Temperature-dependent dynamics of Staphylococcus aureus killing by endolysin LysRODI in milk

Bacteriophage endolysins are promising antimicrobials for targeted control of Gram-positive pathogens in dairy matrices, but their efficacy is highly sensitive to processing conditions. Here, we investigated how temperature shapes the killing dynamics of the staphylococcal endolysin LysRODI against Staphylococcus aureus in UHT skim milk, and developed a mechanistic model to predict bacterial behavior across relevant dairy temperature ranges. Bacterial growth, endolysin killing rates, and enzyme stability were quantified at relevant temperatures and used to parameterize a system of ordinary differential equations. While a simple model accounting only for bacterial growth and thermal enzyme inactivation overestimated long-term killing, experimental assays revealed a significant loss of LysRODI activity after incubation under lytic conditions, even at temperatures where thermal inactivation was negligible. Incorporation of an activity-dependent decay term to the model markedly improved its performance and enabled accurate prediction of bacterial behavior under static and dynamic temperature scenarios. These results demonstrate that LysRODI efficacy depends on the combined effects of bacterial growth, thermal stability, and activity-associated enzyme loss, providing a framework for optimizing LysRODI treatment against S. aureus Sa9 in UHT skim milk.