What makes them stick? A genetic analysis of biofilm formation of an infective endocarditis-causing Streptococcus canis strain using transposon directed insertion-site sequencing

Background Streptococcus canis is an emerging zoonotic pathogen capable of causing infective endocarditis (IE) in animals and humans. In IE, bacterial biofilms form vegetations on heart valves, protecting microbes from antibiotics and immune responses, which complicates treatment and promotes chronic infection. Methods To identify genes required for biofilm development, we performed transposon-directed insertion site sequencing (TraDIS) in combination with a biofilm formation assay on a fibrin matrix under physiologically relevant flow conditions. Mutant libraries were screened for deficiencies in biofilm formation, followed by pathway enrichment and targeted functional assays. Results Mutants impaired in biofilm formation were enriched for disruptions in carbohydrate metabolism, cell wall biogenesis, and quorum sensing pathways. The rfb operon and galE , genes essential for extracellular polysaccharide synthesis, were identified as key contributors. Pathway analysis highlighted quorum-sensing and HIF-1 signaling as regulators of metabolic adaptation and matrix production under flow. Biofilm formation by the IE strain was inhibited by carvacrol, an inhibitor of LuxS-dependent quorum sensing. Deletion of the fibronectin-binding serum opacity factor (ScSOF) significantly reduced biofilm formation on fibronectin-coated surfaces and altered matrix composition, demonstrating its role in host matrix–dependent adhesion. Discussion These findings provide the first genome-wide characterization of biofilm-associated gene networks in S. canis , revealing how metabolic pathways, quorum sensing, and host adhesion factors interact to promote endocardial biofilm formation.