High-Throughput Desorption Electrospray Ionization Mass Spectrometry for Untargeted Antibiotic Susceptibility Testing

Abstract Antibiotic susceptibility testing (AST) is critical in combating the rise of antimicrobial resistance, as it quantitively assesses the efficacy of antibiotics by estimating minimum inhibitory concentrations (MICs) thus defining the susceptibility of pathogens to treatment. Rapid AST provides timely patient care while supporting antibiotic stewardship. Here we demonstrate an approach based on high-throughput desorption electrospray ionization mass spectrometry (HT-DESI-MS) for rapid quantitative AST via untargeted lipid profiling (1 sample per second) within 4 h of antimicrobial exposure. Using a model E. coli strain, we demonstrate that bacterial growth inhibition can be monitored via total lipid intensities against nonexposed controls, and that the EC50 values estimated from this analysis are in close agreement with MICs determined via gold-standard AST. Furthermore, the untargeted nature of the lipidomic data collected, together with the large sample size accessible via this HT method, allow for further evaluation of downstream metabolic effects of antibiotic exposure as shown here using unsupervised and supervised classification methods. Overall, this study establishes the capability of a fully automated HT-DESI-MS platform for rapid untargeted AST, potentially offering a reduced turnaround time to assess susceptibility and improve clinical outcomes.