Sequential Dual-Ion MALDI Glycotyping Enables Rapid Phenotypic O-Antigen Typing of Escherichia coli and Shigella

Accurate O-antigen typing of Gram-negative bacteria is important for surveillance, outbreak investigation, and quality control of reference strains. However, commonly used typing approaches, including serological agglutination assays and molecular methods, do not always resolve structural variation in expressed O-antigen phenotypes. Here, we describe an improved MALDI glycotyping workflow based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that enables rapid phenotypic characterization of O-antigen repeating units (RUs). The workflow uses a sequential dual-ion acquisition strategy in which positive-ion spectra are obtained first and negative-ion analysis is triggered only when RU signals are absent, enabling detection of both neutral and acidic O-antigen structures from the same sample spot. Applied to a diverse panel of 71 Escherichia coli and Shigella strains, RU-derived signals were detected in more than 80% of isolates. The approach resolved modification-level structural variation and discriminated isobaric O-antigen phenotypes, enabling scalable phenotypic profiling of O-antigen composition and inference of candidate O-antigen identities from RU-level information. Integration with agglutination testing further revealed discrepancies between archived serotype annotations and expressed O-antigen phenotypes, enabling reassignment of several strains to alternative O-antigen types. Because the workflow can be implemented on MALDI-TOF MS platforms already widely used for microbial identification, this method provides a practical phenotypic complement to conventional O-antigen typing in clinical microbiology laboratories and remains compatible with rapid single-colony MALDI workflows used in routine microbial identification. ImportanceAccurate O-antigen characterization is essential for pathogen surveillance and quality control of reference strain integrity. However, existing methods often rely on genetic or serological proxies rather than direct structural analysis of the expressed O-antigen. We established a sequential dual-ion MALDI glycotyping method that enables rapid phenotypic characterization of both neutral and acidic O-antigen repeating units in E. coli and Shigella. This approach detected O-antigen signals in over 80% of a diverse strain panel and identified fine structural modifications that conventional tests miss. Importantly, our method uncovered discrepancies in archived serotype data, allowing for the corrected reassignment of several reference strains. By integrating glycan phenotyping into existing MALDI-TOF MS workflows already common in clinical settings, this method offers a robust and scalable tool for high-resolution bacterial typing and quality control.