Realplex4

Fecal samples were collected from offspring “humanized” mice and homogenized in 10 μL/mg sterile 1X PBS. Suspensions were serially diluted and plated on Tryptic-Soy Agar plates with 5% sheep blood (TSA; Teknova; Hollister, CA, USA) and Brucella Agar with 5% Sheep blood plates (Teknova; Hollister, CA, USA). Subsequently, plates were incubated aerobically (TSA plates) or anaerobically (Brucella plates) at 37 °C for 48–72 hours. For total 16S rRNA gene quantification, DNA was extracted from 200 l of fecal suspensions using Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo; Irvine, CA, USA), following manufacturer’s protocol. Fecal DNA from samples were eluted in 100 μL EB buffer, and concentrations were quantified using NanoDrop One (Thermo; Waltham, MA, USA). Femto Bacterial DNA Quantification Kit (Zymo; Irvine, CA, USA) was subsequently used for absolute quantification of 16S copies in fecal samples, according to the manufacturer’s instructions. qPCR reactions were prepared in duplicates in 96-well plate format and performed on an Eppendorf RealPlex4 (Eppendorf; Germany) with the following conditions: 10 mins at 95 °C, followed by 40 cycles of 30 sec at 95 °C, 30 sec at 50 °C and 1 min at 72 °C, then final extension of 7 mins at 72 °C. 16S concentrations were determined using a calibration curve of known concentrations of E. coli.