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A protocol for characterization of extremely preterm infant gut microbiota in double-blind clinical trials.

ABSTRACT: 16S rRNA gene sequencing enables microbial community profiling, but recovering fecal DNA from extremely premature infants is challenging. Here, we describe an optimized protocol for fecal DNA isolation, library preparation for 16S rRNA gene sequencing, taxonomy assignation, and statistical analyses. The protocol is complemented with a quantitative PCR for probiotic L. reuteri identification. This protocol describes how to characterize preterm infant gut microbiota and relate it to probiotic supplementation and clinical outcomes. It is customizable for other clinical trials. For complete details on the use and execution of this protocol, please refer to Martí et al. (2021) and Spreckels et al. (2021).

SUBMITTER: Marti M

PROVIDER: S-EPMC8283139 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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A protocol for characterization of extremely preterm infant gut microbiota in double-blind clinical trials.

Martí Magalí M Spreckels Johanne E JE Jenmalm Maria C MC Abrahamsson Thomas T

STAR protocols 20210709 3

16S rRNA gene sequencing enables microbial community profiling, but recovering fecal DNA from extremely premature infants is challenging. Here, we describe an optimized protocol for fecal DNA isolation, library preparation for 16S rRNA gene sequencing, taxonomy assignation, and statistical analyses. The protocol is complemented with a quantitative PCR for probiotic <i>L. reuteri</i> identification. This protocol describes how to characterize preterm infant gut microbiota and relate it to probiot ...[more]

PMID: 34308378

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Project description:BackgroundEstablishment of the human gut microbiota begins at birth. This early-life microbiota development can impact host physiology during infancy and even across an entire life span. However, the functional stability and population structure of the gut microbiota during initial colonization remain poorly understood. Metaproteomics is an emerging technology for the large-scale characterization of metabolic functions in complex microbial communities (gut microbiota).ResultsWe applied a metagenome-informed metaproteomic approach to study the temporal and inter-individual differences of metabolic functions during microbial colonization of preterm human infants' gut. By analyzing 30 individual fecal samples, we identified up to 12,568 protein groups for each of four infants, including both human and microbial proteins. With genome-resolved matched metagenomics, proteins were confidently identified at the species/strain level. The maximum percentage of the proteome detected for the abundant organisms was ~45%. A time-dependent increase in the relative abundance of microbial versus human proteins suggested increasing microbial colonization during the first few weeks of early life. We observed remarkable variations and temporal shifts in the relative protein abundances of each organism in these preterm gut communities. Given the dissimilarity of the communities, only 81 microbial EggNOG orthologous groups and 57 human proteins were observed across all samples. These conserved microbial proteins were involved in carbohydrate, energy, amino acid and nucleotide metabolism while conserved human proteins were related to immune response and mucosal maturation. We identified seven proteome clusters for the communities and showed infant gut proteome profiles were unstable across time and not individual-specific. Applying a gut-specific metabolic module (GMM) analysis, we found that gut communities varied primarily in the contribution of nutrient (carbohydrates, lipids, and amino acids) utilization and short-chain fatty acid production.ConclusionsOverall, this study reports species-specific proteome profiles and metabolic functions of human gut microbiota during early colonization. In particular, our work contributes to reveal microbiota-associated shifts and variations in the metabolism of three major nutrient sources and short-chain fatty acid during colonization of preterm infant gut.

Project description:Microbiota in early life is closely associated with the health of infants, especially premature ones. Probiotics are important drivers of gut microbiota development in preterm infants; however, there is no consensus regarding the characteristics of specific microbiota in preterm infants receiving probiotics. In this study, we performed a meta-analysis of 5 microbiome data sets (1816 stool samples from 706 preterm infants) to compare the gut microbiota of preterm infants exposed to probiotics with that of preterm infants not exposed to probiotics across populations. Despite study-specific variations, we found consistent differences in gut microbial composition and predicted functional pathways between the control and probiotic groups across different cohorts of preterm infants. The enrichment of Acinetobacter, Bifidobacterium, and Lactobacillus spp and the depletion of the potentially pathogenic bacteria Finegoldia, Veillonella, and Klebsiella spp. were the most consistent changes in the gut microbiota of preterm infants supplemented with probiotics. Probiotics drove microbiome transition into multiple preterm gut community types, and notably, preterm gut community type 3 had the highest α-diversity, with enrichment of Bifidobacterium and Bacteroides spp. At the functional level, the major predicted microbial pathways involved in peptidoglycan biosynthesis consistently increased in preterm infants supplemented with probiotics; in contrast, the crucial pathways associated with heme biosynthesis consistently decreased. Interestingly, Bifidobacterium sp. rather than Lactobacillus sp. gradually became dominant in gut microbiota of preterm infants using mixed probiotics, although both probiotic strains were administered at the same dosage. Taken together, our meta-analysis suggests that probiotics contribute to reshaping the microbial ecosystem of preterm infants at both the taxonomic and functional levels of the bacterial community. More standardized and relevant studies may contribute to better understanding the crosstalk among probiotics, the gut microbiota, and subsequent disease risk, which could help to give timely nutritional feeding guidance to preterm infants. This systematic review and meta-analysis was registered at PROSPERO (https://www.crd.york.ac.uk/PROSPERO/) as CRD42023447901.

Dataset Information

A protocol for characterization of extremely preterm infant gut microbiota in double-blind clinical trials.

Publications

A protocol for characterization of extremely preterm infant gut microbiota in double-blind clinical trials.

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