Species-level verification of Phascolarctobacterium association to colorectal cancer

Background and aims: We have previously demonstrated an association between increased abundance of Phascolarctobacterium 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 Phascolarctobacterium species and their interaction with other microbial species. Methods: We analyze Phascolarctobacterium 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 dataset, Curatedmetagenomedata. Using metagenome assembled genomes from the CRCbiome study we explore genomic characteristics and functional potential of the Phascolarctobacterium pangenome. Results: Three species of Phascolarctobacterium associated with adenoma/CRC were consistently detected by qPCR and sequencing. Positive associations with adenomas/CRC were verified for P. succinatutens and negative associations were shown for P. faecium and adenoma in Curatedmetagenomedata. Men show higher prevalence of P. succinatutens across cohorts. Co-occurrence among Phascolarctobacterium species was low (<6%). Each of the three species show distinct microbial composition and form distinct correlation networks with other bacterial taxa, although Dialister invisus was negatively correlated to all investigated Phascolarctobacterium species. Pangenome analyses showed P. succinatutens to be enriched for genes related to porphyrin metabolism and degradation of complex carbohydrates, whereas glycoside hydrolase enzyme 3 was specific to P. faecium. Conclusion: We have verified that P. succinatutens is increased in adenoma and CRC and this species should therefore be recognised among the most important CRC-associated bacteria. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was funded by the South-Eastern Norway Regional Health Authority (project number 2020056 and 2022067), Oslo Metropolitan University (project number 202401) and Akershus University Hospital. The CRCbiome study was funded by grants from the Norwegian Cancer Society (project number 190179 and 198048). Sequencing of the NORCCAP samples was funded by the Cancer Registry of Norway funds. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The CRCAhus study, BCSN trial, the CRCbiome study and the NORCCAP trial have been approved by the Regional Committee for Medical and Health-related Research Ethics in Southeast Norway (REK ref: 2012/1944, 2011/1272, 63148, and 22337 respectively). The CRCAhus study also received approval from the data protection manager at Akershus University Hospital. The BCSN trial is registered at clinicaltrials.gov (Clinical Trial (NCT) no.: 01538550). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data from the CRCbiome project have been deposited in the database Federated EGA under accession code EGAS50000000170 (https://ega-archive.org/studies/EGAS50000000170) and the Curatedmetagenomedata is available here: https://waldronlab.io/curatedMetagenomicData/index.html. Due to the sensitive nature of the data derived from human subjects, including personal health information, analyses and sharing of data from cohorts in this project must comply with the General Data Protection Regulation (GDPR). Data processors must have approval from the Regional Committee for Medical Research in Norway (REC), legal basis according to GDPR Article 6 and 9 and the need for a Data Protection Impact Assessment (DPIA) according to GDPR article 35 must be considered. Requests for data access can be directed to corresponding author Trine B Rounge. The custom R scripts used in this study are available at: https://github.com/Rounge-lab/Phascolarctobacterium_CRC.