Biofilm-forming ability of Staphylococcus lugdunensis clinical isolates associates with sequence type and virulence in a Galleria mellonella model.

Staphylococcus lugdunensis, one of the most virulent members of coagulase-negative staphylococci, exhibits a strong biofilm-forming capacity, raising the question of whether this trait directly contributes to its virulence in vivo. Here, we used crystal violet staining to assess the biofilm-forming capacity of 202 S. lugdunensis and examined its association with sample source, multilocus sequence types (STs), agr types, oxacillin susceptibility, and virulence in a larval infection model. Among the 202 isolates, 135 (66.8%) formed strong biofilms, 46 (22.8%) moderate, and 21 (10.4%) weak biofilms. Most S. lugdunensis isolates were obtained from blood, pus, and deep tissue samples, accounting for 47.5%, 15.3%, and 12.4% of cases, respectively, with only a weak association between sample source and biofilm-forming ability. Bacteremia isolates were classified as primary infections (n = 36) or contaminants (n = 40), with primary infection isolates forming significantly stronger biofilms. Biofilm formation was not strongly associated with oxacillin susceptibility, SCCmec, or agr types; however, ST3 isolates exhibited higher biofilm formation than ST6 and ST27. In addition, gene annotation of whole-genome sequences from 19 S. lugdunensis isolates revealed that biofilm-associated genes are highly conserved across all strains regardless of their biofilm phenotype. Larvae infected with extremely weak biofilm formers showed 100% survival up to 7 days, whereas strong biofilm formers caused markedly reduced survival, with 75% showing <50% survival compared to 30% among weak biofilm formers. Therefore, despite the phenotypic variation in biofilm formation, our work demonstrated an association between biofilm phenotype, bacteremia classification, sequence types, and in vivo virulence.