Abstract
BACKGROUND: Although the gut microbiome plays a crucial role for maintaining overall host homeostasis and metabolism, it is significantly influenced by dietary changes, leading to substantial temporal variations in microbial composition within and between individuals. Despite this, incidental fecal sampling remains the standard method for microbiome assessment. Recently, the blood microbiome, defined by microbial DNA (cmDNA) circulating in the bloodstream, has emerged as a potentially more stable and integrated alternative. Preliminary data suggest that blood microbiome analysis may offer more consistent insights than fecal-based approaches, although the methodological validity of the approach has been questioned. METHOD/DESIGN: This study aims to establish or rule-out cmDNA as a representative of the gut microbiome. In a prospective, single-arm crossover trial, effects of dairy product withdrawal and reintroduction of a yoghurt with a known consortium of bacteria will be assessed in healthy volunteers aged 18-65. Participants will first abstain from all dairy products, a phase expected to reduce yogurt-associated cmDNA in the bloodstream. Yogurt will then be reintroduced, during which reappearance of cmDNA of specific bacteria (especially LGG, LA-5 and BB-12) is anticipated. Shotgun metagenomic sequencing will be used to track cmDNA dynamics over time. This longitudinal sampling approach will provide experimental evidence supporting the existence and responsiveness of the circulating microbiome, while also revalidating the bioinformatic pipeline used for its analysis. CONCLUSION: This pilot study will test whether blood-derived microbial DNA can serve as a valid surrogate for gut microbiome composition. If successful, this approach may provide a more stable and integrative alternative to fecal sampling and support future biomarker development and mechanistic research. CLINICAL TRIAL REGISTRATION: NCT06944002.