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Spus et al. Microbiome Res Rep 2023;2:33 https://dx.doi.org/10.20517/mrr.2023.20 Page 3 of 15

change) which is a factor pushing a microbial community towards a tipping point of the shift. The shift in
community composition leads to an alternative state that is difficult to reverse. Often, catastrophic shifts are
[13]
caused by a stochastic event, which can lead to, e.g., a wipeout of a part of a population . In the particular
case of a complex starter culture, such a wipeout of a sub-population can be caused by bacteriophage
predation. The negative impact of phage predation on acidification during the production of dairy products
has previously been covered by various publications [14,15] . Nevertheless, complex starter cultures, in general,
present better resilience to phage predation as compared to defined starters . Several studies describe the
[16]
impact of bacteriophages on individual bacterial strains [17-19] , but in the case of complex (multi-strain)
microbial communities, empirical data that could support the PS and/or CD dynamic community models
[9]
are completely lacking. Rodriguez-Valera et al. suggested experimentally measuring the individual fitness of
different bacterial strains isolated from a natural habitat to determine whether fitter variants are selected
[9]
against environments under phage pressure .
Therefore, in this study, we used complex blends of well-characterized strains from the Ur starter as a model
system to investigate the role of bacteriophages in population dynamics. These blends were designed
according to the following criteria. Firstly, all eight genetic lineages of the complex dairy starter culture
called Ur were represented by multiple strains. Secondly, all strains were mixed at equal initial relative
[3]
[5]
abundance. Thirdly, we deliberately included strains with different bacteriophage resistance profiles .
Consequently, we obtained a blend of strains that resembled the diversity of the natural complex starter
culture at the level of (i) genetic lineages; (ii) at the strain level; and (iii) at the different levels of
bacteriophage resistance. Our blends were sequentially propagated in milk for more than 500 generations
without (control) or with the addition of a phage cocktail composed of three lytic phages isolated previously
[3]
from the Ur culture . Throughout the propagation experiment, we monitored the abundance of genetic
lineages and the presence of bacteriophages. Our study generated empirical data on phage predation and
microbial population dynamics that were subsequently analyzed in the context of PS and CD dynamic
community models.

METHODS
Preparation of the multi-strain blend
The starter culture Ur (obtained from foundation BOZ, Ede, the Netherlands) is comprised of three species
of lactic acid bacteria (LAB): Lactococcus lactis, Lactococcus cremoris, and Leuconostoc mesenteroides. Many
[20]
single colony isolates of the Ur culture were previously characterized using AFLP-typing and ascribed to
eight genetic lineages . Five genetic lineages (1, 3, 5, 6, and 7) belong to the species L. cremoris, two lineages
[3]
(2 and 4) belong to the species L. lactis (L. lactis ssp. lactis biovar diacetylactis), and one (lineage 8) is
identified as Lc. mesenteroides ssp. cremoris. Genetic lineages of the Ur starter possess traits that are relevant
for their function in converting milk into cheese. For instance, strains of L. cremoris lineages 1, 3 and 5 have
caseinolytic activity linked to the presence of a functional extracellular protease, which cleaves caseins into
peptides (prt ) . Moreover, strains of L. lactis lineages 2, 4 and Lc. mesenteroides lineage 8 are able to
+ [21,22]
[23]
convert citrate (cit ) into acetoin and/or diacetyl , two compounds that have a profound impact on cheese
+
aroma. The main function of the remaining two L. cremoris lineages 6 and 7 is the fast conversion of lactose
causing rapid acidification of the cheese milk .
[3]
When the genomes of representative strains of L. cremoris lineages 1 and 5 and L. lactis lineages 2 and 4
were compared, only a limited number of unique gene sequences was found between the strains in these
two genome pairs. None of these unique genes met the criteria for specific primer design [to be potentially
used to differentiate them by (q)PCR], and thus L. cremoris lineages 1 and 5 (1 & 5) and L. lactis lineages 2
and 4 (2 & 4) were further identified and enumerated together .
[3]