Phage lysates are reservoirs of biologically active quorum sensing signals

Bacteriophage lysates are widely used to study phage-bacterial interactions and are typically applied after filtration without further purification. As a result, host-derived metabolites produced during phage propagation are transferred alongside phage particles, yet the biological consequences of this carryover remain largely unexplored. Here, we show that the Pseudomonas aeruginosa quorum sensing signal PQS persists in a bioactive state in phage lysates for at least one year. In contrast, the quorum sensing molecules 3OC12-HSL and C4-HSL are rapidly degraded. We demonstrate that PQS stability is caused by its association with outer membrane vesicles, which protect the signal from degradation. PQS from phage lysates induces production of the virulence factor pyocyanin and increases mortality in a Galleria mellonella model of P. aeruginosa infection. Importantly, PQS carryover does not explain previous reports of phage mediated activation of the PQS system, as this response also occurs in PQS free phage lysates, establishing phage lysate-mediated signal transfer and phage-induced signaling as distinct mechanisms. Because hydrophobic signaling molecules are associated with outer membrane vesicles in diverse Gram-negative bacteria, including Vibrio harveyi (CAI-1) and Paracoccus (C16-HSL), our findings suggest that phage lysates may serve as reservoirs and overlooked vectors of host-derived communication signals in a broad range of phage-host systems. Our results reveal a previously overlooked layer of phage-host interactions in which biologically active bacterial signals persist beyond phage lysate preparation and influence recipient-cell behavior independently of phage infection.