Isolation and identification of gut microbes by liquid-liquid co-culture method

Abstract

Gut microbes are of crucial in various industrial fields including drug development; therefore, the isolation, cultivation, and classification of difficult-to-culture microorganisms and new species are essential for the development of new live biotherapeutic products. We performed liquid-liquid co-cultures to obtain isolates using a previously unreported method. Selectively filtered fecal samples were anaerobically cultured under the influence of supporting bacteria through a membrane filter. 16S rRNA gene and genomic analyses showed that isolates were identified as either Waltera intestinalis or a new species closely related to W. intestinalis. Isolates in broth medium had short and thin cells during the early stages of growth, but became longer and thicker with longer duration of incubation. Therefore, the cells during the early growth stage may have passed through the filter owing to their small size and elongated at a later stage. Filter-treated isolates grew only in the co-culture, whereas untreated isolates grew better in the mono-culture than in the co-culture. This suggests that the growth of the filter-treated isolates was enhanced by the co-culture, whereas the growth of the elongated cells of the isolates was inhibited by the co-culture. Furthermore, all isolates had genes involved in sporulation and germination. This suggested that the small spores that passed through the filter germinated under the influence of the metabolites produced by the supporting bacteria; however, the growth of the spores was inhibited by the metabolites.