Impact of the calcium concentration on the efficacy of phage phiIPLA-RODI, LysRODI¿Ami and nisin for the elimination of Staphylococcus aureus during lab-scale cheese production

Staphylococcus aureus is a Gram-positive human opportunistic pathogen that may also cause food poisoning because of the ability of some strains to produce heat stable enterotoxins that can persist in food even after the pathogen is successfully eliminated. In this context, biopreservation may be a forward-looking strategy to help eliminate staphylococcal contamination in dairy products by using natural compounds. However, these antimicrobials exhibit individual limitations that may be overcome by combining them. This work investigates the combination of a virulent bacteriophage, phiIPLA-RODI, a phage-derived engineered lytic protein, LysRODIΔAmi, and the bacteriocin nisin for the elimination of S. aureus during lab-scale cheese production at two CaCl2 concentrations (0.2 % and 0.02 %), and subsequent storage at two different temperatures (4 °C and 12 °C). In most of the assayed conditions, our results demonstrate that the combined action of the antimicrobials led to a greater reduction of the pathogen population than the compounds individually, albeit this effect was additive and not synergistic. However, our results did show synergy between the three antimicrobials for reducing the bacterial load after 14 days of storage at 12 °C, temperature at which there is growth of the S. aureus population. Additionally, we tested the impact of the calcium concentration on the activity of the combination treatment and observed that higher CaCl2 levels led to a notable increase in endolysin activity that allowed the utilization of approximately 10-times less protein to attain the same efficacy. Overall, our data show that the combination of LysRODIΔAmi with nisin and/or phage phiIPLA-RODI, and an increase in the calcium concentration are successful strategies to decrease the amount of protein required for the control of S. aureus contamination in the dairy sector with a low potential for resistance selection, thereby reducing costs.

This work was funded by grants 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, FICYT and FEDER EU, Principado de Asturias, Spain). S.A. has an FPI fellowship (Ministry of Science and Innovation, Spain).