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Fitness cost constrains the spectrum of marR mutations in ciprofloxacin-resistant Escherichia coli: Multiple Antibiotic-Resistance, Gram-Negative Bacteria, Multidrug Efflux Pump, Urinary-Tract-Infections, Fluoroquinolone Resistance, Quinolone Resistance, Mechanisms, Expression, Sequence, Soxs
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Diarmaid Hughes)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Diarmaid Hughes)
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.
2017 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 2, no 11, p. 3016-3024Article in journal (Refereed) Published
Abstract [en]

Objectives: To determine whether the spectrum of mutations in marR in ciprofloxacin-resistant clinical isolates of Escherichia coli shows evidence of selection bias, either to reduce fitness costs, or to increase drug resistance. MarR is a repressor protein that regulates, via MarA, expression of the Mar regulon, including the multidrug efflux pump AcrAB-TolC. Methods: Isogenic strains carrying 36 different marR alleles identified in resistant clinical isolates, or selected for resistance in vitro, were constructed. Drug susceptibility and relative fitness in growth competition assays were measured for all strains. The expression level of marA, and of various efflux pump components, as a function of specific mutations in marR, was measured by qPCR. Results: The spectrum of genetic alterations in marR in clinical isolates is strongly biased against inactivating mutations. In general, the alleles found in clinical isolates conferred a lower level of resistance and imposed a lower growth fitness cost than mutations selected in vitro. The level of expression of MarA correlated well with the MIC of ciprofloxacin. This supports the functional connection between mutations in marR and reduced susceptibility to ciprofloxacin. Conclusions: Mutations in marR selected in ciprofloxacin-resistant clinical isolates are strongly biased against inactivating mutations. Selection favours mutant alleles that have the lowest fitness costs, even though these cause only modest reductions in drug susceptibility. This suggests that selection for high relative fitness is more important than selection for increased resistance in determining which alleles of marR will be selected in resistant clinical isolates.

Place, publisher, year, edition, pages
2017. Vol. 2, no 11, p. 3016-3024
National Category
Microbiology in the medical area
Research subject
Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-320003DOI: 10.1093/jac/dkx270ISI: 000413464200006PubMedID: 28962020OAI: oai:DiVA.org:uu-320003DiVA, id: diva2:1089964
Funder
Swedish Research Council, 2013-02904Swedish Research Council, 2016-04449Available from: 2017-04-21 Created: 2017-04-21 Last updated: 2018-02-02Bibliographically approved
In thesis
1. Constraints on up-regulation of drug efflux in the evolution of ciprofloxacin resistance
Open this publication in new window or tab >>Constraints on up-regulation of drug efflux in the evolution of ciprofloxacin resistance
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The crucial role of antibiotics in modern medicine, in curing infections and enabling advanced medical procedures, is being threatened by the increasing frequency of resistant bacteria. Better understanding of the forces selecting resistance mutations could help develop strategies to optimize the use of antibiotics and slow the spread of resistance.

Resistance to ciprofloxacin, a clinically important antibiotic, almost always involves target mutations in DNA gyrase and Topoisomerase IV. Because ciprofloxacin is a substrate of the AcrAB-TolC efflux pump, mutations causing pump up-regulation are also common.

Studying the role of efflux pump-regulatory mutations in the development of ciprofloxacin resistance, we found a strong bias against gene-inactivating mutations in marR and acrR in clinical isolates. MIC and fitness measurements revealed that amino acid substitutions conferred smaller susceptibility reductions and smaller fitness costs than gene-inactivating mutations, suggesting that resistance mutations in clinical isolates are selected for high fitness rather than high resistance (Paper I and II).

We asked whether the high fitness costs of marR-inactivating mutations could be ameliorated without affecting the resistance phenotype. Multiple independent lineages were experimentally evolved to select for improved growth fitness. Whole genome sequencing revealed mutations affecting marA, lon and arcA as potential compensatory pathways. For the marA and lon mutations the improved growth rate was associated with an increased susceptibility (arcA is being investigated). (Paper III).

An evolution experiment selecting for ciprofloxacin resistance revealed upon whole genome sequencing the expected mutations in drug target and efflux-regulatory genes, but also in genes encoding aminoacyl-tRNA synthetases. We investigated two independently selected leuS mutations, and concluded that they contributed to ciprofloxacin resistance by activating the stringent response that in turn caused up-regulation of genes involved in efflux. However, these leuS mutations incur a high fitness cost (Paper IV).

To summarize, the research findings in this thesis suggest that the potential ciprofloxacin resistome may include more genes than previously thought, but a strong selection for high fitness selectively purifies many resistance mutations from clinical isolates. In conclusion, selection for high relative fitness constrains the spectrum of mutations that survive and get fixed in clinical populations of bacteria.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. p. 48
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1335
Keywords
ciprofloxacin, antibiotic resistance, drug efflux, bacterial fitness, marR, acrR, marA, experimental evolution
National Category
Natural Sciences Medical and Health Sciences
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-320580 (URN)978-91-554-9923-5 (ISBN)
Public defence
2017-06-09, B22, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2017-05-17 Created: 2017-04-22 Last updated: 2017-06-07

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