Comparison of culture-based strategies for isolating 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 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 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, as well as ethanol and Tyndallization treatment of samples to select for endospore-forming taxa. A total of 234 taxa (91 previously uncultured) comprising 80 genera and seven 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 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 a lower diversity than the ethanol method. However, the isolation frequency of previously uncultured bacteria was greatest 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, 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.IMPORTANCE This work determined that using a combination of isolation methods is necessary to increase the diversity of bacteria recovered from the intestines of monogastric mammals. Direct plating methods have traditionally been used to isolate enteric bacteria, and recent methods (e.g. diffusion methods [i.e. Ichip] or differential isolation of endospore-forming bacteria) have been suggested to be superior at increasing diversity, including the recovery of previously uncultured taxa. We showed that long-term enrichment of samples using a variety of media isolated the most diverse and novel bacteria. Application of the Ichip method delivered similar diversity of bacteria to enrichment and direct plating methods. Methods that selected for endospore-forming bacteria generated collections that differed in composition to other methods with reduced diversity. However, the ethanol treatment frequently isolated novel bacteria. By using a combination of methods in concert, a diverse collection of enteric bacteria was generated for ancillary experimentation.