Anaerobic Stool Preparation Method for Fecal Microbiota Transplantation Is Not Superior to Conventional Aerobic Method in Preserving Anaerobic Bacteria

BACKGROUND: Fecal Microbiota Transplantation (FMT) has shown to be effective in the treatment of recurrent Clostridium difficile infection, but its efficacy in treating ulcerative colitis has not been promising to date. In several studies, FMT solution is usually prepared using a household blender under aerobic conditions. However, bacterial counts of most anaerobes, which are major components of human microbiota, might be decreased during this procedure. In this study, we prepared FMT suspensions under both anaerobic and aerobic conditions, and compared the live gut microbiota composition by 16S rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR). METHODS: We collected 20 fecal samples from 20 Japanese healthy adult donors. Feces were collected and transferred under anaerobic conditions using air tight fecal container and Anaeropack® (deoxidizer). In an anaerobic glove box (where air is substituted by nitrogen and oxygen concentration is maintained at ≤1%), we divided each sample into two portions. Aliquots were diluted with normal saline, and blended using a house hold blender for 30 seconds in an anaerobic glove box (“Anaero-group”) or under aerobic conditions (“Aero-group”). Fecal suspensions were transferred into tubes containing 2 mL of RNAlater® (RNA stabilization solution) and stored at 4°C. Using Yakult Intestinal Flora Scan (YIF-SCAN), the number of live gut bacteria per sample were obtained. RESULTS: The percent change in the number of bacteria after fecal preparation was evaluated for total bacteria, obligate anaerobic bacteria, facultative anaerobic bacteria, and aerobic bacteria. The percent changes in the number of bacteria between the “Anaero-group” and “Aero-group” were compared by Wilcoxon rank sum test. There was no significant difference, between the “Anaero-group” and “Aero-group”, in the percent changes of total bacteria (median 74%, interquartile range 28% vs. median 83%, IQR 41%, respectively), obligate anaerobic bacteria (median 74%, IQR 28% vs. median 85%, IQR 41%, respectively), facultative anaerobic bacteria (median 95%, IQR 26% vs. median 103%, IQR 57%, respectively), and aerobic bacteria (median 100%, IQR 432% vs. median 100%, IQR 513%, respectively). CONCLUSION(S): In this study, the anaerobic preparation method was not superior to conventional aerobic preparation method in preserving live gut microbiota in FMT suspensions. Limitations of this study include potential sampling bias. Further studies are essential to develop better techniques for preparation of FMT suspension.