Pre-digest of unprotected DNA by Benzonase improves the representation of living skin bacteria and efficiently depletes host DNA

Abstract

Background: The identification of microbiota based on next-generation sequencing (NGS) of extracted DNA has drastically improved our understanding of the role of microbial communities in health and disease. However, DNA-based microbiome analysis cannot per se differentiate between living and dead microorganisms. In environments such as the skin, host defense mechanisms including antimicrobial peptides and low cutaneous pH result in a high microbial turnover, likely resulting in high numbers of dead cells present and releasing substantial amounts of microbial DNA. NGS analyses may thus lead to inaccurate estimations of microbiome structures and consequently functional capacities.

Results: We investigated in this study the feasibility of a Benzonase-based approach (BDA) to pre-digest unprotected DNA, i.e., of dead microbial cells, as a method to overcome these limitations, thus offering a more accurate assessment of the living microbiome. A skin mock community as well as skin microbiome samples were analyzed using 16S rRNA gene sequencing and metagenomics sequencing after DNA extraction with and without a Benzonase digest to assess bacterial diversity patterns. The BDA method resulted in less reads from dead bacteria both in the skin mock community and skin swabs spiked with either heat-inactivated bacteria or bacterial-free DNA. This approach also efficiently depleted host DNA reads in samples with high human-to-microbial DNA ratios, with no obvious impact on the microbiome profile. We further observed that low biomass samples generate an α-diversity bias when the bacterial load is lower than 10⁵ CFU and that Benzonase digest is not sufficient to overcome this bias.

Conclusions: The BDA approach enables both a better assessment of the living microbiota and depletion of host DNA reads. Video abstract.

Keywords: 16S rRNA; Benzonase; DNA extraction; Diversity; Live/dead; Low biomass; Next-generation sequencing; Skin; Skin microbiome.

Fig. 1

Benzonase digest efficiently depletes DNA from dead bacteria and free bacterial DNA in skin mock community samples. Microbial DNA was extracted using a a Benzonase-digest approach (BDA) or b without Benzonase pre-digest (NDA). Relative OTU abundance of reads obtained from amplified 16S rRNA genes are shown. The mock community consists of living bacteria (live, left), including heat-inactivated (hi, middle) bacteria (P. aeruginosa and P. mirabilis) and additional free B. simplex DNA (hi DNA, right). c Relative abundances of P. aeruginosa and d P. mirabilis subjected (hi) or not (live) to heat inactivation before DNA extraction based on 16S rRNA gene sequencing of mock communities. p values were calculated using Wilcoxon-Mann-Whitney test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. e As panel c, but showing free DNA of B. simplex, spiked in before DNA extraction. f Principal coordinate analysis (PCoA) plot of β-diversities for different skin mock communities extracted using BDA or NDA

Fig. 2

Human DNA is efficiently depleted by the BDA approach with no impact on the microbiome profile. a DNA yields from skin mock community samples with heat-inactivated bacteria and added free DNA (hi, DNA), further supplemented with 10⁵ PBMCs and processed either with BDA or NDA. p values were calculated using Wilcoxon-Mann-Whitney test *** p ≤ 0.001. b Percentage of reads related to humans or bacteria based on metagenomic sequencing. *** p ≤ 0.001. c Relative OTU abundance obtained from amplified 16S rRNA genes from mock community members following BDA or d NDA approaches

Fig. 3

Serial dilutions of skin mock community samples display increased α-diversity. The original mock community (10⁸ CFU) was diluted to 10⁷, 10⁵, and 10³ CFU/sample. Relative OTU abundance obtained from amplified 16S rRNA genes upon serial dilutions followed by DNA extraction using a BDA or b NDA. The α-diversity increased upon dilution when expressed as either richness for c BDA and d NDA or Shannon diversity index for e BDA and f NDA. p values were calculated using Wilcoxon-Mann-Whitney test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. PCoA plots show shifting of β-diversity of diluted samples prepared by g BDA or h NDA

Fig. 4

Changes of OTU abundance in mock community samples upon serial dilutions. Relative OTU abundance obtained from amplified 16S rRNA gene sequencing from mock communities processed by a BDA or b after NDA extraction decreased for most mock community strains but increased for S. epidermidis. p values were calculated using Wilcoxon-Mann-Whitney test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001