Milk promotes the development of complex, protein-rich biofilms in Staphylococcus aureus and limits the efficacy of phage-based eradication strategies

The ability of Staphylococcus aureus to establish strong biofilms in milk poses a threat to consumers’ health and a challenge for the dairy industry. Indeed, biofilms are the main bacterial reservoir along the food chain. Here, we evaluate the efficacy of phage Kayvirus rodi and lytic protein LysRODIΔAmi for combating biofilm formation on polystyrene and stainless steel surfaces submerged in UHT skim milk compared to culture medium. Biofilms grown in milk exhibited a denser and more complex matrix, mainly composed of proteins, presumably casein. Additionally, cells exhibited an adaptive transcriptional response intended to metabolize lactose and to increase biofilm formation. These changes in metabolism, matrix composition and structure conferred milk-grown biofilms a higher resistance to both LysRODIΔAmi (4 μM) and phage Kayvirus rodi (10⁸ PFU/ml) individual and combined treatments, although the combination was more efficacious. Both antimicrobials were more successful at biofilm inhibition, exhibiting a synergistic interaction when used together. Unexpectedly, the bacteriophage treatment displayed enhanced efficacy in milk compared to fresh medium, likely due to the buffering capacity and ions in milk, which help stabilize pH and promote phage stability. Our results demonstrate the importance of testing antimicrobials under conditions that reflect the real-life application as much as possible.

This work was funded by grants PID2022-140988OB-I00 (MCIN/AEI/10.13039/501100011033/FEDER, UE) to P.G and L.F, PID2019-105311RB-I00 (MICIU/AEI/FEDER, UE, Spain) to P.G. and A.R.; and AYUD/2021/52120 (Program of Science, Technology and Innovation 2021–2023 and FEDER, EU, Principado de Asturias, Spain). S.A. has an FPI fellowship (Ministry of Science and Innovation, Spain). DairySafe group is part of the Spanish Network of Bacteriophages and Transducer Elements-FAGOMA.

Peer reviewed