Advanced methods in detection of pathogenic microorganisms and analysis the antimicrobials’ effect using Raman spectroscopy and other methods at the molecular level in optofluidic systems

The project addresses design, development and use of physical methods for research of microbial pathogens and their interactions with the environment. The metabolic processes of these microorganisms are regulated by various influences, such as oxygen availability, temperature, culture age, the presence of biofilm, other bacterial species, bacteriophages, or antimicrobial drugs. These interactions remain largely unexplored due to lack of techniques that would allow long-term non-destructive chemical analysis of individual cells under strictly regulated conditions. Understanding of metabolic changes during the bacterial cell cycle under various conditions is essential for determination of an effective course of treatment for bacterial infections including sepsis. Therefore, the central themes of this project are design, development and testing of optofluidic systems enabling safe and effective cultivation of pathogenic microorganisms in optimal conditions and simultaneous long-term microscopic observation, optical trapping of individual cells and their automated Raman or fluorescence spectroscopy, growth curve measurement, etc. Microspectroscopic Raman analysis of microbes at the level of individual cells will allow monitoring of changes in respiration, cell wall and protein composition, DNA, RNA, individual nucleotides, etc. We will monitor the interaction of different bacterial strains with antibiotics under different conditions, especially in bacterial consortia and biofilms, at elevated temperatures, with reduced oxygen access, etc. The specific output of the project will be a set of optofluidic devices for systematic automated spectroscopic analysis of bacterial colonies, biofilms and individual cells in suspension. These systems will be used to characterize selected collection strains of pathogenic microorganisms, and will allow parallel validation of the Raman spectroscopic analyses by means of mass spectrometry, capillary electrophoresis and other techniques.