A study uncovered significant differences in the intestinal microbiome of infants with cow's milk-induced food protein–induced enterocolitis syndrome compared with healthy controls.
In the study, published in the Journal of Pediatric Gastroenterology and Nutrition, researchers combined 16S rDNA amplicon and shotgun metagenomic sequencing to examine the intestinal microbiome of patients with and without milk-induced food protein–induced enterocolitis syndrome (CM-FPIES).
They recruited 12 infantile patients with CM-FPIES (mean age = 4 ± 2 months) and 14 age-matched healthy controls (mean age = 5 ± 1 months). All of the patients were born at term (≥ 37 weeks gestation). There were no significant differences between groups in gender distribution, mode of delivery, or type of feeding before diagnosis. Fecal samples were collected and analyzed using various techniques:
Microbiome Analysis:
- 16S rDNA amplicon sequencing was performed on all 26 samples using an Illumina MiSeq System.
- Shotgun metagenomic sequencing was conducted on 6 CM-FPIES samples and 14 control samples using an Illumina NovaSeq system.
The researchers found that patients with CM-FPIES exhibited a depletion of beneficial bifidobacteria and an enrichment of potentially pathogenic enterobacteria, particularly Klebsiella and Escherichia coli. The study reported higher fecal excretion of fatty acids in those with CM-FPIES. Additionally, the researchers observed reduced levels of certain fecal immune factors in these infants.
Among the key findings were:
- No statistically significant differences in α-diversity (Shannon and Simpson indexes) between groups
- Statistically significant differences in β-diversity (P < .05) using Bray-Curtis dissimilarity method and PERMANOVA
- Lower abundance of Actinomycetota (P = .05) and Bifidobacteriaceae in patients with CM-FPIES
- Higher abundance of Pseudomonadota (P < .05), Enterobacteriaceae (P < .05), Klebsiella, and Escherichia (P < .05) in patients with CM-FPIES
- Gene count from shotgun sequencing: control group = 17,829 ± 5,775, patients with FPIES = 17,086 ± 2,547
- Functional analysis revealed 709 gene families and 21 metabolic pathways associated with Enterobacteriaceae in patients with CM-FPIES
Specific microbial gene families and metabolic pathways:
- Higher abundances in control group: Bifidobacterium bifidum = 2,447 gene families, 48 metabolic pathways; Bifidobacterium longum = 255 gene families; Eggerthella lenta = 7 metabolic pathways
- Higher abundances in patients with FPIES: Escherichia coli = 689 gene families, 11 metabolic pathways; Klebsiella pneumoniae = 20 gene families, 10 metabolic pathways.
Carbohydrate degradation pathways:
- D-fructuronate and D-galactarate pathways: Predominant in patients with FPIES
- Sucrose degradation pathways: More abundant in controls.
Gas chromatography was used to measure fecal fatty acid concentrations.
Among the results were:
- Significantly higher total fatty acid excretion in patients with CM-FPIES (P < .001)
- Acetic acid levels were 10 times higher in patients with CM-FPIES compared with controls: Controls = ~20 μg/g (median), patients with FPIES = ~200 μg/g (median)
- Propionic acid: Controls = ~5 μg/g (median), patients with FPIES = ~20 μg/g (median)
- Butyric acid: Controls = ~5 μg/g (median), patients with FPIES = ~10 μg/g (median)
- Significantly higher BCFA levels in patients with CM-FPIES (P = .04): Controls = ~1 μg/g (median), patients with FPIES = ~3 μg/g (median).
Immunological Markers:
-
- Fecal calprotectin (FC) was measured using ELISA.
- 27 immune factors were analyzed using a Bio-Plex Pro Human Cytokine 27-plex Assay Kit.
The results showed:
- Higher FC levels in patients with CM-FPIES (292.88 μg/g) compared with controls (92.83 μg/g), although not statistically significant (P = .06)
- Significantly lower levels of interleukin (IL)-1ra, IP-10, and PDGF-bb in patients with CM-FPIES (P = .01): IL-1ra = Controls 2275.62 pg/mL, patients with FPIES 698.05 pg/mL (median); IP-10 = Controls 140.39 pg/mL, patients with FPIES 18.82 pg/mL (median); PDGF-bb = Controls 32.60 pg/mL, patients with FPIES 7.30 pg/mL (median).
Correlation Analysis:
-
- Positive correlations were found between Bifidobacteria abundance and levels of IL-1ra, IP-10, and PDGF-bb (Pearson correlation coefficient ~0.5).
- Negative correlations were observed between these immune factors and E coli abundance.
- Strong positive correlations were found between Enterobacteriaceae and Klebsiella abundance and propionic acid levels (Pearson correlation coefficient ~0.8).
- Negative correlations between Actinomycetota abundance and propionic and butyric acids.
- No significant associations were found for acetic acid levels.
The researchers hypothesized that the increased fecal fatty acid levels in patients with CM-FPIES may have been caused by reduced absorption and altered colonocyte metabolism, potentially linked to the observed shift in the obligate to facultative anaerobic ratio.
They acknowledged the study's limited sample size as a potential constraint as well as emphasized the need for larger cohort studies to confirm these findings and further investigate the role of diet in shaping the gut microbiome of patients with CM-FPIES.
This study provided new data on the gut microbiome alterations and associated metabolic and immune changes in infants with CM-FPIES. The findings suggested potential avenues for future research into diagnostic biomarkers and therapeutic interventions for this condition.
Two authors are Scientific Founder and Member of the Scientific Advisory Board of MicroViable Therapeutics S.L. The remaining authors declared no conflict of interest.