Colibactin targets the gut microbiota at the onset of intestinal colonization by genotoxic E. coli.
To understand whether the genotoxin colibactin may also target host gut microbiota, beyond the effects observed on host cells[7, 8], we applied a protocol of vertical mother-to-pups transmission (Supplementary Figure 1). Briefly, pregnant mothers were given by gavage either a non-genotoxic E. coli commensal strain (MG1655, phylogroup A, control group) or both MG1655 and the genotoxic E. coli SP15 strain (phylogroup B2, SP15clb+) or its non-genotoxic mutant (SP15clb-)). The molecular strategy applied to generate the two isogenic E. coli SP15 strains is reported in Supp.Fig.2. We analysed the overall putative changes in the gut microbiota of mice pups at 15 days after birth. The mother-to-pup transfer of the non-genotoxic E. coli SP15clb- strain was associated to a higher relative abundance of Proteobacteria. By contrast, the mother-to-pup transfer of the genotoxic E. coli SP15clb+ strain was associated to a higher relative abundance of family Lachnospiraceae (Fig.1A). Both the non-genotoxic and the genotoxic E. coli SP15 strains significantly affected the overall gut microbiota profile (Fig.1B). However, the overall gut microbiota diversity was unaffected, despite the significant reduction in the Menhininck index, regardless of genotoxicity of the E. coli SP15 strain (Fig.1C). Then, considering the putative antibiotic activity of colibactin[17], we focused on microbial taxa whose abudance was lower following the colonization with the genotoxic E. coli SP15 strain. As reported in Fig.2A-F, the phylum- Proteobacteria and all the other related taxa displayed a significantly lower abundance in mice colonized with the genotoxic E. coli SP15clb+ strain compared to the non-genotoxic E. coli SP15clb-. Next, we analysed the gut microbiome by performing a PICRUSt[18]-based functional analysis. As reported in Fig.3A-B, we identified microbial pathways significantly enriched in the control and in the group of mice pups co-colonized with the non-genotoxic E. coli SP15clb- strain, but not with group co-colonized with the genotoxic E. coli SP15clb+ strain. Overall, these data show that 15 days after birth E. coli genotoxic activity exerts an intra-species taxonomical but not functional impact on the gut microbiota.
Colibactin targets the gut microbiota and microbiome following intestinal colonization by genotoxic E. coli.
Then, to investigate whether the early impact of colibactin had long lasting consequences on gut microbiota composition, we analysed the overall putative changes in the gut microbiota of mice 35 days after birth. Differently from what observed above at day 15, the co-infection with the non-genotoxic E. coli SP15clb- strain was associated to a higher relative abundance of Firmicutes. By contrast, the co-infection with the genotoxic SP15clb+ strain was associated to a higher relative abundance of genus Alistipes and family Rikenellaceae (Fig.4A). A PCA showed a complete separation between the gut microbiota profile of mice co-colonized with the genotoxic E. coli SP15clb+ strain compared to the the other groups of mice (Fig.4B). In addition, the calculation of several diversity indices showed a precise cluster separation among the three gut microbiota profiles, with a general significant higher diversity induced by the genotoxic strain compared to the non-genotoxic one, except for the Berger-Parker index (Fig.4C). As for the reduction in microbial taxa the phylum Firmicutes and all the other related taxa displayed a significantly lower abundance in mice colonized with the genotoxic E. coli SP15clb+ strain compared to the non-genotoxic E. coli SP15clb- strain (Fig.5A-F). Next, we analysed the gut microbiome by performing a PICRUSt[18]-based functional analysis. As reported in Fig.6A-B, we identified microbial pathways significantly enriched in all the three groups. In detail, microbial pathways related to replication and repair, DNA repair and recombination proteins and DNA replication, among the totality identified, were found significantly enriched in the mice co-colonized with the genotoxic E. coli SP15clb+ strain (Fig.6B).
Overall, these data show that 35 days after birth, the E. coli genotoxic activity profoundly affect the gut microbiota at a both taxonomical and functional level and that it exerts an inter-species impact on the gut microbiota.
Evolution of gut microbiota and microbiome following intestinal colonization by genotoxic or non-genotoxic E. coli SP15 strain in mice.
Next, we evaluated the effects of the infection with the above reported E. coli strains on the evolution of gut microbiota and microbiome, by comparing the two time points at day 15 and day 35 after birth. On a taxonomical level, mice that received E. coli MG1655 strain displayed increased abundance of the family Lachnospiraceae at day 35 after birth (Suppl.Fig.3A); in terms of overall diversity, the infection with E. coli MG1655 did not induce a net separation between the two time points of 15 and 35 days (Suppl.Fig.3B), albeit some microbial functions were found significantly enriched (Suppl.Fig.3C). Mice that received the non-genotoxic E. coli SP15clb- strain displayed increased abundance of the phyla Firmicutes and Deferribacteres at day 35 after birth (Suppl.Fig.4A). The overall microbial diversity between the two time points of day 15 and day 35 was not affected (Suppl.Fig.4B), albeit some microbial functions were found significantly enriched (Suppl.Fig.4C). By contrast, mice that received the genotoxic E. coli SP15clb+ strain displayed increased level of the phyla Deferribacteres and Tenericutes at day 35 after birth (Suppl.Fig.5A) and a net microbial diversity separation between the two time points of dy 15 and day 35, with cellular processes related to signalling as the identified microbial function found significantly enriched (Suppl.Fig.5C). Overall, these data show that the genotoxic E. coli SP15clb+ strain affected the gut microbiota diversity to a greater extent compared to the other E. coli strains.