Unraveling bacterial biofilm assembly from the single cell to the extracellular matrix level via Raman microspectroscopy – ULTIM

ULTIM aims at a unique understanding of the principles behind bacterial biofilm formation, the major bacterial lifestyle on Earth with significant impact on human activities and geochemical cycles. Its goal is to achieve a time-resolved functional mapping of a growing biofilm based on a combination of conventional and compressive Raman microspectroscopies assisted by stable isotope probing (SIP) and multiparametric computational analysis. The label-free Raman imaging will provide high chemical selectivity and excellent optical resolution. Recently, Partner 2 has developed methods in compressive Raman microspectroscopy, leading to more sensitive and faster detection in turbid biological specimens but requiring strong enough signal levels. Conversely, conventional Raman imaging is slower and has a high data-throughput (too computationally intensive for large-scale imaging and kinetics analysis) but is more sensitive at lower signal levels. With ULTIME, we will develop a hybrid strategy which will allow to address the challenging question of the biofilm spatio-temporal metabolic kinetics. Notably, this methodology will resolve cells AND the stealth extracellular matrix (ECM), a key organ which remains poorly understood and difficult to image. We also expect the coupling between cell metabolic activity and ECM to emerge from the results. The project accomplishment will provide a new comprehensive vision of bacterial biofilms by linking molecular, functional and structural properties. As well, the work will bring about a novel Raman microspectroscopy methodology to investigate complex biological systems.
We will start imaging a simple model biofilm of E. coli, followed by strains of medical and environmental interest such as Pseudomonas aeruginosa or Bacillus thuringiensis. In the longer term, the achievement of the project should allow addressing the more complex question of the multi-species communities functioning, a key issue of modern microbiology.