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

Albumin is the most abundant protein in blood, and thus, its presence in faecal samples has been suggested
[23]
to be an indication of increased gut permeability . Albumin increased in association with Gram-negative
organisms, including Haemophilus, but lowered with Collinsella and Bifidobacterium. These results are in
line with previous findings from preterm infants, where increased gut permeability measured by the
lactulose-mannitol test was associated with opportunistic pathogens, such as Staphylococcus and
[43]
enterobacteria, while permeability was low in infants dominated by bifidobacteria . Albumin was
correlated with IAP and BPI, suggesting a shared regulatory pattern or the possibility that increased
permeability stimulates the secretion of antibacterial compounds. However, zonulin, a protein known to
increase tight junction permeability and often used as a marker of gut permeability , did not correlate with
[44]
albumin. In most infants, zonulin levels were below the detection level. Our results suggest that albumin
may be a more sensitive marker of gut permeability in healthy infants than zonulin.

Secretory IgA is the most abundant antibody in the intestine and is considered the first line of defence

against pathogens in the gut [21,45,46] , but is also secreted into breastmilk. IgA is thought to help support
favourable microbiota [21,46] . We found that changes in IgA correlated with changes in IAP, lysozyme, and
albumin. Like albumin, IgA was positively associated with Haemophilus at 6 months and at 12 months by
Phascolarctobacterium. Putative inhibition or degradation of IgA by specific bacterial populations was
observed at 6 months but not at 12 months.

IAP is produced by intestinal epithelial cells in response to bacterial lipopolysaccharide (LPS) produced by
Gram-negative bacteria, and it serves to neutralise LPS and thus limit LPS-induced inflammation [15,47,48] .
Alkaline phosphatase is also produced by bacteria, and it has been estimated that 20%-30% of faecal alkaline
phosphatase activity is derived from bacteria [49,50] . Our IAP measurement may have suffered from neutral
pH, as its activity is optimal at pH 10, but dilution with PBS rather than an alkaline buffer enabled the
simultaneous analysis of multiple biomarkers from the same faecal water sample. In our data, IAP levels
correlated with other markers of inflammation, but IAP increased together with IgA and LTF - molecules
whose role is to limit inflammation by binding and eliminating microbes and antigens. A correlation
[50]
between IAP and IgA has been shown before in mice and humans , suggesting that they may be regulated
by common factors, or as suggested by Lassenius et al., immunoglobulin secretion may be stimulated by
[50]
IAP . Bacteroides appeared as the main putatively stimulatory bacterium of IAP at 6 months, and members
of Proteobacteria and Negativicutes at 12 months. IAP counters the inhibitory effect of ATP on bacterial
growth and is believed to affect the balance of gut microbes, as IAP KO mice are not colonised with
Escherichia coli and have increased Clostridia levels [47,51,52] . Our data confirm similar associations in human
infants. IAP thus emerges as a potential regulator of gut microbiota in human infants, appearing to be
stimulated by Gram-negative bacteria and to inhibit the growth of Gram-positive bacteria.

BPI is a microbicidal protein with endotoxin-neutralising abilities produced by neutrophils . We found
[16]
BPI to be generally stimulated by the overall bacterial load, specifically by Proteobacteria, while its
expression appeared to be attenuated by Lactobacillus at 6 months and by members of Clostridia at 12
months. BPI appeared to inhibit bacterial growth only at 12 months, when it was especially effective against
Bilophila, known for its inflammatory effects . BPI’s generally negative associations with bacterial growth
[53]
fit its function as a microbicidal peptide. Our data indicate that BPI activity may mature after the
introduction of solid foods, as it is not stimulated by high bacterial abundance nor inhibitory against
bacteria at 6 months.