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Mechanisms of Resistance

Selection and Characterization of β-Lactam–β-Lactamase Inactivator-Resistant Mutants following PCR Mutagenesis of the TEM-1 β-Lactamase Gene

Sergei B. Vakulenko, Bruce Geryk, Lakshmi P. Kotra, Shahriar Mobashery, Stephen A. Lerner
Sergei B. Vakulenko
Departments of Medicine and
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Bruce Geryk
Departments of Medicine and
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Lakshmi P. Kotra
Department of Chemistry, Wayne State University, Detroit, Michigan 48202
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Shahriar Mobashery
Department of Chemistry, Wayne State University, Detroit, Michigan 48202
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Stephen A. Lerner
Departments of Medicine and
Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, and
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DOI: 10.1128/AAC.42.7.1542
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    Fig. 1.

    TEM-1 β-lactamase with clavulanic acid bound in the active site. The side chains of the residues Met69, Ser130, Arg244, and Asn276 are shown as a ball and stick representation; Clav, clavulanic acid. The figure was prepared by using the MOLSCRIPT program (17).

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    Fig. 2.

    (A) Preacylation complex of TEM-1 β-lactamase and clavulanic acid. (B) The active site is shown for the regions within the vicinity of the acyl-enzyme intermediate of TEM-1 β-lactamase and clavulanic acid. Clavulanic acid (Clav), water molecules, and Ser70 are shown as ball and stick representations. The arrow indicates the B3 strand, which forms a portion of the active-site surface. The figure was prepared by using the MOLSCRIPT program (17).

Tables

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  • Table 1.

    Types of mutant β-lactamases selected by growth of transformants in the presence of ampicillin plus clavulanate

    Mutant typeaAmino acidb substitutions for the following amino acids of the wild-type TEM-1 β-lactamase:Silent mutation(s)c
    A42N52M69dK111S130H153K192T195L201G238R244T272N276
    1N52YM69LT272AA42A, T281T
    2M69LK192RR9R, Q206Q
    3S130G
    4A42GS130GT195AG238ST265T
    5R244C
    6A42GL201PR244CL73L
    7A42GH153DR244CR9R, K34K, L49L, R61R, Q88Q, K111K
    8A42GK111RL201PR244CR9R, K34K, L49L, R61R, G87G, I260I
    9R244SG45G, P257P
    10L201PR244SR65R, P67P, I260I
    11N276D
    • ↵a The wild type is TEM-1.

    • ↵b Amino acids are indicated by conventional single-letter nomenclature.

    • ↵c Mutations in the base sequence of the gene that do not alter the amino acid sequence of the enzyme.

    • ↵d The boldface font indicates sites at which mutations have previously been reported to confer resistance to β-lactam–clavulanate combinations.

  • Table 2.

    MICs of ampicillin, β-lactam inhibitors, and their combinations conferred by the TEM-1 β-lactamase and various types of mutant derivatives for E. coli JM83

    β-Lactamase or mutant derivativeMIC (μg/ml)a
    ApClAp + Cl 1bAp + Cl 2Ap + Cl 5Ap + Cl 10SbAp + Sb 5Ap + Sb 10Ap + Sb 20TbAp + Tb 5Ap + Tb 10Ap + Tb 50
    −c43244423221NG256444
    TEM-116,0003225012564166416,0008,0004,00025632164
    18,000321,000500250125324,0002,000NG25664324
    31,000321,0001,0005002503225064NG2561256416
    4500325005002501253212532NG2561256416
    52,000322,0001,00050012532500125NG25664328
    62,000322,0001,00050012532500125NG256125648
    750032500250125643212532NG25632164
    82,000322,0001,00050012532500125NG256125648
    98,000324,0002,0001,000250322,000500NG256500648
    102,000322,0001,00050012532500125NG25664328
    1116,000324,0002,0001,0002506416,00016,0008,0002562,000648
    • ↵a Abbreviations: Ap, ampicillin; Cl, clavulanate; Sb, sulbactam; Tb, tazobactam.

    • ↵b The numbers indicate the fixed concentration (in micrograms per milliliter) of the indicated inhibitor in combination with ampicillin for determination of ampicillin MICs.

    • ↵c −, E. coli JM83 without a plasmid.

    • d NG, no growth in the presence of the lowest concentration of ampicillin tested (0.06 μg/ml for E. coliJM83 and 8 μg/ml for the transformants bearing a β-lactamase).

  • Table 3.

    MICs of cephalosporins and aztreonam conferred by TEM-1 β-lactamase and various types of mutant derivatives forE. coli JM83

    β-Lactamase or mutant derivative MIC (μg/ml)
    CephalothinCephaloridineCeftazidimeCeftriaxoneAztreonam
    −a420.1250.030.06
    TEM-1321280.250.030.06
    1420.1250.030.06
    3420.1250.030.06
    4420.1250.030.06
    5420.1250.030.06
    6420.1250.030.06
    7420.1250.030.06
    8420.1250.030.06
    9420.1250.030.06
    10420.1250.030.06
    11161280.50.030.06
    • ↵a −, E. coli JM83 without a plasmid.

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Selection and Characterization of β-Lactam–β-Lactamase Inactivator-Resistant Mutants following PCR Mutagenesis of the TEM-1 β-Lactamase Gene
Sergei B. Vakulenko, Bruce Geryk, Lakshmi P. Kotra, Shahriar Mobashery, Stephen A. Lerner
Antimicrobial Agents and Chemotherapy Jul 1998, 42 (7) 1542-1548; DOI: 10.1128/AAC.42.7.1542

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Selection and Characterization of β-Lactam–β-Lactamase Inactivator-Resistant Mutants following PCR Mutagenesis of the TEM-1 β-Lactamase Gene
Sergei B. Vakulenko, Bruce Geryk, Lakshmi P. Kotra, Shahriar Mobashery, Stephen A. Lerner
Antimicrobial Agents and Chemotherapy Jul 1998, 42 (7) 1542-1548; DOI: 10.1128/AAC.42.7.1542
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KEYWORDS

Drug Resistance, Multiple
Escherichia coli
beta-lactam resistance
beta-lactamases

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