Bacterial DNA on the skin surface overrepresents the viable skin microbiome

Abstract

The skin microbiome provides vital contributions to human health. However, the spatial organization and viability of its bacterial components remain unclear. Here, we apply culturing, imaging, and molecular approaches to human and mouse skin samples, and find that the skin surface is colonized by fewer viable bacteria than predicted by bacterial DNA levels. Instead, viable skin-associated bacteria are predominantly located in hair follicles and other cutaneous invaginations. Furthermore, we show that the skin microbiome has a uniquely low fraction of viable bacteria compared to other human microbiome sites, indicating that most bacterial DNA on the skin surface is not associated with viable cells Additionally, a small number of bacterial families dominate each skin site and traditional sequencing methods overestimate both the richness and diversity of the skin microbiome. Finally, we performed an in vivo skin microbiome perturbation-recovery study using human volunteers. Bacterial 16S rRNA gene sequencing revealed that, while the skin microbiome is remarkably stable even in the wake of aggressive perturbation, repopulation of the skin surface is driven by the underlying viable population. Our findings help explain the dynamics of skin microbiome perturbation as bacterial DNA on the skin surface can be transiently perturbed but is replenished by a stable underlying viable population. These results address multiple outstanding questions in skin microbiome biology with significant implications for future efforts to study and manipulate it.

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“…This stands in contrast to the recent findings reported by Acosta et al that bacterial DNA on the skin surface overrepresents the viable skin microbiome. 27 Once activated PMA and its close derivative PMAxx are highly reactive nitrene compounds that irreversibly bind to double stranded DNA and prevent it from being amplified or sequenced. 34,35 However, too high a concentration of PMA or PMAxx for the sample biomass can result in false negatives, either due to being toxic to live cells or PMA/PMAxx remaining active beyond the duration of photoactivation and subsequent interaction with DNA released during the extraction process from live cells.…”

“…Together, Figure 2B-C highlight the importance of identifying the appropriate PMAxx concentration for the intended study community type and contradict the recent claim that under normal circumstances the majority of bacterial DNA on the skin are from diseased microbes. 27 Across all experiments and sampled body sites, the percentage of live bacteria on unperturbed skin ranges from 43-86% for dry body sites (95% confidence interval of 58-65%) and from 43-99% for moist sites (95% confidence interval of 70-76%; Table S2, Fig. 2B-C, Fig.…”

“…To selectively evaluate DNA from live microorganisms within the skin microbiome pre-and post-antiseptic exposure, we optimized a propidium monoazide (PMAxx) based viability assay. Previous work has used PMAxx to estimate the viable portion of the skin microbiome, 27 concluding that viable bacterial cells are very low. Here, we show that the concentrations of PMAxx previously reported are toxic towards live bacteria.…”