Species-level verification of <i>Phascolarctobacterium</i> association with colorectal cancer

We have previously demonstrated an association between increased abundance of <i>Phascolarctobacterium</i> and colorectal cancer (CRC) and adenomas in two independent Norwegian cohorts. Here we seek to verify our previous findings using new cohorts and methods. In addition, we characterize lifestyle and sex specificity, the functional potential of the <i>Phascolarctobacterium</i> species, and their interaction with other microbial species. We analyze <i>Phascolarctobacterium</i> with 16S rRNA sequencing, shotgun metagenome sequencing, and species-specific qPCR, using 2350 samples from three Norwegian cohorts-CRCAhus, NORCCAP, and CRCbiome-and a large publicly available data set, curatedMetagenomicData. Using metagenome-assembled genomes from the CRCbiome study, we explore the genomic characteristics and functional potential of the <i>Phascolarctobacterium</i> pangenome. Three species of <i>Phascolarctobacterium</i> associated with adenoma/CRC were consistently detected by qPCR and sequencing. Positive associations with adenomas/CRC were verified for <i>Phascolarctobacterium succinatutens</i> and negative associations were shown for <i>Phascolarctobacterium faecium</i> and adenoma in curatedMetagenomicData. Men show a higher prevalence of <i>P. succinatutens</i> across cohorts. Co-occurrence among <i>Phascolarctobacterium</i> species was low (<6%). Each of the three species shows distinct microbial composition and forms distinct correlation networks with other bacterial taxa, although <i>Dialister invisus</i> was negatively correlated to all investigated <i>Phascolarctobacterium</i> species. Pangenome analyses showed <i>P. succinatutens</i> to be enriched for genes related to porphyrin metabolism and degradation of complex carbohydrates, whereas glycoside hydrolase enzyme 3 was specific to <i>P. faecium</i>.IMPORTANCEUntil now <i>Phascolarctobacterium</i> has been going under the radar as a CRC-associated genus despite having been noted, but overseen, as such for over a decade. We found not just one, but two species of <i>Phascolarctobacterium</i> to be associated with CRC-<i>Phascolarctobacterium succinatutens</i> was more abundant in adenoma/CRC, while <i>Phascolarctobacterium faecium</i> was less abundant in adenoma. Each of them represents distinct communities, constituted by specific microbial partners and metabolic capacities-and they rarely occur together in the same patients. We have verified that <i>P. succinatutens</i> is increased in adenoma and CRC and this species should be recognized among the most important CRC-associated bacteria.