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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  E test. 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 OD 450 .    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.

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