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Mechanisms and fitness costs of tigecycline resistance in Escherichia coli
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Dan I. Andersson)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Dan I. Andersson)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Dan I. Andersson)
2013 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 12, 2809-2819 p.Article in journal (Refereed) Published
Abstract [en]

Objectives: To stepwise select tigecycline-resistant Escherichia coli mutants in vitro, determine the mutation rates, identify the resistance mechanisms, determine the resistance level and cross-resistance to other antibiotic classes, evaluate the fitness costs of tigecycline resistance mechanisms and investigate if the same in vitro-identified target genes were mutated in clinical isolates.

Methods: Spontaneous mutants with reduced susceptibility to tigecycline were selected on agar plates supplemented with tigecycline. Resistance levels and cross-resistance were evaluated by performing MIC assays and determining mutation rates using Luria-Delbruck fluctuation tests. Mutant fitness was estimated by measuring exponential growth rates, lag phase and total yield. Illumina whole-genome sequencing was used to identify mutations increasing MICs of tigecycline.

Results: Spontaneous mutants with reduced susceptibility to tigecycline were selected at a rate of similar to 10-8 to 10-6 per cell per generation; however, the clinical MIC breakpoint was not reached. The resistance level of tigecycline was low and some of the mutants had elevated MICs of hydrophobic drugs (chloramphenicol, erythromycin and novobiocin) or decreased MICs of SOS response inducers (ciprofloxacin and nitrofurantoin). Mutations were identified in efflux regulatory network genes (lon, acrR and marR) or lipopolysaccharide core biosynthesis pathway genes (lpcA, rfaE, rfaD, rfaC and rfaF). Mutations in the same target genes were found in clinical isolates.

Conclusions: Tigecycline selects for low-level resistance mutations with relatively high mutation rates and the majority of them come with a substantial fitness cost. Further in vivo experiments are needed to evaluate how these mutations affect bacterial virulence and ability to establish a successful infection.

Place, publisher, year, edition, pages
2013. Vol. 68, no 12, 2809-2819 p.
Keyword [en]
E. coli, bacterial resistance, LPS core biosynthesis, AcrAB efflux system
National Category
Medical and Health Sciences Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-212839DOI: 10.1093/jac/dkt263ISI: 000326980000015OAI: oai:DiVA.org:uu-212839DiVA: diva2:680441
Available from: 2013-12-18 Created: 2013-12-16 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Evolution and Mechanisms of Tigecycline Resistance in Escherichia coli
Open this publication in new window or tab >>Evolution and Mechanisms of Tigecycline Resistance in Escherichia coli
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antibiotic resistance is an ongoing global medical crisis and we are in great need of new antibacterial agents to combat rapidly emerging resistant pathogens. Tigecycline is one of few drugs that have been introduced into medicine during the last two decades. It is a broad-spectrum third generation tetracycline that is active against multidrug-resistant bacteria that cause complicated infections.

In this thesis I examined the development of tigecycline resistance in Escherichia coli and associated in vitro and in vivo fitness effects. Selections of spontaneous E. coli mutants revealed relatively high accumulation rates of changes in the multidrug efflux system AcrAB-TolC regulation network and in heptose biosynthesis and transport pathways important for lipopolysaccharide (LPS) synthesis. Both groups of mutations led to reduced susceptibility to tigecycline and slower growth compared to the wild-type bacteria. Additional in vitro fitness assays and in vivo competitions showed that LPS mutants were less fit than efflux mutants, providing a possible explanation for why up-regulation of multidrug efflux pumps is the main tigecycline resistance mechanism reported in clinical isolates.

Tigecycline was designed to evade the two most common tetracycline resistance mechanisms conferred by Tet proteins, efflux and ribosomal protection. However, tigecycline is a substrate for the tetracycline modifying enzyme Tet(X). Screening of Tet protein mutant libraries showed that it is possible to select Tet mutants with minimal inhibitory concentrations of tigecycline that reach clinically relevant levels. Mutations in Tet proteins that permitted a better protection from tigecycline frequently exhibited reduced activity against earlier generations of tetracyclines, except for the Tet(X) enzyme mutants, which were better at inactivating all tested tetracyclines. This is particularly worrisome because different variants of Tet(X) have recently spread to multidrug-resistant pathogens through horizontal gene transfer. Therefore, Tet(X) mutants with improved activity threaten the medical future of tetracyclines.

Multidrug resistance is easily disseminated through horizontally spreading conjugative plasmids. pUUH239.2 is an example of a successful conjugative plasmid that caused the first clonal outbreak of extended spectrum β-lactamase-producing Klebsiella pneumoniae in Scandinavia. This plasmid was formed after rearrangements between two different plasmid backbones and it carries resistance genes to multiple antibiotic classes, heavy metals, and detergents.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 58 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1121
Keyword
Tigecycline, Bacterial resistance, Fitness, Escherichia coli, AcrAB, LPS, Tet proteins, Tet(A), Tet(K), Tet(M), Tet(X), tet genes, pUUH239.2
National Category
Medical and Health Sciences Natural Sciences
Research subject
Microbiology
Identifiers
urn:nbn:se:uu:diva-259226 (URN)978-91-554-9285-4 (ISBN)
Public defence
2015-09-25, B42, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2015-08-26 Created: 2015-07-30 Last updated: 2015-10-01

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Linkevicius, MariusSandegren, LinusAndersson, Dan I.

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