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Horwell et al. Microbiome Res Rep 2025;4:1 https://dx.doi.org/10.20517/mrr.2024.32 Page 9 of 20

urban dwellers are more likely to be born via CS, less likely to have been EBF, and more likely to have been
exposed to microbiome-altering medications (e.g., antibiotics) during childhood, making the distinction
[147]
between their rural counterparts more complicated .

It is, however, clear that when looking at the granular level, i.e., intranational, the geography of where
children live has significant impacts on the microbiome and health outcomes of children. Numerous
epidemiological studies have shown that children who grow up in rural locations have a significantly lower
probability of IBD, asthma, and diabetes in later life [148-150] . The mechanism of this association has only
recently started to become causally linked to the microbiome. As an example, one study looked at children
growing up near (< 500 m) rural environments and compared their microbiome to those in urban areas,
finding that the proximity to nature increases the abundance of Proteobacteria and Verrucomicrobiales,
[151]
genera known to be beneficial symbionts and from the microbial rich soil . These findings have been
[14]
replicated with children growing up on farms . A Finnish study found that children who grow up with this

rural microbiome have reduced inflammatory response to bacterial cell wall antigens, with reduced
expression of the pro-inflammatory cytokines IL-1β, IL-6, and IL-12 . Moreover, an elegantly designed
[152]
study has demonstrated a linear “dose response” to a child’s proximity to greenspaces, showing that infants
growing up in urban areas near a greenspace (e.g., a park) have a degree of protection to IBD, and as the
[153]
distance to urban greenspaces increases, this protective effect decreases .
The soil found in rural areas, particularly forest floors, appears to be a crucial reservoir for beneficial
microbial species that modulate the immune system, particularly in terms of atopic disease and IBD [154,155] .
[156]
Indeed, soil is the most microbially rich substrate to be described and the soil-dwelling species (e.g.,
Bacillus) have been identified as candidates for Rook’s old friends hypothesis . This theory posits that we
[157]
have co-evolved with environmental microbiomes typically encountered as forest hunter-gatherer
omnivores, and these microbial species drive immunoregulatory and metabolic pathways . Without these
[158]
microbial populations, our GIT has an increased risk of dysregulation and results in the chronic
inflammatory conditions often seen in the urbanised West. This does not explain the whole pathogenesis,
but the mounting evidence does suggest it at least explains part of it . Furthermore, animal studies have
[159]
shown that exposure to soil during early life impacts the metabolome of the microbiome, increasing
beneficial SCFAs, an effect that persists into adulthood . Our group has recently shown that exposure to a
[160]
strain of Bacillus velezensis, a prototypical soil dweller that produces bioactive cyclic-lipopeptides, can
modulate the immune system in a beneficial way that ameliorates acute ulcerative colitis . One group
[161]
from Finland has gone one step further and designed an interventional trial for children aged 3-5 growing
up in urban day care centres. These children were exposed to a specific rural microbiome by covering their
outdoor areas with soil transplanted from a forest. They found this modulated the children’s microbiome
with a particular increase in Proteobacteria, and noted an increase in plasma IL-10, a potent anti-
inflammatory cytokine . How these soil bacteria colonise the human GIT is poorly understood. It has
[162]
been hypothesised that we are exposed to them by eating unwashed food, and by direct contact with the soil
(e.g., playing in a field) [163,164] . The recently characterised aerobiome may also be responsible, at least in part,
for colonisation dynamics in infants. As air blows over the ground and vegetation, microbial species are
picked up and transported, and studies have shown significant differences in urban and rural aerobiomes,
with rural aerobiomes having a greater abundance particularly in Bacillus and Proteobacteria than urban
ones [165,166] . One group has experimentally demonstrated how exposure to a soil-based aerobiome might
modulate the microbiome . This might explain the linear proximity effect described earlier for children
[167]
living in cities but near greenspaces, although more research on this new science is needed.