研究背景：生命早期肠道微生物群落是调控宿主代谢和免疫稳态的关键因子，据报道，肠道微生物群落失调已在各种儿科危重疾病人群中得到了很好的研究，包括早产儿和营养不良的新生儿等。先天性心脏病(Congenital heart disease ,CHD) 是最常见的出生缺陷，其中重症先心病(critical congenital heart disease,CCHD)新生儿常常面临着多种应激因素如低氧血症，异常肠道灌注等，这些因素均可诱导患儿肠道微生物群落失调。然而，CCHD新生儿生命早期肠道微生物组对宿主代谢健康、免疫稳态及临床预后的潜在影响在很大程度上仍然未知。因此，本研究旨在探究CCHD新生儿生命早期肠道微生物组特征及其与宿主代谢之间的生物学联系。同时，我们进一步探索CCHD新生儿中紊乱失调的宿主-微生物相互作用对患儿免疫稳态及临床预后的影响。

方法学：我们对来自 95 名新生儿（45名CCHD新生儿和50名健康足月新生儿）的配对粪便样本进行了深度宏基因组学和代谢组学的测序分析。 我们从微生物的组成、功能和基因组遗传结构变异这三个维度来系统性揭示CCHD新生儿生命早期肠道菌群变化特征及其对宿主代谢健康的潜在影响。此外，我们通过对研究队列肠道病毒组的深度分析进一步揭示了CCHD新生儿肠道内“病毒-细菌群落”的生态互作联系。最后，我们测量了CCHD患儿队列的炎症细胞因子谱与肠道屏障损伤的血液生物标志物，并基于多组学数据进行了亚组联合分析，从而进一步探索CCHD新生儿中“宿主-微生物”相互作用是否可通过调控患儿术前免疫炎症状态进而影响患儿的临床预后。

结果：首先，我们证明了与健康足月新生儿相比，CCHD新生儿存在肠道微生物组紊乱失调（包括肠道细菌、病毒和真菌群落）。有趣的是，CCHD新生儿肠道细菌组成紊乱主要表现为双歧杆菌(Bifidobacterium)的耗竭缺失和肠球菌(Enterococcus)的过度生长扩张，该肠道细菌组成紊乱与患儿粪便代谢组中芳香乳酸(aromatic lactic acids)、人乳寡糖(human milk oligosaccharide, HMO)、多不饱和脂肪酸(polyunsaturated fatty acids, PUFAs)和 B族维生素(B vitamins)等有益代谢物水平的降低呈现高度相关性，而这些代谢物都是被广泛报道的可调节肠道稳态的益生元。同时，我们发现双歧杆菌的耗竭缺失、HMO利用基因的缺失、以及双歧杆菌与肠球菌的基因组遗传结构变异可共同介导CCHD新生儿生命早期的代谢稳态失衡。此外，我们发现以长尾噬菌体科病毒（Siphoviridae）为核心的温和噬菌体病毒群落可通过改变微生物适应性来影响宿主肠道菌群组成，从而进一步介导了CCHD新生儿肠道中肠球菌的过度生长扩张。最后在亚组分析中，我们发现在CCHD新生儿肠道中：肠球菌的过度生长扩张可通过改变微生物相关代谢产物水平来调控患儿术前免疫炎症状态与肠屏障功能，进而影响患儿的临床预后。

结论：我们表征了CCHD新生儿生命早期肠道微生物群落的特征，并破译了患儿的肠道菌群紊乱与宿主代谢扰动之间的内在生态学联系。此外，我们进一步揭示了CCHD新生儿中异常的肠道菌群可通过改变微生物相关代谢产物水平来调控患儿术前免疫炎症状态与肠屏障功能，进而影响患儿的临床预后。

Backgrounds: Early-life gut microbiome is a crucial modulator of host metabolic and immune homeostasis, and gut microbiome dysbiosis has been well studied in a variety of paediatric critical illness populations, including preterm infants and malnourished neonates. Congenital heart disease (CHD) is the most common birth defect, among which neonates with critical congenital heart disease (CCHD) usually face   numerous stressors including hypoxemia and abnormal gut perfusion, leading to abnormalities in the intestinal microbiome. However, the potential implications of early-life gut microbiome in neonates with CCHD on host metabolic health, immunologic homeostasis and clinical outcomes remain largely unknown. Herein we aimed to investigate the characteristics of early-life gut micriobiome in neonates with CCHD and their ecological association with host metabolism. Simultaneously, we further explore the influence of disrupted host-microbe interactions in neonates with CCHD on immune homeostasis and clinical prognosis.

Methods: We performed deep metagenomic sequencing and metabolomic profiling of paired fecal samples from 95 neonates (45 CCHD and 50 healthy controls (HCs)). We systematically investigated characteristics of early-life gut microbiome in neonates with CCHD from three dimensions (microbial composition, functionality and genetic variations), and its potential implications in host metabolic health. Additionally, ecological interaction between gut viral and bacterial communities was explored based on in-depth analysis of virome. Finally, we measured inflammatory cytokine profiles and blood biomarkers of intestinal barrier damage in patient cohorts and performed a combined subgroup analysis based on multi-omics data to further explore whether the host-microbe interaction in neonates with CCHD can affect the clinical prognosis of those patients by regulating their preoperative immune inflammatory state.

Results: Firstly, we demonstrate that gut microbiome dysbiosis, including intestinal bacterial, viral, and fungal communities, is observed in CCHD neonates compared to HCs. Interestingly, intestinal bacterial community dysbiosis of CCHD neonates is mainly characterized by depletion of Bifidobacterium and overgrowth of Enterococcus, which is highly correlated with a panel of fecal metabolites (including aromatic lactic acids, human milk oligosaccharides (HMOs), polyunsaturated fatty acids and B vitamins), which are well-recognized prebiotics mediating intestinal homeostasis. Meanwhile, we found that the depletion of Bifidobacterium and HMO-utilization genes as well as genetic variations in Bifidobacterium and Enterococcus could orchestrate the metabolomic perturbations in neonates with CCHD. Additionally, temperate core virome represented by Siphoviridae is identified to implicate in shaping host gut bacterial composition by modifying microbial adaptation, enabling the excessive overgrowth of Enterococcus in neonates with CCHD. Finally, in the subgroup analysis, we found that the overgrowth of Enterococcus in neonates with CCHD could regulate the preoperative immune inflammatory state and intestinal barrier function of the patients by changing the level of microbial-related metabolites, thus affecting the clinical prognosis of the patients.

Conclusions: We characterized the early-life intestinal microbiome in neonates with CCHD and deciphered the intrinsic ecological connections between gut microbiome disturbances and metabolomic perturbations. In addition, we further revealed that aberrant gut microbiome in neonates with CCHD could regulate preoperative immune-inflammatory state and intestinal barrier function by changing the level of microbial-related metabolites, thus affecting the clinical prognosis of the patients.