Benchmarking gut microbiome health indices by their sensitivity to subclinical risk in 12,561 adults

ABSTRACT A growing number of indices claim to summarize gut “microbiome health,” yet they are routinely treated as interchangeable, even though their relative sensitivity to host risk remains undefined. In 12,561 adults from the American Gut Project (16S rRNA gene profiles), we benchmarked five indices spanning distinct constructs—taxonomic composition (Gut Microbiome Wellness Index 2 [GMWI2] and ZOE), diversity (Shannon), functional redundancy index (FRI), and reference-based structural deviation (microbiome deviation index [MDI])—along a cumulative lifestyle-risk gradient and against four chronic disease clusters. Shannon diversity, abundance-weighted ZOE, and MDI each shifted dose-dependently from the earliest risk increments (Jonckheere–Terpstra P < 0.001), and pathway-level composites moved in the expected directions; FRI and GMWI2 did not. Disease associations were index specific and cluster specific: Shannon (protective direction) and ZOE (risk direction) were associated with all four clusters, MDI with metabolic/systemic disease, and FRI with gastrointestinal disease, whereas GMWI2 showed no association throughout. The five indices were only moderately intercorrelated (Spearman | ρ | ≤0.52). Shannon and ZOE—the most consistent performers—showed only modest effects (per standard deviation, odds ratios 0.84–0.94 and 1.08–1.22, respectively), and neither captured the structural-deviation signal indexed by MDI. Associations between environmental stressors (including PM 10 , heptachlor, and ozone) and microbiome outcomes were concentrated in higher-risk hosts, consistent with host vulnerability gating the microbiome’s environmental stress response. Together, these results show that widely used microbiome health indices are not interchangeable: they detect overlapping but non-identical facets of dysbiosis, and their sensitivity to subclinical risk depends on index formulation and data resolution. An interpretable, complementary panel rather than any single score may therefore best support early stratification. IMPORTANCE Gut microbiome “health” is increasingly summarized using single numerical scores, yet these scores are often treated as equivalent despite measuring different biological features. By comparing five widely used indices in 12,561 adults, we show that they differ substantially in their ability to detect early, subclinical changes associated with cumulative lifestyle risk. Measures of microbial diversity, abundance-weighted taxonomic composition, and deviation from a healthy reference changed progressively from the earliest risk increments, whereas a functional redundancy measure and a presence/absence-based taxonomic index were comparatively insensitive, and pairwise correlations between indices were only moderate (| ρ | ≤0.52). We also found that associations between environmental pollutants and microbiome disruption were strongest among individuals with higher baseline lifestyle risk. These findings argue against relying on a single universal microbiome health score and support the use of complementary indices for early risk stratification and future microbiome-based prevention research.