Supplementary Material for: Routine Use of Probiotics in Preterm Infants: Longitudinal Impact on the Microbiome and Metabolome

<b><i>Background:</i></b> Probiotics are live microbial supplements that colonize the gut and potentially exert health benefit to the host. <b><i>Objectives:</i></b> We aimed to determine the impact of a probiotic (Infloran®: <i>Lactobacillus acidophilus</i>-NCIMB701748 and <i>Bifidobacterium bifidum</i>-ATCC15696) on the bacterial and metabolic function of the preterm gut while in the neonatal intensive care unit (NICU) and following discharge. <b><i>Methods:</i></b> Stool samples (n = 88) were collected before, during, and after probiotic intake from 7 patients, along with time-matched controls from 3 patients. Samples were also collected following discharge home from the NICU. Samples underwent bacterial profiling analysis by 16S rRNA gene sequencing and quantitative PCR (qPCR), as well as metabolomic profiling using liquid chromatography mass spectrometry. <b><i>Results:</i></b> Bacterial profiling showed greater <i>Bifidobacterium </i>(15.1%) <i>and Lactobacillus</i> (4.2%) during supplementation compared to the control group (4.0% and 0%, respectively). While <i>Lactobacillus</i> became reduced after the probiotic had been stopped, <i>Bifidobacterium</i> remained high following discharge, suggestive of successful colonisation. qPCR analysis showed a significant increase (p ≤ 0.01) in <i>B. bifidum</i> in infants who received probiotic treatment compared to controls, but no significant increase was observed for <i>L. acidophilus</i> (p = 0.153). Metabolite profiling showed clustering based on receiving probiotic or matched controls, with distinct metabolites associated with probiotic administration. <b><i>Conclusions:</i></b> Probiotic species successfully colonise the preterm gut, reducing the relative abundance of potentially pathogenic bacteria, and effecting gut functioning. <i>Bifidobacterium</i> (but not <i>Lactobacillus</i>) colonised the gut in the long term, suggesting the possibility that therapeutically administered probiotics may continue to exert important functional effects on gut microbial communities in early infancy.

背景：益生菌是一类可定植于肠道的活微生物补充剂，能够对宿主产生潜在健康益处。
研究目的：本研究旨在探究益生菌制剂Infloran®（嗜酸乳杆菌<i>Lactobacillus acidophilus</i>-NCIMB701748与两歧双歧杆菌<i>Bifidobacterium bifidum</i>-ATCC15696）对新生儿重症监护病房（neonatal intensive care unit, NICU）住院期间及出院后早产儿的肠道菌群与代谢功能的影响。
研究方法：本研究纳入7名服用该益生菌的早产儿，分别在益生菌摄入前、摄入期间及摄入后采集粪便样本共88份，同时为3名受试者采集时间匹配的对照样本；此外还采集了这些早产儿从NICU出院回家后的粪便样本。所有样本均通过16S rRNA基因测序、定量聚合酶链反应（quantitative PCR, qPCR）进行细菌谱分析，并采用液相色谱-质谱联用法（liquid chromatography mass spectrometry）开展代谢组谱分析。
研究结果：菌群谱分析结果显示，与对照组（双歧杆菌相对丰度4.0%，乳杆菌相对丰度0%）相比，益生菌补充期间受试者的双歧杆菌<i>Bifidobacterium</i>相对丰度达15.1%、乳杆菌<i>Lactobacillus</i>相对丰度达4.2%，均显著升高。停用益生菌后，乳杆菌相对丰度有所下降，但双歧杆菌相对丰度在出院后仍维持较高水平，提示定植成功。qPCR分析显示，与对照组相比，接受益生菌治疗的婴儿体内两歧双歧杆菌<i>B. bifidum</i>的丰度显著升高（p≤0.01），但嗜酸乳杆菌<i>L. acidophilus</i>的丰度无显著升高（p=0.153）。代谢物谱分析显示，样本可根据是否接受益生菌干预形成明显聚类，且存在与益生菌施用相关的特异性代谢物。
研究结论：益生菌菌株可成功定植于早产儿肠道，降低潜在致病菌的相对丰度，并改善肠道功能。双歧杆菌<i>Bifidobacterium</i>（而非乳杆菌<i>Lactobacillus</i>）可在肠道中长期定植，提示治疗性施用的益生菌可能在婴儿早期仍可对肠道微生物群发挥重要的调控功能。