Document Type

Thesis - Open Access

Award Date

2022

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Xiuqing Wang

Abstract

Plant-based diets contain numerous flavonoid compounds that produce antibacterial effects and gut health improvement. Genistein is among the most abundant isoflavones present in a plant-based diet and can be found in high amounts in soy products (up to 1g Kg-1). We describe here a robust method to identify genistein tolerant and metabolizing bacteria in swine gut microbiota and to screen the strains that have antibacterial and immunestimulatory properties. Such strains could be developed as non-antibiotic alternatives to prevent enteric infections in pigs and improve gut immunity. To this end, a mini bioreactorbased system was used to enrich genistein metabolizing bacteria in swine microbiota from pooled pig fecal samples. Pooled pig fecal samples were supplemented with 0.5mg/mL genistein and run in a mini bioreactor model in six replicates for 21 days. At the end of the run, the six replicates were pooled together and were used for isolating genisteinmetabolizing bacteria. Bacterial species were isolated by micro cultivation array and routine anaerobic culture using modified BHI media and were identified by MALDI-TOF MS and 16S rRNA analysis. The genistein biotransformation capacity of the strains was determined using the DPH assay. We also determined their hemolytic and invasive capabilities. Our culture method was able to isolate a large number of strains belonging to 19 species. These include Streptococcus lutetiensis, Streptococcus equinus, Enterococcus faecalis, Streptococcus alactolyticus, Streptococcus gallolyticus, Acidaminococcus fermentans, Lactobacillus salivarius, Peptostreptococcus russellii, Mitsuokella jalaludinii, Faecalicoccus pleomorphus, Lactobacillus agilis, Bacteroides vulgatus, Bacteroides uniformis, Bacteroides fluxus, Enterococcus avium, Sharpea azabuensis, Collinsella phocaeensis, Selenomonas montiformis and Syntrophococcus sucromutans. Of the 19 species isolated, Lactobacillus salivarius is the only species that was completely hemolytic (𝛽 hemolysis). Eleven species namely Streptococcus gallolyticus, Acidaminococcus fermentans, Streptococcus equinus, Streptococcus alactolyticus, Bacteroides vulgatus, Bacteroides fluxus, Mitsuokella jalaludinii, Bacteroides uniformis, Sharpea azabuensis, Selenomonas montiformis and Syntrophococcus sucromutans were non-invasive. Mitsuokella jalaludinii and Peptostreptococcus russellii were negative for the DPH assay. 16S rRNA analysis revealed the abundance of six phyla namely Firmicutes, Bacteroidota, Actinobacteria, Proteobacteria, Synergistota and Euryarcheota and several genera belonging to Firmicutes. All the genera identified with culturomics were present in the taxa data from the 16S analysis, however, 16S analysis revealed other genera that could not be captured with culturomics. Hence, combining culturomics with 16S analysis is the best shot at trying to cover as much diversity as possible. We have been able to isolate ten nonhemolytic, non-invasive genistein-metabolizing species, which are potential antibiotic alternatives. Future studies will be focused on the species’ properties regarding their in vitro adhesion and pathogen exclusion, in vivo colonization and pathogen exclusion and acid and bile tolerance.

Number of Pages

109

Publisher

South Dakota State University

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Rights Statement

In Copyright