Highly Multiplexed Spatial Mapping of Microbial Communities

Mapping the complex biogeography of microbial communities in situ with high taxonomic and spatial resolution poses a major challenge because of the high density(1) and rich diversity(2) of species in environmental microbiomes and the limitations of optical imaging technology(3–6). Here, we introduce High Phylogenetic Resolution microbiome mapping by Fluorescence in situ Hybridization (HiPR-FISH), a versatile technology that uses binary encoding, spectral imaging, and machine learning based decoding to create micron-scale maps of the locations and identities of hundreds of microbial species in complex communities. We demonstrate the ability of 10-bit HiPR-FISH to distinguish 1023 E. coli isolates, each fluorescently labeled with a unique binary barcode. HiPR-FISH, in conjunction with custom algorithms for automated probe design and single-cell image analysis, reveals the disruption of spatial networks in the mouse gut microbiome in response to antibiotic treatment and the longitudinal stability of spatial architectures in the human oral plaque microbiome. Combined with super-resolution imaging, HiPR-FISH reveals the diverse ribosome organization strategies of human oral microbial taxa. HiPR-FISH provides a framework for analyzing the spatial ecology of environmental microbial communities at single-cell resolution.