Gut microbiota development in very preterm infants following fortification of human milk

Background Very preterm infants (VPIs) are born with an immature gut, being sensitive to gut microbiota dysbiosis-related disease like necrotizing enterocolitis. While human milk is the best source of nutrition for VPIs, it requires fortification to meet their nutrient requirements for optimal growth. However, the optimal type of fortifier remains uncertain. Bovine colostrum (BC), rich in protein and bioactive components, may be an alternative to conventional fortifiers (CF). We aimed to investigate the distinct impacts of different bovine fortifiers, BC and CF, on the gut microbiota of VPIs. The gut microbiota of 225 VPIs who were fed human milk fortified with either BC or CF, were profiled by 16S rRNA gene amplicon sequencing of fecal samples collected before, one and two weeks of fortification. Results Fortifier type affected the microbial community structure to a modest extent, but only explaining 1% of the variance, and no specific taxa differed between the BC and CF groups. This fortifier-derived impact was predominantly observed in VPIs born via caesarean section. Birth mode exhibited transient effects on microbial community structure shortly after birth, with caesarean section-born VPIs dominated by Firmicutes , while vaginally-born VPIs were dominated by Proteobacteria . This birth mode-derived difference diminished with age and disappeared around one month after birth. The fecal pH, increased by BC, was positively correlated with Staphylococcus and Corynebacterium , and negatively with Bifidobacterium abundance. The change in relative abundance of Staphylococcus was negatively correlated with weight gain. Conclusion Collectively, fortification of human milk with BC or CF does influence the gut microbiota of VPIs but only to a modest extent during early life. Conversely, birth mode appears to be a significant temporary factor influencing the gut microbiota during this period. Our findings are consistent with existing literature and support the idea that the choice of fortifier has limited effects on gut microbiota development in the first month of life of VPIs. ### Competing Interest Statement The University of Copenhagen holds a patent on the use of colostrum for human infants (PCT/DK2013/050184). Biofiber Damino A/S is given options to license this patent. P.T.S. is listed as a sole inventor but has declined any share of potential revenue arising from commercial exploitation of such a patent. P.T.S. did not take part in any clinical work in the units involved in this study. Other authors declare no competing interests. ### Clinical Trial NCT03537365 ### Funding Statement This study was funded by the Innovation Fund Denmark (NEOCOL 6150-00004B) in collaboration with Biofiber-Damino, Denmark. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Scientific Ethical Committee of the Region of Southern Denmark (S-20170095) and the Danish Data Protection Agency (17/33672) gave ethical approval for this work. An independent data safety monitoring board (DSMB) followed and reviewed trial data and safety. Parental consent was obtained in writing from both parents of participated infants prior to intervention. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The 16S sequencing data produced in the present study is available in the Sequence Read Archive at NCBI with the accession number PRJNA1090537 in an anonymized form. Clinical data containing personally identifiable information of individual infants participating in the cohort cannot be freely available to protect the privacy of the participants and their families. This is in accordance with the Danish Data Protection Act and European Regulation 2016/679 of the European Parliament and of the Council (GDPR), which prohibit distribution even in pseudo-anonymized form.