Dead or Alive? Molecular life-dead distinction in human stool samples reveals significantly different composition of the microbial community

The gut microbiome is strongly interwoven with human health. To analyse the composition of the gut microbiome, stool samples are subjected to 16S rRNA gene targeting next generation sequencing (NGS) and results are correlated with clinical parameters. However, some microorganisms might not be alive at the time point of sampling, and thus could have less impact on the human. As conventional NGS methods lack the opportunity to differentiate between viable and dead microbial components, retrieved results give only limited information. Propidium monoazide (PMA) is used in food safety monitoring to discriminate living from dead cells. It binds to free DNA and masks it for subsequent procedures, such as PCR reaction and sequencing. Our goal was to show the impact of PMA on the results of 16S rRNA gene-targeting NGS from human stool samples, and to determine the optimal concentration and protocol.We treated fresh stool samples with a concentration series (0-300 µM PMA) and subjected the samples to NGS. The results show that substantial part of the human microbial community is dead at the time point of sampling, and PMA treatment significantly reduces the diversity and richness of the sample. Concentrations of 100 µM PMA were sufficient to discriminate signals from disrupted microbial cells. The application of PMA in complex microbiome samples is possible, if the PMA concentration is adapted accordingly. We prove, that the classical NGS approach, compared to our PMA-NGS, reveals significant differences in microbial composition and the abundance of certain important microorganisms (i.e. Akkermansia, Bacteroides).