Comprehensive Cultivation of the Swine Gut Microbiome Reveals High Bacterial Diversity and Guides Bacterial Isolation in Pigs.

Despite the substantial progress made in human gut culturomics, little is known about the culturability of the swine gut microbiota. In this study, we cultured swine gut microbiota using 53 bacterial cultivation methods with different medium and gas combinations from three pigs at four different growth stages. Both culture-dependent (CD; colony mixtures from each method) and culture-independent (CI; original fecal suspensions) samples were subjected to 16S rRNA gene amplicon sequencing. Increasing microbial diversities were observed in both CI and CD samples from successive growth stages. While a total of 378, 482, 565, and 555 bacterial amplicon sequence variants (ASVs) were observed in the CI samples, higher microbial diversities (415, 675, 808, and 823 observed ASVs) were detected using the CD methods at the lactation, nursery, growing, and finishing stages, respectively. We constructed reference culture maps showing the preferred cultivation conditions for specific bacterial taxa and examined the effects of culturing factors such as oxygen, medium, donor pig age, antibiotics, and blood culture preincubation on swine gut microbiota cultivation. We focused on a wide range of beneficial bacteria, chose 1,299 colonies based on the reference map, and Sanger sequenced their 16S rRNA genes. These isolates clustered into 148 different bacterial taxa covering 28 genera. We observed 11, 19, 33, and 25 pairs of cooccurring ASVs in both CD and CI samples at four successive growth stages. This study provides guidance in culturing the swine gut microbiota of interest, which is critical when characterizing their functions in this important animal species.