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Mechanisms of Resistance

Diversity of TEM Mutants in Proteus mirabilis

R. Bonnet, C. De Champs, D. Sirot, C. Chanal, R. Labia, J. Sirot
R. Bonnet
Laboratoire de Bactériologie, Faculté de Médecine, 63001 Clermont-Ferrand Cedex, and
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C. De Champs
Laboratoire de Bactériologie, Faculté de Médecine, 63001 Clermont-Ferrand Cedex, and
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D. Sirot
Laboratoire de Bactériologie, Faculté de Médecine, 63001 Clermont-Ferrand Cedex, and
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C. Chanal
Laboratoire de Bactériologie, Faculté de Médecine, 63001 Clermont-Ferrand Cedex, and
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R. Labia
UMR 175, CNRS-MNHN, 29000 Quimper, France
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J. Sirot
Laboratoire de Bactériologie, Faculté de Médecine, 63001 Clermont-Ferrand Cedex, and
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DOI: 10.1128/AAC.43.11.2671
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    Fig. 1.

    (A) Agarose (0.7%) electrophoresis of plasmid DNA from ESBL-producing E. coli transconjugants and from IRT-producing P. mirabilis. (B) Hybridization with TEM-specific DNA probes. Lanes: 1, plasmid RSa (39 kb); 2, plasmid TP114 (61 kb); 3, pCFF04 (TEM-3; 85 kb); 4, pCFF74 (TEM-24; 85 kb); 5, pCFF14 (TEM-5; 180 kb); 6, pCFF134 (TEM-3); 7, TrCF669 (TEM-66); 8, TrCF39 (TEM-3); 9, TrCF249 (TEM-24); 10, CF449 (TEM-44); 11, CF659 (TEM-65).

Tables

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  • Table 1.

    β-Lactam MICs for TEM-producingP. mirabilis strains

    β-LactamMIC (μg/ml) for the following strains (enzymes):
    CF19 (TEM-1)CF29 (TEM-2)CF579 (TEM-57)
    Amoxicillin5124,0964,096
    Amoxicillin + CAa41616
    Ticarcillin128512512
    Ticarcillin + CA222
    Piperacillin8256256
    Piperacillin + TAZb0.511
    Cephalothin888
    • ↵a CA, clavulanic acid (2 μg/ml).

    • ↵b TAZ, tazobactam (4 μg/ml).

  • Table 2.

    β-Lactam MICs for ESBL-producing P. mirabilis strains and their E. coli transconjugants

    β-LactamsMIC (μg/ml) for the following strains (enzymes):
    P. mirabilis CF249 (TEM-24)E. coli TrCF249 (TEM-24)P. mirabilis CF39 (TEM-3)E. coliTrCF39 (TEM-3)P. mirabilis CF669 (TEM-66)E. coli TrCF669 (TEM-66)
    Cefotaxime0.1212484
    Cefotaxime + CAa0.030.030.060.060.060.03
    Ceftazidime4640.5884
    Ceftazidime + CA0.120.250.120.250.120.12
    Aztreonam0.0640.0620.252
    Aztreonam + CA0.010.030.010.060.060.003
    • ↵a CA, clavulanic acid (2 μg/ml).

  • Table 3.

    β-Lactam MICs for IRT-producingP. mirabilis strains

    β-LactamMIC (μg/ml) for the following strains (enzymes):
    CF739 (TEM-73 [IRT-18])CF749 (TEM-74 [IRT-19])CF449 (TEM-44 [IRT-13])CF659 (TEM-65 [IRT-16])
    Amoxicillin1,0242,0481,02464
    Amoxicillin + CAa1,02451251264
    Ticarcillin832161
    Ticarcillin + CA4421
    Piperacillin161682
    Piperacillin + TAZb410.50.5
    Cephalothin2442
    • ↵a CA, clavulanic acid (2 μg/ml).

    • ↵b TAZ, tazobactam (4 μg/ml).

  • Table 4.

    pIs and kinetic parameters inhibitors for TEM-type and IRT-type β-lactamases

    DrugKinetic parameters for the following strains (enzymes; pIs):
    CF19 (TEM-1; 5.4)CF29 (TEM-2; 5.6)CF579 (TEM-57; 5.2)CF739 (TEM-73 [IRT-18]; 5.2)CF749 (TEM-74 [IRT-19]; 5.2)CF449 (TEM-44 [IRT-13]; 5.4)CF659 (TEM-65 [IRT-16]; 5.4)
    VmaxaKm(μM)VmaxKm(μM)VmaxKm(μM)VmaxKm(μM)VmaxKm(μM)VmaxKm(μM)VmaxKm(μM)
    Benzylpenicillin100251002010040100334100330100375100350
    Amoxicillin772695257338207315140400145360150400
    Ticarcillin10101091015281027652.34602600
    Cephalothin1025025500179001.51,000130023122400
    • ↵a Vmax values are given as a percentage of the Vmax for benzylpenicillin, which was taken as 100%.

  • Table 5.

    IC50s of inhibitors for TEM-type and IRT-type β-lactamases

    Strain (enzyme)IC50 (μM)
    ClavulanateTazobactam
    CF19 (TEM-1)0.180.05
    CF29 (TEM-2)0.090.04
    CF579 (TEM-57)NDaND
    CF739 (TEM-73 [IRT-18])3112
    CF749 (TEM-74 [IRT-19])9.42.9
    CF449 (TEM-44 [IRT-13])103.0
    CF65a [TEM-65 [IRT-16])9.03.0
    • ↵a ND, not done.

  • Table 6.

    pI and kinetic parameters for ESBL-type β-lactamases

    DrugKinetic parameters for the following strains (enzymes; pIs):
    CF249 (TEM-24; 6.5)CF39 (TEM-3; 6.3)CF669 (TEM-66; 6.0)
    VmaxaKm(μM)VmaxKm(μM)VmaxKm(μM)
    Benzylpenicillin100510041005
    Amoxicillin6243725705
    Cephalothin281431072211030
    Cefotaxime13050445100400100
    Ceftazidime1,4103774040040450
    • ↵a Vmax values are given as a percentage of the Vmax for benzylpenicillin, which was taken as 100%.

  • Table 7.

    Nucleotide and amino acid substitutions in theblaTEM genes fromP. mirabilis strains

    β-Lactamase and strain or enzymeMutation at the following nucleotide (substituted amino acid)a:
    32b226 (8)263 (21)317 (39)346 (48)436 (78)477 (92)512 (104)604 (134)682 (160)692 (164)911 (237)914 (238)917 (240)925 (242)929 (244)990 (265)
    TEM-like
     TEM-1 (blaTEM-1A)cGCC-TAC (Phe)C (Leu)C (Gln)A (Glu)C (Gly)G (Gly)G (Glu)G (Ala)T (Thr)C (Arg)G (Ala)G (Gly)G (Glu)G (Gly)C (Arg)C (Thr)
     TEM-1 (blaTEM-1B)cGCC-TATTT
     TEM-2 (blaTEM-2)cGCT-TAA (Lys)GTCA
     CF579 (TEM-57)GCTATAGTA (Asp)CA
    ESBL
     CF249 (TEM-24)GCT-TAA (Lys)GTA (Lys)CA (Ser)A (Thr)A (Lys)A
     CF39 (TEM-3)GCT-TAA (Lys)GTA (Lys)A (Ser)A
     CF669 (TEM-66)GCT-TAA (Lys)GTA (Asp)A (Lys)A (Ser)
    IRT-like
     CF449 (TEM-44 [IRT-13])GCT-TAA (Lys)GTCAA (Ser)
     CF659 (TEM-65 [IRT-16])GCT-TAA (Lys)GCAT (Cys)
     CF739 (TEM-73 [IRT-18])GCT-TAT (Phe)CGTCAT (Cys)T (Met)
     CF749 (TEM-74 [IRT-19])GCT-TAT (Phe)CGTCAA (Ser)T (Met)
    • ↵a Nucleotide positions are from Sutcliffe (37), and amino acids positions are from Ambler et al. (1).

    • ↵b The nucleotide at position 32 is given in boldface type.

    • ↵c The sequences are from Chen and Clowes (12) and Goussard and Courvalin (15).

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Diversity of TEM Mutants in Proteus mirabilis
R. Bonnet, C. De Champs, D. Sirot, C. Chanal, R. Labia, J. Sirot
Antimicrobial Agents and Chemotherapy Nov 1999, 43 (11) 2671-2677; DOI: 10.1128/AAC.43.11.2671

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Diversity of TEM Mutants in Proteus mirabilis
R. Bonnet, C. De Champs, D. Sirot, C. Chanal, R. Labia, J. Sirot
Antimicrobial Agents and Chemotherapy Nov 1999, 43 (11) 2671-2677; DOI: 10.1128/AAC.43.11.2671
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KEYWORDS

Proteus mirabilis
beta-lactamases

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