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Free radical generation plays a key role in killing bacteria by antibiotics. However, radicals are short-lived and reactive, and thus difficult to detect for the state of the art. Here we use a technique which allows optical nanoscale magnetic resonance imaging (MRI) to detect radical generation on the scale of single bacteria. We demonstrate that the radical generation in Staphylococcus aureus increases in the presence of UV irradiation as well as vancomycin and is dependent on the antibiotic's dose. With a method based on ensembles of nitrogen vacancy (NV) centers in diamond, we were able to follow the radical formation near individual bacteria over the whole duration of the experiment to reveal the dynamics of radical generation. Using this new approach, we observed free radical concentrations within nanoscale voxels around the diamond particles and determined its exact timing depending on the antibiotic dose. Since changes in the response to antibiotics emerge in only a few bacteria of the entire population, such a single-cell approach can prove highly valuable for research into drug resistance.

Obesity and diabetes are established comorbidities for COVID-19. Adipose tissue demonstrates high expression of ACE2 which SARS- CoV-2 exploits to enter host cells. This makes adipose tissue a reservoir for SARS-CoV-2 viruses and thus increases the integral viral load. Acute viral infection results in ACE2 downregulation. This relative deficiency can lead to disturbances in other systems controlled by ACE2, including the renin-angiotensin system. This will be further increased in the case of pre-conditions with already compromised functioning of these systems, such as in patients with obesity and diabetes. Here, we propose that interactions of virally-induced ACE2 deficiency with obesity and/or diabetes leads to a synergistic further impairment of endothelial and gut barrier function. The appearance of bacteria and/or their products in the lungs of obese and diabetic patients promotes interactions between viral and bacterial pathogens, resulting in a more severe lung injury in COVID-19.

Bacterial bloodstream infections (BSIs) are a major healthcare problem causing high mortality and economic cost. BSIs require an immediate initiation of antibiotic therapy as any delay is associated with a mortality increase. With the emergence of antimicrobial resistance, the choice of the appropriate antibiotic becomes increasingly difficult, thus creating an urgent need for new diagnostics, ideally to be done at the point of care. The current gold standard is blood culture with subsequent susceptibility testing although several molecular methods have recently entered the market. However, in many instances there is a discrepancy between the in-vitro data provided by the test and the outcome of antimicrobial therapy in-vivo because current diagnostics fail to take into account the impact of the environment in the patient such as the immune system, pharmacokinetics and pharmacodynamics or bacterial fitness. In this thesis, it was hypothesised that the measurement of the bacterial gene expression after the beginning of antibiotic therapy might be a more accurate indicator of the therapy outcome because it reflects the bacterial response under in-vivo conditions. In the first part of the thesis the expression of a set of pre-defined mRNA markers was investigated under various conditions. Experiments conducted with clinical E. coli isolates incubated in human whole blood revealed an excellent correlation between the gene expression, the treatment outcome, the antibiotic susceptibility and the genetic background for three different classes of antimicrobial drugs. The second part of the thesis describes the extraction of bacterial RNA from human whole blood specimen. The effect of different agents for the lysis of human blood cells and the impact of co-purified human RNA were analysed and a method for high yield extraction of undegraded bacterial RNA was established. The third part of the thesis investigates two methods for the sensitive measurement of the bacterial gene expression. This is relevant because the bacterial loads in BSI patients are extremely low. For genes with high gene expression levels both methods yielded reliable results but were unable to quantify the expression of the previously investigated mRNA markers due to their low copy numbers. Other approaches, especially those based on single cell measurements, might be able to overcome the problem in the future and should be explored in greater detail. Overall, the foundations for a future diagnostic test based on the measurement of the bacterial gene expression have been laid in this work. Future work should address the mRNA quantification and further evaluate the connection between gene expression and therapy outcome, e.g. in animal models. A future diagnostic test should also fulfil point-of-care requirements. This will include integrated sample preparation and quantification as well as a time-to-result in the range of a few minutes.