Effect of bacterial DNA enrichment on detection and quantification of bacteria in an infected tissue model by metagenomic next-generation sequencing

Before implementing metagenomic next-generation sequencing (mNGS) in the routine diagnostic laboratory, several challenges need to be resolved. To address strengths and limitations of mNGS in bacterial detection and quantification in samples with overwhelming host DNA abundance, we used the pig muscle tissue spiked with a home-made bacterial mock community, consisting of four species from different phyla. From the spiked tissue, we extracted DNA using: (i) a procedure based on mechanical/chemical lysis (no bacterial DNA enrichment); (ii) the Ultra-Deep Microbiome Prep (Molzym) kit for bacterial DNA enrichment; and (iii) the same enrichment kit but replacing the original proteinase K treatment for tissue solubilization by a collagenases/thermolysin digestion and cell filtration. Following mNGS, we determined bacterial: ‘host’ read ratios and taxonomic abundance profiles. We calculated the load of each mock-community member by combining its read counts with read counts and microscopically-determined cell counts of other co-spiked bacteria. In unenriched samples, bacterial quantification and taxonomic profiling were fairly accurate but at the expense of the sensitivity of detection. The removal of ‘host’ DNA by the modified enrichment protocol substantially improved bacterial detection in comparison to the other two extraction procedures and generated less distorted taxonomic profiles as compared to the original enrichment protocol.