Development and validation of the first species-specific MALDI-TOF MS main spectra profile database for rapid identification of Aeromonas schubertii associated with white nodular disease in freshwater fish

Abstract

Background and Aim: Aeromonas schubertii is an emerging aquatic bacterial pathogen associated with distinctive white nodular disease in freshwater fish and is increasingly relevant to aquaculture health and disease surveillance. Accurate species-level identification remains challenging because commercial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) databases have limited species-specific spectral representation of A. schubertii. This study aimed to develop and validate the first species-specific main spectra profile (MSP) database for rapid and reliable identification of A. schubertii. Materials and Methods: Six representative A. schubertii isolates were used to construct MSPs following ethanol–formic acid–acetonitrile protein extraction. A minimum of 20 high-quality spectra per isolate were retained for MSP generation. The customized database was evaluated using 19 A. schubertii isolates, including the six isolates used for database construction and 13 additional isolates. Identification reliability and repeatability were evaluated, and discriminatory performance was assessed against four closely related non-target Aeromonas species. Principal component analysis (PCA), spectral profiling, and dendrogram analyses were used to evaluate spectral relatedness and species differentiation. Results: Using the commercial database alone, 15/19 (78.95%) isolates achieved reliable species-level identification (log scores of 2.01–2.22), whereas 4/19 (21.05%) were identified only at the genus level (scores of 1.95–1.98). After incorporating the customized MSP database, all 19 isolates (100%) were correctly identified as A. schubertii, with highly reliable log scores ranging from 2.49 to 2.68. Technical replicates showed high spectral reproducibility, with Pearson correlation coefficients of 0.95–0.97. PCA and dendrogram analyses clearly separated A. schubertii from the tested non-target Aeromonas species, supporting the discriminatory performance of the customized database. Molecular analysis of 16S rRNA and gyrB sequences further supported the taxonomic identification of the isolates. Conclusion: The species-specific MSP database substantially improved MALDI-TOF MS identification of A. schubertii, increasing species-level identification from 78.95% to 100%. The database provides a rapid and reliable framework for improving diagnosis and surveillance of this emerging aquaculture pathogen. Further expansion with geographically diverse isolates and multicenter validation is warranted to strengthen database transferability and broader diagnostic application. Keywords: Aeromonas schubertii, aquaculture diagnostics, bacterial identification, freshwater fish, MALDI-TOF MS, main spectra profile, spectral database, white nodular disease.