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Mechanisms of Resistance

Mechanisms of Decreased Susceptibility to Cefpodoxime in Escherichia coli

Antonio Oliver, Linda M. Weigel, J. Kamile Rasheed, John E. McGowan Jr., Patti Raney, Fred C. Tenover
Antonio Oliver
1Department of Microbiology, Ramon y Cajal Hospital, Madrid, Spain
2Division of Healthcare Quality Promotion, Centers for Disease Control and Prevention
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  • For correspondence: aoliver@hsd.es
Linda M. Weigel
2Division of Healthcare Quality Promotion, Centers for Disease Control and Prevention
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J. Kamile Rasheed
2Division of Healthcare Quality Promotion, Centers for Disease Control and Prevention
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John E. McGowan Jr.
3Rollins School of Public Health, Emory University, Atlanta, Georgia
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Patti Raney
2Division of Healthcare Quality Promotion, Centers for Disease Control and Prevention
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Fred C. Tenover
2Division of Healthcare Quality Promotion, Centers for Disease Control and Prevention
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DOI: 10.1128/AAC.46.12.3829-3836.2002
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    FIG. 1.

    OMP profiles of selected E. coli isolates and control strains grown in nutrient broth. Proteins were separated on 10% polyacrylamide-8 M urea gels and stained with Coomassie blue. (A) Comparison of isolates with phenotypes designated as A (EC3589, EC3437, EC922, and EC3095) or B (EC689, EC3194, EC3076) and control strains E. coli C600 (wild type), E. coli MH225 (OmpC−), and CDC3100 (enhanced AmpC producer). (B) Comparison of isolates with phenotypes designated as C (EC251), D (EC4081), A (EC3006), or E (EC1552) and E. coli C600 and E. coli MH225 (OmpC−).

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    FIG. 2.

    DNA sequence of the ampC promoter and attenuator regions from selected E. coli isolates compared with analogous sequences of K12 and DH5α (4, 5). The −35 and −10 consensus promoter sequences and the translation initiation codon for AmpC β-lactamase are underlined. Arrows indicate the transcription attenuator region. The amino acid sequence of the initial segment of the AmpC leader region is shown below the corresponding nucleotide sequence.

Tables

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  • TABLE 1.

    Primers used for PCR amplification of β-lactamase genes

    GenePrimerSequence (5′→3′)PCR product size (pb)
    E. coli ampC promoterAB1GATCGTTCTGCCGCTGTG271
    AmpC2GGGCAGCAAATGTGGAGCAA
    TEM ForwardATGAGTATTCAACATTTCCG867
    ReverseCTGACAGTTACCAATGCTCTC
    SHV ForwardGGTTATGCGTTATATTCGCC867
    ReverseTTAGCGTTGCCAGTGCTC
    OXA-1 OXA-1FACACAATACATATCAACTTCGC814
    OXA-1RAGTGTGTGTTTAGAATGGTGATC
    OXA-2 OXA-2FTTCAAGCCAAAGGCACGATAG704
    OXA-2RTCCGAGTTGACTGCCGGGTTG
    OXA-10 OXA-10FCGTGCTTTGTAAAAGTAGCAG651
    OXA-10RCATGATTTTGGT GGGAATGG
  • TABLE 2.

    β-Lactam resistance phenotypes for 59 E. coli isolates for which the MIC of cefpodoxime is ≥2 and that of ceftazidime is ≤1 μg/mla

    MIC (μg/ml)
    Major phenotypesMinor phenotypes
    ABCDE
    n strains (%)32 (54.2%)18 (30.5%)7 (11.9%)1 (1.7%)1 (1.7%)
    β-Lactamase pI5.4≥8.57.25.4, 5.7, 6.8
    AMX>6432->64>644>64
    AMX-CLAV16-3216-3232->64432
    TIC>1288-32>1288>128
    PIP>128<4-8>12816>128
    PIP-TZ8->128<4-8≥1281616
    CFZ16->2564-6416-32216
    CFZ-CLAV2-42-164-812
    CXM16-328-3232-12816128
    CPD2-42-44-842
    FOX16->328-328-321616
    CAZ0.5-10.5-10.25-10.51
    CAZ-CLAV0.50.5-10.12-0.50.50.5
    CTX0.50.25-1110.5
    CTX-CLAV0.25-0.50.12-0.50.250.50.5
    FEP0.12-0.50.06-0.252-40.250.5
    FEP-CLAV0.120.06-0.120.12-0.50.120.12
    • ↵ a NCCLS breakpoints (S/I/R): AMX, CAZ, CXM, CFZ, FEP, FOX, ≤8/16/≥32; CPD, ≤2/4/≥8; CTX, ≤8/16-32/≥64; PIP, TIC, ≤16/32-64/≥128

  • TABLE 3.

    MICs for EC410 and EC3437 and transconjugants

    AntibioticMIC (μg/ml) for:
    HB101EC410HB101/410TCEC3437HB101/3437TC
    AMX4>64>64>64>64
    AMX-CLAV4168164
    TIC≤4>128>128>128>128
    PIP≤4>128>128>128>128
    PIP-TZ≤4≤4≤432≤4
    CFZ2164324
    CFZ-CLAV14141
    CXM4324164
    CXM-CLAV2162162
    FOX4324324
    CPD12141
    CPD-CLAV0.520.520.5
    CAZ0.50.50.50.50.5
    CAZ-CLAV0.250.250.250.50.25
    CTX0.120.50.120.250.12
    CTX-CLAV0.120.250.060.250.12
    ATM0.250.50.250.50.25
    ATM-CLAV0.120.50.120.250.25
    FEP0.060.120.060.120.12
    FEP-CLAV0.060.060.060.120.06
    • a Strains EC410, EC3437 and their respective transconjugants harbored a pI 5.4 TEM beta-lactamase (see characterization of phenotype A).

  • TABLE 4.

    Resistance profiles of EC251 and EC3006 and their respective transconjugants and clonesa

    AntibioticMIC (μg/ml)
    HB101DH5αEC251HB101 251TCDH5α (pBG251)EC3006HB101 3006TCDH5α(pBG3006)
    AMX4≤2>64>64>64>64>64>64
    AMX-CLAV4≤2321632321632
    TIC≤4≤4>128>128>128>128>128>128
    PIP≤4≤4>12864>128>128>128>128
    PIP-TZ≤4≤412816>128>128128>128
    CZ2116432>25632256
    CZ-CLAV11411211
    CXM423286432816
    CXM-CLAV2116441642
    FOX4232421642
    CPD10.54116412
    CPD-CLAV0.50.520.50.520.50.25
    CAZ0.50.250.50.250.2510.50.5
    CAZ-CLAV0.250.250.250.250.120.50.250.25
    CTX0.120.0610.540.50.120.12
    CTX-CLAV0.120.060.250.120.120.50.060.06
    ATM0.250.060.50.120.50.50.250.12
    ATM-CLAV0.120.060.50.120.060.250.120.12
    FEP0.060.0621>320.50.120.5
    FEP-CLAV0.060.030.120.120.250.120.060.12
    • ↵ a EC251 and its corresponding transconjugant (HB101 251TC) and clone [DH5α (pBG251)] harbored an OXA-30 β-lactamase (see characterization of phenotype C) and EC3006 and its corresponding transconjugant (HB101 3006TC) and clone [DH5α (pBG3006)] harbored a TEM-1 β-lactamase.

  • TABLE 5.

    Mutations in ampC promoter and attenuator regions and cephalosporinase specific activity of selected E. coli isolates

    StrainMutations in promoter and attenuatoraCephalo- sporinase sp actb
    DH5αSame as E. coli K-12 control1.00
    EC689−73, −66, −28, +172.59 ± 0.26
    EC3076−76, +22, +26, +27, +322.04 ± 0.13
    EC3194−76, +22, +26, +27, +322.67 ± 0.12
    EC3358No mutations detected2.40 ± 0.09
    Control strains
        CDC10−73, −28, 1-bp insert between −35 and −10, +1713.6 ± 2.2
        CDC110−73, −32, +5814.6 ± 1.3
        CDC3100−76, 2-bp insert between −35 and −10, +22, +26, +27, +3217.2 ± 3.7
    • ↵ a Key mutations associated with enhanced AmpC production are indicated by bold type.

    • ↵ b Specific cephalosporinase activities are relative values based on the activity in the wild type strain DH5α.

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Mechanisms of Decreased Susceptibility to Cefpodoxime in Escherichia coli
Antonio Oliver, Linda M. Weigel, J. Kamile Rasheed, John E. McGowan Jr., Patti Raney, Fred C. Tenover
Antimicrobial Agents and Chemotherapy Dec 2002, 46 (12) 3829-3836; DOI: 10.1128/AAC.46.12.3829-3836.2002

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Mechanisms of Decreased Susceptibility to Cefpodoxime in Escherichia coli
Antonio Oliver, Linda M. Weigel, J. Kamile Rasheed, John E. McGowan Jr., Patti Raney, Fred C. Tenover
Antimicrobial Agents and Chemotherapy Dec 2002, 46 (12) 3829-3836; DOI: 10.1128/AAC.46.12.3829-3836.2002
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KEYWORDS

Ceftizoxime
Escherichia coli
beta-lactamases

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