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Page 8 of 19 van Beek et al. Microbiome Res Rep 2025;4:13 https://dx.doi.org/10.20517/mrr.2024.45

was higher in the caesarean-born infants (P = 0.02, linear mixed model, Table 1), and Bristol score, which
was higher at 6 months (P < 0.001).

Associations between faecal biomarkers
As a first exploratory step, we assessed the raw correlations between faecal biomarkers normalised within
each time series without adjusting for repeated sampling. At both 6 and 12 months, total bacteria load was
positively associated with pH and total protein [Figure 1A and B]. The concentrations of most measured
biomarkers - IAP, LTF, Cal, Muc2, HD5, lysozyme, and ECP - were positively correlated with each other
and with total protein and total bacterial load [Figure 1A]. BPI was positively correlated with albumin and
negatively correlated with lysozyme and Muc2 [Figure 1A]. At 12 months, total protein was still
significantly correlated with IAP, LTF, HD5, IgA, and ECP and with Cal, but not with Cal [Figure 1B]. Cal
correlated only with zonulin and vice versa [Figure 1B]. Total bacteria were not significantly associated with
any biomarker at 12 months [Figure 1B].

At both time points, LCN2 concentrations were inversely correlated with the protein and bacteria content of
the stool and with pH, but only significantly at 6 months [Figure 1A and B]. Muc2 was negatively associated
with pH at 6 months and with total bacteria at both time points [Figure 1A and B].

To obtain a more detailed understanding of the interrelated dynamics between the biomarkers, we analysed
the associations between their daily changes [Figure 1A and B upper right triangle]. Increases in total
bacterial load were associated with increasing pH and total protein content at both time points. Changes in
HD5 were positively correlated with changes in ECP, BPI, and zonulin. So too were changes in albumin and
IgA, and LTF, IAP, and Cal [Figure 1A and B].

Changes in LCN2 were negatively correlated with changes in IAP, total protein, and pH at 6 months. At 6
months, muc2 decreased when total bacteria or pH increased [Figure 1A]. These associations were still
present but not significant at 12 months [Figure 1B].

Because bifidobacteria have been previously associated negatively with faecal pH in infants, we tested these
associations specifically, and discovered a significant negative association at 6 months (P ≤ 0.001), but a
positive one at 12 months (P ≤ 0.001).

Associations between bacterial population growth and faecal biomarker changes
General association patterns
We attempted to identify bacterial stimulation of immune biomarkers by predicting the daily change in
biomarker concentrations with the daily changes in microbial absolute abundances. We identified both
negative and positive associations between bacterial population growth and biomarker changes, potentially
indicative of bacterial stimulation or inhibition of biomarker expression [Figure 2]. Overall, Collinsella
emerged as the most consistent and the strongest potential inhibitor of many immune-related biomarkers,
especially ECP. Bifidobacterium and Akkermansia stood out due to their weak and exclusively negative
associations with the biomarkers [Figure 2]. Both had a negative association with HD5 and Cal at 6 months
(although not significant for Bifidobacterium), and Bifidobacterium with albumin at 12 months.

Indicators of gut homeostatic regulation
Levels of muc2 at 6 months increased with an increase in Haemophilus, Veillonella, Lachnospira, and
Dorea, and decreased with increasing levels of Sellimonas and Collinsella [Figure 2A]. At 12 months, muc2
decreased with an increase in many bacterial genera, including members of Clostridia, but was positively