Adult-sourced fecal microbiota transplantation ameliorates early-life antibiotic-induced intestinal immune stress in juvenile mice associated with the modulation of the TLR4/MyD88 signaling pathway

In a controlled animal study, adult-sourced fecal microbiota transplantation was associated with reduced intestinal immune markers after early-life antibiotic exposure in juvenile mice. Following transplantation, colonic TLR4 protein abundance and IL-17 and IL-22 transcript levels were lower than in antibiotic-only controls, while IL-10 remained elevated. The treatment also produced a microbiota that differed from both the untreated juvenile baseline and the antibiotic-disrupted state.

The experiment involved 18 specific-pathogen-free male C57BL/6 mice aged 2–3 weeks. After a week of acclimatization, the mice were assigned to three groups of six: an untreated control group, an antibiotic group and a fecal microbiota transplantation group. The latter two groups received a broad-spectrum oral antibiotic cocktail containing ampicillin, gentamicin and vancomycin twice daily for eight consecutive days. Antibiotic-only mice then received sterile phosphate-buffered saline by gavage for four days, while transplantation recipients received a fecal microbiota suspension by the same route once daily for that period.

The donor suspension was prepared from pooled feces collected from five healthy, untreated 8-week-old adult C57BL/6J mice. It was administered at 0.1 mL per mouse and used within two hours of preparation. The investigators did not sequence the donor inoculum, so the analyses could not directly establish the origin or engraftment rate of specific donor strains. They also did not include a comparison group receiving age-matched juvenile donor material.

Microbial composition was assessed by sequencing the V3–V4 regions of the 16S rRNA gene, with six mice per group. Antibiotic exposure sharply reduced the Chao1 richness and Shannon diversity indices compared with controls, with P values below 0.001 for both measures. After transplantation, the Shannon index recovered to a level that did not differ significantly from the control group, while the Chao1 index showed a partial, statistically significant improvement over the antibiotic group. Principal coordinate analysis based on unweighted UniFrac distance showed distinct community structures among the three groups, with an Adonis R-squared value of 0.582 and P=0.001.

The transplanted microbiota was not simply a return to the pre-antibiotic juvenile composition. Antibiotic-treated mice showed marked depletion of taxa including Muribaculaceae and Lachnospiraceae_NK4A136_group and a substantial relative expansion of Bacteroides. Fecal microbiota transplantation was associated with reduced Bacteroides abundance and recovery of Muribaculaceae to levels not significantly different from controls. It also increased the relative abundance of low-abundance taxa and produced biomarker profiles that included Parabacteroides, Lachnoclostridium, Tannerellaceae and members of Desulfobacterota. The investigators characterized these findings as a distinct community state, although they could not confirm that the detected taxa originated from the adult donor because the inoculum itself was not sequenced.

Colonic tissue was collected on day 13 from three randomly selected mice per group for molecular analyses. Quantitative reverse-transcription polymerase chain reaction showed that antibiotics increased colonic transcript levels of IL-10, IL-17 and IL-22 relative to controls, with P values of 0.01 or lower. Transplantation was associated with lower IL-17 and IL-22 expression than antibiotic treatment alone, with P=0.036 and P=0.024, respectively. IL-10 expression in the transplantation group did not differ significantly from the antibiotic group, at P=0.068, and remained higher than in controls, at P=0.014.

Western blot analysis provided a partial measure of changes in the TLR4/MyD88 signaling axis. Antibiotic-treated mice had higher colonic TLR4 and MyD88 protein abundance than controls, with P values below 0.001. After transplantation, TLR4 abundance was significantly lower than in the antibiotic group, at P=0.011. MyD88 showed a downward trend but did not differ significantly from the antibiotic-only group. These measurements therefore associated fecal microbiota transplantation with attenuation of TLR4 expression and IL-17 and IL-22 transcription, rather than demonstrating a complete normalization of the pathway or the immune state.

Several limitations constrain the findings. The molecular endpoints were based on three mice per group, limiting the study’s ability to detect smaller differences in immune responses. Quantitative body-weight and food-intake data were not systematically recorded, and intestinal barrier function and tissue architecture were not assessed. The study also lacked luminal lipopolysaccharide and short-chain fatty acid measurements, direct sequencing of the pooled donor material, and an age-matched donor comparison. The findings consequently establish changes in mouse gut microbial composition and selected colonic immune markers over a short follow-up; they do not demonstrate a human clinical benefit, long-term stability of the transplanted community or restoration of mucosal homeostasis.

Overall, the mouse experiment indicates that a four-day course of adult-sourced fecal microbiota transplantation following early-life antibiotic exposure was associated with a partly restored microbial diversity index, suppression of antibiotic-associated Bacteroides expansion, a distinct taxonomic profile and lower selected intestinal immune markers. The authors said the results warrant further investigation of donor-age effects on microbial and mucosal immune recovery, while the absence of donor sequencing, a juvenile-donor comparator, functional barrier assays and a larger molecular sample limits stronger mechanistic conclusions.

Subject of Research: Effects of adult-sourced fecal microbiota transplantation on antibiotic-disrupted gut microbiota and colonic immune markers in juvenile mice

Article Title: Adult-sourced fecal microbiota transplantation ameliorates early-life antibiotic-induced intestinal immune stress in juvenile mice associated with the modulation of the TLR4/MyD88 signaling pathway

Article References: Qian, X., Shen, Y., Luo, X., Zhang, S., Wang, L., He, H., & Wang, B. (2026). Adult-sourced fecal microbiota transplantation ameliorates early-life antibiotic-induced intestinal immune stress in juvenile mice associated with the modulation of the TLR4/MyD88 signaling pathway. PLOS One, 21(10), e0359634. https://doi.org/10.1371/journal.pone.0359634

Image Credits: AI Generated

DOI: 10.1371/journal.pone.0359634

Keywords: fecal microbiota transplantation, early-life antibiotics, juvenile mice, gut microbiota, TLR4, MyD88, IL-17, IL-22, IL-10, 16S rRNA sequencing, Adult-sourced, fecal