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

Abstract

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 Escherichia coli (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 E. coli. 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.

1

Representative mass spectra of an E. coli MG1655 cultured in LB (A) and LB containing 10% D₂O (B), showing a mass range of m/z 50–1200 with an emphasis on complex lipids (m/z 600–900). Different colors indicate growth conditions: LB (light blue) and LB with 10% D₂O (dark blue).

2

3D PCA scores plot of REIMS data for E. coli MG1655 at the 1 h time point (TEV = 47.63%), cultured in LB with varying concentrations of D₂O (A). Colored diamonds indicate the different concentrations of D₂O used in the culture medium, with numbers denoting the percentage of D₂O. Gray arrow illustrates the trend in deuterium incorporation levels. PCA scores plot (TEV: 65.78%) of REIMS data for E. coli MG1655 grown in LB (yellow pentagrams) and LB containing 10% D₂O (red pentagrams) after 1 h (B). PC1 loadings plot of REIMS data for E. coli MG1655 at the 1 h time point, showing bins that contributed most to the clustering patterns observed (C). Colored bins indicate binned values that fall within the expected ranges for deuterium-labeled phospholipids such as phosphatidic acid (PA; blue), phosphatidylethanolamine (PE; green), and phosphatidylglycerol (PG; orange), based on prior literature. These assignments remain tentative as no structural confirmation was performed. The legend provides a color key for these putative phospholipid groupings.

3

PCA scores plot (TEV: 35.10%) displays REIMS data for UPEC isolates grown in MMCAA with (A) or without (B) 10% D₂O and with (T) or without (U) TMP treatment. Colors represent sampling time points: Blue for 0 min, green for 30 min, and red for 60 min. Experimental conditions are distinguished by color shading: darker shades indicate growth in D₂O, while lighter shades indicate growth in H₂O. TMP-treated samples are marked as empty symbols. Diamonds correspond to TMP-resistant isolates (R), and circles to TMP-susceptible isolates (S). The legend provides a color key to interpret the figure.

4

PCA scores plot of REISM data of UPEC isolates (TEV: 35.10%) grown in MMCAA with (A and B) or without (C and D) 10% D₂O and with (T) or without (U) TMP. Colors represent sampling time points: Blue for 0 min, green for 30 min, and red for 60 min. Experimental conditions are distinguished by color shading: darker shades indicate growth in D₂O (A and B), while lighter shades indicate growth in H₂O (C and D). TMP-treated samples are marked as empty symbols. Diamonds correspond to TMP-resistant isolates (R), and circles to TMP-susceptible isolates (S). The legend provides a color key to interpret the figure.