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van Beek et al. Microbiome Res Rep 2025;4:13 https://dx.doi.org/10.20517/mrr.2024.45 Page 15 of 19

low pH, as well as with increasing abundances of small intestine organisms (Veillonellaceae and
[75]
[75]
Streptococcacae ) and decreasing abundances of colon-dwelling organisms (Roseburia ), we suggest that
LCN2 may be a potential indicator of fast gut transit in infants.
ECP is an eosinophil granulocyte-secreted protein that mediates the inflammatory response of the host to
microbes and parasites and is elevated in the serum of individuals with atopic diseases such as allergic
[18]
[76]
rhinitis and asthma . Levels of ECP also correlate with the disease severity of ulcerative colitis . It is
cytotoxic by inducing apoptosis yet is also involved in immune modulation and tissue repair . ECP has
[18]
[18]
antibacterial effects . In our data, it correlated with IAP and HD5. ECP appeared to be stimulated by
Streptococcaceae and Clostridioides, while Collinsella seemed to reduce its expression at 6 months.
Associations at 12 months were weaker, but Enterococcus appeared to stimulate it and some members of
Clostridia and Haemophilus to inhibit it. ECP seemed to inhibit Akkermansia growth at 12 months.
Lysozyme is an antimicrobial peptide that cleaves peptidoglycan, the major component of Gram-positive

[77]
bacterial cell walls . It is an important component of human breastmilk, yet it has seemingly conflicting
properties: both increased and decreased levels have offered protection against colitis in previous
studies [78-80] . The effect of lysozyme levels is, therefore, likely to depend on the microbiota. Changes in
lysozyme levels were associated with bacterial population growth. At 6 months, bacterial growth appeared
to inhibit lysozyme, while at 12 months, we observed both stimulatory and inhibitory associations.
Lysozyme has been linked with increased IgA in piglets , which we confirmed. While lysozyme has been
[81]
associated with increasing lactobacilli in piglets, we did not find lysozyme to be a strong regulator of the
microbiota in human infants [79,81,82] .

Zonulin is a protein that reversibly increases the permeability of tight junctions and serves as a biomarker
[83]
for intestinal barrier integrity . Elevated zonulin levels have been associated with many intestinal diseases
where barrier dysfunction is involved, such as celiac disease, IBD, and necrotising enterocolitis . Its
[84]
correlation with gut microbiota has been studied in a variety of different settings, and it is elevated in
combination with higher levels of gram-negative strains or opportunistic pathogens, such as Clostridium,
while a higher prevalence of gram-positive bacteria is linked to lower zonulin levels . In our data, zonulin
[84]
appeared to be stimulated by several Gram-positive genera at 12 months, but not at 6 months. On the other
hand, zonulin appeared to stimulate the growth of Flavonifractor, Bilophila, and Blautia, suggesting that
these bacteria may benefit from increased permeability or inflammation.

Limitations
This research has analysed 216 samples so far. According to power calculation, with a sample size of 216, we
could detect a correlation coefficient of 0.19 or larger at the 0.05 P-value cut-off and with a 0.2 type II error
rate. The sample size is thus sufficient to detect modest associations. However, these samples are derived
from 8 faecal time series of infants from the same area in Finland, meaning that the correlations found here
might not be representative of infants in general and would need to be replicated in an independent cohort
to test reproducibility and generalisability of the found associations. Most (3 out of 4) 11-12-month-olds
were still breastfed, and we, therefore, do not have sufficient representative data for infants who solely
consume solid food or formula milk. We did not control diet, and thus, infants present with a natural
variation in microbiome based on variations in their diet in addition to any inherent variation between
infants. Future research will expand the variety of infants sampled, enabling a more robust correlation of
biomarkers, microbiome, and gut health. While we were able to observe consistent associations between
microbial population growth and biomarkers’ abundances and changes, these do not enable causal
inference.