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

Donà, Valentina; Scheidegger, Maximilian; do Couto Pires, João Pedro; Furrer, Hansjakob; Atkinson, Andrew; Babouee Flury, Baharak (2019). Gradual in vitro Evolution of Cefepime Resistance in an ST131 Escherichia coli Strain Expressing a Plasmid-Encoded CMY-2 β-Lactamase. Frontiers in Microbiology, 10, p. 1311. Frontiers 10.3389/fmicb.2019.01311

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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 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. 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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases
04 Faculty of Medicine > Service Sector > Institute for Infectious Diseases > Research
04 Faculty of Medicine > Department of Haematology, Oncology, Infectious Diseases, Laboratory Medicine and Hospital Pharmacy (DOLS) > Clinic of Infectiology

Graduate School:

Graduate School for Cellular and Biomedical Sciences (GCB)

UniBE Contributor:

Donà, Valentina; Scheidegger, Maximilian; do Couto Pires, João Pedro; Furrer, Hansjakob; Atkinson, Andrew and Babouee Flury, Baharak

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health
600 Technology > 630 Agriculture

ISSN:

1664-302X

Publisher:

Frontiers

Language:

English

Submitter:

Annelies Luginbühl

Date Deposited:

13 Aug 2019 12:01

Last Modified:

18 Aug 2019 02:39

Publisher DOI:

10.3389/fmicb.2019.01311

PubMed ID:

31244817

Uncontrolled Keywords:

CMY-2 CMY-69 ST131 WGS cefepime resistance evolution

BORIS DOI:

10.7892/boris.131697

URI:

https://boris.unibe.ch/id/eprint/131697

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