Deuterium Labeling Mass Spectrometry for Rapid Antimicrobial Susceptibility Testing - PROJECT SUMMARY Antimicrobial resistance (AMR) is a significant challenge for human health, especially with the emergence of multi-drug resistance bacteria and the lack of new antibiotics development. In the United States alone there are over 2.8 million cases each year with over 35,000 deaths. Worldwide annual deaths are over 700,000 and this number is expected to grow to 10 million by 2050. There is a great concern that the COVID pandemic may have increased AMR risk. While the next generation ASTs have been actively under development in the last decade and some automatic tools were approved by the FDA, they are mostly based on morphological changes with limitations in sensitivity and selectivity, mostly requiring overnight cultures and long turn-around-time. Additionally, a separate capital investment is necessary for automatic AST instruments. In vivo isotope labeling with heavy water (D2O) has been used to monitor microscale cellular responses such as protein synthesis and turnover, DNA replication, and de novo lipogenesis. When bacteria are cultured in D2O, newly synthesized bacterial lipids are labeled by deuterium and can be readily detected by mass spectrometry (MS). We are developing a new antibiotic susceptibility test (AST) using deuterium-labeling mass spectrometry (DLMS), in which AMR bacteria can be rapidly detected by a bench top matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF) that is commonly available in many clinical labs for bacterial species identification. Based on previous success on resistant E. coli and methicillin-resistant Staphylococcus aureus (MRSA), our DLMS AST will be now applied to all ESKAPE (Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, and Enterobacter) pathogens using a bench top MALDI-TOF, the Bruker Biotyper. The minimum inhibitory concentration (MIC) measured by DLMS will be compared with a traditional broth dilution method, which is expected to be comparable but achieved in only a few hours instead of one or two days. To further accelerate the adoption of this DLMS AST assay by clinical bacteriologists, an on-agar assay will also be developed using ETESTÒ and D2O agar. Furthermore, we will explore the discovery of new AMR metabolite biomarkers using DLMS, which will not only shorten the turn- around-time but also provide information about resistant mechanisms. Our long-term vision is to have both global markers (deuterated lipids) and specific AMR biomarkers (deuterated small metabolites) simultaneously detected by DLMS using a bench-top MALDI-TOF, allowing prompt prescription of the most appropriate antibiotics and reducing antibiotic overuse.
