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Mueller et al. Microbiome Res Rep 2024;3:33 https://dx.doi.org/10.20517/mrr.2024.09 Page 15 of 18

phylogroup is most strongly associated with the health benefit of interest. Additionally, if one or more
phylogroups of Akkermansia are determined to be negatively associated with health outcomes in a
particular context, knowing what strains to avoid during therapeutic development will decrease the risk of
failure or development of adverse effects.

Our study presents certain limitations. First, the novel species described in our analysis of the Akkermansia
pangenome are relatively rare in the human populations we have characterized and appear to be present in
lower abundance than other Akkermansia species. There are currently only five isolates of A. ignis and two
of A. durhamii. Two strains, BAA-2860 and CSUN-56, may also represent novel species. Prescreening of
MAGs generated from metagenomic sequencing of fresh stool samples would allow for more efficient,
targeted isolation of these rare species. The functional characterization of Akkermansia strain by standard
methods like the API 20A system is of limited use as the composition of the assay medium is not compatible
with culturing Akkermansia. Indeed, the species identification table provided by bioMérieux does not
include Akkermansia. There are also some limitations to the use of StrainR in reliably distinguishing
between the A. muciniphila AmIa and AmIb subspecies.

Finally, the rarity of the non-A. muciniphila species can decrease the statistical power of any associations
between these species and human health conditions. As illustrated in cases of pediatric obesity, the
proportion of individuals with a detectable level of A. biwaensis was low, which, compounded with the low
prevalence of these new species compared to A. muciniphila, makes determining relevant disease
associations more challenging. Thus, much larger datasets will be necessary to properly investigate the
importance of these new Akkermansia species to human health.

DECLARATIONS
Authors’ contributions
Conception, data acquisition, data analysis and interpretation, and writing: Mueller KD
Akkermansia isolation and genome sequencing: Davey L, Panzetta ME
Research guidance and manuscript revision: Valdivia RH
Manuscript revision: Rawls JF, McCann JR, Flores GE

Availability of data and materials
Genomes retrieved from NCBI and their accession numbers are listed in Supplementary Table 1. 16S and
metagenomic datasets for method validation were provided by the POMMS . Metagenomic sequencing
[40]
samples case studies were obtained from the SRA using Bioproject accessions PRJNA398089,
PRJNA400072, PRJEB42151, PRJEB42155, PRJNA751792, and PRJNA782662 [41-44] . Sequences for the
Akkermansia durhamii isolates have been deposited in GenBank under BioProject accession number
PRJNA1066260.

Financial support and sponsorship
The work described here was supported by NIH grants (AI42376 to R.H.V and R24-DK110492 to Rawls JF).
Additional support from the HHMI EPI program to Valdivia RH and Flores GE was provided by the
National Institutes of Health through the National Institute of General Medical Sciences (NIGMS) (grant
number SC1GM136546). Mueller KD was partially supported by the National Science Foundation under
Grant Number DGE 1545220.

Conflicts of interest
Valdivia RH is a co-founder of Bloom Sciences (San Diego, CA). The company was not involved in
sponsoring or analyzing and interpreting the data presented. Other authors declared that there are no
conflicts of interest.