Comparing the effects of microbiota-directed therapeutic foods in gnotobiotic mice, gnotobiotic piglets and undernourished children

We illustrate an approach for integrating preclinical gnotobiotic animal models with human studies to understand the contributions of perturbed gut microbiota development to childhood undernutrition, and to identify new microbiota-directed therapeutic concepts/leads. Combining metabolomic and proteomic analyses of serially collected plasma samples with metagenomic analyses of serially collected fecal samples, we characterized the biological state of Bangladeshi children with severe acute malnutrition (SAM) as they transitioned to moderate acute malnutrition (MAM) after standard treatment. Gnotobiotic mice were subsequently colonized with a defined consortium of bacterial strains representing different stages of microbiota development in healthy children from Bangladesh. Administering different combinations of Bangladeshi complementary food ingredients to colonized mice and germ-free controls revealed diet-dependent changes in representation and metabolism of targeted weaning-phase strains, including accompanying increases in branched-chain amino acids, plus diet- and colonization-dependent augmentation of IGF-1/mTOR signaling. Host and microbial effects of microbiota-directed complementary food (MDCF) prototypes were subsequently examined in gnotobiotic mice colonized with post-SAM MAM microbiota and in gnotobiotic piglets colonized with a defined consortium of targeted age- and growth-discriminatory bacteria. Finally, ar andomized, double-blind study revealed a lead MDCF that affected the representation of targeted bacterial taxa and increased levels of biomarkers and mediators of growth, bone formation, neurodevelopment, and immune function. 191 plasma samples total were measured from non-fasted venous blood, collected in EDTA-coated tubes, and rapidly cryoperserved before transfer to Washington University School of Medicine in St. Louis. The different groups include healthy (n = 21), ready-to-use supplementary food (RUSF) pre- (n = 14) and post-treatment (n = 14), microbiota-directed complementary food formulation 1 (MDCF1) pre- (n = 16) and post-treatment (n = 16), MDCF2 pre- (n = 16) and post-treatment (n = 16), MDCF3 pre- (n = 16) and post-treatment (n = 16), severe acute malnutrition (SAM, n = 30), post-SAM moderate acute malnutrition (post-SAM MAM) immediately after discharge from acute stabilizaiton (n = 8), and post-SAM MAM 6 months after discharge from acute stabilization (n = 8).

本研究阐述了一种整合临床前限菌动物模型（gnotobiotic animal model）与人体研究的研究策略，旨在解析肠道菌群发育紊乱对儿童营养不良的贡献，并挖掘新型菌群靶向治疗理念与候选方案。研究团队将连续采集的血浆样本的代谢组学（metabolomic）、蛋白质组学（proteomic）分析，与连续采集的粪便样本的宏基因组学（metagenomic）分析相结合，对孟加拉国重症急性营养不良（severe acute malnutrition, SAM）患儿在标准治疗后转为中度急性营养不良（moderate acute malnutrition, MAM）的生物学状态进行了表征。

随后，研究人员将代表孟加拉健康儿童肠道菌群发育不同阶段的特定细菌菌株联合体定植于限菌小鼠（gnotobiotic mice）体内。通过向定植小鼠与无菌对照组（germ-free control）饲喂不同组合的孟加拉辅食原料，研究发现靶向断奶期菌株的占比与代谢谱存在饮食依赖性变化，伴随支链氨基酸（branched-chain amino acids）水平升高，同时观察到饮食与定植状态依赖性的胰岛素样生长因子1/雷帕霉素靶蛋白（IGF-1/mTOR）信号通路激活增强。

后续，研究团队在定植有SAM后中度急性营养不良菌群的限菌小鼠，以及定植有特定年龄与生长特征性细菌联合体的限菌仔猪中，评估了菌群靶向辅食（microbiota-directed complementary food, MDCF）原型制剂的宿主与微生物效应。最后，一项随机双盲试验证实，一款先导MDCF制剂可改变靶向细菌类群的占比，并提升生长、骨形成、神经发育与免疫功能相关的生物标志物及调控因子水平。

本研究共采集非空腹静脉血样本191份，采用乙二胺四乙酸（EDTA）抗凝管采集后快速冷冻保存，随后转运至圣路易斯华盛顿大学医学院开展检测。

研究分组如下：健康对照组（n=21）、即食补充食品（ready-to-use supplementary food, RUSF）治疗前组（n=14）与治疗后组（n=14）、菌群靶向辅食配方1（MDCF1）治疗前组（n=16）与治疗后组（n=16）、MDCF2治疗前组（n=16）与治疗后组（n=16）、MDCF3治疗前组（n=16）与治疗后组（n=16）、重症急性营养不良组（SAM, n=30）、急性稳定治疗出院后即刻的SAM后中度急性营养不良组（post-SAM MAM, n=8），以及急性稳定治疗出院后6个月的SAM后中度急性营养不良组（n=8）。