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Page 8 of 15 Spus et al. Microbiome Res Rep 2023;2:33 https://dx.doi.org/10.20517/mrr.2023.20

Figure 3. Susceptibility of the original strains used in the blend to phages present in the supernatants at different time points (after 6.6,
139, 272, 405 and 538 generations) of the propagation experiment. Blends were sequentially propagated in milk for up to 538
generations. Replicates A-1 and A-2 were used as control where no phage cocktail was added. Replicates B-1 and B-2 were challenged
at the onset of the experiment with a cocktail of lactococcal phages ϕTIFN1, ϕTIFN5 and ϕTIFN7.

susceptible to any of the phages used in the cocktail, showing the development of phage sensitivity in some
strains during the propagation experiment or induction of a lysogenic phage, which is a known
phenomenon for the Ur culture strains .
[28]
Strains isolated from blend B-2 at the end time point were all shown to be resistant to the three phages.
None of the strains isolated from B-2 was shown to belong to L. cremoris lineage 1 & 5 and most of them
were ascribed to L. cremoris lineage 3, and did not exhibit any obvious phage sensitivity.

In terms of the strains isolated from the control blends, most of them belong to L. cremoris lineage 1 & 5,
which in the case of blend A-1 was 43/50 strains and for blend A-2 42/50. Those 85 out of 100 strains were
susceptible to all three phages used in the cocktail at the onset of propagation.

These results confirm the dynamic character of interactions of strains in blends with bacteriophages from
the cocktail. In cases of control blends, strains present after 538 generations were still susceptible to the
three phages used in the cocktail. Susceptible strains can thrive in the community in the absence of phage
predation pressure. In contrast, in the case of the phage-challenged blend B-1, after prolonged propagation,
most of the isolated strains were resistant to phages used in the cocktail at the onset of the experiment
[Figure 4], demonstrating an important competitive advantage of phage resistant strains in the culture. In
blend B-2, no susceptibility to phages used in the cocktail was found among strains isolated at the end time
point, suggesting a lack of susceptible host and subsequent “wash out” of the lytic phages.