Development of a Protocol for Anaerobic Preparation and Banking of Fecal Microbiota Transplantation Material: Evaluation of Bacterial Richness in the Cultivated Fraction

Abstract

Fecal microbiota transplantation (FMT) has shown highly variable results in indications beyond recurrent Clostridioides difficile infection. Microbiota dysbiosis in many diseases is characterized by the depletion of strictly anaerobic bacteria, which may be crucial for FMT efficacy. We developed a protocol to ensure anaerobic conditions during the entire transplant preparation and banking process, from material collection to administration. The protocol necessitates an anaerobic cabinet, i.e., a non-standard laboratory equipment. We analyzed the population of viable anaerobes by combining cultivation and 16S rRNA gene profiling during the transplant preparation, and after 4, 8, and 12 months of anaerobic or aerobic storage at -80 °C, 78% of fecal species were captured via cultivation. Our findings suggest that strictly anaerobic transplant preparation and storage may preserve species richness better than oxic conditions, but the overall difference was not significant. However, specific anaerobes such as Neglecta and Anaerotruncus were affected by the oxygen exposure. A storage time of up to 12 months did not affect the presence of cultivated taxa. Noteworthy, our analysis focused on the richness of cultivated anaerobes rather than their abundance, which may have been affected. The benefits of the developed anaerobic protocol in FMT for specific indications remain to be demonstrated in clinical trials.

Keywords: Clostridium difficile; anaerobic conditions; dysbiosis; fecal microbiota transplantation (FMT); inflammatory bowel disease; short-chain fatty acid (SCFA).

Figure 1

Protocol for the anaerobic preparation of frozen fecal inoculum for fecal microbiota transplantation (FMT) (a) from anaerobic packaging by the donors to (b) the anaerobic preparation of transplants in the laboratory; donor samples are preserved from oxygen by anaerobic packaging and transported to the laboratory after defecation. A total of 30 g of feces are suspended into 150 mL of sterile saline by mixing with a spatula in a 250 mL screw cap container and 10% glycerol (final concentration) is added inside the anaerobic cabinet. The package is sealed hermetically in a vacuum sealing bag inside the anaerobic chamber and freeze-stored at −80 °C. For FMT administration, samples are allowed to thaw inside the sealed bag at +37 °C or room temperature, mixed briefly, and aliquoted to FMT syringes. If necessary, a pre-sterilized and stainless-steel strainer can be used to remove non-suspended particles present in the suspension before loading the syringes.

Figure 2

Protocol for the anaerobic preparation and preservation study of frozen fecal inoculum. Donor samples were packed anaerobically and transported to the laboratory. Samples were taken from fresh feces and cultivations were conducted, and further DNA extraction and 16S rRNA sequencing was performed. Feces were prepared into suspensions and for storage using three conditions; anaerobic (ANA), aerobic/anaerobic (AER/ANA), and aerobic (AER), according to their oxygen exposure during preparation and storage at −80 °C, as described in detail in the main text. Cultivations from the freeze-stored samples were conducted at time points of 4, 8, and 12 months on GAM, FAA, and YCFA plates, bacterial lawns were collected, DNA was extracted, and 16S rRNA gene amplicons were sequenced.

Figure 3

Species richness estimates in the fecal samples from the three healthy fecal donors and the cultivated bacterial lawns after the storage of fecal suspensions: (a) all samples from all three donors combined and (b) the samples from individual donors. Oxygen exposure degree during the preparation and freeze storage of fecal suspension is indicated as ANA (anaerobic), AER/ANA (aerobic/anaerobic), and AER (aerobic) (see main text for further details). The time points refer to the baseline (T₀) and 4-, 8- and 12-month storage at −80 °C before cultivation (T₄, T_8, and T₁₂). Asterisks indicate a statistically significant difference (NS = not significant, ** = p < 0.01, *** = p < 0.001) between the samples.

Figure 4

Presence of bacterial taxa in the fecal and cultivated bacterial lawn samples of the three donors. Color gray = present, color white = absent. Dx = donor’s fresh feces, Tx = time point cultivations. All taxa with over 0.01% relative abundance of the total microbiota and over 10% prevalence in all samples are included.