Abstract
BackgroundIn 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.MethodsFEP-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.ResultsThe 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, blaCMY 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 blaCMY-harboring IncI1 plasmid. All mutants showed a slight growth defect but no significant difference in relative growth rates compared to Ec-1.ConclusionIn 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.
Highlights
Cefepime (FEP) consumption in Swiss hospitals is among the highest in Europe (Pluss-Suard et al, 2012)
MIC values were interpreted in accordance with the 2016 European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria, besides for cephalothin, cefoxitin and polymyxin B, for which clinical and laboratory standards institute (CLSI) criteria (M100-S26, 2016) were applied
PCR amplification and Sanger sequencing of the blaCMY-coding region indicated that in EC-4, EC16 and EC-32, the CMY-2 had evolved to a CMY-69 due to the single nucleotide polymorphism (SNP) G940C resulting in an Ala294Pro substitution [based on the amino acid numbering scheme of Enterobacter cloacae P99 (Galleni et al, 1988)] in the H-10 helix (Supplementary Figure S1)
Summary
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 extendedspectrum 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
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have