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Increased expression of Qnr is sufficient to confer clinical resistance to ciprofloxacin 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.
2018 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 73, no 2, p. 348-352Article in journal (Refereed) Published
Abstract [en]

Background: Ciprofloxacin, a fluoroquinolone, targets two essential bacterial enzymes, DNA gyrase and topoisomerase IV. Plasmid-borne qnr genes, encoding proteins that protect DNA gyrase and topoisomerase IV from inhibition by fluoroquinolones, contribute to resistance development. However, the presence of a plasmid-borne qnr gene alone is insufficient to confer clinical resistance. Objectives: We asked whether the level of expression of qnr was a limiting factor in its ability to confer clinical resistance and whether expression could be increased without reducing fitness or viability. Methods: qnrB and qnrS were recombineered onto the chromosome of Escherichia coli under the control of constitutive promoters of various strengths. Expression was measured by qPCR, MIC and relative fitness as a function of expression level were determined. Results: For both qnr genes there was a positive relationship between the level of qnr mRNA and the MIC of ciprofloxacin. The highest MICs achieved with qnrB or qnrS as the sole resistance determinant were 0.375 and 1 mg/L, respectively, and were reached at expression levels that did not affect growth rate or viability. The qnrS-mediated MIC is above the EUCAST clinical breakpoint for resistance to ciprofloxacin. In the absence of Lon protease activity, overexpression of qnr genes was associated with high fitness cost, possibly explaining observations of toxicity in other genetic backgrounds. Conclusions: The ability to generate a high MIC without incurring a fitness cost shows that, in an appropriate genetic context, qnrS has the potential to generate clinical resistance to ciprofloxacin in one step.

Place, publisher, year, edition, pages
2018. Vol. 73, no 2, p. 348-352
National Category
Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-361199DOI: 10.1093/jac/dkx375ISI: 000424144300010PubMedID: 29106520ISBN: 1460-2091 (Electronic) 0305-7453 (Linking) OAI: oai:DiVA.org:uu-361199DiVA, id: diva2:1250021
Funder
Swedish Research Council, 2013-02904Swedish Research Council, 2016-04449Available from: 2018-09-21 Created: 2018-09-21 Last updated: 2019-06-24Bibliographically approved
In thesis
1. Exploring the Ciprofloxacin Resistome
Open this publication in new window or tab >>Exploring the Ciprofloxacin Resistome
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis presents an exploration of the resistance evolution in Escherichia coli towards the antibiotic ciprofloxacin. High level ciprofloxacin resistance is typically acquired by an accumulation of mutations and plasmid borne genes reducing drug target binding, increasing drug efflux, and modifying the drug.

Paper I describes the finding that novel mutations in tRNA synthetase gene leuS conferred resistance to ciprofloxacin. We also provided evidence for a mechanism, where the leuS mutations induced global changes in transcription that generated a net effect of increased drug efflux.

In Paper II we observed that the evolutionary trajectory towards high level ciprofloxacin resistance in E. coli is repeatable and predictable in in vitro evolution experiments. However, the types and order of appearance of selected mutations was highly dependent on the bottleneck size used. In addition to the findings in Paper I, we found that mutations involved in transcription and translation were repeatedly selected upon subjection to high concentrations of ciprofloxacin.

Paper III explored the resistance capacity of the plasmid-borne gene qnr, which reduces ciprofloxacin susceptibility by a target protection mechanism. We found that upon increased expression, the gene qnrS was able to bring E. coli to clinically resistant levels of ciprofloxacin without the addition of other resistance elements.  

In Paper IV we aimed for a similar study as described above but with another plasmid-borne gene, the inner-membrane efflux pump qepA. However, we ran into the interesting finding of a potentially undescribed regulatory mechanism of qepA expression, which we are currently investigating.

The work in this thesis presents a new addition of mutations causing ciprofloxacin resistance, and evidence that the dogma of accumulative mutations being a requirement to develop clinical resistance to ciprofloxacin in E. coli can be circumvented. This shows that there is still much to explore, even with a drug used for several decades with an already well documented resistome. We need to learn more about the evolutionary trajectories leading to antibiotic resistance, in order to slow down its development towards existing and future antibiotics to the furthest extent possible.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 61
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1495
Keywords
Antibiotic resistance, Experimental evolution, Ciprofloxacin, Escherichia coli
National Category
Microbiology
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-361204 (URN)978-91-513-0448-9 (ISBN)
Public defence
2018-11-09, B42, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2018-10-16 Created: 2018-09-21 Last updated: 2018-11-19

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