Masters Thesis

Investigation of Clostridia Isolated from Animal Feces for Potential use as a Probiotic

Clostridium species make up a large portion of the intestinal microbiota in humans and animals with strong implications that they are necessary for overall gut function. Dysbiosis due to decreased abundances and diversity of Clostridium often cause major health consequences due to the resulting lack of intestinal short chain fatty acids such as butyrate provided by these organisms. Safe and effective results have been reported upon oral administration of Clostridium probiotic supplements for symptom reduction and disease prevention indicating their prospective use as a therapeutic agent. We hypothesized that intestinal butyrate producing Clostridium can be isolated from the feces of animals and that some of these isolates will have the potential to be used safely as a probiotic dietary supplement. Enrichment and isolation of Clostridium from horse, cattle, sheep, pig, and goat fecal samples were identified and characterized by their colony morphology on egg yolk agar, microscopic morphology by staining, biochemical reactions, and 16S rRNA sequencing results. A total of 6 species from 134 Clostridia isolates were identified and the butyrate kinase gene was confirmed in all of these isolates implicating the ability to produce butyrate. Based on hemolysis and antibiotic susceptibility tests outlined by the European Food and Safety Authority, 6 isolates were considered to be potentially safe as a probiotic and were further biochemically characterized by API microbial identification kits. Of these probiotic candidates, Paeniclostridium sordellii isolates P1F and P2E were determined to be unsafe for use as a probiotic due to the high risk of virulence factors and horizontal gene transfer of toxins. Clostridium senegalense isolate C4H was potentially the safest for use as a probiotic followed by Clostridium tepidum isolates C4B, P2D, and P4D.