Studies on the efficient lysis of bacterial endospores for the development of a photonic detection method

Abstract

Endospore-forming bacteria pose a relevant risk in both clinical and biosecurity settings due to their high resistance and persistence. The aim of this thesis was to develop a rapid detection method based on the release and quantification of dipicolinic acid (DPA), a molecule highly specific to bacterial spores. A laser-based assay was established to induce spore disruption and DPA release within 15 minutes. To assess the efficiency of this approach, germination and chemical lysis assays were conducted to determine the maximum expected DPA content from a fully disrupted spore suspension. The laser irradiation assay showed a rapid decrease in spore viability, while DPA release occurred more gradually, reaching approximately 35-40% after 15 minutes. This suggests that laser exposure causes early functional damage before complete structural lysis. Supporting microscopic and PCR analyses confirmed partial disruption and DNA damage. Temperature measurements indicated that thermal effects alone were insufficient to explain the observed lysis. To enhance the DPA release, enzymatic additives were tested. The addition of lysozyme and mutanolysin significantly accelerated DPA release and reduced spore viability when combined with laser treatment. Control experiments with enzymes alone resulted in inactivation without full DPA release, indicating that the synergy between laser and enzyme treatment is essential for effective disruption. Furthermore, native cortex-lytic enzymes from Clostridium pasteurianum and Bacillus subtilis (SleC, SleB, CwlJ) were cloned, recombinantly expressed, and tested. General protein production in E. coli was successful, but the right protein size was not detected via SDS-PAGE. Also, activity assays revealed only minimal effects, likely due to insufficient folding or lack of activation. A lysozyme-based activity assay using Micrococcus luteus was established and used to benchmark enzyme kinetics. Based on these results, a concentration of 10 µg/ml was selected for functional testing. Overall, this study presents a fast and adaptable detection system based on laserinduced spore disruption and DPA quantification, which can be further improved by enzymatic and chemical additives. Future work should aim to optimize native enzyme activity and explore the integration of species-specific detection tools such as ultrafast PCR.