Avirulent T3SS-negative strains belonging to Clade 5 produce variable quantities of secondary metabolites.

species are ubiquitous in the environment and serve as valuable source of enzymes and secondary metabolites for industrial applications. secretes metalloproteases, such as elastase LasB and produces bioactive small molecules, including pyocyanin, rhamnolipids, and pyoverdine, with potential biotechnological applications. However, the interest in for industrial use has been limited due to the virulence-associated Type III Secretion System (T3SS), a key factor in host-pathogen interactions. In this study, we genotypically and phenotypically characterized a collection of strains naturally lacking T3SS-encoding genes. Phylogenetic analysis revealed that these strains belong to two distinct clades. Several strains exhibited low or no cytotoxicity on epithelial cell lines and were avirulent in the infection model. The level of LasB and the three metabolites-pyocyanin, rhamnolipids, and pyoverdine-varied independently of virulence profiles. Notably, we identified avirulent strains capable of producing at least two secondary metabolites, including mono-rhamnolipids, highlighting their potential for biotechnological applications.