Transposon Mutagenesis Identifies lon, csgF, and rfbF as Critical Regulators of Biofilm Architecture and Adhesion in Multidrug-Resistant Salmonella Typhimurium
Salmonella Typhimurium relies on biofilm formation to survive environmental stressors, resist antimicrobial agents, and establish persistent host infections. Despite its clinical importance, the genetic networks governing biofilm development remain incompletely understood. In this study, we profiled the biofilm-forming capacity of 708 S. Typhimurium isolates from human and animal sources, revealing that 88.56% were biofilm producers, with 3.81% classified as strong producers exhibiting extensive multidrug resistance. To elucidate the underlying genetic mechanisms, we subjected a strong biofilm-producing clinical isolate (15E341) to EZ-Tn5 transposon mutagenesis, generating a library of approximately 5,000 mutants. Phenotypic screening and whole-genome sequencing identified three distinct loci--lon, csgF, and rfbF--whose disruption significantly impaired biofilm formation without affecting planktonic growth rates. Microscopic evaluation via LSCM and FESEM demonstrated severely compromised 3D biofilm architecture and defective surface colonization in these mutants. Furthermore, the mutants exhibited significantly reduced adhesion to Caco-2 epithelial cells, glass, and silicone surfaces, though cellular invasion remained unaffected. Transcriptomic analysis (RNA-Seq) revealed a distinct regulatory shift in the mutants, characterized by the marked upregulation of flagellar assembly pathways and the downregulation of fimbrial and sugar metabolism genes. Notably, while the roles of lon and csgF are documented, the involvement of the O-antigen biosynthesis gene rfbF represents a novel determinant in S. Typhimurium biofilm regulation. These findings highlight complex, multi-gene coordination in biofilm development and identify potential therapeutic targets for disrupting persistent S. Typhimurium infections.