The immature gut: Microbes and nutrition orchestrate maturation of the preterm gastrointestinal tract

Preterm birth interrupts the natural, intrauterine growth path of infants that occurs during the third trimester. As a result of preterm birth, there is a discrepancy between the maturation status of the gastrointestinal tract and the process of microbial colonization in early life. Yet, the concordant maturation of the gastrointestinal tract and the microbiome is pivotal for growth and health of the preterm infant. Neonatal support in the early life of preterm infants offers the opportunity to orchestrate the maturation of the immature gastrointestinal tract and the colonizing microbes. Despite continuous improvements in preterm infant care, optimal feeding is challenging due to the infant’s immaturity.

The intestinal bacteria and fungi are pivotal in health and disease. In preterm infants, variation in the gut microbiota (in this thesis the bacterial community) and mycobiota (the fungal community of the microbiota) is introduced due to a unique set of environmental conditions. Within this thesis, we hypothesized that prematurity of the gut microbiota may be an inconspicuous challenge in nutritional neonatal care. Moreover, we expected the intestinal fungi to be affected by clinical variables in early life. A better understanding of the development of the gastrointestinal tract and the microbes in preterm infants is key for optimal nutritional support. The work in this thesis therefore studied the maturation of the gastrointestinal tract and of the intestinal microbes in preterm infants with their implications for infant growth, development and health.

The infants studied in this thesis participated previously in a single-center, observational study in which they were admitted to the neonatal intensive care unit or the pediatric ward of the Isala Women and Children’s Hospital (Zwolle, The Netherlands). The preterm and full-term infants were born between 24 and 42 weeks of gestation and were followed in the first six postnatal weeks, during which clinical variables and feces were collected weekly. Gastric aspirates were additionally collected daily in preterm infants during the first two postnatal weeks. Within this thesis, infants were selected from this cohort based on gestational age and antibiotic use of the mother and the infant. The infant’s gastrointestinal and microbial functionality was assessed by enzyme activity analyses of host proteins and by metaproteomics with LC-MS/MS analyses. The mycobiota’s composition was characterized by sequencing the internal transcribed spacer region 2 (ITS2).

Functional analyses on the host and the microbiota were performed to improve the understanding of gastrointestinal maturation during the early life of preterm infants. We identified host and microbial marker proteins for digestion and barrier defense, which were indicative for gastrointestinal maturation in the first six postnatal weeks. The combination of enzyme activity analyses and metaproteomics showed that preterm infants were capable to digest human milk, albeit to a lesser extent than full-term infants. Moreover, gastrointestinal barrier proteins were compromised in preterm infants compared to full-term infants in the first six postnatal weeks. The maturation status of the infant was additionally found to have implications on the microbiome. Despite the immature status, human milk offers a protective function as shown by multiple bioactive proteins detected in the preterm gastrointestinal tract.

Within this thesis we also present a clinical study design for the “From Mum to Bum” study,  in which we aim to investigate the effect of preterm birth on the microbiota’s functionality and its relation to anthropometric outcomes. This pilot study will be an observational, single-center study performed at the neonatal intensive care unit at Isala Women and Children’s Hospital. A cohort of preterm and full-term mother–infant pairs will be followed during the first six postnatal weeks with follow-up at three- and six-months postnatal age. Compositional and functional methods will be combined to analyze multiple samples along the length of the gastrointestinal tract. As such, we follow digestion of human milk from the breast of the mother throughout the gastrointestinal tract of the infant, or “From Mum to Bum”.

Besides the preterm intestinal bacteria, the work in this thesis also describes preterm intestinal fungi. Like bacteria, the colonization and development of the mycobiota in the preterm intestine was hypothesized to be affected by clinical variables. We detected fungi and other eukaryotic kingdoms in the intestinal tract of preterm and full-term infants in the first six postnatal weeks. The gut mycobiota composition and development was influenced by gestational and postnatal age patterns, individuality and mode of delivery. Our data support the hypothesis of vertical transmission of fungi and underpin the role of the mode of delivery in the development of the mycobiota in preterm infants.

The research described within this thesis contributes to current knowledge of the preterm gastrointestinal maturation and its intestinal microbes during the early life of preterm infants, as well as the clinical influences on their development. The intestinal barrier proves to be an important environment where microbes interact with the intestinal epithelium and the immune system to drive growth, development and health of the preterm infant. In light of its clinical relevance, future research should consider the functionality of the preterm microbiota in human milk digestion coupled to anthropometric outcomes as well as the interkingdom interactions in the (preterm) infant intestine. Based on the research described in this thesis, the microbiome and nutrition hold promising applications for preterm infant care that help to orchestrate maturation of the gastrointestinal tract. Microbiota modulation offers hope for future improvements in preterm infant care that pave the way for systemic and lifelong effects. Before nutritional therapies targeting the microbiome can be implemented in preterm infant care, the mechanisms by which microbes are involved in preterm infant health need to be thoroughly assessed. As such, the preterm infant gut microbiome remains a research priority.