysis of beta -lactam susceptibility. The MICs of beta -lactams for the CTX-M-18- or CTX-M-19-producing strains are shown in Table 2. These results indicate that CTX-M-18- and CTX-M-19-producing strains were resistant to penicillins and restricted- and expanded-spectrum cephalosporins. For the CTX-M-18-producing strains (E. coli ILT-1, K. pneumoniae ILT-2, their E. coli transconjugants, E. coli JM109 harboring pMA-1, and E. coli JM109 harboring pMA-2), the MICs of cefotaxime and aztreonam were higher than those of ceftazidime. On the contrary, the MICs of ceftazidime were higher than the MICs of cefotaxime and aztreonam for the CTX-M-19 producers [K. pneumoniae ILT-3, its transconjugant, and E. coli JM109(pMA-3)]. Moreover, the MICs of cefepime and cefpirome for the CTX-M-18 producers were higher than those for the CTX-M-19 producers. The addition of clavulanic acid and tazobactam strongly reduced the MICs of penicillins and cephalosporins for the CTX-M-18 and CTX-M-19 producers. The differences in the MICs of beta -lactams for the CTX-M-18 and CTX-M-19 producers could be related not only to the catalytic activities of the enzymes but also to differences in folding or the stabilities of the proteins. Indeed, although the kinetic parameters for CTX-M-18 and CTX-M-19 were very similar for piperacillin (Table 3), the specific activities of nonpurified extracts of cultures of E. coli JM109(pMA-1/CTX-M-18) and of E. coli JM109(pMA-3/CTX-M-19) were 840 and 433 mU · mg of protein-1, respectively, with 100 µM piperacillin as the substrate.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The work described here further characterized biochemically two CTX-M-type enzymes, CTX-M-18 in E. coli and K. pneumoniae and CTX-M-19 in K. pneumoniae. CTX-M enzymes have been identified mostly from E. coli and Salmonella sp. strains and rarely from K. pneunomiae, whereas the TEM- and SHV-type extended-spectrum derivatives have been identified mostly from K. pneumoniae (22, 32). Identification of blaCTX-M-18 in E. coli and K. pneumoniae signals the interspecies transfer of a plasmid-mediated CTX-M gene between isolates in the fecal flora of the same patient.

The CTX-M beta -lactamases have been reported in Europe, mostly in Eastern Europe. CTX-M-9 was recently identified in an E. coli isolate in Spain and was also likely present in 22 additional E. coli strains and in 1 S. enterica serotype Virchow strain in the same hospital in Barcelona, Spain (28). Although CTX-M-1 (MEN-1) was identified from a clinical sample of an Italian patient hospitalized in France (4), CTX-M-3-producing Enterobacter cloacae was the first CTX-M-producing isolate recovered in France from a French patient (11). In the present study, the enterobacterial isolates were from a Vietnamese patient hospitalized in France several times and consecutively in different French hospitals. Thus, it is difficult to define reliably the geographical origins of the isolated strains.

On the basis of their amino acid sequences, CTX-M enzymes may be divided into three clusters (28, 32) that differ by 20 to 25%. The first cluster groups CTX-M-1, CTX-M-3, CTX-M-10, and CTX-M-12; the second cluster groups CTX-M-2, CTX-M-4 to CTX-M-7, and Toho-1; and the third cluster groups Toho-2, CTX-M-9, and now CTX-M-18 and CTX-M-19. The plasmid-mediated CTX-M-18 and CTX-M-19 enzymes are related to the chromosomal penicillinase of K. ascorbata, underlining their possible origin. All characteristic substitutions assumed to be implicated in expanded-spectrum cephalosporin hydrolysis of the CTX-M enzymes are also present in CTX-M-18 and CTX-M-19, for example, Ser237, Thr244, and Arg276 (32). Our work has characterized the biochemical properties of CTX-M-18, which were found to be similar to those of the other cefotaxime-hydrolyzing CTX-M enzymes and which may correspond to those partially reported for CTX-M-9 (28). Tazobactam exhibited a stronger inhibitory activity than clavulanic acid against CTX-M-18 and CTX-M-19, as previously reported for the CTX-M enzymes (32). The catalytic efficiencies of CTX-M-18 and CTX-M-19 for several beta -lactams remained lower than those reported for CTX-M-1 and CTX-M-5, the only other two CTX-M enzymes for which detailed kinetic data have been reported (4, 9).

Analysis of the crystal structure of Toho-1 shows that the amino acids of the omega loop play the main role in its substrate profile for cephalosporins (15). The omega loop amino acids (residues 161 to 179) of CTX-M-9 and CTX-M-18 are identical to those of Toho-1. Thus, results of analysis of the crystal structure of the Glu166Ala mutant of Toho-1 (15) may be applied to analysis of CTX-M-9 and CTX-M-18. The Toho-1 beta -lactamase has fewer hydrogen bond interactions between the omega loop and the alpha -beta domain in the vicinity of Asn170 and Asp240 compared to the numbers of interactions for the restricted-spectrum beta -lactamase of Bacillus licheniformis (15). Moreover, no hydrogen bond connects both the N and the C termini of the omega loop between the amino acid at position 160 and the threonine at position 181. The CTX-M-9, CTX-M-18, and CTX-M-19 beta -lactamases, like the Toho-1 beta -lactamase, have a Phe residue at position 160 that cannot form a hydrogen bond with the threonine at position 181. The same Phe residue cannot interact with Asp157. The CTX-M-9, CTX-M-18, and CTX-M-19 beta -lactamases, like the Toho-1 beta -lactamase, retain a hydrogen bond between Asp179 and Arg164, as in some narrow-spectrum class A beta -lactamases; in addition, they retain the interactions between Lys73 and Glu166 and between Asn136 and Glu166 that help to maintain the structural integrity of the omega loop. The expanded-spectrum hydrolytic activity of the CTX-M-9, CTX-M-18, and CTX-M-19 beta -lactamases, like that of the Toho-1 beta -lactamase, may be related to an increased flexibility of the omega loop.

The most interesting aspect of our work is the CTX-M-19-mediated resistance to ceftazidime. The proline-to-serine change in CTX-M-19 occurs in the omega loop structure of class A beta -lactamases (Fig. 1). No natural class A beta -lactamases have a serine residue at position 167 like CTX-M-19 does. Amino acid substitutions in the omega loop of extended-spectrum TEM- and SHV-type enzymes selected in vivo were identified only at position 164 or 179, breaking the hydrogen bond between Arg164 and Asp179 (2, 8, 19, 20, 33). Site-specific mutagenesis experiments showed that a Pro-to-Gly substitution at position 167 in TEM-1 and PSE-4 increased the hydrolytic activities of the enzymes against ceftazidime (25, 30). It is possible that Ser167 in CTX-M-19 may enlarge the binding site for ceftazidime in the catalytic site of the enzyme. Interestingly, Ser167 in CTX-M-19 is located next to Glu166, an amino acid residue that is known to promote activation of the hydrolytic water molecule for hydrolysis of the acyl enzyme intermediate in class A beta -lactamases (3).

The substrate profile of CTX-M-19 was extended to ceftazidime but its catalytic efficiency for the other extended-spectrum cephalosporins was lower than that obtained for the other CTX-M-type enzymes. The variabilities of the substrate profiles of expanded-spectrum cephalosporins (except ceftazidime) have been reported for CTX-M enzymes such as CTX-M-2 and its derivative with a point mutation, CTX-M-4 (12). The kinetic parameters of CTX-M-19 against ceftazidime revealed a surprisingly low catalytic efficiency. A similar discrepancy between MIC results and kinetic data has been reported in other cases, such as for ceftazidime and OXA-10 derivatives of Ambler class D (10). Interestingly, misfolding was found for a TEM-1 beta -lactamase with a Pro167Thr mutation (34). Similarly, the Pro167Ser change in CTX-M-19 may the explain instability of this protein and the rapid loss of its activity (data not shown)

The blaCTX-M-18 and blaCTX-M-19 genes were located on self-transferable and structurally related plasmids of enterobacterial isolates from the same patient, thus suggesting the in vivo selection of a CTX-M-19 producer that conferred resistance to ceftazidime. The blaCTX-M-18 and blaCTX-M-19 genes were associated with blaTEM-1 on structurally related plasmids. Although clinical strains that produce two beta -lactamase genes have been reported, plasmids that carry two beta -lactamases genes have not been extensively described. Interestingly, the probes for blaCTX-M and blaTEM also hybridized at the chromosomal position (Fig. 2B and C). This result may indicate a transposon location of the blaCTX-M-18 and blaCTX-M-19 genes, as suggested for Toho-1-like genes (35), which is being studied in our laboratory.

Finally, this work showed once again that novel ESBLs can be identified from clinical specimens of patients hospitalized in ICUs and that the substrate profiles of the CTX-M enzymes may be extended to ceftazidime. Thus, detection of CTX-M producers should not be based solely on the fact that the MICs of cefotaxime are higher than those of ceftazidime.


    ACKNOWLEDGMENTS

This work was financed by a grant from the Ministères de l'Education Nationale et de la Recherche (grant UPRES, JE-2227), Université Paris XI, Paris, and the French network on beta -lactamase research, "Les beta -Lactamases; de l'Observation Clinique à la Structure."


    FOOTNOTES

* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France. Phone: 33-1-45-21-36-32. Fax: 33-1-45-21-63-40. E-mail: nordmann.patrice{at}bct.ap-hop-paris.fr.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

1. Ambler, R. P., A. F. W. Coulson, J.-M. Frère, J. M. Ghuysen, B. Joris, M. Forsman, R. C. Lévesque, G. Tiraby, and S. G. Waley. 1991. A standard numbering scheme for the class A beta -lactamases. Biochem. J. 276:269-270.
2. Arlet, G., M. Rouveau, and A. Philippon. 1997. Substitution of alanine for aspartate at position 179 in the SHV-6 extended-spectrum beta-lactamase. FEMS Microbiol. Lett. 152:163-167[Medline].
3. Barnerjee, S., U. Pieper, G. Kapadia, L. K. Pannell, and O. Herzberg. 1998. Role of omega-loop in the activity, substrate specificity, and structure of class A beta -lactamase. Biochemistry 37:3286-3296[CrossRef][Medline].
4. Barthélémy, M., J. Peduzzi, H. Bernard, C. Tancrède, and R. Labia. 1992. Close amino acid sequence relationship between the new plasmid-mediated extended-spectrum beta -lactamase MEN-1 and chromosomally encoded enzymes of Klebsiella oxytoca. Biochim. Biophys. Acta 1122:15-22[CrossRef][Medline].
5. Bauernfeind, A., H. Grimm, and S. Schweighart. 1990. A new plasmidic cefotaximase in a clinical isolate of Escherichia coli. Infection 18:294-298[CrossRef][Medline].
6. Bauernfeind, A., I. Stemplinger, R. Jungwirth, S. Ernst, and J. M. Casellas. 1996. Sequences of beta-lactamase genes encoding CTX-M-1 (MEN-1) and CTX-M-2 and relationship of their amino acid sequences with those of other beta -lactamases. Antimicrob. Agents Chemother. 40:509-513[Abstract].
7. Bonnet, R., J. L. M. Sampaio, R. Labia, C. De Champs, D. Sirot, C. Chanal, and J. Sirot. 2000. A novel CTX-M beta -lactamase (CTX-M-8) in cefotaxime-resistant Enterobacteriaceae isolated in Brazil. Antimicrob. Agents Chemother. 44:1936-1942[Abstract/Free Full Text].
8. Bonomo, R. A., S. D. Rudin, and D. M. Shlaes. 1997. OHIO-1 beta -lactamase mutants: Asp179Gly mutation confers resistance to ceftazidime. FEMS Microbiol. Lett. 152:275-278[Medline].
9. Bradford, P. A., Y. Yang, D. Sahm, I. Grope, D. Gardovska, and G. Storch. 1998. CTX-M-5: a novel cefotaxime-hydrolyzing beta -lactamase from an outbreak of Salmonella typhimurium in Latvia. Antimicrob. Agents Chemother. 42:1980-1984[Abstract/Free Full Text].
10. Danel, F., L. M. C. Hall, B. Duke, D. Gür, and D. M. Livermore. 1999. OXA-17, a further extended-spectrum variant of OXA-10 beta -lactamase isolated from P. aeruginosa. Antimicrob. Agents Chemother. 43:1362-1366[Abstract/Free Full Text].
11. Doucet-Populaire, F., J. C. Ghnassia, R. Bonnet, and J. Sirot. 2000. First isolation of a CTX-M-3-producing Enterobacter cloacae in France. Antimicrob. Agents Chemother. 44:3239-3240[Free Full Text].
12. Gazouli, M., E. Tzelepi, S. V. Sidorenko, and L. S. Tzouvelekis. 1998. Sequence of the gene encoding a plasmid-mediated cefotaxime-hydrolyzing class A beta -lactamase (CTX-M-4): involvement of serine 237 in cephalosporin hydrolysis. Antimicrob. Agents Chemother. 42:1259-1262[Abstract/Free Full Text].
13. Girlich, D., A. Karim, L. Poirel, M.-H. Cavin, C. Verny, and P. Nordmann. 2000. Molecular epidemiology of an outbreak due to IRT-2 beta -lactamase-producing strains of Klebsiella pneumoniae in a geriatric department. J. Antimicrob. Chemother. 45:467-473[Abstract/Free Full Text].
14. Gniadkowski, M., I. Schneider, A. Palucha, R. Jungwirth, B. Mikiewicz, and A. Bauernfeind. 1998. Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaxime-hydrolyzing beta -lactamase that is closely related to the CTX-M-1/MEN-1 enzyme. Antimicrob. Agents Chemother. 42:827-832[Abstract/Free Full Text].
15. Ibuka, A., A. Taguchi, M. Ishiguro, S. Fushinobu, Y. Ishii, S. Kamitori, K. Okuyama, K. Yamaguchi, M. Konno, and H. Matsuzawa. 1999. Crystal structure of the E166A mutant of extended-spectrum beta -lactamase Toho-1 at 1.8 Å resolution. J. Mol. Biol. 285:2079-2087[CrossRef][Medline].
16. Jarlier, V., M.-H. Nicolas, G. Fournier, and A. Philippon. 1988. Extended broad-spectrum beta -lactamases conferring transferable resistance to newer beta -lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev. Infect. Dis. 10:867-878[Medline].
17. Joris, B., P. Ledent, O. Dideberg, E. Fonze, J. Lamotte-Brasseur, J. A. Kelly, J. M. Ghuysen, and J.-M. Frère. 1991. Comparison of the sequences of class A beta -lactamases and of the secondary structure elements of penicillin-recognizing proteins. Antimicrob. Agents Chemother. 35:2294-2301[Abstract/Free Full Text].
18. Kariuki, S., J. E. Corkill, G. Revathi, R. Musoke, and C. A. Hart. 2001. Molecular characterization of a novel plasmid-encoded cefotaximase (CTX-M-12) found in clinical Klebsiella pneumoniae isolates from Kenya. Antimicrob. Agents Chemother. 45:2141-2143[Abstract/Free Full Text].
19. Kurokawa, H., T. Yagi, N. Shibata, K. Shibayama, K. Kamichi, and Y. Arakawa. 2000. A new SHV-derived extended-spectrum beta -lactamase (SHV-24) that hydrolyzes ceftazidime through a single-amino-acid substitution (D179G) in the omega loop. Antimicrob. Agents Chemother. 44:1725-1727[Abstract/Free Full Text].
20. Matagne, A., J. Lamotte-Brasseur, and J.-M. Frère. 1998. Catalytic properties of class A beta -lactamases: efficiency and diversity. Biochem. J. 330:581-598.
21. National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed. Approved standard M7-A4. In National Committee for Clinical Laboratory Standards, Wayne, Pa.
22. Nordmann, P. 1998. Trends in beta -lactam resistance among Enterobacteriaceae. Clin. Infect. Dis. 27:S100-S106.
23. Oliver, A., J. C. Pérez-Diaz, T. M. Coque, F. Baquero, and R. Canton. 2001. Nucleotide sequence and characterization of a novel cefotaxime-hydrolyzing beta -lactamase (CTX-M-10) isolated in Spain. Antimicrob. Agents Chemother. 45:616-620[Abstract/Free Full Text].
24. Palucha, A., B. Mikiewicz, W. Hryniewicz, and M. Gniadkowski. 1999. Concurrent outbreaks of extended-spectrum beta -lactamase-producing organisms of the family Enterobacteriaceae in a Warsaw hospital. J. Antimicrob. Chemother. 44:489-499[Abstract/Free Full Text].
25. Palzkill, T., Q. Q. Le, K. V. Venkatachalam, M. LaRocco, and H. Ocera. 1994. Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of beta -lactamase. Mol. Microbiol. 12:217-229[CrossRef][Medline].
26. Poirel, L., I. Le Thomas, T. Naas, A. Karim, and P. Nordmann. 2000. Biochemical sequence analyses of GES-1, a novel class A extended-spectrum beta -lactamase, and the class 1 integron In52 from Klebsiella pneumoniae. Antimicrob. Agents Chemother. 44:622-632[Abstract/Free Full Text].
27. Poirel, L., T. Naas, M. Guibert, E. B. Chaibi, R. Labia, and P. Nordmann. 1999. Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum beta -lactamase encoded by an Escherichia coli integron gene. Antimicrob. Agents Chemother. 43:573-581[Abstract/Free Full Text].
28. Sabaté, M., R. Tarrago, F. Navarro, E. Miro, C. Vergés, J. Barbé, and G. Prats. 2000. Cloning and sequence of the gene encoding a novel cefotaxime-hydrolyzing beta -lactamase (CTX-M-9) from Escherichia coli in Spain. Antimicrob. Agents Chemother. 44:1970-1973[Abstract/Free Full Text].
29. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
30. Therrien, C., F. Sanschagrin, T. Palzkill, and R. C. Lévesque. 1998. Roles of amino acids 161 to 179 in the PSE-4 omega loop in substrate specificity and in resistance to ceftazidime. Antimicrob. Agents Chemother. 42:2576-2583[Abstract/Free Full Text].
31. Tzouvelekis, L. S., M. Gazouli, A. Markigiannakis, E. Paraskaki, N. J. Legakis, and E. Tzelepi. 1998. Emergence of resistance to third-generation cephalosporins amongst Salmonella typhimurium isolates in Greece: report of the first three cases. J. Antimicrob. Chemother. 42:273-275[Free Full Text].
32. Tzouvelekis, L. S., E. Tzelepi, P. T. Tassios, and N. J. Legakis. 2000. CTX-M-type beta -lactamases: an emerging group of extended-spectrum enzymes. Int. J. Antimicrob. Agents 14:137-142[CrossRef][Medline].
33. Vakulenko, S. B., P. Taibi-Tronche, M. Toth, I. Massova, S. A. Lerner, and S. Mobashery. 1999. Effects on substrate profile by mutational substitutions at positions 164 and 179 of the class A TEMpUC19 beta -lactamase from Escherichia coli. J. Biol. Chem. 274:23052-23060[Abstract/Free Full Text].
34. Vanhove, M., X. Raquet, T. Palzkill, R. H. Pain, and J. M. Frère. 1996. The rate limiting step in the folding of the cis-Pro167Thr mutant of TEM-1 beta -lactamase is the trans to cis isomerization of a non-proline peptide bond. Proteins 25:104-111[CrossRef][Medline].
35. Yagi, T., H. Kurokawa, K. Senda, S. Ichiyama, H. Ito, S. Ohsuka, K. Shibayama, K. Shimokata, N. Kato, M. Ohta, and Y. Arakawa. 1997. Nosocomial spread of cephem-resistant Escherichia coli strains carrying multiple Toho-1-like beta -lactamase genes. Antimicrob. Agents Chemother. 41:2606-2611[Abstract].

Antimicrobial Agents and Chemotherapy, December 2001, p. 3355-3361, Vol. 45, No. 12
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.12.3355-3361.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Yin, J., Cheng, J., Sun, Z., Ye, Y., Gao, Y.-F., Li, J.-B., Zhang, X.-J. (2009). Characterization of two plasmid-encoded cefotaximases found in clinical Escherichia coli isolates: CTX-M-65 and a novel enzyme, CTX-M-87. J Med Microbiol 58: 811-815 [Abstract] [Full Text]  
  • Zong, Z., Partridge, S. R., Thomas, L., Iredell, J. R. (2008). Dominance of blaCTX-M within an Australian Extended-Spectrum {beta}-Lactamase Gene Pool. Antimicrob. Agents Chemother. 52: 4198-4202 [Abstract] [Full Text]  
  • Kiratisin, P., Apisarnthanarak, A., Laesripa, C., Saifon, P. (2008). Molecular Characterization and Epidemiology of Extended-Spectrum- {beta}-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Isolates Causing Health Care-Associated Infection in Thailand, Where the CTX-M Family Is Endemic. Antimicrob. Agents Chemother. 52: 2818-2824 [Abstract] [Full Text]  
  • Novais, A., Canton, R., Coque, T. M., Moya, A., Baquero, F., Galan, J. C. (2008). Mutational Events in Cefotaximase Extended-Spectrum {beta}-Lactamases of the CTX-M-1 Cluster Involved in Ceftazidime Resistance. Antimicrob. Agents Chemother. 52: 2377-2382 [Abstract] [Full Text]  
  • Perez-Llarena, F. J., Cartelle, M., Mallo, S., Beceiro, A., Perez, A., Villanueva, R., Romero, A., Bonnet, R., Bou, G. (2008). Structure-function studies of arginine at position 276 in CTX-M {beta}-lactamases. J Antimicrob Chemother 61: 792-797 [Abstract] [Full Text]  
  • Stepanova, M. N., Pimkin, M., Nikulin, A. A., Kozyreva, V. K., Agapova, E. D., Edelstein, M. V. (2008). Convergent In Vivo and In Vitro Selection of Ceftazidime Resistance Mutations at Position 167 of CTX-M-3 {beta}-Lactamase in Hypermutable Escherichia coli Strains. Antimicrob. Agents Chemother. 52: 1297-1301 [Abstract] [Full Text]  
  • Fang, H., Ataker, F., Hedin, G., Dornbusch, K. (2008). Molecular Epidemiology of Extended-Spectrum {beta}-Lactamases among Escherichia coli Isolates Collected in a Swedish Hospital and Its Associated Health Care Facilities from 2001 to 2006. J. Clin. Microbiol. 46: 707-712 [Abstract] [Full Text]  
  • Lartigue, M.-F., Zinsius, C., Wenger, A., Bille, J., Poirel, L., Nordmann, P. (2007). Extended-Spectrum {beta}-Lactamases of the CTX-M Type Now in Switzerland. Antimicrob. Agents Chemother. 51: 2855-2860 [Abstract] [Full Text]  
  • Bae, I. K., Lee, Y.-N., Lee, W. G., Lee, S. H., Jeong, S. H. (2007). Novel Complex Class 1 Integron Bearing an ISCR1 Element in an Escherichia coli Isolate Carrying the blaCTX-M-14 Gene. Antimicrob. Agents Chemother. 51: 3017-3019 [Abstract] [Full Text]  
  • Fernandez, A., Gil, E., Cartelle, M., Perez, A., Beceiro, A., Mallo, S., Tomas, M. M., Perez-Llarena, F. J., Villanueva, R., Bou, G. (2007). Interspecies spread of CTX-M-32 extended-spectrum {beta}-lactamase and the role of the insertion sequence IS1 in down-regulating blaCTX-M gene expression. J Antimicrob Chemother 59: 841-847 [Abstract] [Full Text]  
  • Naas, T., Oxacelay, C., Nordmann, P. (2007). Identification of CTX-M-Type Extended-Spectrum-{beta}-Lactamase Genes Using Real-Time PCR and Pyrosequencing. Antimicrob. Agents Chemother. 51: 223-230 [Abstract] [Full Text]  
  • Gonzalez-Lopez, J. J., Sabate, M., Lavilla, S., Larrosa, M. N., Bartolome, R. M., Prats, G. (2006). In Vivo Reversion to the Wild-Type {beta}-Lactam Resistance Phenotype Mediated by a Plasmid Carrying ampR and qnrA1 in Enterobacter cloacae.. Antimicrob. Agents Chemother. 50: 3175-3178 [Abstract] [Full Text]  
  • Jin, Y., Ling, J. M. (2006). CTX-M-producing Salmonella spp. in Hong Kong: an emerging problem.. J Med Microbiol 55: 1245-1250 [Abstract] [Full Text]  
  • Bae, I. K., Lee, B. H., Hwang, H. Y., Jeong, S. H., Hong, S. G., Chang, C. L., Kwak, H.-S., Kim, H. J., Youn, H. (2006). A novel ceftazidime-hydrolysing extended-spectrum {beta}-lactamase, CTX-M-54, with a single amino acid substitution at position 167 in the omega loop. J Antimicrob Chemother 58: 315-319 [Abstract] [Full Text]  
  • Sauvage, E., Fonze, E., Quinting, B., Galleni, M., Frere, J.-M., Charlier, P. (2006). Crystal Structure of the Mycobacterium fortuitum Class A {beta}-Lactamase: Structural Basis for Broad Substrate Specificity.. Antimicrob. Agents Chemother. 50: 2516-2521 [Abstract] [Full Text]  
  • Lartigue, M.-F., Poirel, L., Aubert, D., Nordmann, P. (2006). In Vitro Analysis of ISEcp1B-Mediated Mobilization of Naturally Occurring {beta}-Lactamase Gene blaCTX-M of Kluyvera ascorbata.. Antimicrob. Agents Chemother. 50: 1282-1286 [Abstract] [Full Text]  
  • Delmas, J., Robin, F., Carvalho, F., Mongaret, C., Bonnet, R. (2006). Prediction of the Evolution of Ceftazidime Resistance in Extended-Spectrum {beta}-Lactamase CTX-M-9. Antimicrob. Agents Chemother. 50: 731-738 [Abstract] [Full Text]  
  • Paterson, D. L., Bonomo, R. A. (2005). Extended-Spectrum {beta}-Lactamases: a Clinical Update. Clin. Microbiol. Rev. 18: 657-686 [Abstract] [Full Text]  
  • Delmas, J., Robin, F., Bittar, F., Chanal, C., Bonnet, R. (2005). Unexpected Enzyme TEM-126: Role of Mutation Asp179Glu. Antimicrob. Agents Chemother. 49: 4280-4287 [Abstract] [Full Text]  
  • Poirel, L., Van De Loo, M., Mammeri, H., Nordmann, P. (2005). Association of Plasmid-Mediated Quinolone Resistance with Extended-Spectrum {beta}-Lactamase VEB-1. Antimicrob. Agents Chemother. 49: 3091-3094 [Abstract] [Full Text]  
  • Moubareck, C., Daoud, Z., Hakime, N. I., Hamze, M., Mangeney, N., Matta, H., Mokhbat, J. E., Rohban, R., Sarkis, D. K., Doucet-Populaire, F. (2005). Countrywide Spread of Community- and Hospital-Acquired Extended-Spectrum {beta}-Lactamase (CTX-M-15)-Producing Enterobacteriaceae in Lebanon. J. Clin. Microbiol. 43: 3309-3313 [Abstract] [Full Text]  
  • Pitout, J. D. D., Nordmann, P., Laupland, K. B., Poirel, L. (2005). Emergence of Enterobacteriaceae producing extended-spectrum {beta}-lactamases (ESBLs) in the community. J Antimicrob Chemother 56: 52-59 [Abstract] [Full Text]  
  • Kim, J., Lim, Y.-M., Jeong, Y.-S., Seol, S.-Y. (2005). Occurrence of CTX-M-3, CTX-M-15, CTX-M-14, and CTX-M-9 Extended-Spectrum {beta}-Lactamases in Enterobacteriaceae Clinical Isolates in Korea. Antimicrob. Agents Chemother. 49: 1572-1575 [Abstract] [Full Text]  
  • Welsh, K. J., Barlow, M., Tenover, F. C., Biddle, J. W., Rasheed, J. K., Clark, L. A., McGowan, J. E. Jr. (2005). Experimental Prediction of the Evolution of Ceftazidime Resistance in the CTX-M-2 Extended-Spectrum Beta-Lactamase. Antimicrob. Agents Chemother. 49: 1242-1244 [Abstract] [Full Text]  
  • Ishii, Y., Kimura, S., Alba, J., Shiroto, K., Otsuka, M., Hashizume, N., Tamura, K., Yamaguchi, K. (2005). Extended-Spectrum {beta}-Lactamase-Producing Shiga Toxin Gene (stx1)-Positive Escherichia coli O26:H11: a New Concern. J. Clin. Microbiol. 43: 1072-1075 [Abstract] [Full Text]  
  • Poirel, L., Lartigue, M.-F., Decousser, J.-W., Nordmann, P. (2005). ISEcp1B-Mediated Transposition of blaCTX-M in Escherichia coli. Antimicrob. Agents Chemother. 49: 447-450 [Abstract] [Full Text]  
  • Pitout, J. D. D., Hossain, A., Hanson, N. D. (2004). Phenotypic and Molecular Detection of CTX-M-{beta}-Lactamases Produced by Escherichia coli and Klebsiella spp.. J. Clin. Microbiol. 42: 5715-5721 [Abstract] [Full Text]  
  • Wachino, J.-i., Doi, Y., Yamane, K., Shibata, N., Yagi, T., Kubota, T., Arakawa, Y. (2004). Molecular Characterization of a Cephamycin-Hydrolyzing and Inhibitor-Resistant Class A {beta}-Lactamase, GES-4, Possessing a Single G170S Substitution in the {Omega}-Loop. Antimicrob. Agents Chemother. 48: 2905-2910 [Abstract] [Full Text]  
  • Sturenburg, E., Kuhn, A., Mack, D., Laufs, R. (2004). A novel extended-spectrum {beta}-lactamase CTX-M-23 with a P167T substitution in the active-site omega loop associated with ceftazidime resistance. J Antimicrob Chemother 54: 406-409 [Abstract] [Full Text]  
  • Cartelle, M., del Mar Tomas, M., Molina, F., Moure, R., Villanueva, R., Bou, G. (2004). High-Level Resistance to Ceftazidime Conferred by a Novel Enzyme, CTX-M-32, Derived from CTX-M-1 through a Single Asp240-Gly Substitution. Antimicrob. Agents Chemother. 48: 2308-2313 [Abstract] [Full Text]  
  • Kimura, S., Ishiguro, M., Ishii, Y., Alba, J., Yamaguchi, K. (2004). Role of a Mutation at Position 167 of CTX-M-19 in Ceftazidime Hydrolysis. Antimicrob. Agents Chemother. 48: 1454-1460 [Abstract] [Full Text]  
  • Eckert, C., Gautier, V., Saladin-Allard, M., Hidri, N., Verdet, C., Ould-Hocine, Z., Barnaud, G., Delisle, F., Rossier, A., Lambert, T., Philippon, A., Arlet, G. (2004). Dissemination of CTX-M-Type {beta}-Lactamases among Clinical Isolates of Enterobacteriaceae in Paris, France. Antimicrob. Agents Chemother. 48: 1249-1255 [Abstract] [Full Text]  
  • Bonnet, R. (2004). Growing Group of Extended-Spectrum {beta}-Lactamases: the CTX-M Enzymes. Antimicrob. Agents Chemother. 48: 1-14 [Full Text]  
  • Walckenaer, E., Poirel, L., Leflon-Guibout, V., Nordmann, P., Nicolas-Chanoine, M.-H. (2004). Genetic and Biochemical Characterization of the Chromosomal Class A {beta}-Lactamases of Raoultella (formerly Klebsiella) planticola and Raoultella ornithinolytica. Antimicrob. Agents Chemother. 48: 305-312 [Abstract] [Full Text]  
  • Edelstein, M., Pimkin, M., Palagin, I., Edelstein, I., Stratchounski, L. (2003). Prevalence and Molecular Epidemiology of CTX-M Extended-Spectrum {beta}-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae in Russian Hospitals. Antimicrob. Agents Chemother. 47: 3724-3732 [Abstract] [Full Text]  
  • Mammeri, H., Poirel, L., Nordmann, P. (2003). In Vivo Selection of a Chromosomally Encoded {beta}-Lactamase Variant Conferring Ceftazidime Resistance in Klebsiella oxytoca. Antimicrob. Agents Chemother. 47: 3739-3742 [Abstract] [Full Text]  
  • Nagano, N., Shibata, N., Saitou, Y., Nagano, Y., Arakawa, Y. (2003). Nosocomial Outbreak of Infections by Proteus mirabilis That Produces Extended-Spectrum CTX-M-2 Type {beta}-Lactamase. J. Clin. Microbiol. 41: 5530-5536 [Abstract] [Full Text]  
  • Poirel, L., Decousser, J.-W., Nordmann, P. (2003). Insertion Sequence ISEcp1B Is Involved in Expression and Mobilization of a blaCTX-M {beta}-Lactamase Gene. Antimicrob. Agents Chemother. 47: 2938-2945 [Abstract] [Full Text]  
  • Aumeran, C., Chanal, C., Labia, R., Sirot, D., Sirot, J., Bonnet, R. (2003). Effects of Ser130Gly and Asp240Lys Substitutions in Extended-Spectrum {beta}-Lactamase CTX-M-9. Antimicrob. Agents Chemother. 47: 2958-2961 [Abstract] [Full Text]  
  • Tribuddharat, C., Moore, R. A., Baker, P., Woods, D. E. (2003). Burkholderia pseudomallei Class A {beta}-Lactamase Mutations That Confer Selective Resistance against Ceftazidime or Clavulanic Acid Inhibition. Antimicrob. Agents Chemother. 47: 2082-2087 [Abstract] [Full Text]  
  • Bonnet, R., Recule, C., Baraduc, R., Chanal, C., Sirot, D., De Champs, C., Sirot, J. (2003). Effect of D240G substitution in a novel ESBL CTX-M-27. J Antimicrob Chemother 52: 29-35 [Abstract] [Full Text]  
  • Poirel, L., Kampfer, P., Nordmann, P. (2002). Chromosome-Encoded Ambler Class A {beta}-Lactamase of Kluyvera georgiana, a Probable Progenitor of a Subgroup of CTX-M Extended-Spectrum {beta}-Lactamases. Antimicrob. Agents Chemother. 46: 4038-4040 [Abstract] [Full Text]  
  • Poirel, L., Gniadkowski, M., Nordmann, P. (2002). Biochemical analysis of the ceftazidime-hydrolysing extended-spectrum {beta}-lactamase CTX-M-15 and of its structurally related {beta}-lactamase CTX-M-3. J Antimicrob Chemother 50: 1031-1034 [Abstract] [Full Text]  
  • Ho, P. L., Cheung, T. K. M., Yam, W. C., Yuen, K. Y. (2002). Characterization of a laboratory-generated variant of BPS {beta}-lactamase from Burkholderia pseudomallei that hydrolyses ceftazidime. J Antimicrob Chemother 50: 723-726 [Abstract] [Full Text]  
  • Baraniak, A., Fiett, J., Hryniewicz, W., Nordmann, P., Gniadkowski, M. (2002). Ceftazidime-hydrolysing CTX-M-15 extended-spectrum {beta}-lactamase (ESBL) in Poland. J Antimicrob Chemother 50: 393-396 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Poirel, L.
Right arrow Articles by Nordmann, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poirel, L.
Right arrow Articles by Nordmann, P.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»