Transcriptomic insights into cultivation-driven virulence in Aeromonas spp.: a new approach to optimizing autogenous...

Autogenous vaccines are a critical tool in aquaculture for managing bacterial diseases when commercial vaccines are unavailable or ineffective. To improve vaccine efficacy, this study explored how different cultivation conditions influence virulence gene expression in two major fish pathogens, Aeromonas salmonicida subsp. salmonicida and Aeromonas hydrophila. Isolates were cultured in nutrient-rich (tryptic soy broth [TSB]) and nutrient-limited (Mueller-Hinton broth [MH]) media, with and without supplementation of 1% fetal bovine serum (tryptic soy broth supplemented with 1% FBS [TSB1] and Müller-Hinton supplemented with 1% FBS [MH1]), to mimic environmental and host-like conditions. Total RNA was sequenced using the Oxford Nanopore MinION platform, and gene expression was quantified using featureCounts and Salmon, followed by differential expression analysis with DESeq2. Results revealed that culture conditions significantly shaped transcriptomic profiles. TSB1 promoted the highest and most consistent expression of classical virulence genes such as aerA, exeC, and fliP, due to serum-derived host signals. In contrast, MH induced higher expression of genes linked to motility and early host interaction, including flpI and exeB, despite overall lower transcriptional activity. These findings highlight the complementary expression of virulence factors under distinct nutritional conditions. Heatmaps and principal component analysis (PCA) confirmed clustering of expression profiles across media types. In relation to our findings, TSB1 is therefore recommended as the primary medium for bacterin production in autogenous vaccine development. However, combining cultures grown in both TSB1 and MH may capture a broader antigen repertoire, enhancing immune recognition and protection. This transcriptomics-based strategy presents as a rational framework for designing next-generation autogenous vaccines in aquatic veterinary medicine.