Evaluación de probióticos y sus metabolitos en huevos y larvas de trucha como modelo One Health para su aplicación en salud humana y animal

The growing threat posed by antimicrobial resistance underscores the urgent need to develop effective and safe prophylactic strategies for both human and animal health. Within the One Health framework, this Doctoral Thesis aims to evaluate the potential of probiotic strains and their postbiotic metabolites as preventive agents through their application in models based on embryonated eggs and trout larvae (Salmo trutta and Oncorhynchus mykiss). These models were used for the early screening of candidates applicable to aquaculture and, by extension, to veterinary and human medicine, contributing to the development of integrated solutions against infections resistant to conventional treatments. Initially, a comprehensive health diagnosis was carried out in Spanish trout farms using RT-qPCR, MALDI-TOF, and 16S rRNA and ITS-1 sequencing. From these data, an Integrated Health Index (IHI) was generated, allowing the selection of broodstock and eggs free of relevant pathogens, as well as the isolation of lactic acid bacteria (LAB). The LAB isolates obtained were subjected to in vitro antagonism assays, using both live bacteria and their cell-free supernatants, against bacterial pathogens of fish and humans. Subsequently, trout eggs and larvae were exposed by immersion to selected live LAB or their supernatants in order to assess toxicity, survival, and hatching rate. The experimental results revealed the existence of a critical embryonic sensitivity window between 0 and 450 degree-days in S. trutta eggs, during which exposure to certain bacterial strains can compromise embryonic viability. In this context, some Lactiplantibacillus strains caused high embryonic mortality, whereas Pediococcus acidilactici and Lactococcus lactis displayed safety profiles comparable to the control. Moreover, neutralized postbiotic supernatants (pH 7–6.5) did not cause adverse effects at any developmental stage evaluated and maintained specific inhibitory activity, particularly against Carnobacterium maltaromaticum and Vagococcus salmoninarum. In addition, the combined diagnostic approach (qPCR + MALDI-TOF) was confirmed as a rapid, reliable, and accurate tool for the etiological identification of infectious outbreaks in aquaculture environments. Overall, this Doctoral Thesis validates embryonated eggs and yolk-sac trout larvae as an ethical, sensitive, and low-cost vertebrate model for probiotic and postbiotic screening, aligned with the 3Rs principle (Replacement, Reduction, and Refinement) and framed within the One Health perspective. The findings demonstrate that (1) toxicity is strain-specific, (2) postbiotics exhibit a superior safety profile, and (3) certain LAB strains show strong antagonistic activity against relevant aquaculture pathogens. These results lay the groundwork for the development of probiotic or postbiotic formulations aimed at reducing antibiotic use in aquaculture, with direct implications for improving food safety and contributing to the global fight against antimicrobial resistance.