Development and validation of a custom Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) peptide database for the rapid and accurate identification of Aeromonas spp. isolated from diseased fish

Aeromonas spp. are key pathogens responsible for motile Aeromonas septicemia (MAS) in freshwater fish. This research is focused on identifying Aeromonas spp. by employing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in conjunction with a custom peptide database developed from reference strains such as A. veronii, A. jandaei, A. schubertii, A. hydrophila, A. diversa, and A. punctata isolated from diseased fish. Peptides from these strains were used to construct main spectra profiles (MSPs), which were integrated into the MALDI Biotyper database. Validation involved 38 bacterial isolates. Distinct MSPs were observed, with species-specific clustering. Identification accuracy was assessed by comparing the MALDI Biotyper log scores to gyrB gene sequencing as the reference standard. Using the standard Biotyper library, 89.4% of isolates were properly recognized at the genus level (log scores: 2.00–2.29), 5.3% at the species level (2.30–3.00), and 5.3% with probable genus matches (1.70–1.99). The custom database significantly improved performance, yielding 100% accurate genus-level identification with log scores > 2.30. The outcomes revealed that MALDI-TOF MS, enhanced with a custom database, offers a fast, reliable, and efficient technique for identifying Aeromonas in aquaculture, supporting its application in diagnostics and microbial research.