Screening of Human and Environmental Microbiota-Derived Extracts Reveals Pathogen-Specific Anti-Virulence Activity

Aims Targeting bacterial virulence is a promising alternative to traditional antibiotics. In this study, we aimed to identify microbiome-derived factors capable of suppressing virulence traits in pathogenic bacteria. Methods and Results Eighty-two cell-free extracts (CFEs) applied at 10% (v/v), from human (n = 56) and soil (n = 26) bacterial isolates were screened for effects on planktonic growth, biofilm formation, and virulence using a Galleria mellonella infection model. Growth inhibition was rare, observed only for S. aureus exposed to a Bacillus flexus extract. Anti-biofilm activity was frequent against P. aeruginosa (65/82 CFEs, 79%), less so for S. aureus (20/82, 24%), and absent or reversed for E. coli. Eight CFEs improved larval survival in P. aeruginosa infections; one partly protected against S. aureus, while (47/82, 57%) increased mortality in E. coli-infected larvae. Thirty-eight of 65 anti-biofilm CFEs (58%) for P. aeruginosa tested positive (i.e. observed) in a violacein-based quorum-sensing inhibition assay. One extract (Streptococcus australis) also protected keratinocytes from P. aeruginosa-induced cell loss. Proteomic analysis of P. aeruginosa exposed to S. australis CFE indicated modulation of proteins associated with biofilm regulation and quorum-sensing pathways (e.g. the RsmA/CsrA family), consistent with anti-virulence activity. Conclusions Of 82 microbiome-derived CFEs, (65/82, 79%) partially inhibited P. aeruginosa biofilm formation, (20/82, 24%) affected S. aureus, and none inhibited E. coli biofilms. Ten % improved in vivo survival in P. aeruginosa infections, while (47/82, 57%) increased mortality in E. coli-infected larvae. Anti-virulence effects were pathogen-specific and occurred without measurable impacts on planktonic growth under the conditions tested.