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

Valentina Donà, Maximilian Scheidegger, João Pires, Hansjakob Furrer, Andrew Atkinson & Baharak Babouee Flury

abstract

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.

Conclusion
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.
   
citation Donà V, Scheidegger M, Pires J, Furrer H, Atkinson A, Babouee Flury B. Gradual Evolution of Cefepime Resistance in an ST131 Strain Expressing a Plasmid-Encoded CMY-2 β-Lactamase. Front Microbiol 2019; 10:1311.
   
type journal paper/review (English)
date of publishing 12-06-2019
journal title Front Microbiol (10)
ISSN print 1664-302X
pages 1311
PubMed 31244817
DOI 10.3389/fmicb.2019.01311