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Mueller et al. Microbiome Res Rep 2024;3:33 https://dx.doi.org/10.20517/mrr.2024.09 Page 11 of 18

isolate their sequences clustered. We then compared the predicted phylogroups to the known species in
those same samples. Out of 34 samples with both 16S rRNA sequencing data and an isolated strain, the
predicted and isolated species matched in 10 samples. In 21 samples, an additional phylogroup was
identified. While there may be more than one Akkermansia strain present in the original stool, we mostly
isolated one strain from any one sample and the predicted phylogroups from 16S rRNA gene sequences
[20]
matched in 31/34 (91%) of cases. Overall, our validation of both 16S rRNA and metagenomic analysis
suggests a high accuracy in identifying the most prevalent human Akkermansia species.

Correlations between Akkermansia species and health in metagenomic studies
We reanalyzed metagenomic datasets of patients with obesity, inflammatory bowel disease, and
responsiveness to cancer immunotherapies - instances where Akkermansia has been reported to influence
health - to determine if there are correlations between specific species of Akkermansia and the health status
of their host. Among all the samples analyzed where Akkermansia sequences were detectable, we found
A. muciniphila to be the most prevalent species (354/1,088, 32.54%), followed by A. massiliensis (11.86%)
and A. biwaensis (5.15%). In some of those samples (78/1,088, 7.17%), more than one Akkermansia was
present. Most samples (648/1,088, 59.56%) did not have measurable levels of Akkermansia.

There is a negative correlation between Akkermansia abundance and obesity in adult humans and mouse
models of diet-induced obesity [3-5,7,71] . We performed Mann-Whitney U tests to compare the relative
abundances of A. muciniphila, A. massiliensis, or A. biwaensis between children with obesity (case) and
control samples from this same dataset [Figure 3B]. There was no significant difference in the relative
abundance of A. muciniphila (U = 6,971, P = 0.1569) or A. massiliensis between cases and controls
(U = 7,367, P = 0.3087). There was a significant difference in A. biwaensis between case and control samples
(U = 7,262, P = 0.0368); however, the low prevalence of A. biwaensis (8.16% of control samples and only
2.22% of case samples had detectable levels of A. biwaensis) limits the power of such analysis.

Akkermansia abundance has been linked to varying risk for IBD. Akkermansia has been found to be
depleted in adult human cases of Crohn’s disease (CD) and ulcerative colitis (UC), while other studies have
found Akkermansia to only be depleted in the case of CD (in pediatric patients), or to be associated with
responsiveness to treatment rather than with disease occurrence [31,72-74] . Other multi-omics studies have
found no significant association between Akkermansia and IBD [43,44] . We combined three of the largest
adult-IBD metagenomic studies [42-44] and processed them to make Akkermansia species and subspecies
assignments. In this combined dataset, 34.64% of samples (124/358) contained detectable levels of
Akkermansia.

Kruskal-Wallis H tests were performed to compare the relative abundances of A. muciniphila,
A. massiliensis, or A. biwaensis between CD, UC, and control samples [Figure 3C], indicating that there is a
statistically significant difference in the relative abundance of A. muciniphila between disease groups
[H(3) = 20.08, P < 0.0001]. Pairwise comparisons using Dunn’s multiple comparisons test indicated that
A. muciniphila is significantly decreased in the UC group relative to healthy controls (control mean
rank = 207.1, UC mean rank = 157.8, P < 0.0001). Similarly, there is a significant difference in A. massiliensis
relative abundance between groups [H(3) = 10.30, P = 0.0058]. Pairwise comparisons did not yield any
statistically significant results, possibly due to the low prevalence of A. massiliensis in all groups (16.46% in
controls, 8.66% in CD, and 8.33% in UC). We also found no significant difference in A. biwaensis relative
abundance between groups [H(3) = 5.075, P = 0.0791].