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Microbes interact in natural communities in a spatially structured manner, particularly in biofilms and polymicrobial infections. While next generation sequencing approaches provide powerful insights into diversity, metabolic capacity, and mutational profiles of these communities, they generally fail to recover in situ spatial proximity between distinct genotypes in the interactome. Hi-C is a promising method that has assisted in analysing complex microbiomes, by creating chromatin cross-links in cells, that aid in identifying adjacent DNA, to improve de novo assembly. This study explored a modified Hi-C approach involving an initial lysis phase prior to DNA cross-linking, to test whether adjacent cell chromatin can be cross-linked, anticipating that this could provide a new avenue for study of spatial-mutational dynamics in structured microbial communities. An artificial polymicrobial mixture of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli was lysed for 1-18 h, then prepared for Hi-C. A murine biofilm infection model was treated with sonication, mechanical lysis, or chemical lysis before Hi-C. Bioinformatic analyses of resulting Hi-C interspecies chromatin links showed that while microbial species differed from one another, generally lysis significantly increased links between species and increased the distance of Hi-C links within species, while also increasing novel plasmid-chromosome links. The success of this modified lysis-Hi-C protocol in creating extracellular DNA links is a promising first step toward a new lysis-Hi-C based method to recover genotypic microgeography in polymicrobial communities, with potential future applications in diseases with localized resistance, such as cystic fibrosis lung infections and chronic diabetic ulcers., (© 2021 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)