| 1. |
- Garoff, Linnéa, et al.
(författare)
-
Effect of aminoacyl-tRNA synthetase mutations on susceptibility to ciprofloxacin in Escherichia coli
- 2018
-
Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press (OUP). - 0305-7453 .- 1460-2091. ; 73:12, s. 3285-3292
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 2. |
- Garoff, Linnéa, 1989-
(författare)
-
Exploring the Ciprofloxacin Resistome
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
|
|
| 3. |
- Garoff, Linnéa, et al.
(författare)
-
Increased expression of Qnr is sufficient to confer clinical resistance to ciprofloxacin in Escherichia coli
- 2018
-
Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press (OUP). - 0305-7453 .- 1460-2091. ; 73:2, s. 348-352
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 4. |
- Garoff, Linnéa
(författare)
-
The qepA Gene is Dependent on Upstream Sequences to Reduce Susceptibility to Ciprofloxacin
- 2018
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The plasmid-borne qepA gene encodes a quinolone efflux pump that confers resistance to ciprofloxacin in clinical isolates of Escherichia coli. According to published data qepA is transcribed from a promoter 31 bp upstream of the coding sequence. We asked whether ciprofloxacin MIC associated with qepA in E. coli would vary as a function of mRNA expression level. To our surprise we found that the annotated qepA coding sequence was not sufficient to increase MIC. We decided to investigate the role played by surrounding sequences in determining resistance to ciprofloxacin. The qepA region was engineered onto the E. coli chromosome and mutations were generated to test the significance of surrounding sequences for expression of resistance. MIC was measured by broth microdilution. A 3.2 kb fragment from a resistance plasmid, including the annotated qepA coding sequence (1.5 kb), generated an 8-fold ciprofloxacin MIC increase when recombined into the E. coli chromosome. Deletion analysis revealed that the MIC increase was dependent on sequences upstream of qepA, annotated as ∆int1/groEL and ∆dfr2. Combining sequence analysis and mutagenesis, we identified a promoter within the ∆int1/groEL sequence, required for expression of resistance. The predicted transcript included two open reading frames (orf1 261 bp, orf2 189 bp) upstream of qepA. Deletion analysis revealed the essentiality of orf2 for the MIC increase. In conclusion, we have identified a new promoter for qepA, provided evidence that expression of the qepA coding sequence is not sufficient for resistance, and that resistance is influenced by sequences upstream of the qepA coding sequence. Details of the resistance mechanism remain to be elucidated.
|
|
| 5. |
- Pietsch, Franziska, et al.
(författare)
-
Evolutionary Trajectories Dependent on Bottleneck Size and a New Class of Genes Selected During the Development of Ciprofloxacin Resistance in Escherichia coli
- 2018
-
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.
|
|
