Fecal virome transfer improves proliferation of commensal gut Akkermansia muciniphila and unexpectedly enhances the fertility rate in laboratory mice

Figures & data

Figure 1. Experimental setup of the animal model. In total 24 male and 24 female C57BL/6NTac mice (4 weeks old) were divided into six groups: LGG+FVT, AKM+FVT, Pro-sham+FVT, LGG+Saline, and AKM+Saline, control (Pro-sham+Saline). The Saline consisted of SM buffer and Pro-Sham of Intralipid. The mice were administered 1 M sodium bicarbonate prior oral gavage of FVT/Saline to protect the viral community against the acidic environment in the stomach. The day after, the mice were inoculated with probiotic solutions of LGG/AKM/Pro-sham suspended in Intralipid which constituted the 1^st inoculation. The same procedure was repeated as the 2^nd inoculation one week after. The mice were fed ad libitum low-fat diet (LFD) for the entire study (6 weeks) until termination at age 9 weeks. Fecal samples from baseline, after intervention, and termination were analyzed in this study. Abbreviations: Lacticaseibacillus rhamnosus GG = LGG, Akkermansia muciniphila = AKM, fecal virome transplantation = FVT, Pro-sham = probiotic sham.

Figure 2. qPCR with L. rhamnosus and A. muciniphila specific primers were used to assess the abundance of gene copies per gram feces over the time span of baseline, after intervention, and termination. A) the development of the A. muciniphila abundance, B) L. rhamnosus abundance. C) the abundance development of native A. muciniphila strains for mice receiving FVT and no probiotic A. muciniphila (Pro-sham+FVT and LGG+FVT) compared to not receiving FVT or A. muciniphila (Lgg+saline and control). D) the abundance development of native L. rhamnosus strains for mice receiving FVT and no L. rhamnosus (Pro-sham+FVT and AKM+FVT) compared to not receiving FVT or L. rhamnosus (Akm+saline and control). Abbreviations: Lacticaseibacillus rhamnosus GG = LGG, Akkermansia muciniphila = AKM, fecal virome transplantation = FVT, Pro-sham = probiotic sham.

Figure 3. Gut bacteriome analysis of A) the bacterial diversity (Shannon diversity index) and B) PCoA plots of the bacterial composition (Bray-Curtis dissimilarity) at baseline, after intervention, and termination. C) relative abundance of ASVs with significant differential abundance (p < 0.05) between FVT treated mice and mice not receiving FVT, summarized at genus level. Abbreviations: Lacticaseibacillus rhamnosus GG = LGG, Akkermansia muciniphila = AKM, fecal virome transplantation = FVT, Pro-sham = probiotic sham, ASV = amplicon sequence variant.

Figure 4. Gut virome analysis of A) the viral diversity (Shannon diversity index) and B) PCoA plots of the viral composition (Bray-Curtis dissimilarity) at baseline, after intervention, and termination. C) relative abundance of viral contigs with significant differential abundance (p < 0.05) between FVT treated mice and mice not receiving FVT, summarized at family level. Abbreviations: Lacticaseibacillus rhamnosus GG = LGG, Akkermansia muciniphila = AKM, fecal virome transplantation = FVT, Pro-sham = probiotic sham.

Figure 5. The ileum tissue was investigated for changes in the expression levels of genes associated to inflammatory responses at termination. In A) to M) the expression levels of these selected genes are shown, and the different treatment groups are compared with the control mice. The relative abundance of total number of T cells N) and cytotoxic T cells (CD8+ T cells) P) were measured. abbreviations: “*” = (p < 0.05), “.” = (p < 0.1), Lacticaseibacillus rhamnosus GG = LGG, Akkermansia muciniphila = AKM, fecal virome transplantation = FVT, Pro-sham = probiotic sham.

Figure 6. Bar plots of the fertility rate (number of pups) and pregnancy rate. A) The observed number of pups (born or as fetuses) based on a linear model (y~FVT+probiotics). B) The mean distribution of the of the binary event of pregnancy. Only the 23 female mice that received either FVT/Saline along with probiotic solutions of AKM/LGG/Pro-sham was included in the statistical analysis that was based on a generalized logistic regression model. Abbreviations: Lacticaseibacillus rhamnosus GG = LGG, Akkermansia muciniphila = AKM, fecal virome transplantation = FVT, Pro-sham = probiotic sham, “*” = (p < 0.05).

Data availability statement

Raw sequencing data can be accessed at ENA with project ID: PRJEB52388 (https://www.ebi.ac.uk/ena/browser/).