Longitudinal Study of Lactococcus Phages in a Canadian Cheese Factory

Abstract

The presence of virulent phages is closely monitored during cheese manufacturing, as these bacterial viruses can significantly slow down the milk fermentation process and lead to low-quality cheeses. From 2001 to 2020, whey samples from cheddar cheese production in a Canadian factory were monitored for the presence of virulent phages capable of infecting proprietary strains of Lactococcus cremoris and Lactococcus lactis used in starter cultures. Phages were successfully isolated from 932 whey samples using standard plaque assays and several industrial Lactococcus strains as hosts. A multiplex PCR assay assigned 97% of these phage isolates to the Skunavirus genus, 2% to the P335 group, and 1% to the Ceduovirus genus. DNA restriction profiles and a multilocus sequence typing (MLST) scheme distinguished at least 241 unique lactococcal phages from these isolates. While most phages were isolated only once, 93 of them (out of 241, 39%) were isolated multiple times. Phage GL7 was isolated 132 times from 2006 to 2020, demonstrating that phages can persist in a cheese factory for long periods of time. Phylogenetic analysis of MLST sequences showed that phages could be clustered based on their bacterial hosts rather than their year of isolation. Host range analysis showed that Skunavirus phages exhibited a very narrow host range, whereas some Ceduovirus and P335 phages had a broader host range. Overall, the host range information was useful in improving the starter culture rotation by identifying phage-unrelated strains and helped mitigating the risk of fermentation failure due to virulent phages. IMPORTANCE Although lactococcal phages have been observed in cheese production settings for almost a century, few longitudinal studies have been performed. This 20-year study describes the close monitoring of dairy lactococcal phages in a cheddar cheese factory. Routine monitoring was conducted by factory staff, and when whey samples were found to inhibit industrial starter cultures under laboratory conditions, they were sent to an academic research laboratory for phage isolation and characterization. This led to a collection of at least 241 unique lactococcal phages, which were characterized through PCR typing and MLST profiling. Phages of the Skunavirus genus were by far the most dominant. Most phages lysed a small subset of the Lactococcus strains. These findings guided the industrial partner in adapting the starter culture schedule by using phage-unrelated strains in starter cultures and removing some strains from the starter rotation. This phage control strategy could be adapted for other large-scale bacterial fermentation processes.

Keywords: Lactococcus; MLST; bacteriophages; cheese; dairy; food microbiology; lactic acid bacteria; microbiology; milk; phage; virology; whey.

FIG 1

Schematic of the protocol performed at the cheese factory (top) and at the university laboratory (bottom). Whey is represented by the yellow-green color. Growth medium is shown in brown, bacterial growth in beige, and phage buffer in light blue.

FIG 2

Number of lactococcal phage isolations in a 20-year period from cheddar cheese whey samples and the most frequent Lactococcus strains used as a host. (A) Distribution of unique and recurrent phages isolated per year. The 2020 bar* represents half the year since the phage monitoring program ended in June 2020. (B) The number of times the 30 most dominant lactococcal phages were isolated. All of the phages belong to the Skunavirus genus, except BM13*, which belongs to the P335 group. (C) Lactococcus strains used as a host more than five times.

FIG 3

Skunavirus phylogenetic analysis of the MLST profiles highlighting the clustering according to the bacterial hosts. The neighbor-joining phylogenetic tree of the Skunavirus phage isolates was constructed from the concatenated MLST sequence data. Branch support values greater than 90% are indicated by gray circles. Labels are color coded based on the hosts used for phage isolation. Striped labels account for phages isolated on more than one host. *, for clarity, phages that share the sequence type 13 with GL7 are listed on the right.

FIG 4

Host specificity of selected lactococcal phages. Bars represent the number of phages that infect a specified number of Lactococcus strains. Eighty-eight Skunavirus phages (orange), nine P335 phages (teal), and six Ceduovirus phages (black) were tested on 54 industrial Lactococcus strains.