Human Milk and Dietary Supplements Differentially Modulate Microbial Communities of Stool Isolated from Term and Preterm Infants

Abstract

Background: After birth, the term neonatal gut becomes colonized by commensal bacteria that benefit host health. Premature infants, however, are prone to abnormal colonization that increases the risk for neonatal intestinal disease such as necrotizing enterocolitis (NEC). Breastfeeding confers a lower risk for NEC, and this is likely due to prebiotics, probiotics, and anti-inflammatory cytokines present in breast milk. Despite the known associations of NEC with gut microbiota and breastfeeding, the exact interactions between preterm gut microbes and various milk types remains relatively unexplored. Furthermore, Lactobacillus are frequently included in probiotic supplements, but the interplay between Lactobacillus, milk, and NEC is not fully understood. Hypothesis: We hypothesize that in in vitro models of preterm gut microbiota, human breast milk exerts an overall beneficial modulatory effect on bacterial composition. We also suspect that the anti-inflammatory properties of Lactobacillus are enhanced when treated with fresh breast milk compared to formula. Methods: Stool samples from preterm infants were incubated anaerobically in mini-bioreactors overnight at 37°C to establish complex communities. The next day, they were supplemented with fresh milk with or without fortifier, donor milk with or without fortifier, formula, vitamin mix, iron mix, or water and incubated for another 24 hrs. Bacterial gDNA was examined by qPCR and back-calculated to colony-forming units (CFUs) to quantify bacterial composition. To examine the effect of milk on Lactobacillus metabolites, we monocultured six Lactobacillus ( L. acidophilus, L. breve, L. johnsonii, L. paracasei, L. reuteri, and L. rhamnosus) in a defined medium supplemented with fresh milk, formula, or water. After overnight incubation, supernatants were collected for nontargeted metabolomics. The supernatant was also applied to preterm intestinal organoids to examine epithelial responses. Results: Formula significantly elevated Bifidobacterium and Streptococcus, while fresh and donor milk caused upward trends in Lactobacillus and Bifidobacterium. LC-MS/MS data revealed that in the presence of fresh breast milk but not formula, at least one Lactobacillus strain demonstrated increased levels of anti-inflammatory metabolites such as γ-glutamylphenylalanine, ascorbic acid, vaccenic acid, hydroxyphenylacetic acid, or indoleacrylic acid. Conclusions: These data indicate that different milk types and supplements variably affect bacterial growth and metabolites in the preterm gut. This study was supported in part by the NIH K01DK123195 (MAE), NATS NIH KL2TR001452 (KEC) and UL1TR001450 (KEC) grants. This study does not represent the offcial view of the NIH. This study was also supported in part by the David and Laura Stone Endowment for Advancement in Neonatal Medicine (KEC). The Texas Children’s Hospital Department of Pathology provides salary support to the Texas Children’s Microbiome Center-Metabolomics and Proteomics Mass Spectrometry Laboratory staff, and purchased all of the reagents, the consumables and durable supplies, and the LC-MS/MS equipment described herein (TH). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.