Identification of Salmonella enterica serovar Enteritidis mutants overproducing OMVs as vaccine candidates.

Salmonella enterica serovar Enteritidis (S. Enteritidis) remains one of the predominant serotypes causing infections in both humans and animals. In poultry farms, it is frequently reported as the leading cause of infection. Therefore, the development of vaccines against S. Enteritidis is considered a key strategy to control bacterial infections in poultry. Outer membrane vesicles (OMVs) have emerged as promising vaccine candidates for preventing bacterial infections in both animals and humans. However, the natural yield of OMVs from wild-type Salmonella is low, limiting large-scale application. FM1-43 is a lipophilic fluorescent dye that emits fluorescence upon specific binding to cell membranes. In this study, we established an FM1-43-based screening method to identify high-OMVs-producing mutants from a constructed S. Enteritidis Tn5 transposon mutant library. Using this approach, we successfully recovered previously reported Tol-Pal system mutants (ΔtolQ, ΔtolR, and ΔtolA), thereby validating the utility of this method. In addition, we identified five novel high-OMVs-producing mutants: ΔftsK, ΔxerD, ΔoxyR, ΔdegS, and ΔwzxE. Among the identified 28 mutants, ΔtolQ, ΔtolR, ΔtolA, and ΔftsK displayed significantly increased protein levels in OMVs compared with the wild type, while the ΔdegS mutant accumulated the highest lipid levels. Phenotypic analysis revealed that seven mutants exhibited reduced motility, with ΔxerD completely lacking motility, whereas ΔtolQ and ΔxerD showed enhanced biofilm formation. Antimicrobial susceptibility testing further demonstrated that ΔtolQ, ΔtolR, and ΔtolA were more sensitive to ciprofloxacin and polymyxin. Together, these findings suggest that genes involved in bacterial membrane integrity play key roles in OMVs production. The high-OMVs-yielding mutants identified here provide promising candidates for the development of subunit vaccines against S. Enteritidis infections in poultry.