The bovine mammosphere-derived epithelial cell secretome inhibits bacterial growth in vitro and contains peptidoglycan recognition protein 1

in planktonic and biofilm assays. Secretome analysis by heat treatment, size fractionization, and extracellular vesicle isolation suggested that soluble proteins of different sizes, or in complexes, drive the anti-bacterial effects. We identified the antimicrobial protein peptidoglycan recognition protein 1 (PGLYRP1) in MDECs and detected it in the secretome. As knock down of PGLYRP1 expression in MDECs was unsuccessful using RNA interference and CRISPR-Cas9 gene editing, a direct link between the antimicrobial activity of the secretome and this protein could not be confirmed. The newly identified antimicrobial activity of the bovine MDEC secretome supplements its tissue reparative features, poising it as a potential adjunct or alternative therapy for bovine mastitis that may reduce acute milk loss and improve long term milk production.