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

ABSTRACT: 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.IMPORTANCE This work determined that using a combination of anaerobic 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 a diversity of bacteria similar to those of enrichment and direct plating methods. Methods that selected for endospore-forming bacteria generated collections that differed in composition from those of 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.

SUBMITTER: Moote PE

PROVIDER: S-EPMC8091012 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Blastocystis is an ubiquitous, enteric protozoan of humans and many other species. Human infection has been associated with gastrointestinal disease such as irritable bowel syndrome, however, this remains unproven. A relevant animal model is needed to investigate the pathogenesis/pathogenicity of Blastocystis. We concluded previously that pigs are likely natural hosts of Blastocystis with a potentially zoonotic, host-adapted subtype (ST), ST5, and may make suitable animal models. In this study, we aimed to characterise the host-agent interaction of Blastocystis and the pig, including localising Blastocystis in porcine intestine using microscopy, PCR and histopathological examination of tissues. Intestines from pigs in three different management systems, i.e., a commercial piggery, a small family farm and a research herd (where the animals were immunosuppressed) were examined. This design was used to determine if environment or immune status influences intestinal colonisation of Blastocystis as immunocompromised individuals may potentially be more susceptible to blastocystosis and development of associated clinical signs. Intestines from all 28 pigs were positive for Blastocystis with all pigs harbouring ST5. In addition, the farm pigs had mixed infections with STs 1 and/or 3. Blastocystis organisms/DNA were predominantly found in the large intestine but were also detected in the small intestine of the immunosuppressed and some of the farm pigs, suggesting that immunosuppression and/or husbandry factors may influence Blastocystis colonisation of the small intestine. No obvious pathology was observed in the histological sections. Blastocystis was present as vacuolar/granular forms and these were found within luminal material or in close proximity to epithelial cells, with no evidence of attachment or invasion. These results concur with most human studies, in which Blastocystis is predominantly found in the large intestine in the absence of significant organic pathology. Our findings also support the use of pigs as animal models and may have implications for blastocystosis diagnosis/treatment.

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

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