MALDI-TOF MS Using a Custom-Made Database, Biomarker Assignment, or Mathematical Classifiers Does Not Differentiate spp. and .

spp. and are closely related and cannot be distinguished using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) with commercially available databases. Here, three alternative approaches using MALDI-TOF MS to identify and distinguish spp., , and its pathotype EIEC were explored and evaluated using spectra of 456 spp., 42 , and 61 EIEC isolates. Identification with a custom-made database resulted in >94% identified at the genus level and >91% and at the species level, but the distinction of , , and was poor. With biomarker assignment, 98% isolates were correctly identified, although specificity was low. Discriminating markers for , , and were not assigned at all. Classification models using machine learning correctly identified in 96% of isolates, but most isolates were also assigned to . None of the proposed alternative approaches were suitable for clinical diagnostics for identifying spp., , and EIEC, reflecting their relatedness and taxonomical classification. We suggest the use of MALDI-TOF MS for the identification of the spp./ complex, but other tests should be used for distinction.