Gaun, N. | bioRxiv Science

bioRxiv2025

Micro-scale spatial metagenomics: revealing high-resolution spatial biogeography of gut microbiomes

Spatial organisation is a fundamental yet poorly resolved aspect of gut microbial ecology. Conventional shotgun metagenomics provides rich functional information but relies on homogenised, macro-scale samples that obscure the micron-scale distributions critical for understanding microbial community dynamics. Here, we introduce Micro-Scale Spatial Metagenomics (MSSM), a new methodological framework that couples laser micro-dissection of tissue sections, ultra-low-input library preparation, and genome-resolved bioinformatics to reconstruct microbial communities from intestinal microsamples measuring as little as [~]500 {micro}m{superscript 2} ({approx}100 bacterial cells). We describe a fully optimised laboratory and computational pipeline that enables quantitative, strain-resolved, and functionally informed spatial profiling directly from intact gut tissue. Using chicken intestinal samples, we validated MSSM through combinatorial single-cell fluorescence in situ hybridisation (FISH) imaging and comparisons with macro-scale metagenomics, demonstrating its robustness and accuracy. MSSM captured fine-scale heterogeneity in taxonomic and functional composition across intestinal cryosections, hinting at spatially structured assemblages and segregation of metabolic capacities. Strain-level analyses uncovered coexisting Lawsonibacter lineages exhibiting distinct spatial distributions and host-specific occurrence patterns, while SNP-level microdiversity analyses showed that genetically coherent clonal populations cluster at spatial scales below [~]200 {micro}m. By enabling shotgun metagenomics at micron resolution, MSSM closes a longstanding methodological gap and provides a scalable platform for studying microbial ecosystems in situ. This approach unlocks a previously inaccessible view of microbial biogeography, offering new opportunities to investigate host-microbe and microbe-microbe interactions, and the spatial principles governing gut ecosystems. Significance statementUnderstanding how microbial communities are organised in space is essential to explaining their ecological and functional roles, yet microbiome research still relies overwhelmingly on bulk, spatially averaged measurements. We introduce micro-scale spatial metagenomics (MSSM), the first method that brings shotgun metagenomics to the microscale, enabling direct measurement of functional and taxonomic variation across regions containing as few as [~]100 cells. Unlike existing spatial approaches, MSSM reconstructs complete genomes and resolves strain-level diversity within intact tissue, allowing researchers to map metabolic potential, microdiversity, and community structure in situ. By coupling high-resolution sequencing with spatial context, MSSM reveals a previously inaccessible layer of microbial organisation, transforming how host-associated ecosystems can be studied.

microbiology↗

bioRxiv2024

Dietary intervention in captive-bred hares fails to enrich gut microbiomes with wild-like functions

Reintroducing captive-bred animals into the wild often faces limited success, with the underlying causes frequently unclear. One emerging hypothesis is that maladapted gut microbiota may play a significant role in these challenges. To investigate this possibility, we employed genome-resolved metagenomics to analyse the taxonomic and functional differences in the gut microbiota of wild and captive European hares (Lepus europaeus), as well as to assess the impact of a dietary switch to grass aimed at pre-adapting captive hares to wild conditions. Our analyses recovered 860 metagenome-assembled genomes, with 87% of them representing novel species. We found significant taxonomic and functional differences between the gut microbiota of wild and captive hares, notably the absence of Spirochaetota in captive animals and differences in amino acid and sugar degradation capacities. While the dietary switch to grass induced some minor changes in the gut microbiota, it did not result in a shift towards a more wild-like microbial community. The increased capacity for degrading amino acids and specific sugars observed in wild hares suggest that, instead of bulk grass, dietary interventions tailored to their specific dietary preferences might be necessary for pre-adapting hare gut microbiota to wild conditions. ImportanceThis study sheds light on the critical role of gut microbiota in the success of reintroducing captive-bred animals into the wild. By comparing the gut microbiota of wild and captive European hares, we identified significant taxonomic and functional differences, including the absence of key microbial groups in captive hares. Dietary interventions, such as switching to grass, showed limited success in restoring a wild-like microbiota, highlighting the need for tailored approaches to mimic natural diets. With 87% of recovered microbial genomes representing novel species, this research also enriches our understanding of microbial diversity in wildlife. These findings emphasise that maladapted gut microbiota may hinder the survival and adaptation of reintroduced animals, suggesting that microbiome-targeted strategies could improve conservation efforts and the success of animal rewilding programs.

ecology↗