Office for Risk Assessment and Research Coordination

Utrecht, Netherlands

Office for Risk Assessment and Research Coordination

Utrecht, Netherlands
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Feng Y.,University of Amsterdam | Jonker M.J.,University of Amsterdam | Moustakas I.,University of Amsterdam | Brul S.,University of Amsterdam | And 2 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2016

Pseudomonas aeruginosa is an opportunistic pathogen that causes considerable morbidity and mortality, specifically during intensive care. Antibiotic-resistant variants of this organism are more difficult to treat and cause substantial extra costs compared to susceptible strains. In the laboratory, P. aeruginosa rapidly developed resistance to five medically relevant antibiotics upon exposure to stepwise increasing concentrations. At several time points during the acquisition of resistance, samples were taken for whole-genome sequencing. The increase in the MIC of ciprofloxacin was linked to specific mutations in gyrA, parC, and gyrB, appearing sequentially. In the case of tobramycin, mutations in fusA, HP02880, rplB, and capD were induced. The MICs of the beta-lactam compounds meropenem and ceftazidime and the combination of piperacillin and tazobactam correlated linearly with beta-lactamase activity but not always with individual mutations. The genes that were mutated during the development of beta-lactam resistance differed for each antibiotic. A quantitative relationship between the frequency of mutations and the increase in resistance could not be established for any of the antibiotics. When the adapted strains are grown in the absence of the antibiotic, some mutations remained and others were reversed, but this reversal did not necessarily lower the MIC. The increased MIC came at the cost of moderately reduced cellular functions or a somewhat lower growth rate. In all cases except ciprofloxacin, the increase in resistance seems to be the result of complex interactions among several cellular systems rather than individual mutations. Copyright © 2016, American Society for Microbiology. All Rights Reserved.


Handel N.,University of Amsterdam | Schuurmans J.M.,University of Amsterdam | Feng Y.,University of Amsterdam | Brul S.,University of Amsterdam | And 2 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2014

Bacteria can become resistant not only by horizontal gene transfer or other forms of exchange of genetic information but also by de novo by adaptation at the gene expression level and through DNA mutations. The interrelationship between changes in gene expression and DNA mutations during acquisition of resistance is not well documented. In addition, it is not known whether the DNA mutations leading to resistance always occur in the same order and whether the final result is always identical. The expression of > 4, 000 genes in Escherichia coli was compared upon adaptation to amoxicillin, tetracycline, and enrofloxacin. During adaptation, known resistance genes were sequenced for mutations that cause resistance. The order of mutations varied within two sets of strains adapted in parallel to amoxicillin and enrofloxacin, respectively, whereas the buildup of resistance was very similar. No specific mutations were related to the rather modest increase in tetracycline resistance. Ribosome-sensed induction and efflux pump activation initially protected the cell through induction of expression and allowed it to survive low levels of antibiotics. Subsequently, mutations were promoted by the stress-induced SOS response that stimulated modulation of genetic instability, and these mutations resulted in resistance to even higher antibiotic concentrations. The initial adaptation at the expression level enabled a subsequent trial and error search for the optimal mutations. The quantitative adjustment of cellular processes at different levels accelerated the acquisition of antibiotic resistanceCopyright © 2014, American Society for Microbiology. All Rights Reserved.


Handel N.,University of Amsterdam | Hoeksema M.,University of Amsterdam | Mata M.F.,University of Amsterdam | Brul S.,University of Amsterdam | And 2 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2016

Strategies to prevent the development of antibiotic resistance in bacteria are needed to reduce the threat of infectious diseases to human health. The de novo acquisition of resistance due to mutations and/or phenotypic adaptation occurs rapidly as a result of interactions of gene expression and mutations (N. Handel, J. M. Schuurmans, Y. Feng, S. Brul, and B. H. Ter Kuile, Antimicrob Agents Chemother 58:4371-4379, 2014, http://dx.doi.org/10.1128/AAC.02892-14). In this study, the contribution of several individual genes to the de novo acquisition of antibiotic resistance in Escherichia coli was investigated using mutants with deletions of genes known to be involved in antibiotic resistance. The results indicate that recA, vital for the SOS response, plays a crucial role in the development of antibiotic resistance. Likewise, deletion of global transcriptional regulators, such as gadE or soxS, involved in pH homeostasis and superoxide removal, respectively, can slow the acquisition of resistance to a degree depending on the antibiotic. Deletion of the transcriptional regulator soxS, involved in superoxide removal, slowed the acquisition of resistance to enrofloxacin. Acquisition of resistance occurred at a lower rate in the presence of a second stress factor, such as a lowered pH or increased salt concentration, than in the presence of optimal growth conditions. The overall outcome suggests that a central cellular mechanism is crucial for the development of resistance and that genes involved in the regulation of transcription play an essential role. The actual cellular response, however, depends on the class of antibiotic in combination with environmental conditions. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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