Evaluating small extracellular vesicle-based vaccination across heterologous strains isolated from wastewater.

infections pose significant public health challenges worldwide. The diversity of strains, particularly those isolated from environmental and clinical sources, necessitates innovative approaches to prevention and treatment. Previous research has shown that small extracellular vesicles (sEVs) produced by macrophages during Typhimurium infection can induce robust immune responses when used as a vaccine, offering complete protection in systemic infection models. In this study, we isolated 120 strains from qPCR -positive wastewater samples collected in Gainesville, FL. These strains underwent enrichment, selection, and biochemical confirmation, followed by serotyping and whole genome sequencing. Two isolates, subsp. diarizonae (Diarizonae) and serovar Enteritidis, were selected for further analysis based on community prevalence and clinical severity. We also assessed the ability of sEVs produced by . Typhimurium-infected macrophages to induce immune responses against these heterologous and circulating strains in mice. Immunization with sEVs induced robust antigen-specific SIgA and IgG responses against . Typhimurium, Enteritidis, and Diarizonae, with high titers observed in sera and fecal samples. Proteomic analysis revealed differential expression of proteins in these strains, including antigenic proteins present in sEVs such as OmpA, FliC, or OmpD. Moreover, this study highlights the role of wastewater-based epidemiology (WBE) as a tool for environmental surveillance, offering a complementary perspective on dynamics within a population. Integrating WBE with traditional surveillance methods, along with the promising results of sEV-based vaccination, provides a pragmatic strategy for developing effective preventative measures against infections, addressing the diversity of non-typhoidal strains.