Distinct shifts in bacteriophage diversity and abundance during various stages of Gouda-type cheese production

Bacteriophage (or phage) infection is known to disrupt milk fermentation processes involving lactic acid bacteria (LAB). This highlights the need to understand the dynamics of phage infection, particularly in fermentations involving undefined complex starter cultures, where conventional phage detection methodologies that rely on host bacteria come with limitations. Here, a metagenomic approach combined with microbiological methods was employed to assess the presence, diversity, and compositional changes in lactococcal phages within an industrial cheese production process. The virome data sets of ingredients and samples taken at various stages of the fermentation process were analyzed, revealing 12 presumptive lactococcal phage genomes belonging to either the Skunavirus genus or the P335 group phages. Phylogenetic analysis of the receptor-binding protein (RBP) sequence from these identified Skunavirus phages enabled us to predict the cell wall polysaccharide (CWPS) types of their bacterial hosts. This prediction was partially validated through host-range assays of three isolated phages, which matched phage genome contigs identified in the virome. Furthermore, alignment of their RBP-encoding gene sequences against virome data, along with real-time quantitative PCR targeting the Skunavirus genus, revealed a significant shift in the abundance and diversity of Skunavirus phages infecting different CWPS type strains throughout the production regime. Overall, our study revealed fluctuations in phage abundance and diversity during a cheese production cycle, underscoring the potential risk of inconsistent product quality due to phage proliferation and the need for effective strategies to mitigate phage contamination.IMPORTANCEThis study investigated dairy samples collected during a full fermentation cycle in a Gouda-type cheese facility to assess the prevalence, abundance, diversity, and dynamics of bacteriophages (phages) infecting starter culture components using a combination of metagenomic and microbiological assays. Also, by predicting the cell wall polysaccharide (CWPS) type of the host bacteria based on receptor-binding protein (RBP) sequences, the possible impact of bacteriophages on starter culture composition was evaluated. These findings highlight the need for tracking strategies to monitor bacteriophage dynamics in order to ensure robust and reliable fermentations.