Multiple indicators of gut dysbiosis predict all-cause and cause-specific mortality in solid organ transplant recipients

Objective Gut microbiome composition is associated with multiple diseases, but relatively little is known about its relationship with long-term outcome measures. While gut dysbiosis has been linked to mortality risk in the general population, the relation with overall survival in specific diseases has not been extensively studied. In the current study, we present in-depth analyses regarding the relationship between gut dysbiosis and all-cause and cause-specific mortality in the setting of solid organ transplant recipients (SOTR). Design We analyzed 1,337 metagenomes derived from fecal samples of 766 kidney, 334 liver, 170 lung and 67 heart transplant recipients from the TransplantLines Biobank and Cohort; a prospective cohort study including extensive phenotype data with 6.5 years of follow up. To quantify gut dysbiosis, we included additional 8,208 metagenomic samples from a general population from the same geographical location. Multivariable Cox regression and a machine learning algorithm were used to analyze the association of indicators of gut dysbiosis and species abundances, with all-cause and cause-specific mortality. Results We identified two patterns representing overall microbiome community variation that were associated with both all-cause and cause specific mortality. Gut microbial distance to the average of the general population was associated with all-cause mortality and infection-, malignancy- and cardiovascular disease related mortality. Using multivariable Cox regression, we identified 23 species that were associated with all-cause mortality. By using a machine learning algorithm, we identified a log-ratio of 19 species predictive of all-cause mortality, all of which were also independently associated in the multivariable Cox-regression analysis. Conclusion Gut dysbiosis is consistently associated with mortality in SOTR. Our results support the observations that gut dysbiosis is predictive of long-term survival. Since our data do not provide causative evidence, further research needs to be done to see determine whether gut-microbiome targeting therapies might improve long term outcomes What is already known on this subject? What are the new findings? ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement We would like to thank all participants from the TransplantLines- and Lifelines cohort and biobank study. We would like to thank the Center for Information Technology of the University of Groningen (RUG) for support and for providing access to the Peregrine high-performance computing cluster and the Genomic Coordination Center (UMCG and RUG) for support and for providing access to Calculon and Boxy high-performance computing clusters. The TransplantLines Biobank and Cohort study received funding from Astellas BV (TransplantLines Biobank and Cohort study) and Chiesi Pharmaceuticals BV (PA-SP/PRJ-2020-9136) and was co-financed by the Dutch Ministry of Economic Affairs and Climate Policy by means of the PPP-allowance made available by the Top Sector Life Sciences & Health to stimulate public-private partnerships. Sequencing of the kidney part of the TransplantLines cohort was funded by a grant from the Dutch NWO/TTW/DSM partnership program Animal Nutrition and Health (project number 14939) to S.J.L.B. R.K.W. is supported by the Seerave Foundation, the Netherlands Organization for Scientific Research (NWO), and the EU Horizon Europe Program grant miGut-Health: personalized blueprint of intestinal health (101095470). ### 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: All solid organ transplantation recipients (SOTR) cross-sectional gut microbiome data from the TransplantLines Biobank and Cohort study (Trial registration number [NCT03272841][1]) was included. The TransplantLines study has been previously described in detail and aimed to include all potential adult solid organ transplant recipients and kidney donors at the University Medical Center Groningen (UMCG), The Netherlands, starting from June 2015. We included 1337 fecal samples from SOTR. 8,208 subjects from the Dutch Microbiome Project were included to quantify the extent of dysbiosis and per species dysbiosis analysis. All participants signed an informed consent form prior to sample collection. TransplantLines (METc 2014/077) and Lifelines (METc 2017/152) were approved by the local institutional ethics review board (IRB) from the UMCG. Both studies adhere to the UMCG Biobank Regulation and are in accordance with the World Medical Association (WMA) Declaration of Helsinki and the Declaration of Istanbul. 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 The raw microbiome sequencing data and basic phenotypes used in this study are available at the European Genome-Phenome Archive under accession numbers EGAD00001008907 (), EGAS00001006257 () and EGAS00001006258 (). Due to patient confidentiality, the clinical datasets associated with the metagenomic datasets are available upon request to the University Medical Centre Groningen. Access to this clinical dataset requires a minimal access procedure consisting of a request per email (datarequest.transplantlines{at}umcg.nl) for a data access form. A response will be provided within two working weeks. This access procedure is to ensure that the data are being requested for research/scientific purposes only and thus complies with the informed consent signed by TransplantLines participants, which specifies that the collected data will not be used for commercial purposes. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT03272841&atom=%2Fmedrxiv%2Fearly%2F2023%2F10%2F29%2F2023.10.28.23297709.atom