The Application of Molecular Imaging Techniques to Better Understand Antimicrobial Resistance in Pseudomonas Biofilms

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Antimicrobial resistance (AMR) is among the most notable challenges of the modern age, it poses a significant threat to biosecurity and global healthcare. AMR is pervasive and evolving across a multitude of settings, including: natural reservoirs, industrial facilities and clinics. Industrially, drug-resistant bacteria cause losses in productivity due to: investment in failing decontamination methodologies, diminished outputs and subsequent loss of profits. Water treatment facilities highlight this as the accumulation of drug resistant biofilms on water filters is proving increasingly difficult to tackle. Within clinics, AMR infections cause the loss of human life; unless a paradigm shift occurs, by 2050 it is forecasted that more than 10 million avoidable global deaths will occur. AMR is well studied within bacteria; many key resistance mechanisms are well understood, yet our understanding of biofilms in this context is limited. Biofilm formation is a universal feature of bacteria; current understanding dictates that the primary function of biofilms is to reduce a bacteria's exposure to a given environmental stressor via the secretion of extracellular polymetric substances (EPS). EPS work by preventing the accumulation of biocidal agents to lethal levels within the matrix. Over 80% of human bacterial infections are linked to complex biofilms. Evidently, a better understanding of biofilm's role in AMR is necessary to combat its global issue. The current project aims to address this gap in knowledge by unearthing the microbial response to antimicrobial exposure and adaptive evolution, as well as drug targets, penetration and distribution within and across Pseudomonas biofilms.Through the application and development of molecular imaging techniques and cryo-sectioning methodologies, the current Biotechnology and Biological Sciences Research Council (BBSRC) funded project will provide insight into AMR in biofilms using Pseudomonas as the model organism. A primary aim of the project will involve studying the communal and cellular response of Pseudomonas to antibacterial treatment through the optimisation, scrutiny and establishment of novel methodologies associated with label-free 3D imaging of bacterial cells. The project will determine the antibiotic resistance profile of Pseudomonas aeruginosa isolates. Through the optimisation and development of biofilm embedding and cryo-sectioning P. aeruginosa biofilms will be cultivated for analysis. Subsequently, bacterial-drug interaction dynamics and drug distribution will be monitored over time, utilising: stimulated Raman scattering (SRS), coherent anti-stokes Raman scattering (CARS) and optical photothermal infrared (O-PTIR) spectroscopy. Stable isotope probing (SIP) will also be used synergistically with molecular imaging techniques [Raman and Infrared micro-spectroscopy, and Desorption Electrospray Ionisation (DESI) mass spectrometry imaging] to produce biofilm depth profiles and monitor metabolic activity at various depths upon antibiotic exposure. Using DESI mass spectrometry in tandem with SIP will enable bacterial metabolic activity to be probed, and for drug distribution and drug metabolites to be monitored. This work will be conducted in collaboration with Waters Corp Wilmslow Laboratories. The application of: Raman and Infrared Micro-spectroscopy, and DESI mass spectrometry as complementary molecular imaging techniques will facilitate the detection of a wider range of biomolecules.