Integrated matrix-assisted laser desorption ionization time-of-flight mass spectrometry serotype-specific peak analysis and trans-cinnamic acid antimicrobial activity against Salmonella spp. from the poultry chain

Background and Aim: Salmonella spp. are major foodborne pathogens within the poultry chain and pose a substantial public health risk. Rapid and accurate serotype identification is essential for effective surveillance, outbreak investigation, and control strategies. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides rapid species-level identification; however, its reliability for serotype differentiation remains limited by overlapping spectral profiles and incomplete reference databases. Concurrently, rising antimicrobial resistance (AMR) necessitates exploration of alternative antimicrobial agents. This study aimed to (i) evaluate the accuracy of MALDI-TOF MS for Salmonella serotype identification using specific peak analysis compared with conventional serotyping and (ii) assess the antimicrobial effectiveness of trans-cinnamic acid (TCA) against poultry-associated Salmonella isolates. Materials and Methods: A total of 63 Salmonella isolates representing six serotypes were analyzed, including Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Kentucky, Salmonella Newjersey, Salmonella Fresno, and Salmonella Weltevreden, obtained from poultry production environments in Thailand. MALDI-TOF MS performance was evaluated against conventional serotyping using overall percentage agreement (OPA), positive percentage agreement (PPA), negative percentage agreement (NPA), and Cohen’s kappa statistic. Serotype-specific mass spectral peaks were identified through comparative analysis with reference strains. The antimicrobial activity of TCA was evaluated using broth dilution assays to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results: MALDI-TOF MS showed high concordance with conventional serotyping, with OPA, PPA, and NPA values ranging from 97.3% to 100%. Cohen’s kappa values indicated substantial to perfect agreement, with minor discordance observed for S. Enteritidis (κ = 0.65). Serotype-associated peaks were consistently detected at 6,094 ± 2 mass-to-charge ratio (m/z) for S. Enteritidis, 7,156 ± 2 m/z for S. Typhimurium, and 5,370 ± 2 m/z for S. Kentucky. TCA exhibited uniform antimicrobial activity against all tested serotypes, with MIC and MBC values of 10 mM and 20 mM, respectively, and no significant differences among serotypes (p > 0.05). Conclusion: MALDI-TOF MS combined with specific peak analysis provides a reliable and rapid approach for Salmonella serotype identification in the poultry chain, although database expansion remains necessary for uncommon serotypes. TCA demonstrated consistent inhibitory and bactericidal activity, supporting its potential role as a complementary, non-antibiotic intervention for Salmonella control and AMR mitigation in poultry production systems. Keywords: antimicrobial resistance, food safety, MALDI-TOF MS, poultry chain, Salmonella, serotype identification, specific peak analysis, trans-cinnamic acid.