Zebrafish inoculation with a human gut microbiota

Zebrafish larvae were inoculated at 3 dpf with a fecal gut microbiota from a normal-weight (BMI < 22) 35 years old human volunteer. The fecal sample was transported to the laboratory in anaerobiosis and processed before 2 hours, as previously reported (Goodman et al., 2011). In brief, 3g of fecal sample was homogenized for 5 min with 45 ml of pre-reduced sterile PBS with 0,1 % cysteine and allowed to stand for 5 min. Zebrafish larvae were inoculated with a 1/10 dilution of the supernatant in sterile E3 medium. After 30 min, the bacterial inoculum was removed and larvae were washed at least 4 times with sterile E3 and maintained with sterile E3 at 28ºC. In colonization studies with human bacterial isolates, zebrafish larvae (3, 4 or 5 dpf) were inoculated by immersion with a suspension of each bacterium at a final concentration of 108 CFU/ml in sterile E3 medium, as described above. To evaluate the bacterial colonization, the microbiota of the inoculated and non-inoculated larvae was identified from 3 to 7 or 9 dpf larvae with molecular and culture methods, as described below. All colonization experiments were performed 6 times.