Application of Rapid Evaporative Ionization Mass Spectrometry (REIMS) to Identify Antimicrobial Resistance in Uropathogenic <i>Escherichia coli</i> (UPEC) Isolates via Deuterium Isotope Probing

Antimicrobial resistance (AMR) continues to pose a significant threat to global health, undermining advances in modern medicine and increasing mortality from previously treatable infections. Rapid and accurate antimicrobial susceptibility testing (AST) is critical, both for effective judicious treatment and controlling the spread of AMR. For the first time, we demonstrate the application of rapid evaporative ionization mass spectrometry (REIMS), combined with deuterium isotope probing (DIP), as a novel approach for identifying AMR in uropathogenic <i>Escherichia coli</i> (UPEC) isolates within only a 1 h incubation period. By directly analyzing bacterial samples without extensive preparation, REIMS serves as a rapid fingerprinting tool, employing DIP and multivariate statistical analysis to provide AST profiling of UPEC isolates. Distinct clustering patterns were observed between trimethoprim-susceptible and trimethoprim-resistant UPEC isolates grown in media containing 10% deuterium oxide (D₂O). TMP-susceptible isolates treated with trimethoprim displayed no significant deuterium incorporation, serving as an indicator of a lower metabolic activity resulting from antimicrobial action. We also demonstrated the ability to differentiate the origin of heavy water, confirming that deuterium incorporation was a biological process rather than of extracellular origin resulting from chemical processes. Several mass spectral bins showed patterns consistent with deuterated phospholipid species, including those in the expected mass range for phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), which are the most abundant phospholipids in <i>E. coli</i>. However, these annotations remain tentative, as no structural confirmation (e.g., MS/MS) was performed. These findings suggest that REIMS, combined with DIP and multivariate statistical analysis, serves as an efficient fast workflow for the rapid detection of AMR.