Development of a rapid screening method for Carbapenem-Resistant Enterobacteriaceae using high resolution mass spectrometry

世界卫生组织(WHO)将耐碳青霉烯类肠杆菌列为开发新药和治疗监测策略的关键优先事项。包括大肠埃希菌和肺炎克雷伯氏菌在内的物种都产生了多重耐药菌株。碳青霉烯被世卫组织称为最后的抗菌药，如果不立即采取行动，到2050年，抗菌素耐药性将成为每年的主要死亡原因，导致1000万人死亡，造成超过100万亿美元的经济损失。该项目将有助于开发新的筛选方法，以便能够快速识别碳青霉烯耐药生物体或耐药基因的存在。目前已知的碳青霉烯类耐药酶有三类(A类碳青霉烯酶、B类金属-B-内酰胺酶和D类OXA碳青霉烯酶)，根据微生物的不同，这些酶可进一步分为几个亚类。MALDI-MS、LC-MS和GC-MS等技术已经被证明在细菌识别中是有用的，并已被微生物实验室广泛采用。在这项研究项目中，学生将使用高分辨率质谱仪从细胞外产生蛋白质、脂类和代谢物(挥发性和非挥发性)来定位和表征耐药机制的分子表型，目的是发现具有诊断潜力的代谢物生物标记物。学生将从同一物种的无菌培养和共培养中提取样本，以确定核心和耐药微生物足迹，以及在与药物敏感菌株共培养时，在获得耐药性基因的过程中动态种群变化所产生的代谢物。为了确认与新陈代谢途径的关联，学生还将通过培养基同位素浓缩来进行功能分析。然后，学生将进行复杂的单变量和多变量分析，以开发稳健的分类算法，并将这些算法与常用的微生物学分析进行比较。然后，预期的代谢物生物标志物将直接有助于形成可用于临床鉴定、环境分析(例如农业或废水)和食品工业应用的快速质谱仪或预制鉴定测试，从而有助于对AMR的全球监测。

Carbapenem-resistant Enterobacteriaceae are categorised by the World Health Organisation (WHO) as a critical priority for the development of novel drugs and treatment surveillance strategies. Species include Escherichia coli and Klebsiella pneumoniae both of which have developed multi-drug resistant strains. Carbapenems are referred to as the last resort antimicrobial by the WHO, and without immediate action antimicrobial resistance will be the leading cause of death per year by 2050 costing 10 million lives and causing economic damage in excess of $100 trillion. This project will contribute towards the development of novel screening methods to enable rapid identification of carbapenem-resistant organism or presence of resistance genes. There are currently three classes of carbopenem-resistance enzymes known (Class A carbapenemases, Class B Metallo-B-lactamases, and Class D OXA carbapenemases), which are further divided into several subgroups dependant on the microorganism. Techniques such as MALDI-MS, LC-MS, and GC-MS have already shown to be useful in discriminating bacteria and have been widely adopted by microbiology laboratories. In this research project, the student will use high resolution mass spectrometry to target and characterise the molecular phenotype of drug resistance mechanisms from the extracellular production of proteins, lipids, and metabolites (both volatile and non-volatile) with the aim of discovering metabolite biomarkers with diagnostic potential. The student will sample from axenic cultures and co-cultures of the same species to identify core and resistant microbial footprint, and metabolites produced from dynamic population change in acquiring resistance genes when in co-culture with drug sensitive strains. To confirm association with metabolic pathways, the student will also perform functional analysis by isotopic enrichment of culture media. The student will then perform complex univariate and multivariate analyses to develop robust classification algorithms and compare these to commonly used microbiology assays. Prospective metabolite biomarkers will then directly contribute to the formation of rapid mass-spectrometry or pre-cast identification tests which can be used for clinical identification, environment analysis (e.g. agriculture or wastewater), and applications in the food industry, thereby contributing to global surveillance of AMR.