| dc.description.abstract | Antibiotics revolutionized medicine during the 1900s. However, a void in new discoveries in the last decades and increased antibiotic resistance are major threats to society. New antibiotics are highly needed to cope with this challenge. Research on already established antibiotics is also very valuable and may provide input to new target points, adjuvant therapies, and strategies to enhance antibiotic activity. Metabolomics is the systematic study of metabolites and is important in understanding bacterial physiology. A better understanding of bacterial physiology in different growth conditions and under different antibiotic stressors may contribute to solving the antibiotic crisis.
The first study in this thesis, published as Paper I, presents reference metabolomes for eight commonly applied model systems, both prokaryotes and eukaryotes. This was a basic study for familiarizing and validating the core metabolite profiling methodology. We used three different quantitative mass spectrometry-based analyses and reported absolute intracellular concentrations of 68 metabolites in the central carbon metabolism. The metabolites included constituents in the glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle, in addition to nucleotides, deoxynucleotides, and amino acids.
While working with the first study, we discovered that our methods were insufficient for metabolite profiling of the important model bacterium Escherichia coli. Therefore, we performed a comprehensive optimization of sampling and extraction of E. coli, where we thoroughly validated an optimized protocol. Further, we employed this method to present a reference metabolome for E. coli similar to the ones in Paper I. This was published as Paper II.
For the two remaining studies, published as Paper III and IV, we finally turned to the main objective of the PhD project: to study antibiotic treatment in E. coli, with high and low antibiotic doses, respectively. For paper III, we investigated the impact of different growth conditions and medium compositions before and during treatment with high doses of three different antibiotics: ampicillin, ciprofloxacin, and streptomycin. The surviving fractions of E. coli depended on antibiotic type, growth phase before treatment, and media composition during treatment. Furthermore, we performed metabolite profiling in two different growth phases (exponential and nitrogen-limited stationary phase) and immediately after resuspension into growth media with or without nitrogen. Here, we observed a stronger effect in a ΔrelA mutant, where it seems as if this mutant is less fit to cope with sudden changes in the growth conditions.
For Paper IV, we investigated low doses of antibiotics, where we wanted to avoid too much death after treatment. We included five antibiotics: ampicillin, kanamycin, ciprofloxacin, colistin, and trimethoprim, two doses of each antibiotic, and four time points. We included several assays to follow how the treatments affected the bacteria. These included optical density (OD), bacterial counts by flow cytometry and colony forming units (CFU), assessment of morphological changes, and development of reactive oxygen species (ROS). We also included a thorough assessment of transcriptional and metabolic responses after the treatments, with RNA sequencing and mass spectrometry-based metabolite profiling. The responses to the antibiotics differed in kinetics, with, for example, colistin having a strong initial effect that decreased over time, while most of the other antibiotics caused a gradual increase in response over time. The comparability between the different assays also varied between the antibiotics, where, for example, ampicillin and ciprofloxacin resulted in elongation, leading to discrepancies between OD and CFU/flow cytometry results.
In conclusion, this PhD has established cultivation and analytical workflows that have paved the way for more mechanistic studies. Additionally, it has contributed to increased knowledge of bacterial responses to different cultivation conditions and antibiotics. | en_US |
