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Mechanisms of Resistance

Characterization and Nucleotide Sequence of CARB-6, a New Carbenicillin-Hydrolyzing β-Lactamase from Vibrio cholerae

Danièle Choury, Gérald Aubert, Marie-France Szajnert, Kemal Azibi, Marc Delpech, Gérard Paul
Danièle Choury
Laboratoire de Biologie Moléculaire des Cellules Eucaryotes,
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Gérald Aubert
Laboratoire de Bactériologie, CHU, Saint-Etienne, France, and
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Marie-France Szajnert
INSERM U129, and
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Kemal Azibi
INSERM U129, and
CHU Alger-Ouest, Algiers, Algeria
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Marc Delpech
Laboratoire de Biologie Moléculaire des Cellules Eucaryotes,
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Gérard Paul
Laboratoire de Recherche en Microbiologie, UFR Cochin Port-Royal, 75014 Paris, and
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DOI: 10.1128/AAC.43.2.297
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  • Fig. 1.
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    Fig. 1.

    Sequencing strategy for CARB-6 β-lactamase gene. The CARB-6 gene is represented by the black box. The arrows indicate the oligonucleotide positions.

  • Fig. 2.
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    Fig. 2.

    Gene sequence and deduced amino acid sequence of theV. cholerae β-lactamase (CARB-6). The deduced amino acid sequence is designated by the one-letter code. The active site,STFK, is boxed, and the differences relative to CARB-1 to CARB-3 are underlined.

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    Fig. 3.

    Multiple sequence alignment of the amino acid sequences of CARB-1, CARB-2, CARB-3, CARB-4, P. mirabilis N29,P. mirabilis GN79, and CARB-6 β-lactamases. The shadowed boxes (I to VII) correspond to amino acid boxes conserved in all penicillin-recognizing enzymes, as identified by Joris et al. (14). Alpha-helix and beta-barrel motifs are indicated from the PC-1 crystal structure (4, 12). Asterisks indicate the conserved residues specific for class A β-lactamases. Amino acid changes are written as black letters in white boxes. Sequences are numbered as described by Ambler (2).

Tables

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  • Table 1.

    Oligonucleotide primers used for PCR amplification

    Primer pairLocation (5′-3′)aSequence (5′-3′)
    V144–65TTGATGTTATGGAGCAGCAACG
    V1′266–248CAATTGCCTTAACGTCTTG
    V275–88AGCAGGGCAGTCGC
    V2′254–236CGTCTTGTTCAACTTGCTG
    V3235–254TCAGCAAGTTGAACAAGACG
    V3′899–878CAACTGCTGTAATACTCCGAGC
    V4684–708TGAGGGATACGACAACTCCTAAGGC
    V4′1047–1027TGTTAGCCTTATCAGCGCGAC
    • ↵a Oligonucleotide positions are given according to their location on the CARB-3 gene sequence as described elsewhere (4).

  • Table 2.

    Physicochemical properties of the CARB-6 β-lactamase ofV. cholerae and comparison with TEM-1 and other carbenicillin-hydrolyzing enzymes

    β-LactamaseMol wtpIa
    CARB-633,000b5.35
    SAR-133,700b4.9
    TEM-128,911c5.4
    CARB-131,405c5.3
    CARB-231,347c5.7
    CARB-331,313c5.75
    • ↵a pI was determined in a polyacrylamide gel with an Ampholine gradient (pH 3.5 to 9.5; Pharmacia).

    • ↵b Molecular weight determined by electrophoresis in an SDS–12% polyacrylamide gel by the method of Laemmli (19) with low-range prestained SDS-polyacrylamide gel electrophoresis standards.

    • ↵c Molecular weight as estimated according to the amino acid sequence. The molecular mass of CARB-6 calculated according to the amino acid composition is 31,429 Da (Bio-Oriented Web Server Facilities, ABIM-Aix Marseille University).

  • Table 3.

    Kinetic parameters for V. cholerae CARB-6 β-lactamase in comparison with the published values for β-lactamases TEM-1, SAR-1, and CARB-1 to CARB-3a

    AntibioticKm(μM)Vm(relative)b
    CARB-6SAR-1TEM-1CARB-1CARB-2CARB-3CARB-6SAR-1TEM-1CARB-1CARB-2CARB-3
    Penicillin G964224137.39.8100100100100100100
    Amoxicillin157 —c4313.616.421.8266—846312897
    Ampicillin—68353020.924—6311194.593100
    Ticarcillin55—104018.82159—38812697
    Carbenicillin—190141227295—12210126343147
    Piperacillin7—43———15.9—86———
    Mezlocillin58.5—24615.51420100—1008683111
    Oxacillin37—6583124.55.25—55.411.412.5
    Cephaloridine5269395040.520841859.5217614.61644.2
    Cephalothin——————<0.5—201.250.80.5
    • ↵a Data for TEM-1 (21), SAR-1 (29), and CARB-1 to CARB-3 (16, 18) have been published previously.

    • ↵b Vm is expressed as a percentage of hydrolysis of benzylpenicillin (for which the Vm is equal to 100), which was used as a reference.

    • ↵c —, not determined.

  • Table 4.

    Effects of chemical and β-lactam inhibitors on CARB-6, SAR-1, and TEM-1 β-lactamasesa

    Substrates% Inhibition (concn [μM] of inhibitor)a
    CARB-6SAR-1TEM-1
    NaCl3 (5.104) —b0 
    Cloxacillin100 (100)50 (7)70 (100)
    Oxacillin50 (100)——
    Methicillin100 (100)——
    Tazobactam50 (0.06)—50 (0.74)
    Sulbactam50 (1.4)—50 (19)
    Clavulanic acid50 (0.075)50 (0.005)50 (0.034)
    PCMBc52 (20)50 (>100)50 (200)
    • ↵a Data for SAR-1 (29) and TEM-1 (21) have been published previously. Results are expressed as percentage of inhibited activity at the indicated concentration of inhibitor in the presence of benzylpenicillin. The inhibitory effects of clavulanic acid and sulbactam were determined after preincubation with the enzyme for 10 min at 37°C.

    • ↵b —, not determined.

    • ↵c PCMB, p-hydroxymercuribenzoate.

  • Table 5.

    Amino acid point mutations in β-lactamases CARB-1, CARB-2, CARB-3, and CARB-6

    Amino acid positionaAmino acid changeb
    CARB-6CARB-1CARB-2CARB-3
    192GlnLysLysLys
    193LysPhePheLys
    198ThrAlaAlaAla
    202AlaMetMetMet
    203SerAsnAsnAsn
    227ValAlaAlaAla
    228LysGlyGlyGly
    230SerAsnAsnAsn
    249IleValValVal
    255GluHisHisHis
    256LysGlnGlnGln
    257LysAlaAlaAla
    258ThrProProPro
    269GluGlnGlnGln
    273AlaGluAlaAla
    284ArgHisHisHis
    288GluAspAspAsp
    • ↵a The numbering of the positions of the mutations is according to Ambler (2).

    • ↵b Boldface italics indicate mutated amino acids relative to CARB-3 β-lactamase.

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Characterization and Nucleotide Sequence of CARB-6, a New Carbenicillin-Hydrolyzing β-Lactamase from Vibrio cholerae
Danièle Choury, Gérald Aubert, Marie-France Szajnert, Kemal Azibi, Marc Delpech, Gérard Paul
Antimicrobial Agents and Chemotherapy Feb 1999, 43 (2) 297-301; DOI: 10.1128/AAC.43.2.297

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Characterization and Nucleotide Sequence of CARB-6, a New Carbenicillin-Hydrolyzing β-Lactamase from Vibrio cholerae
Danièle Choury, Gérald Aubert, Marie-France Szajnert, Kemal Azibi, Marc Delpech, Gérard Paul
Antimicrobial Agents and Chemotherapy Feb 1999, 43 (2) 297-301; DOI: 10.1128/AAC.43.2.297
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KEYWORDS

Carbenicillin
penicillins
Vibrio cholerae
beta-lactamases

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