Fig. 4: Integration of fecal microbial and metabolic profiles of infants at elevated- and low-likelihood of ASD and in vitro studies.

a Network constructed on CLR transformed OTUs and log transformed metabolites at 5 months of age using Spearman correlations. The full network was initially generated using all available OTUs and metabolites and correlations with adjusted p values < 0.25 were retained (full network available in Supplementary Fig. 6). To improve visualization, only Bifidobacterium and Clostridium species of interest and their correlated metabolites are displayed. GABA and acetate were positively correlated to the Bifidobacterium species of interest, while the Clostridium related species correlated with butyrate and glutamate. Red edges indicate positive correlations and blue edges indicate negative correlations. Edge width scaled on the absolute correlation values. b GABA can be produced through the GABA shunt, a closed-loop process that converts the α-ketoglutarate from the TCA cycle into glutamate, then GABA and finally into succinate, which re-enter the TCA cycle. GABA can also be produced from polyamines (e.g., spermidine and putrescine). Three different substrates (glutamate, putrescine, and spermidine) that can be converted into GABA were used in the Bifidobacterium and Clostridium cultures. c Bacterial isolates were cultured for 24 h in monocultures with the added substrate. B. breve and B. scardovii produced glutamate. GABA was produced by B. breve, B. adolescentis and B. scardovii, only when already present in the medium, and consumed by C. difficile and C. bolteae. Putrescine was consumed by Clostridium related species and produced by Bifidobacterium species, only when already present in the medium. No interactions with spermidine were observed. d A positive correlation was found between Bifidobacterium/Clostridium ratio and GABA concentration in fecal samples. e To determine whether the ratio of Bifidobacterium to Clostridium species. influenced the abundance of GABA, different growth conditions were investigated in vitro (Bifidobacterium spp.: Clostridium spp.; 2:1, 1:1, 1:2). GABA abundance was reduced to values comparable to that of the negative control (NC) when a greater proportion (2:1 Clostridium spp.: Bifidobacterium spp.) of Clostridium spp. were present in the cultures. Group means are plotted as line.