Plasmid encoded mrk gene cluster promotes biofilm formation, environmental persistence, and invasiveness in Salmonella Thompson
Biofilm formation is a fundamental survival strategy that enables bacterial persistence across diverse environments and hosts. While the mrk gene cluster encoding type 3 fimbriae is a well-established determinant of biofilm formation in Klebsiella pneumoniae, its presence and functional significance in Salmonella enterica remains poorly defined. In this study, we identified a plasmid-encoded mrkABCDF gene cluster carried on a highly conjugative IncX1 plasmid in a clinical Salmonella Thompson isolate. Using CRISPR/Cas9-mediated knockout of the mrk-containing Tn6011 transposon, plasmid curing, targeted gene deletion, and genetic complementation, we systematically dissected the contribution of mrkABCDF genes to biofilm development and associated phenotypes. Loss of the mrk gene cluster resulted in a profound reduction in biofilm biomass and a concomitant increase in bacterial motility. Type 3 fimbriae were detected exclusively on the surface of mrk-positive cells, confirming their structural role in surface attachment. The mrk operon was strongly expressed from an IS1-associated promoter, bypassing canonical MrkH-dependent regulation. Functionally, mrk expression enhanced bacterial tolerance to desiccation and oxidative stresses, and reduced susceptibility to macrophage phagocytosis. In vivo, mrk-positive strains exhibited enhanced gastrointestinal colonization and tissue invasion. Notably, carbapenems exhibited exceptional efficacy in inhibiting mrk-mediated biofilm formation, indicating their superior potential for treating biofilm-associated infections. Our findings demonstrate that plasmid-encoded mrkABCDF genes can act as key architectural and functional determinants of biofilm formation in Salmonella enterica. The horizontal dissemination of mrk-carrying IncX1 plasmids may promote the emergence of biofilm-adapted Salmonella lineages with enhanced environmental persistence and host colonization potential.