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Updated: May 5, 2026
A Tandem Liquid Chromatography–Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus
Published on: March 28, 2017
Untargeted Metabolomics Reveals Metabolic Reprogramming During Viable but Non-Culturable State Formation in Aeromonas
Gururaj Moorthy1, Jatuphol Pholtaisong2,3, Anusara Wongkotsila4
1International Center of Excellence in Seafood Science and Innovation, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai 90110, Songkhla, Thailand.
Abstract:
Aeromonas hydrophila is a major seafood-borne pathogen capable of persisting under preservative-associated stress by entering a viable but non-culturable (VBNC) state, thereby evading culture-based detection. Here, untargeted metabolomics was applied as the primary analytical approach to elucidate metabolic reprogramming during VBNC formation under seafood-relevant preservation conditions. Cells were incubated at 4 °C for 30 days in sodium benzoate-supplemented saline, comparing 0.85% NaCl (culturable condition) and 4% NaCl (VBNC-inducing condition), with sampling every 6 days. Under 4% NaCl with sodium benzoate, culturability declined from 6.18 log CFU/mL at day 0 to undetectable levels by day 30, while cell viability was retained, confirming VBNC induction. UHPLC-ESI-QTOF-MS profiling detected over 893 intracellular metabolic features, of which 518 metabolites were significantly altered between VBNC and culturable states at day 30. Principal component analysis revealed clear, time-dependent metabolic divergence, with the VBNC trajectory explaining 34.4% (PC1) and 11.5% (PC2) of total variance. Pathway enrichment analysis demonstrated significant remodeling of alanine, aspartate and glutamate metabolism (8/28 hits, FDR = 5.7 × 10-4); arginine biosynthesis (5/14 hits, FDR = 5.44 × 10-3); purine metabolism (10/70 hits, FDR = 8.34 × 10-3); and pyrimidine metabolism (7/39 hits, FDR = 1.35 × 10-2), indicating nitrogen conservation and metabolic downshifting. A robust biomarker panel, including depleted cyclic AMP, aminoadipic acid, hypotaurine, O6-CM-dG, and betaine, and enriched urocanic acid, pipecolic acid, proline, azelaic acid, and orcinol perfectly discriminated VBNC from culturable cells. These findings demonstrate that sodium benzoate-based preservation can induce a metabolically reprogrammed VBNC state in A. hydrophila, highlighting a hidden food safety risk beyond culture-based assessment.
