A Metataxonomic Tool to Investigate the Diversity of Treponema

Abstract

The genus Treponema contains a number of human and animal pathogenic as well as symbiotic bacteria that are found in vastly different anatomical and environmental habitats. Our understanding of the species range, evolution, and biology of these important bacteria is still limited. To explore the diversity of treponemes, we established, validated, and tested a novel metataxonomic approach. As the informative nature of the hypervariable regions of the 16S rRNA gene differ, we first analyzed each variable region independently. Considering the in silico results obtained, we established and validated the sequencing of the V4-region of the 16S rRNA gene using known mixtures of Treponema species as well as a selected number of clinical samples. The metataxonomic approach was able to identify Treponema to a near-species level. We demonstrate that using a spirochete-specific enrichment, our method is applicable to complex microbial communities and large variety of biological samples. The metataxonomic approach described provides a useful method to unravel the full diversity and range of Treponema in various ecosystems.

Keywords: 16S rRNA; Potorous; Treponema; marsupial; metagenomics; metataxonomics; one health; spirochete.

FIGURE 1

Study design of the metataxonomic assay targeting the V4-region of the 16S rRNA gene. The gray boxes show the tested cycle (c) conditions for each step. The blue shading indicates the modular two-step library preparation.

FIGURE 2

Actual and observed proportions of a spirochete mock community to a genus and species-level. Relative abundance of OTUs in percentage of reads for a spirochete microbial mock community amplified using multiple methods. The spirochete enrichment and V4-region-specific PCR cycle numbers are shown below the taxa plots. The expected strain proportion (actual) of the spirochete mock community represents the theoretical composition and was corrected for differential copy number of the 16S rRNA gene. Species-level classification only shown for the genus Treponema.

FIGURE 3

The effect of spirochete enrichment on the detection limit of T. pallidum. Relative abundance of OTUs in percentage of reads for a T. pallidum-spiked microbial mock community (HM-280). The theoretically calculated amount of spike in T. pallidum ranging from 5 to 5,000 16S rDNA copies is shown along the x-axis. The biologically effective range of T. pallidum may differ from theoretically calculated amount (e.g., 5 copies = 4–6 copies). Cycle number for the spirochete enrichment and V4-region-specific PCR is shown above the taxa plots.

FIGURE 4

Detection limit of the metagenomic approach for Treponema. Total sequence reads resulting from different input amounts of T. pallidum. Displayed amounts of T. pallidum (50,000–0.5 16S rDNA copies) represent theoretically calculated amounts. Biologically effective range may differ from theoretically calculated amount (e.g., 0.5 copies = 0–2 copies). Blank control represents the 16S rDNA amplification control using microbial DNA-free water as input. For these samples 20 cycles of enrichment PCR was followed by 20 cycles of V4-region-specific PCR (for more detail see the section “Materials and Methods”).