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Spus et al. Microbiome Res Rep 2023;2:33 https://dx.doi.org/10.20517/mrr.2023.20 Page 7 of 15

Figure 2. Relative abundance in percentage of lineages in blends during the propagation experiment based on DNA copy number values
obtained with unique-gene-based qPCR. Panel A-1 represents the 1st replicate of the blend without phage challenge; Panel A-2
represents the 2nd replicate of the blend without phage challenge; Panel B-1 represents the 1st replicate of the blend with phage
challenge; Panel B-2 represents the 2nd replicate of the blend with phage challenge.

bacteriophages during the course of the experiment [Figure 3]. In the supernatant after 6.6 generations, we
found bacteriophages predating on 10 different L. cremoris lineage 1 & 5 strains. Two strains, which
previously showed susceptibility to phages used in the cocktail, namely 2MS2 (L. cremoris lineage 1 & 5) and
2MS47 (L. cremoris lineage 6), did not show susceptibility to phages from the supernatants of the phage-
challenged blends after 6.6 generations. L. cremoris strain RS20, for which initially no detectable
susceptibility was found to any of the three phages, showed sensitivity to phages from the supernatant of
both replicates of the phage-challenged blends after 6.6 generations. Supernatants collected from the phage-
challenged cultures after prolonged propagation showed lytic activity on two of the original isolates after
272 generations (2RS32 and 2MS50) and on one for each blend after 405 generations (B-1 - 2RS32 and B-2 -
2RS20) [Figure 3]. None of the supernatants collected after 139 and 538 generations caused lysis of any of
the original strains.

Next, we isolated single colonies (50 for each blend, in total 200 colony isolates) from the end time point of
the propagation experiment to check if any of the previously resistant strains had become sensitive to
phages used in the cocktail or to the ones possibly present in the supernatant of the cultures at the end time
point. First, we determined for these 200 strains the genetic lineage identity using colony PCR . Next, all
[5]
these single colony isolates were challenged in a spot assay with individual phages used in the cocktail as
well as with the supernatants of the end time point (538 generations) blends [Figure 4]. We did not find any
susceptibility of strains isolated from any of the four blends A-1, A-2, B-1 and B-2 when the supernatants of
the end time point were used in the spot assay. However, 18 out of 50 strains isolated from blend B-1 at the
end time point were susceptible to at least one of the three phages used in the cocktail at the onset of the
propagation. Most of these susceptible strains belong to L. cremoris lineage 1 & 5 (16/18). Two isolated
strains, namely 4.30 (ascribed to L. cremoris lineage 3) and 4.46 (ascribed to L. cremoris lineage 7), were
found to be susceptible to phage ϕTIFN5 and ϕTIFN7 (strain 4.30) and phage ϕTIFN5 (strain 4.46). At the
onset of the experiment, the strains of L. cremoris lineages 3 and 7 used for the blend design were not