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Page 6 of 14 Kleerebezem et al. Microbiome Res Rep 2024;3:46 https://dx.doi.org/10.20517/mrr.2024.48

Figure 1. In vitro and in situ competitive fitness assessment of 7 L. plantarum strains. (A) The 7 L. plantarum strains used in this study.
The strains were selected based on their discriminating prebiotic utilization phenotypes for GOS and inulin, combined with the
possibility for high-throughput population dynamics based on strain-specific intergenic alleles that can be assessed using next-
generation amplicon sequencing (for details, see ref [41,42] ); (B) The strain-specific L. plantarum population shifts observed relative to the
7-strain inoculum mixture after 72 generations (generations are here defined as divisions of the overall seven strain population, and
thus does not equal the number of generations of an individual strain in the seven strain mixture) of growth in a laboratory medium that
contained GOS and inulin as a sole carbon source for growth, revealing considerable enrichment of at least some of the prebiotic
utilizing L. plantarum strains by outcompeting non-utilizing strains; (C) The L. plantarum strain-specific population compositions
observed in fecal samples obtained 7 days post-gavage, in comparison to the population composition of the 7-strain gavaged mixture,
in rats that were fed a high-calcium control diet or the same diet supplemented with the prebiotic GOS or inulin. No significant
population composition changes occurred during these 7 days in the intestinal tract, irrespective of the diet fed to the rats. L. plantarum:
Lactiplantibacillus plantarum; GOS: galacto-oligosaccharides.

in vitro growth in a simple microbial consortium of strains of the same species. Notably, L. plantarum 299v
and Heal19 were consistently among the most robust growing strains on various substrates, which may
explain their success as cheaters in the inulin cultures and their apparent competitive advantage over
SD5870 and Lp900 in High-DP GOS nutrient competition [Figure 1].

Selective fitness advantages are further complicated when assessed in a complex microbial ecosystem such
as the gut microbiome, where substantial redundancy for prebiotic utilization capacity can be expected in
the members of the endogenous microbiome. In this context, not only the relative abundance of the
endogenous microbiome members that can express such redundant functions and thereby compete for the
same substrates, but also their substrate affinity and utilization rate relative to the administered