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Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2016 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 5, 1188-1198 p.Article in journal (Refereed) Published
Abstract [en]

The worldwide spread of ESBL-producing Enterobacteriaceae has led to an increased use of carbapenems, the group of beta-lactams with the broadest spectrum of activity. Bacterial resistance to carbapenems is mainly due to acquired carbapenemases or a combination of ESBL production and reduced drug influx via loss of outer-membrane porins. Here, we have studied the development of carbapenem resistance in Escherichia coli in the absence of beta-lactamases. We selected mutants with high-level carbapenem resistance through repeated serial passage in the presence of increasing concentrations of meropenem or ertapenem for similar to 60 generations. Isolated clones were whole-genome sequenced, and the order in which the identified mutations arose was determined in the passaged populations. Key mutations were reconstructed, and bacterial growth rates of populations and isolated clones and resistance levels to 23 antibiotics were measured. High-level resistance to carbapenems resulted from a combination of downstream effects of envZ mutation and target mutations in AcrAB-TolC-mediated drug export, together with PBP genes [mrdA (PBP2) after meropenem exposure or ftsI (PBP3) after ertapenem exposure]. Our results show that antibiotic resistance evolution can occur via several parallel pathways and that new mechanisms may appear after the most common pathways (i.e. beta-lactamases and loss of porins) have been eliminated. These findings suggest that strategies to target the most commonly observed resistance mechanisms might be hampered by the appearance of previously unknown parallel pathways to resistance.

Place, publisher, year, edition, pages
2016. Vol. 71, no 5, 1188-1198 p.
National Category
Biochemistry and Molecular Biology Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-221428DOI: 10.1093/jac/dkv475ISI: 000376291300008PubMedID: 26869688OAI: oai:DiVA.org:uu-221428DiVA: diva2:709069
Funder
Swedish Research Council Formas, 2013-5476-25194-9EU, European Research Council, 282004
Available from: 2014-03-31 Created: 2014-03-31 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Mechanisms and Dynamics of Carbapenem Resistance in Escherichia coli
Open this publication in new window or tab >>Mechanisms and Dynamics of Carbapenem Resistance in Escherichia coli
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The emergence of extended spectrum β-lactamase (ESBL) producing Enterobacteriaceae worldwide has led to an increased use of carbapenems and may drive the development of carbapenem resistance. Existing mechanisms are mainly due to acquired carbapenemases or the combination of ESBL-production and reduced outer membrane permeability. The focus of this thesis was to study the development of carbapenem resistance in Escherichia coli in the presence and absence of acquired β-lactamases. To this end we used the resistance plasmid pUUH239.2 that caused the first major outbreak of ESBL-producing Enterobacteriaceae in Scandinavia.

Spontaneous carbapenem resistance was strongly favoured by the presence of the ESBL-encoding plasmid and different mutational spectra and resistance levels arose for different carbapenems. Mainly, loss of function mutations in the regulators of porin expression caused reduced influx of antibiotic into the cell and in combination with amplification of β-lactamase genes on the plasmid this led to high resistance levels. We further used a pharmacokinetic model, mimicking antibiotic concentrations found in patients during treatment, to test whether ertapenem resistant populations could be selected even at these concentrations. We found that resistant mutants only arose for the ESBL-producing strain and that an increased dosage of ertapenem could not prevent selection of these resistant subpopulations. In another study we saw that carbapenem resistance can even develop in the absence of ESBL-production. We found mutants in export pumps and the antibiotic targets to give high level resistance albeit with high fitness costs in the absence of antibiotics. In the last study, we used selective amplification of β-lactamases on the pUUH239.2 plasmid by carbapenems to determine the cost and stability of gene amplifications. Using mathematical modelling we determined the likelihood of evolution of new gene functions in this region. The high cost and instability of the amplified state makes de novo evolution very improbable, but constant selection of the amplified state may balance these factors until rare mutations can establish a new function.

In my studies I observed the influence of β-lactamases on carbapenem resistance and saw that amplification of these genes would further contribute to resistance. The rapid disappearance of amplified arrays of resistance genes in the absence of antibiotic selection may lead to the underestimation of gene amplification as clinical resistance mechanism. Amplification of β-lactamase genes is an important stepping-stone and might lead to the evolution of new resistance genes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 51 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 998
Keyword
carbapenem, antibiotic resistance, fitness cost, ESBLs, penicillin-binding proteins, gene amplification
National Category
Biochemistry and Molecular Biology Genetics Microbiology
Identifiers
urn:nbn:se:uu:diva-221432 (URN)978-91-554-8950-2 (ISBN)
Public defence
2014-06-05, B42, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2014-05-13 Created: 2014-03-31 Last updated: 2014-06-30Bibliographically approved

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Adler, MarlenAndersson, Dan I.Sandegren, Linus

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