Rapid Multiplex Antimicrobial Resistance Profiling and Bacterial Identification by LAP‐MALDI Mass Spectrometry Biotyping

Rapid and accurate characterization of antimicrobial resistance is essential for effective patient treatment and outcomes. Infection-causing microorganisms often harbour multi-drug resistance, requiring multiple tests for identification. Here, we present a multiplex functional assay using liquid atmospheric pressure (LAP) matrix-assisted laser desorption/ionization (MALDI) as next-generation MALDI biotyping technology, which can accurately determine antibiotic resistance/susceptibility within three hours from <5 µL of bacterial culture, employing a beta-lactam antibiotic panel. Strains with common resistance genes, including OXA-48, KPC-3, and VIM-1, as well as susceptible isolates, are easily and reliably classified. Concurrent with multi-drug testing, the same bacterial sample provides species-identifying lipid and protein profiles (up to 100% classification accuracy) and characterization through tandem mass spectrometry (MS/MS) protein sequencing, facilitated by LAP-MALDI's ability to generate multiply charged protein ions. Detection of multiple antibiotics, their degradation products and lipids/proteins with high mass accuracy, along with the possibility of protein sequencing, offers new diagnostic possibilities for clinical microbiology and antimicrobial stewardship that are less probability-based than conventional MALDI biotyping.