Division of General Academic Pediatrics, Department of Pediatrics, Nemours Children's Health, Wilmington, DE, United States and Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA, United States.
Biomedical Research Department, Nemours Children's Health, Wilmington, DE, United States.
Front Cell Infect Microbiol. 2022 Mar 4;12:816601. doi: 10.3389/fcimb.2022.816601. eCollection 2022.
Different feeding regimens in infancy alter the gastrointestinal (gut) microbial environment. The fecal microbiota in turn influences gastrointestinal homeostasis including metabolism, immune function, and extra-/intra-intestinal signaling. Advances in next generation sequencing (NGS) have enhanced our ability to study the gut microbiome of breast-fed (BF) and formula-fed (FF) infants with a data-driven hypothesis approach.
Next generation sequencing libraries were constructed from fecal samples of BF (n=24) and FF (n=10) infants and sequenced on an Illumina HiSeq 2500. Taxonomic classification of the NGS data was performed using the Sunbeam/Kraken pipeline and a functional analysis at the gene level was performed using publicly available algorithms, including BLAST, and custom scripts. Differentially represented genera, genes, and NCBI Clusters of Orthologous Genes (COG) were determined between cohorts using count data and R (statistical packages edgeR and DESeq2).
Thirty-nine genera were found to be differentially represented between the BF and FF cohorts (FDR ≤ 0.01) including , , , , and . A Welch t-test of the Shannon diversity index for BF and FF samples approached significance (=0.061). Bray-Curtis and Jaccard distance analyses demonstrated clustering and overlap in each analysis. Sixty COGs were significantly overrepresented and those most significantly represented in BF vs. FF samples showed dichotomy of categories representing gene functions. Over 1,700 genes were found to be differentially represented (abundance) between the BF and FF cohorts.
Fecal samples analyzed from BF and FF infants demonstrated differences in microbiota genera. The BF cohort includes greater presence of beneficial genus Several genes were identified as present at different abundances between cohorts indicating differences in functional pathways such as cellular defense mechanisms and carbohydrate metabolism influenced by feeding. Confirmation of gene level NGS data PCR and electrophoresis analysis revealed distinct differences in gene abundances associated with important biologic pathways.
婴儿期不同的喂养方式会改变胃肠道（肠道）微生物环境。肠道微生物反过来会影响胃肠道的稳态，包括代谢、免疫功能和肠内外信号传递。下一代测序（NGS）的进步增强了我们使用数据驱动假设方法研究母乳喂养（BF）和配方奶喂养（FF）婴儿肠道微生物组的能力。
从 24 名 BF 和 10 名 FF 婴儿的粪便样本中构建下一代测序文库，并在 Illumina HiSeq 2500 上进行测序。使用 Sunbeam/Kraken 管道对 NGS 数据进行分类，并使用包括 BLAST 和自定义脚本在内的公共可用算法在基因水平上进行功能分析。使用计数数据和 R（统计软件包 edgeR 和 DESeq2）在队列之间确定差异表达的属、基因和 NCBI 直系同源基因（COG）。
在 BF 和 FF 队列之间发现 39 个属存在差异表达（FDR ≤ 0.01），包括 、 、 、 和 。BF 和 FF 样本的 Shannon 多样性指数的 Welch t 检验接近显著（=0.061）。Bray-Curtis 和 Jaccard 距离分析表明，在每种分析中都存在聚类和重叠。60 个 COG 显著过表达，在 BF 与 FF 样本中最显著的代表基因功能的类别存在二分法。在 BF 和 FF 队列之间发现 1700 多个基因存在差异表达（丰度）。
从 BF 和 FF 婴儿的粪便样本中分析表明，微生物群属存在差异。BF 队列中存在更多有益属 的存在。一些基因的丰度在队列之间存在差异，表明喂养方式影响的功能途径存在差异，如细胞防御机制和碳水化合物代谢。基因水平 NGS 数据的确认——PCR 和电泳分析显示，与重要生物学途径相关的基因丰度存在明显差异。
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