P3.035: Breast Milk Xanthine Oxidase is Associated with Composition of the Gut Microbiome in Preterm Infants

Background: Human milk (HM) is the best source of nutrition for all infants, including preterm infants, due to its nutritional and immunological properties. Human milk also contains substantial concentrations of bioactive enzymes with multifaceted roles in iron regulation and innate immunity, including the enzyme xanthine oxidase (XO). XO largely functions as a potent antimicrobial via generation of reactive oxygen species (ROS, e.g., hydrogen peroxide. Generation of ROS by XO also plays a role in release of heme bound iron along the intestinal tract and promotes incorporation of iron into apotransferrin/apoferritin at optimum at the pH of the intestinal mucosa, thus facilitating absorption of iron. Intriguingly, conditional knockout of XO in the murine mammary gland leads to reduced growth rates of nursing pups. While XO concentrations have been described for term breast milk (BM), the presence/concentration of XO in preterm BM and relationships with the nascent preterm infant gut microbiota are uncharacterized. Using a prospective longitudinal study, we aimed to characterize the concentration of XO in BM fed to preterm infants and assess the relationships with the gut microbiome in very low birth weight (VLBW, < 1500 g) preterm infants.

Methods: BM (n=192) and VLBW preterm infant stool samples (n=376) were collected from 84 mother-infant dyads. BM samples were aseptically collected prior to infant feedings. The concentration of BM XO was measured in duplicate via fluorometric assay. DNA was extracted from stool samples using the Qiagen PowerSoil Pro kit and the V4 hypervariable region of the bacterial 16S rRNA gene was amplified and sequenced on the Illumina MiSeq platform. For comparisons of XO concentration in BM and infant gut microbiome data, stool samples were paired to milk samples fed the same day or within 7 days prior to stool collection. Bivariate and multivariate relationships between XO concentrations and the gut microbiome were assessed and statistical significance was declared at p< 0.05.

Results: The average (SD) concentration of XO in BM was 56.936.1 U/L. The preterm infant gut microbiome was dominated by Klebsiella (23.728.9% relative abundance), Escherichia-Shigella (18.227.8%), Enterococcus (10.215.7%), and with lower abundance of Bifidobacterium (4.210.1%). We identified several associations in the concentration of XO and the relative abundance of bacterial genera, including positive associations with Klebsiella and Enterococcus and a negative association with Escherichia-Shigella. There was no relationship with Bifidobacterium (Figure 1A). Permutational multivariate analysis of variance (PERMANOVA) demonstrated a strong positive relationship between XO concentrations and the community structure of the preterm infant gut microbiome (Figure 1B).

Conclusion: The concentration of XO in BM fed to VLBW preterm infants was associated with the relative abundance of specific bacterial taxa and gut microbiome community composition. This highlights the need for further studies on the role of XO in shaping the gut microbiota.