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Mechanisms of Resistance

Amino Acid Substitutions in a Variant of IMP-1 Metallo-β-Lactamase

Shizuko Iyobe, Haruko Kusadokoro, Junko Ozaki, Naoki Matsumura, Shinzaburo Minami, Shin Haruta, Tetsuo Sawai, Koji O'Hara
Shizuko Iyobe
Laboratory of Drug Resistance in Bacteria, Gunma University School of Medicine, Maebashi, 1
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Haruko Kusadokoro
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Chiba, 2 and
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Junko Ozaki
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Chiba, 2 and
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Naoki Matsumura
Research Laboratory, Toyama Chemical Co., Ltd., Toyama, 3 Japan
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Shinzaburo Minami
Research Laboratory, Toyama Chemical Co., Ltd., Toyama, 3 Japan
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Shin Haruta
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Chiba, 2 and
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Tetsuo Sawai
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Chiba, 2 and
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Koji O'Hara
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Chiba, 2 and
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DOI: 10.1128/AAC.44.8.2023-2027.2000
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    Fig. 1.

    Construction of the hybrid and parent metallo-β-lactamase genes. The procedure is explained in Materials and Methods. The horizontal single lines represent multicloning regions of the vector plasmids flanking the inserts. TheEcoRI (E) and HindIII (H) sites located in the multicloning regions are indicated above the lines. Inserts are shown by boxes, in which the regions of metallo-β-lactamase genes are shaded in black. The sites of the restriction enzymes are indicated above the boxes. Abbreviations not used in the text are B (BamHI) and X (XbaI). The sites indicated under the boxes are P (for promoter) (14), G (for guanine), and A (for adenine).

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  • Table 1.

    Resistance levels conferred by the parent and hybrid metallo-β-lactamasesa

    Plasmidβ-LactamaseAmino acid residue at position:MIC (μg/ml) ofb:
    87196LORCEFCTXCAZAMPIMPATM
    pMS401IMP-3GlyGly64512646481<0.5
    pMS402HybridGluGly64512646481<0.5
    pMS502HybridGlySer321283264642<0.5
    pMS501IMP-1GluSer321283264642<0.5
    pHSG398 Δ Hd28<0.5<0.54<0.5<0.5
    None28<0.5<0.54<0.5<0.5
    • ↵a Host strain: E. coli K-12 JM83.

    • ↵b LOR, cephaloridine; CEF, cephalothin; CTX, cefotaxime; CAZ, ceftazidime; AMP, ampicillin; IMP, imipenem; ATM, aztreonam.

  • Table 2.

    Kinetic parameters for hydrolysis of various β-lactam antibiotics by the hybrid and parent metallo-β-lactamases

    PlasmidEnzymeAmino acid at position:Kinetic parameterAntibiotica
    87196LORCEFCTXCAZPENAMPIMP
    pMS401IMP-3GlyGly k cat(s−1)221 ± 15b 223 ± 2240.1 ± 2.64.5 ± 0.4b 14.3 ± 0.8b 7.4 ± 1.2b 92.3 ± 2b
    Km (μM)248 ± 39b 9.9 ± 1.33.1 ± 0.3128 ± 17b 370 ± 59b 464 ± 145b 1140 ± 40b
    k cat/Km (μM−1s−1)0.8922.512.90.0350.0390.0160.08
    pMS402HybridGluGly k cat(s−1)268 ± 5b 384 ± 1154.8 ± 5.33.6 ± 0.2b 21.6 ± 1.0b 11.8 ± 1.4b 132 ± 8b
    Km (μM)215 ± 17b 9.8 ± 0.33.0 ± 0.3118 ± 19b 464 ± 60b 437 ± 105b 1280 ± 120b
    k cat/Km (μM−1s−1)1.2539.218.30.0310.0470.0270.10
    pMS502HybridGlySer k cat(s−1)38.4 ± 1.243.2 ± 1.716.5 ± 1.311.3 ± 0.3282 ± 1987.0 ± 2.790.8 ± 5.3
    Km (μM)7.1 ± 0.12.3 ± 0.11.3 ± 0.231.8 ± 1.5216 ± 13110 ± 4.933.2 ± 1.9
    k cat/Km (μM−1s−1)5.418.812.70.361.30.82.7
    pMS501IMP-1GluSer k cat(s−1)62.0 ± 3.065.0 ± 2.922.5 ± 1.916.3 ± 0.9461 ± 17162 ± 15127 ± 11
    Km (μM)7.2 ± 0.42.0 ± 0.11.4 ± 0.146.0 ± 1.4241 ± 10140 ± 930.0 ± 3.9
    k cat/Km (μM−1s−1)8.632.516.10.351.91.24.2
    • ↵a PEN, benzylpenicillin; see footnoteb of Table 1 for other abbreviations.

    • ↵b Obtained from Michaelis-Menten graph; see Materials and Methods.

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Amino Acid Substitutions in a Variant of IMP-1 Metallo-β-Lactamase
Shizuko Iyobe, Haruko Kusadokoro, Junko Ozaki, Naoki Matsumura, Shinzaburo Minami, Shin Haruta, Tetsuo Sawai, Koji O'Hara
Antimicrobial Agents and Chemotherapy Aug 2000, 44 (8) 2023-2027; DOI: 10.1128/AAC.44.8.2023-2027.2000

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Amino Acid Substitutions in a Variant of IMP-1 Metallo-β-Lactamase
Shizuko Iyobe, Haruko Kusadokoro, Junko Ozaki, Naoki Matsumura, Shinzaburo Minami, Shin Haruta, Tetsuo Sawai, Koji O'Hara
Antimicrobial Agents and Chemotherapy Aug 2000, 44 (8) 2023-2027; DOI: 10.1128/AAC.44.8.2023-2027.2000
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