Dataset Information

Gradual in vitro Evolution of Cefepime Resistance in an ST131 Escherichia coli Strain Expressing a Plasmid-Encoded CMY-2 β-Lactamase.

ABSTRACT:

Background

In a previous report, a clinical ST131 Escherichia coli isolate (Ec-1),producing a plasmid-encoded AmpC β-lactamase CMY-2, evolved in vivo under cefepime (FEP) treatment to the FEP-resistant Ec-2 strain expressing an extended-spectrum β-lactamase CMY-33. To compare factors responsible for in vitro and in vivo FEP resistance, we reproduced in vitro FEP resistance evolution in Ec-1.

Methods

FEP-resistant mutants were generated by subjecting Ec-1 (FEP MIC = 0.125 mg/L) to sub-inhibitory concentrations of FEP. MICs were obtained by broth microdilution or Etest. Strains were sequenced on an Illumina HiSeq platform. Transcriptional levels and plasmid copy numbers were determined by real-time PCR. Outer membrane proteins (OMPs) were extracted and separated by SDS-PAGE. Growth kinetics was evaluated by measuring OD₄₅₀.

Results

The CMY-2 expressed by Ec-1 evolved to a CMY-69 (strain EC-4) by an Ala294Pro substitution after 24 passages. After 30 passages, the FEP MIC increased to 256 mg/L (strain EC-32). SDS PAGE did not reveal any lack of OMPs in the mutant strains. However, bla _CMY transcription levels were up to 14-times higher than in Ec-1, which was partially explained by mutations in the upstream region of repA resulting in a higher copy number of the bla _CMY-harboring IncI1 plasmid. All mutants showed a slight growth defect but no significant difference in relative growth rates compared to Ec-1.

Conclusion

In vitro sub-inhibitory concentrations of FEP resulted in the selection of resistance mutations altering the H-10 helix of the CMY-2 and increasing the plasmid copy number. Appropriate dosing strategies may help preventing resistance evolution during treatments.

SUBMITTER: Dona V

PROVIDER: S-EPMC6581752 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

Gradual in vitro Evolution of Cefepime Resistance in an ST131 Escherichia coli Strain Expressing a Plasmid-Encoded CMY-2 β-Lactamase.

Donà Valentina V Scheidegger Maximilian M Pires João J Furrer Hansjakob H Atkinson Andrew A Babouee Flury Baharak B

Frontiers in microbiology 20190612

<h4>Background</h4>In a previous report, a clinical ST131 Escherichia coli isolate (Ec-1),producing a plasmid-encoded AmpC β-lactamase CMY-2, evolved in vivo under cefepime (FEP) treatment to the FEP-resistant Ec-2 strain expressing an extended-spectrum β-lactamase CMY-33. To compare factors responsible for in vitro and in vivo FEP resistance, we reproduced in vitro FEP resistance evolution in Ec-1.<h4>Methods</h4>FEP-resistant mutants were ...[more]

PMID: 31244817

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Project description:Cefepime is frequently prescribed to treat infections caused by AmpC-producing Gram-negative bacteria. CMY-2 is the most common plasmid-mediated AmpC (pAmpC) β-lactamase. Unfortunately, CMY variants conferring enhanced cefepime resistance have been reported. Here, we describe the evolution of CMY-2 to an extended-spectrum AmpC (ESAC) in clonally identical Escherichia coli isolates obtained from a patient. The CMY-2-producing E. coli isolate (CMY-2-Ec) was isolated from a wound. Thirty days later, one CMY-33-producing E. coli isolate (CMY-33-Ec) was detected in a bronchoalveolar lavage fluid sample. Two weeks before the isolation of CMY-33-Ec, the patient received cefepime. CMY-33-Ec and CMY-2-Ec were identical by repetitive extragenic palindromic-PCR (rep-PCR), being of hyperepidemic sequence type 131 (ST131) but showing different β-lactam MICs (e.g., cefepime MIC, 16 and ≤ 0.5 μg/ml for CMY-33-Ec and CMY-2-Ec, respectively). Identical CMY-2-Ec isolates were also found in a rectal swab. CMY-33 differs from CMY-2 by a Leu293-Ala294 deletion. Expressed in E. coli strain DH10B, both CMYs conferred resistance to ceftazidime (≥ 256 μg/ml), but the cefepime MICs were higher for CMY-33 than CMY-2 (8 versus 0.25 μg/ml, respectively). The kcat/Km or inhibitor complex inactivation (kinact)/Ki app (μM(-1) s(-1)) indicated that CMY-33 possesses an extended-spectrum β-lactamase (ESBL)-like spectrum compared to that of CMY-2 (e.g., cefoxitin, 0.2 versus 0.4; ceftazidime, 0.2 versus not measurable; cefepime, 0.2 versus not measurable; and tazobactam, 0.0018 versus 0.0009, respectively). Using molecular modeling, we show that a widened active site (∼ 4-Å shift) may play a significant role in enhancing cefepime hydrolysis. This is the first in vivo demonstration of a pAmpC that under cephalosporin treatment expands its substrate spectrum, resembling an ESBL. The prevalence of CMY-2-Ec isolates is rapidly increasing worldwide; therefore, awareness that cefepime treatment may select for resistant isolates is critical.

Dataset Information

Gradual in vitro Evolution of Cefepime Resistance in an ST131 Escherichia coli Strain Expressing a Plasmid-Encoded CMY-2 β-Lactamase.

Background

Methods

Results

Conclusion

Publications

Gradual <i>in vitro</i> Evolution of Cefepime Resistance in an ST131 <i>Escherichia coli</i> Strain Expressing a Plasmid-Encoded CMY-2 β-Lactamase.

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