A novel framework for assessing causal effect of microbiome on health: long-term antibiotic usage as pseudo-randomizer

Gut microbiome (MB) has been widely shown to affect human health. Since MB in turn can be altered by various exposures, such as diet and medications, it holds immense potential for future treatments and healthy ageing. On the one hand, faecal microbiota transplantation and Mendelian Randomization have proven a causal link between treatment, MB and diseases. On the other hand, assessing the causality of the MB effects on health has remained challenging, since randomised trials in human subjects are often unethical or difficult to pursue, and Mendelian Randomization lacks valid instruments. Thus, novel analytical approaches are needed for inferring causal associations. To overcome these barriers, we propose a novel framework of antibiotic instrumental variable regression (AB-IVR) for estimating the causal relationships between MB and various diseases. Our inspiration originates from the popular Mendelian Randomization method that uses genetic mutations as instruments in the instrumental variable analysis (IVR). Further, we rely on the recently shown results that antibiotic (AB) treatment has a cumulative long-term effect on MB, consequently pseudo-randomizing individuals with higher AB usage to have more perturbed MB. Thus, we developed a new AB-IVR framework to utilise the long-term AB usage as an instrument in the IVR for assessing the causal effect of MB on health. We pursued a plethora of sensitivity analyses to explore the properties of our method: varying the sample’s age group and maximum number of AB used; using a buffer-time for incident disease outcomes to account for feedback-mechanism; using subgroups of AB as instrument; and simulating data for disease outcomes. We detected several interesting causal effects of MB on health outcomes; some causal effects – such as MB effects on migraine, depression, irritable bowel syndrome, and several more – remain significant irrespective of the sensitivity analysis used. We believe that our AB-IVR framework has promising potential to be the new widely used method for assessing MB effect on health. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was funded by Estonian Research Council grants (PRG1414 to E.O., N.T. and O.A. and PRG1197 to K.F.) and an EMBO Installation grant (No. 3573 to E.O., N.T. and O.A.). ### 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 participants included in the EstBB cohort provided informed consent for the data and samples to be used for scientific purposes. This study was approved by the Research Ethics Committee of the University of Tartu (approval No. 266/T10) and by the Estonian Committee on Bioethics and Human Research (Estonian Ministry of Social Affairs; approval No. 1.1-12/17 and 1.1-12/624). All participants have joined the Estonian Biobank on a voluntary basis and have signed a broad consent form, which allows to receive participant's personal and health data from national registries and databases. Rights of gene donors are regulated by Human Genes Research Act (HGRA) paragraph 9 - Voluntary nature of gene donation (). 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 metagenomic data generated in this study have been deposited in the European Genome-Phenome Archive database () under accession code EGAS00001008448. The phenotype data contain sensitive information from healthcare registers and they are available under restricted access through the Estonian biobank upon submission of a research plan and signing a data transfer agreement. All data access to the Estonian Biobank must follow the informed consent regulations of the Estonian Committee on Bioethics and Human Research, which are clearly described in the Data Access section at . A preliminary request for raw metagenome and phenotype data must first be submitted via the email address releases{at}ut.ee All participants included in the EstBB cohort provided informed consent for the data and samples to be used for scientific purposes. This study was approved by the Research Ethics Committee of the University of Tartu (approval No. 266/T10) and by the Estonian Committee on Bioethics and Human Research (Estonian Ministry of Social Affairs; approval No. 1.1-12/17 and 1.1-12/624). All participants have joined the Estonian Biobank on a voluntary basis and have signed a broad consent form, which allows to receive participant’s personal and health data from national registries and databases. Rights of gene donors are regulated by Human Genes Research Act (HGRA) § 9 – Voluntary nature of gene donation ( current).