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

抗菌素耐药性（AMR）是现代最显著的挑战之一，它对生物安全和全球医疗保健构成重大威胁。抗菌素耐药性在多种环境中普遍存在并不断发展，包括：天然水库、工业设施和诊所。在工业上，耐药细菌造成生产力损失的原因是：对消毒方法失败的投资、产出减少以及随后的利润损失。水处理设施强调了这一点，因为水过滤器上耐药生物膜的积累越来越难以解决。在诊所内，抗菌素耐药性感染造成人命损失；除非发生范式转变，否则预计到2050年将发生1000多万例可避免的全球死亡。抗生素耐药性在细菌中得到了很好的研究；许多关键的抗性机制已经被很好地理解，但我们对生物膜在这种情况下的理解是有限的。生物膜的形成是细菌的普遍特征；目前的理解表明，生物膜的主要功能是通过分泌细胞外多聚物质（EPS）来减少细菌对给定环境应激源的暴露。EPS的作用是防止生物杀灭剂在基质中积累到致死水平。超过80%的人类细菌感染与复杂的生物膜有关。显然，更好地理解生物膜在抗菌素耐药性中的作用对于解决这一全球性问题是必要的。目前的项目旨在通过揭示微生物对抗菌药物暴露和适应性进化的反应，以及假单胞菌生物膜内和之间的药物靶点，渗透和分布来解决这一知识空白。通过分子成像技术和冷冻切片方法的应用和发展，目前由生物技术和生物科学研究委员会（BBSRC）资助的项目将以假单胞菌为模式生物，深入了解生物膜中的AMR。该项目的主要目标是通过优化、审查和建立与细菌细胞无标签3D成像相关的新方法，研究假单胞菌对抗菌治疗的公共和细胞反应。该项目将确定铜绿假单胞菌分离株的抗生素耐药性。通过优化和发展生物膜包埋和冷冻切片，铜绿假单胞菌的生物膜将被培养用于分析。随后，利用受激拉曼散射（SRS）、相干反斯托克斯拉曼散射（CARS）和光学光热红外（O-PTIR）光谱，监测细菌-药物相互作用动力学和药物分布。稳定同位素探测（SIP）也将与分子成像技术（拉曼和红外微光谱，以及解吸电喷雾电离（DESI）质谱成像）协同使用，以产生生物膜深度剖面，并监测抗生素暴露后不同深度的代谢活性。使用DESI质谱与SIP串联将能够探测细菌代谢活性，并监测药物分布和药物代谢物。这项工作将与沃特斯公司威尔姆斯洛实验室合作进行。拉曼光谱和红外微光谱以及DESI质谱作为互补的分子成像技术的应用将有助于更广泛的生物分子的检测。

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.