Development of a Lyophilization Process for Campylobacter Bacteriophage Storage and Transport

Abstract

Bacteriophages are a sustainable alternative to control pathogenic bacteria in the post-antibiotic era. Despite promising reports, there are still obstacles to phage use, notably titer stability and transport-associated expenses for applications in food and agriculture. In this study, we have developed a lyophilization approach to maintain phage titers, ensure efficacy and reduce transport costs of <i>Campylobacter</i> bacteriophages. Lyophilization methods were adopted with various excipients to enhance stabilization in combination with packaging options for international transport. Lyophilization of <i>Eucampyvirinae</i> CP30A using tryptone formed a cake that limited processing titer reduction to 0.35 ± 0.09 log₁₀ PFU mL^-1. Transmission electron microscopy revealed the initial titer reduction was associated with capsid collapse of a subpopulation. Freeze-dried phages were generally stable under refrigerated vacuum conditions and showed no significant titer changes over 3 months incubation at 4 °C (<i>p</i> = 0.29). Reduced stability was observed for lyophilized phages that were incubated either at 30 °C under vacuum or at 4 °C at 70% or 90% relative humidity. Refrigerated international transport and rehydration of the cake resulted in a total phage titer reduction of 0.81 ± 0.44 log₁₀ PFU mL^-1. A significantly higher titer loss was observed for phages that were not refrigerated during transport (2.03 ± 0.32 log₁₀ PFU mL^-1). We propose that lyophilization offers a convenient method to preserve and transport <i>Campylobacter</i> phages, with minimal titer reduction after the drying process.