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

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

Methods
FEP-resistant mutants were generated by subjecting (FEP MIC = 0.125 mg/L) to sub-inhibitory concentrations of FEP. MICs were obtained by broth microdilution or 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.

Results
The CMY-2 expressed by -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, transcription levels were up to 14-times higher than in -1, which was partially explained by mutations in the upstream region of resulting in a higher copy number of the -harboring IncI1 plasmid. All mutants showed a slight growth defect but no significant difference in relative growth rates compared to -1.