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Page 16 of 19 Klaassens et al. Microbiome Res Rep 2024;3:38 https://dx.doi.org/10.20517/mrr.2024.13

breastfed, exclusively formula fed or mixed feeding of HM and IF) would help to elucidate the ecosystem-
specific dynamics in more detail. In the present study, only exclusively breastfed infant donor samples were
used. Therefore, the donor microbiota was adapted to the presence of different HM oligosaccharides from
the previous feeding history. Therefore, the adaptation of a complex bacterial ecosystem from infants fed
exclusively with IF without prebiotic compounds would be of interest for future studies. In addition, both
infant formulae contained the prebiotic compound GOS to support health-associated bacteria in the infant
donor microbiota, as well as the added probiotic compound. A complex IF with the addition of probiotics -
but without the presence of prebiotics - to the respective formulas would have been of interest to see
possibly greater effects of the respective protein matrix. In the present study, the single or combined
supplementation with infant-age-associated probiotics was tested. When the probiotic strains were used in
combination, a 1:1 ratio was used. Future studies should also test the effects of different ratios or total
numbers of probiotics on metabolic activity to further elucidate putative synergistic effects.

While sequencing-based methods have improved significantly in their ability to detect different members of
the microbial ecosystem, their main limitation is still their blindness to the microbial load in the ecosystem
and their inability to discriminate between viable or metabolically inactive and dead bacteria. Therefore,
methods are needed that enable the enumeration of microbial cells with the ability to reliably discriminate
between viable and dead members of the microbiota. These future approaches, such as the combination of
amplicon sequencing and fluorescence-activated cell sorting, will improve the elucidation of the interplay
between the microbial ecosystem and host health.

Microbial colonization of the neonatal gut is an essential event, as the establishment of a healthy gut
microbiota is pivotal in priming the infant’s immune system and modifying the risk for infections or
allergies in later life. When exclusive breastfeeding is not possible, IF with all its different compounds will
have a major impact on the infant’s microbiome maturation. Therefore, a good understanding of the effects
of different IF compounds on the microbiome and supplemented probiotics is important.

The infant gut microbiome database and the bioinformatics pipeline reliably identified the microbiome of
the samples at the strain level. This enables the assessment of the effects of the different formula matrices
and probiotic supplements on the probiotic abundance and overall bacterial composition. With this
experimental setup, i.e., a combination of ex vivo digestive models, shotgun metagenomics analysis, and a
specific bioinformatics pipeline, the presence of the supplemented Bb and Lf strains was detected.

The present work highlights the crucial finding that the infant microbiota is highly variable and may be
influenced in various ways by different infant formulae and their components, in the event that exclusive
breastfeeding is not possible. Further studies are required to demonstrate the effects of different food
compounds on supplemented probiotics and the infant microbiome at this early and pivotal time point for a
healthy microbiome and immune maturation.

DECLARATIONS
Acknowledgments
We thank the parents for collaboration in the stool sampling. We thank Lucia Köberlein for corrections and
suggestions to the manuscript.

Authors’ contributions
Conceptualization: Schaubeck M, Bongoni R