Infant gut microbiota coverage by different culture media

Culture-independent methods, such as sequencing, have been a breakthrough, but do not allow for complete genetic, biochemical, and phenotypic characterization at strain level. To fully characterize the gut microbiota, effective culture-based methods are needed to complement culture-independent methods. This study aims to identify the culture media and supplements to culture a wide range of bacteria from the infant microbiota. Faecal samples from a one-year-old Caesarean section born baby, and a two-year-old vaginally born baby were grown in 40 different combinations of growth media, supplements and atmosphere. Colonies taxonomically annotated based on 16S rRNA gene sequences, and a generalized linear model was used to determine the effect of the growth conditions on colony number and species diversity per family. Rich non-selective media did not support a broad diversity. A combination of selective media preparations targeting different bacterial groups resulted in better coverage of the total community. A combination of 3 formulations was able to capture 80% of the core infant gut genera: modified De Man-Rogosa-Sharpe (mMRS) with mupirocin, Gifu Anaerobic Medium (GAM) with ciprofloxacin and erythromycin and acidified MRS (MRS5.4) in microaerobic conditions. For species-level diversity, MRS5.4 produced the highest diversity of Bifidobacteriaceae and Enterococcaceae, but other base media did not significantly differ in terms of obtained diversity. However, the addition of specific antibiotics was critical in increasing the observed species-level diversity of bacterial families. Within species, some strains show media preference, and often a combination of media was required to capture the full spectrum of strains. Using a range of base media, supplements, and growth conditions, we were able to culture a large fraction of the core infant gut bacteria. We find that media specificity of infant gut bacterial isolates often deviates from existing information, and our findings could aid choice of media and growth conditions in future culture-based studies of the infant gut microbiota.