Our Preclinical Laboratory Animal Researcher, Jonas Mingaila, authored an article discussing Gut Microbiota Transplantation in the context of preclinical studies. At Cureline Baltic, we offer our expertise and support to assist you in the integration of these techniques into your research. Read the article to know what method is best for Gut Microbiota Transplantation.


The experimental details reported in preclinical fecal microbiota transplantation (FMT) protocols are highly inconsistent, variable, and/or incomplete. We therefore evaluated FMT from a human donor to antibiotic-induced microbial-depleted mice by exploring the effects of six techniques based on antibiotic (AB) or antibiotic + antimycotic (AB + T) gut decontamination, different administration routes, and different dosing intervals on the gut microbial population, assessed using 16S and 18S sequencing. In addition, we explored the effectiveness of FMT in terms of inflammation, physiological, and behavioral outcomes. Our results showed that intrarectal FMT at low dosing intervals better preserved the donor’s gut bacterial community at genus level. Furthermore, we showed a lower abundance of several genera of fungi in animals treated with AB + T. In addition, we observed that AB + T gut decontamination followed by per os FMT, once every 3 days, affected behavioral parameters when compared to other FMT techniques. Accordingly, the same FMT groups that showed an association with some of the behavioral tests were also related to specific gut fungal genera, suggesting a possible mediation. Our findings may be useful for optimizing the practice of FMT and also in terms of donor microbiota preservation. This information may help to improve the reproducibility and reliability of FMT studie


In the current study, we aimed to define the differences in terms of the gut bacterial and fungal communities in groups of animals exposed to different FMT techniques, and the possible implications for several parameters.

For all the FMT techniques considered in this study, we observed a significantly lower gut bacterial alpha diversity compared with the donor, highlighting the overall effects of gut bacterial decontamination. It has been observed that mice treated with an antibiotic cocktail of ampicillin, vancomycin, neomycin, and metronidazole, prior FMT via oral gavage, at a low dosage received effective gut decontamination and a successful engraftment of the donor microbiota [18]. Accordingly, we observed that lower dosing intervals reduced the number of differential abundant genera in comparison with the human donor.

Comparing four different methods based on the FMT frequency over a period of 4 weeks (twice a week, once a week, two FMTs, one FMT), Wrzosek and colleagues showed that FMT administration twice a week for four weeks was too frequent and perturbed the stability of the gut microbial ecosystem, whereas FMT once a week allowed bacterial engraftment and a higher diversity [20]. Thus, we observed that animals exposed to less frequent FMT showed higher alpha diversity compared to animals exposed to more frequent FMT. On the other hand, it was observed that mice treated with a 3-week antibiotic regimen followed by 5-daily FMT showed a greater resemblance to the human donor microbiota compared with mice treated with a 3-day antibiotic regimen followed by 1-daily FMT [21]. However, when comparing different FMT frequencies separately, we did not observe any differences, suggesting that, perhaps, inter-group differences may be attributed to other FMT method-related factors. At present, the optimum FMT dosage to sustain the donor microbiota in the recipient is not clear [12], highlighting the lack of studies permitting answers to this question and the importance of our study from this point of view.

We observed that the gut microbiota of animals treated via intrarectal administration at a lower dosage showed a higher capacity to restore the donor’s microbiota and higher alpha diversity compared with the groups of animals exposed to FMT via oral gavage. It has been speculated that intrarectal administration is more effective than oral gavage, as the inoculum does not need to pass through different gastrointestinal barriers. However, the optimum choice of FMT administration remains a debated topic [13]. According to our results, the FMT techniques did not show effects on the outcomes of inflammation, such blood parameters, meaning it was not possible to determine which type of administration was less harmful to the animals.

We observed that the main difference in the gut microbial community across the study groups was related to the mycobiota (the population of fungi inhabiting the gut) identified through 18S sequencing. The importance of this portion of the gut microbial community to host homeostasis has already been highlighted, but there is a lack of studies identifying the best FMT approach to preserve it. In addition, it was observed that components of the gut mycobiota may contribute to disease recurrence in patients with Clostridium difficile infection treated with FMT [19]. It has been demonstrated that intestinal colonization with mucosal fungi increased the responsiveness of mice to social stimuli, highlighting the ability of gut fungi to modulate host behavior [22]. Accordingly, we observed that higher behavioral parameters were associated with a higher abundance of specific genera of fungi in animals treated with AB + T. Animals treated with AB + T and administered FMT PO once ×3 days showed a higher total track length and velocity than animals treated with AB, and animals treated with AB + T with larger FMT dosing intervals. Furthermore, the activity during the experiment was also higher in group E compared to animals treated with AB with larger FMT dosing intervals.

Exploring the potential mediation of the gut microbiota in the association between the FMT groups and behavioral tests, the main signal only came from the 18S dataset when comparing animals exposed to the AB + T treatment vs. animals exposed to the AB treatment. Accordingly, the same FMT groups that showed an association with some of the behavioral tests were also related to specific gut fungal genera.

In conclusion, FMT at low dosing intervals seems to be the best technique to better preserve donors’ gut bacterial population. However, it seems that the main signal that may help to elucidate the best FMT condition comes from the 18S dataset. Animals exposed to AB + T gut decontamination showed a lower abundance of several fungal genera. In addition, behavioral parameters were affected in animals exposed to AB + T gut decontamination, followed by per os FMT once ×3 days, in comparison with animals treated with AB, and animals treated with AB + T with larger FMT dosing intervals. Several fungal genera appear to mediate this association. This study helps to identify the best FMT procedure in mice, considering a number of different modalities not fully described in the literature, by providing useful information for more comprehensive guideline development. We also aimed to help in standardizing methods for FMT in animals and to improve preclinical studies. Our study presents some limitations, especially related to the small sample size and the inability to generalize our results to other routes. However, from this point of view, these limitations can be used as an advantage when making further improvements.

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