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

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

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.01311

DOI=10.3389/fmicb.2019.01311

ISSN=1664-302X

ABSTRACT=<sec><title>Background</title><p>In a previous report, a clinical ST131 <italic>Escherichia coli</italic> isolate (<italic>Ec</italic>-1),producing a plasmid-encoded AmpC β-lactamase CMY-2, evolved <italic>in vivo</italic> under cefepime (FEP) treatment to the FEP-resistant <italic>Ec</italic>-2 strain expressing an extended-spectrum β-lactamase CMY-33. To compare factors responsible for <italic>in vitro</italic> and <italic>in vivo</italic> FEP resistance, we reproduced <italic>in vitro</italic> FEP resistance evolution in <italic>Ec</italic>-1.</p></sec><sec><title>Methods</title><p>FEP-resistant mutants were generated by subjecting <italic>Ec-1</italic> (FEP MIC = 0.125 mg/L) to sub-inhibitory concentrations of FEP. MICs were obtained by broth microdilution or <italic>E</italic>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<sub>450</sub>.</p></sec><sec><title>Results</title><p>The CMY-2 expressed by <italic>Ec</italic>-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, <italic>bla</italic><sub>CMY</sub> transcription levels were up to 14-times higher than in <italic>Ec</italic>-1, which was partially explained by mutations in the upstream region of <italic>repA</italic> resulting in a higher copy number of the <italic>bla</italic><sub>CMY</sub>-harboring IncI1 plasmid. All mutants showed a slight growth defect but no significant difference in relative growth rates compared to <italic>Ec</italic>-1.</p></sec><sec><title>Conclusion</title><p><italic>In vitro</italic> 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.</p></sec>