| 1. |
- Brandis, Gerrit, 1985-, et al.
(författare)
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Comprehensive phenotypic characterization of rifampicin resistance mutations in Salmonella provides insight into the evolution of resistance in Mycobacterium tuberculosis
- 2015
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Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press. - 0305-7453 .- 1460-2091. ; 70:3, s. 680-685
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Tidskriftsartikel (refereegranskat)abstract
- ObjectivesMutations in the β-subunit of RNA polymerase (RNAP), encoded by rpoB, are responsible for rifampicin resistance (RifR). Although many mutations in rpoB can reduce susceptibility, only a few are frequent amongst RifR clinical Mycobacterium tuberculosis (MTB) isolates. It has been suggested that there is a negative correlation between the fitness costs of RifR mutations and their respective clinical frequency, but so far comparable fitness cost measurements have only been conducted for a very limited number of RifR mutations. We tested this hypothesis using Salmonella and Mycobacterium smegmatis as model organisms.MethodsWe constructed 122 different RifR mutations in Salmonella. MICs and relative fitness costs in the presence and absence of rifampicin were determined for each mutant, including for a smaller number of RifRM. smegmatis strains. Results were compared with available mutation frequency data from clinical MTB isolates.Results(i) RifR mutations frequently found in MTB isolates have a fitness cost in Salmonella Typhimurium and M. smegmatis. (ii) Clinically frequent RifR mutations have a high rifampicin MIC. (iii) There is a strong correlation between the magnitude of the fitness cost of a RifR mutation in Salmonella Typhimurium or M. smegmatis and the frequency with which that mutation is associated with secondary (putative compensatory) mutations in RNAP of clinical MTB isolates.ConclusionsThis suggests that the success of RifR mutations in clinical MTB isolates may be dependent not only on a low initial fitness cost, but rather the results of three factors: (i) a high rifampicin MIC; (ii) a relatively low initial fitness cost; and (iii) the ability to additionally acquire compensatory mutations selected to further reduce fitness cost.
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| 2. |
- Garoff, Linnéa, et al.
(författare)
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Population Bottlenecks Strongly Influence the Evolutionary Trajectory to Fluoroquinolone Resistance in Escherichia coli
- 2020
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Ingår i: Molecular biology and evolution. - : Oxford University Press (OUP). - 0737-4038 .- 1537-1719. ; 37:6, s. 1637-1646
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Tidskriftsartikel (refereegranskat)abstract
- Experimental evolution is a powerful tool to study genetic trajectories to antibiotic resistance under selection. A confounding factor is that outcomes may be heavily influenced by the choice of experimental parameters. For practical purposes (minimizing culture volumes), most experimental evolution studies with bacteria use transmission bottleneck sizes of 5 x 10(6) cfu. We currently have a poor understanding of how the choice of transmission bottleneck size affects the accumulation of deleterious versus high-fitness mutations when resistance requires multiple mutations, and how this relates outcome to clinical resistance. We addressed this using experimental evolution of resistance to ciprofloxacin in Escherichia coli. Populations were passaged with three different transmission bottlenecks, including single cell (to maximize genetic drift) and bottlenecks spanning the reciprocal of the frequency of drug target mutations (10(8) and 10(10)). The 10(10) bottlenecks selected overwhelmingly mutations in drug target genes, and the resulting genotypes corresponded closely to those found in resistant clinical isolates. In contrast, both the 10(8) and single-cell bottlenecks selected mutations in three different gene classes: 1) drug targets, 2) efflux pump repressors, and 3) transcription-translation genes, including many mutations with low fitness. Accordingly, bottlenecks smaller than the average nucleotide substitution rate significantly altered the experimental outcome away from genotypes observed in resistant clinical isolates. These data could be applied in designing experimental evolution studies to increase their predictive power and to explore the interplay between different environmental conditions, where transmission bottlenecks might vary, and resulting evolutionary trajectories.
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| 3. |
- Huseby, Douglas L., et al.
(författare)
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Mutation supply and relative fitness shape the genotypes of ciprofloxacin-resistant Escherichia coli
- 2017
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Ingår i: Molecular biology and evolution. - : Oxford University Press (OUP). - 0737-4038 .- 1537-1719. ; 34:5, s. 1029-1039
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Tidskriftsartikel (refereegranskat)abstract
- Ciprofloxacin is an important antibacterial drug targeting Type II topoisomerases, highly active against Gram-negatives including Escherichia coli. The evolution of resistance to ciprofloxacin in E. coli always requires multiple genetic changes, usually including mutations affecting two different drug target genes, gyrA and parC. Resistant mutants selected in vitro or in vivo can have many different mutations in target genes and efflux regulator genes that contribute to resistance. Among resistant clinical isolates the genotype, gyrA S83L D87N, parC S80I is significantly overrepresented suggesting that it has a selective advantage. However, the evolutionary or functional significance of this high frequency resistance genotype is not fully understood. By combining experimental data and mathematical modeling, we addressed the reasons for the predominance of this specific genotype. The experimental data were used to model trajectories of mutational resistance evolution under different conditions of drug exposure and population bottlenecks. We identified the order in which specific mutations are selected in the clinical genotype, showed that the high frequency genotype could be selected over a range of drug selective pressures, and was strongly influenced by the relative fitness of alternative mutations and factors affecting mutation supply. Our data map for the first time the fitness landscape that constrains the evolutionary trajectories taken during the development of clinical resistance to ciprofloxacin and explain the predominance of the most frequently selected genotype. This study provides strong support for the use of in vitro competition assays as a tool to trace evolutionary trajectories, not only in the antibiotic resistance field.
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| 4. |
- Pietsch, Franziska, et al.
(författare)
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Ciprofloxacin selects for RNA polymerase mutations with pleiotropic antibiotic resistance effects
- 2017
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Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press. - 0305-7453 .- 1460-2091. ; 72:1, s. 75-84
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Tidskriftsartikel (refereegranskat)abstract
- ObjectivesResistance to the fluoroquinolone drug ciprofloxacin is commonly linked to mutations that alter the drug target or increase drug efflux via the major AcrAB-TolC transporter. Very little is known about other mutations that might also reduce susceptibility to ciprofloxacin. We discovered that an Escherichia coli strain experimentally evolved for resistance to ciprofloxacin had acquired a mutation in rpoB, the gene coding for the β-subunit of RNA polymerase. The aim of this work was to determine whether this mutation, and other mutations in rpoB, contribute to ciprofloxacin resistance and, if so, by which mechanism.MethodsIndependent lineages of E. coli were evolved in the presence of ciprofloxacin and clones from endpoint cultures were screened for mutations in rpoB. Ciprofloxacin-selected rpoB mutations were identified and characterized in terms of effects on susceptibility and mode of action.ResultsMutations in rpoB were selected at a high frequency in 3 out of 10 evolved lineages, in each case arising after the occurrence of mutations affecting topoisomerases and drug efflux. All ciprofloxacin-selected rpoB mutations had a high fitness cost in the absence of drug, but conferred a competitive advantage in the presence of ciprofloxacin. RNA sequencing and quantitative RT–PCR analysis showed that expression of mdtK, encoding a multidrug efflux transporter, was significantly increased by the ciprofloxacin-selected rpoB mutations. The susceptibility phenotype was shown to depend on the presence of an active mdtK and a mutant rpoB allele.ConclusionsThese data identify mutations in RNA polymerase as novel contributors to the evolution of resistance to ciprofloxacin and show that the phenotype is mediated by increased MdtK-dependent drug efflux.
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| 5. |
- Pietsch, Franziska, 1984-
(författare)
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Evolution of Antibiotic Resistance
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The emergence of antimicrobial resistance is a major global threat to modern medicine. The rapid dissemination of resistant pathogens and the associated loss of efficacy of many important drugs needs to be met with the development of new antibiotics and alternative treatment options. A better understanding of the evolution of resistance could help in developing strategies to slow down the spread of antimicrobial drug resistance.In this thesis we investigated the evolution of resistance to two important antibiotics, rifampicin and ciprofloxacin, paying special attention to the resistance patterns occurring with high frequency in clinical isolates.Rifampicin is a first-line drug in tuberculosis treatment and resistance to this valuable drug limits treatment options. Our work on rifampicin resistance helps to explain the extreme bias seen in the frequency of specific resistance mutations in resistant clinical isolates of M. tuberculosis. We identified an important interplay between the level of resistance, relative fitness and selection of fitness-compensatory mutations among the most common resistant isolates.Fluoroquinlones are widely used to treat infections with Gram-negatives and the frequency of resistance to these important drugs is increasing. Resistance to fluoroquinolones is the result of a multi-step evolutionary process. Our studies on the development of resistance to the fluoroquinolone drug ciprofloxacin provide insights into the evolutionary trajectories and reveal the order in which susceptible wild-type E. coli acquire multiple mutations leading to high level of resistance. We found that the evolution of ciprofloxacin resistance is strongly influenced by the mutation supply rate and by the relative fitness of competing strains at each successive step in the evolution. Our data show that different classes of resistance mutations arise in a particular, predictable order during drug selection. We also uncovered strong evidence for the existence of a novel class of mutations affecting transcription and translation, which contribute to the evolution of resistance to ciprofloxacin.
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| 6. |
- Pietsch, Franziska, et al.
(författare)
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Evolutionary Trajectories Dependent on Bottleneck Size and a New Class of Genes Selected During the Development of Ciprofloxacin Resistance in Escherichia coli
- 2018
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The evolution of resistance to ciprofloxacin (CIP) in Escherichia coli is strongly associated with the accumulation of multiple chromosomal mutations. Mutations are selected in genes encoding subunits of the target enzymes, and genes encoding direct or indirect regulators of drug efflux. We asked whether and how transmission bottleneck size would affect the evolutionary trajectory of chromosomal mutation accumulation. Independent lineages of E. coli were selected for growth at increasing concentrations of ciprofloxacin up to and above the clinical resistance breakpoint. Evolution experiments were made with three different transmission bottlenecks: single cell, ≈ 3x108, and ≈ 3x1010 cfu. Whole genome sequencing was used to analyse selected clones and populations at different stages during evolution. Under all conditions mutations in gyrA were the first to be selected and to approach or reach fixation. Evolution with the largest population bottleneck selected combinations of mutations similar to those found in resistant clinical isolates (gyrA S83, D87, with parC S80). As predicted by population genetics theory, evolution with a single cell bottleneck resulted in a greater diversity of mutations. Mutations were selected in genes directly regulating drug efflux, and in novel genes involved in transcription and translation, at least some of which are known to indirectly affect drug efflux. Evolution with the intermediate bottleneck, ≈ 3x108, also selected for mutations in a wide variety of genes, similar to the profile associated with the single cell bottleneck. The data suggest that the order of chromosomal mutations accumulated under selection for resistance to ciprofloxacin is highly predictable but the precise evolutionary trajectories differ significantly as a function of transmission bottleneck size.
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