The increasing threat of antimicrobial resistance (AMR) necessitates novel and complementary approaches to monitor and mitigate its spread in animal-derived biofluids such as milk. Our research applies omics sciences, particularly proteomics and metaproteomics, to detect AMR-related proteins and antimicrobial peptides (AMPs), providing crucial insights into the microbial consortia present in raw milk. By integrating liquid atmospheric pressure matrix-assisted laser desorption/ionization (LAP-MALDI) mass spectrometry (MS) profiling, bottom-up proteomics, and metaproteomics, we characterized AMR-related proteins in milk from intensive (Pezzata Rossa, Bruna Alpina, and Frisona) and non-intensive (Podolica) farming systems. Our findings revealed the presence of β-lactamases and tetracycline resistance proteins in all samples, confirming raw milk as a potential bioindicator of AMR circulation.

In parallel, our research also explored sustainable antimicrobial strategies by evaluating the inhibitory potential of plant-derived essential oils (EOs) against Staphylococcus xylosus, an opportunistic pathogen associated with bovine mastitis. An EO blend composed of Myrtus communis, Salvia officinalis, and Cistus ladanifer was tested alongside erythromycin for its antimicrobial efficacy. MALDI-TOF MS confirmed strain identification, and disk diffusion assays demonstrated a dose-dependent inhibitory effect of the EO blend, suggesting its potential as a complementary strategy to mitigate AMR.

These findings highlight the importance of integrating proteomics-based AMR surveillance with alternative antimicrobial strategies, such as phytocomplexes, to enhance food safety and reduce reliance on conventional antibiotics. Future studies will further investigate the synergistic potential of plant-derived antimicrobials in combination with existing therapeutic options.