Microbes interact in biofilms and polymicrobial infections in a spatially structured manner, yet next-generation sequencing approaches generally fail to recover in situ spatial proximity between distinct genotypes in the interactome or eDNA matrix. 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 the 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 to 18 hours, 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.

These data derive from a novel application of Hi-C which tests the effects of lysis prior to formaldehyde crosslinking to assess whether Hi-C links can form between species in either (1) mixed-species cell communities prepared, by culturing and partly drying cells of Pseudomonas aeruginosa, Staphylococcus aureus, and E. coli in a thin layer then treating with no lysis (controls) "CNL" or with either 1 hr "CSL" or 18 hr of lysis "CLL" with application of Igepal lysing buffer prior to formaldehyde crosslinking or (2) mouse wound biofilms prepared by inoculating dermal wound models with cultured microbes (Pseudomonas aeruginosa and Staphylococcus aureus) and collecting resulting biofilm+skin after 5 days, then treating with brief sonication (controls) "MS" or with mechanical lysis "MML" or mechanical lysis followed by 18 hr of lysis with Igepal lysing buffer "MLL" prior to formaldehyde crosslinking. After crosslinking, Hi-C libraries were prepared by standard protocols except with several modifications to precipitate and preserve eDNA. After Illumina sequencing of Hi-C libraries, data were analyzed following a modified bioinformatic pipeline using the HiC-Pro software. The output "raw" data deposited are the HiC-Pro Matrix (non-normalized) for links between self (same species) and non-self (other species) or plasmid. The corresponding code is attached.

HiC-Pro matrix link data filenames associated with this entry:

CNL = cells no lysis

CSL = cells short lysis

CLL = cells long lysis

MS = mouse sonication

MML = mouse mechanical disruption

MLL = mouse long lysis