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Effect of aminoacyl-tRNA synthetase mutations on susceptibility 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.
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 12, p. 3285-3292Article in journal (Refereed) Published
Abstract [en]

Background: Chromosomal mutations that reduce ciprofloxacin susceptibility in Escherichia coli characteristically map to drug target genes (gyrAB and parCE), and genes encoding regulators of the AcrAB-TolC efflux pump. Mutations in RNA polymerase can also reduce susceptibility, by up-regulating the MdtK efflux pump.

Objectives: We asked whether mutations in additional chromosomal gene classes could reduce susceptibility to ciprofloxacin.

Methods: Experimental evolution, complemented by WGS analysis, was used to select and identify mutations that reduce susceptibility to ciprofloxacin. Transcriptome analysis, genetic reconstructions, susceptibility measurements and competition assays were used to identify significant genes and explore the mechanism of resistance.

Results: Mutations in three different aminoacyl-tRNA synthetase genes (leuS, aspS and thrS) were shown to re- duce susceptibility to ciprofloxacin. For two of the genes (leuS and aspS) the mechanism was partially dependent on RelA activity. Two independently selected mutations in leuS (Asp162Asn and Ser496Pro) were studied in most detail, revealing that they induce transcriptome changes similar to a stringent response, including up-regulation of three efflux-associated loci (mdtK, acrZ and ydhJK). Genetic analysis showed that reduced susceptibility depended on the activity of these loci. Broader antimicrobial susceptibility testing showed that the leuS mutations also reduce susceptibility to additional classes of antibiotics chloramphenicol, rifampicin, mecillinam, ampicillin and trimethoprim).

Conclusions: The identification of mutations in multiple tRNA synthetase genes that reduce susceptibility to ciprofloxacin and other antibiotics reveals the existence of a large mutational target that could contribute to re- sistance development by up-regulation of an array of efflux pumps.

Place, publisher, year, edition, pages
2018. Vol. 73, no 12, p. 3285-3292
National Category
Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-361197DOI: 10.1093/jac/dky356ISI: 000452916600009PubMedID: 30239743OAI: oai:DiVA.org:uu-361197DiVA, id: diva2:1250025
Funder
Swedish Research Council, 2013-02904Swedish Research Council, 2016-04449Swedish Research Council, 2017-03593Available from: 2018-09-21 Created: 2018-09-21 Last updated: 2019-01-10Bibliographically 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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