Transposon mutagenesis identifies lon , csgF , and rfbF as critical genetic determinants of biofilm architect

Salmonella Typhimurium relies on biofilm formation to withstand environmental stressors, resist antimicrobials, and establish persistent host infections. However, the genetic networks governing biofilm development remain poorly understood. Here, we assessed the biofilm‐forming capacity of 708 S.  Typhimurium isolates. Among them, 88.56% formed biofilms, with 3.81% classified as strong producers associated with extensive multidrug resistance. To elucidate underlying genetic determinants, we performed EZ‐Tn5 transposon mutagenesis on a strong biofilm‐producing clinical isolate (15E341), generating ~ 5000 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. Confocal and electron microscopy (LSCM and FESEM) revealed severely compromised 3D biofilm architecture and defective surface colonization. Furthermore, these mutants exhibited significantly reduced adhesion to Caco‐2 epithelial cells, glass, and silicone. Transcriptomic analysis revealed a distinct regulatory shift, characterized by marked upregulation of flagellar assembly pathways and downregulation of fimbrial genes. In a murine model, the mutants exhibited attenuated virulence, evidenced by increased host survival and significantly reduced bacterial colonization in the cecum, liver, and spleen. Notably, whereas CsgF is an established curli‐assembly factor and Lon is a well‐characterized stress and virulence regulator, RfbF, an O‐antigen biosynthesis enzyme, emerged as a previously undercharacterized determinant of biofilm capacity in S. Typhimurium. Collectively, these findings highlight multi‐gene coordination in biofilm development and identify pathways that warrant further investigation for disrupting persistent Salmonella infections.