16s rRNA sequencing for Mapping Pesticide-Induced Metabolic Alterations in Human Gut Bacteria

In the mono-colonization experiments, B. ovatus was cultured in an anaerobic chamber and their identities were confirmed through phenotype verification using GAM plate streaking and PCR primers. For B. ovatus, the primers used were as follows: Forward: 5'-3' TGCAAACTRAAGATGGC and Reverse: 5'-3' CAAACTAATGGAACGCATC. After culturing for 24 hours, a 10 mL saturated bacterial culture was centrifuged and washed three times with PBS. The bacterial pellets were then resuspended in 10 × 1 mL sterile PBS in 2 mL sterile Eppendorf tubes. In the BO group, mice were colonized with B. ovatus ATCC8483 (approximately 8 × 10^6 colony-forming units (CFU)) obtained from the saturated cultures. This colonization process involved orally administering 200 μL of the respective cultures through gavage. On the other hand, mice in the ABX and Control groups were only orally gavaged with 200 μL of PBS. Fecal samples were collected both before and after mono-colonization for phenotype verification. The sterile water was replaced and documented every 2 days, and sterile food was refreshed and recorded on a weekly basis.Regarding pesticide exposure, once successful mono-colonization was achieved, the mice from the Control, and BO groups were subjected to a 4-week exposure to 0.1 μg/mL of 4,4’-DDE in their drinking water. Meanwhile, the mice in the ABX group received sterile water without pesticide. The drinking water was replaced and documented every 2 days, and sterile food was refreshed and recorded on a weekly basis. Prior to and after mono-colonization, 16S amplicon sequencing was performed on the V4 region (515F, 806R) of microbial populations found in individual mice's feces.