Morphological and Physicochemical Characterization of Single-Species Bacterial Biofilms Probed by SEM, FTIR-ATR, and µ-Raman Techniques

Treatment of biofilm-related infections represents a major challenge in public health management. Therefore, the accurate identification of both the composition and architecture of bacterial biofilms, in terms of microorganisms and surrounding extracellular polymeric substances (EPSs), represents a fundamental pre-requisite for the rapid diagnosis of recurrent/resistant biofilm-based infections as well as for the management of several industrial processes. In this work, the results of a combined approach involving scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy in attenuated total reflectance geometry (FTIR-ATR) and µ-Raman spectroscopy for the morphological and physicochemical characterization of monomicrobial biofilms produced by Pseudomonas aeruginosa (PAO1) and Escherichia coli strains, both proficient in infecting human cells and in colonizing medical devices, are presented. In particular, SEM images revealed, for both producing strains, the presence of densely aggregated rod-shaped bacteria on the surface of an extracellular matrix characterized by a "tree trunk"-like matrix, in the case of PAO1, and a “stone”-like one for E. coli, respectively. In addition, several markers based on FTIR-ATR and µ-Raman spectral features were identified starting from assessing the biochemical content of both investigated biofilms. In particular, absorption and scattering features associated with the genetic content turned out to be suitable markers for the proper discrimination between the PAO1 and E. coli biofilm samples, extremely useful in the context of specific therapeutic scheme to be applied. A further aim of this study was the implementation and development of a classification model based on a detailed comparative analysis for the unambiguous categorization of the different biofilm-producing bacterial strains. The study reported in this paper was developed in the framework of the PRIN 2022 FINI (Future challenges in management of recurrent/resistant Infection: development of antimicrobial Nanoparticulate systems and physical-chemical investigation of their Interactions with biofilm-associated infection) project, funded by the European Union-Next Generation EU.