Functional strain redundancy and persistent phage infection in Swiss hard cheese starter cultures

Abstract

Undefined starter cultures are poorly characterized bacterial communities from environmental origin used in cheese making. They are phenotypically stable and have evolved through domestication by repeated propagation in closed and highly controlled environments over centuries. This makes them interesting for understanding eco-evolutionary dynamics governing microbial communities. While cheese starter cultures are known to be dominated by a few bacterial species, little is known about the composition, functional relevance, and temporal dynamics of strain-level diversity. Here, we applied shotgun metagenomics to an important Swiss cheese starter culture and analyzed historical and experimental samples reflecting 82 years of starter culture propagation. We found that the bacterial community is highly stable and dominated by only a few coexisting strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. Genome sequencing, metabolomics analysis, and co-culturing experiments of 43 isolates show that these strains are functionally redundant, but differ tremendously in their phage resistance potential. Moreover, we identified two highly abundant Streptococcus phages that seem to stably coexist in the community without any negative impact on bacterial growth or strain persistence, and despite the presence of a large and diverse repertoire of matching CRISPR spacers. Our findings show that functionally equivalent strains can coexist in domesticated microbial communities and highlight an important role of bacteria-phage interactions that are different from kill-the-winner dynamics.

Extensive diversity and rapid turnover of phage defense repertoires in cheese-associated bacterial communities

“…18). This observation is in contrast to what has been observed in laboratory studies of phage-bacteria coevolution (36, 45) or in cases of chronic phage infections in S. thermophilus (31), where multiple spacers targeting the same vOTU were often found to be integrated into the CRISPR array.…”

“…However, as we only considered fixed mutations, we may underestimate the time of divergence between these genomes which would result in a lower turnover rate. Indeed, previous estimates of CRISPR acquisition rates based on experimental data were ∼1 magnitude lower (<0.1 spacer/generation in (31) or ∼0.5 spacer/generation in (34)).…”

“…Notably, in our previous study focusing on a single cheese starter culture we had found the opposite pattern. This may be explained by the fact that a single phage dominated this community, causing chronic infections and thereby overcoming CRISPR-based immunity (31). Interestingly, with increasing spacer abundance the ratio of protospacer to spacer abundance decreased (slope=-0.48, R 2 =0.32, Fig.…”

“…This makes them tractable systems to study the evolution of phage defense systems in microbial communities at the strain-level (29, 30). Indeed, our previous study of a single Swiss cheese starter culture has shown that extensive intraspecific CRISPR spacer diversity exists in these otherwise nearly clonal populations of bacteria (31).…”

Antimicrobial Susceptibility of Lactobacillus delbrueckii subsp. lactis from Milk Products and Other Habitats

“…Recent findings have indicated that in cheese, a similar mutualistic interaction may take place between S. salivarius subsp. thermophilus and LDL [ 4 ]. The production of semi-hard (e.g., Appenzeller ® and Tête de Moine PDO), hard (e.g., Emmentaler PDO, Le Gruyère PDO, and Comté PDO), and extra-hard cooked cheeses (e.g., Parmigiano Reggiano PDO, Grana Padano PDO, and Sbrinz PDO) relies on the acidification of milk by thermophilic starter cultures containing a combination of thermophilic LAB.…”