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Page 14 of 19 van Beek et al. Microbiome Res Rep 2025;4:13 https://dx.doi.org/10.20517/mrr.2024.45

Markers of inflammation
Cal is a calcium-, zinc-, and manganese-binding protein secreted by neutrophils. It competes with bacteria
[19]
(and fungi) for metals, thereby inhibiting their growth . So far, it has been shown to inhibit the growth of
diverse pathogens, including S. aureus, Acinetobacter baumannii, Helicobacter pylori, Salmonella enterica,
Staphylococcus epidermidis, Staphylococcus lugdunensis, Enterococcus faecalis, Pseudomonas aeruginosa, and
[54]
[55]
Shigella flexneri . It is also proposed to inhibit bacterial binding of L. monocytogenes and S. enterica . Its
abundance in faeces is a marker of inflammation and IBD [56,57] . Most literature agrees that Cal levels in
healthy infants are higher than in adults due to many factors, including fluctuation of microbiota . In fact,
[58]
lower Cal levels are associated with dysregulated microbiota and feeding intolerance in infants . The levels
[57]
tend to drop with age, stabilising at adult levels around a year of age . We found higher Cal levels in C-
[59]
section-born infants compared to vaginally born infants [Table 1]. Different studies have found higher Cal
levels either in C-section-born infants or vaginally born babies [58,61] . The differences may be due to
[60]
differences in gut microbiota compositions, since we found faecal Cal to respond to microbial populations.

At 6 months, Lachnospiraceae and Akkermansia appeared to inhibit the secretion of Cal, while Akkermansia
growth seemed to be inhibited by high Cal levels. Bifidobacteria were also negatively associated with Cal
levels (albeit not significantly, therefore not shown), which is clinically relevant as supplementation with
bifidobacteria has been proposed as a Cal-lowering intervention in babies . At 12 months,
[62]
Pseudoruminococcus and Prevotella appeared to stimulate Cal, and Cal seemed to have only positive effects
on bacterial growth - most strongly on Pseudoruminococcus and Lactobacillus.

LTF is an iron-sequestering protein with antibacterial and antiviral activity that is found in breastmilk and is
secreted by neutrophils in the gut in response to inflammation [20,63] . Because we found LTF to correlate with
Cal, most of the LTF in our samples is likely gut-derived rather than originating in breastmilk. LTF has been
proposed to reduce inflammation, neutralise endotoxins, and, most importantly, aid in commensal
colonisation [63-65] . However, most of these studies are done in vitro or look at immune biomarkers rather
than bacteria. Apart from Haemophilus at 6 months and Varibaculum at 12 months, we did not observe
significant effects of LTF on gut microbes. This has also been reported after administering oral LTF to
toddlers .
[66]

HD5 is an antimicrobial peptide produced by Paneth cells, known to selectively kill pathogens and preserve
[17]
commensals . It is increased in the inflamed colon of children with IBD and associated with atopic
dermatitis [67,68] . We found that it correlates with ECP, suggestive of a common regulatory system, although
[18]
ECP is produced by eosinophils . Bacteria with known anti-inflammatory properties, such as
Bifidobacterium (6 months) and Roseburia and Prevotella (12 months), appeared to reduce its expression,
while Streptococcus, Megasphaera, and Clostridioides (6 months) appeared to stimulate it. Lactate has been
shown to inhibit HD5 , but we did not find associations between lactic acid bacteria and HD5. At 6
[69]
months, high levels of HD5 appeared to have a negative impact on Akkermansia. The association was not
significant at 12 months, when high HD5 appeared to reduce the growth of Haemophilus, which contains
potential pathogens [70-72] . In addition, at 12 months, HD5 appeared to stimulate the growth of several
commensal bacteria, including Lactobacillus.

LCN2 is an iron-sequestering protein secreted by many cell types in the gut, including neutrophils, that
[73]
inhibits bacterial growth by competing for iron and also has inflammatory effects . LCN2 behaved
generally differently from the other neutrophil-secreted markers, changing inversely with IAP. Previously,
[74]
LCN2 has been shown to correlate with Cal in adult IBD patients , which we did not observe in this
sample of healthy infants. Due to the association of LCN2 with low protein and bacteria concentrations and