Comparison of Strategies for Isolating Anaerobic Bacteria from the Porcine Intestine.

The isolation of bacteria that represent the diversity of autochthonous taxa in the gastrointestinal tract is necessary to fully ascertain their function, but the majority of bacterial species inhabiting the intestines of mammals are fastidious and thus challenging to isolate. The goal of the current study was to isolate a diverse assemblage of anaerobic bacteria from the intestine of pigs as a model animal and to comparatively examine various novel and traditional isolation strategies. Methods used included long-term enrichments, direct plating, a modified ichip method, as well as ethanol and tyndallization treatments of samples to select for endospore-forming taxa. A total of 234 taxa (91 previously uncultured) comprising 80 genera and 7 phyla were isolated from mucosal and luminal samples from the ileum, cecum, ascending colon, and spiral colon removed from animals under anesthesia. The diversity of bacteria isolated from the large intestine was less than that detected by next-generation sequence analysis. Long-term enrichments yielded the greatest diversity of recovered bacteria (Shannon's index [SI] = 4.7). Methods designed to isolate endospore-forming bacteria produced the lowest diversity (SI ≤ 2.7), with tyndallization yielding lower diversity than the ethanol method. However, the isolation frequency of previously uncultured bacteria was highest for ethanol-treated samples (41.9%) and the ichip method (32.5%). The goal of recovering a diverse collection of enteric bacteria was achieved. Importantly, the study findings demonstrate that it is necessary to use a combination of methods in concert to isolate bacteria that are representative of the diversity within the intestines of mammals.