Sensitive Identification of Bacterial DNA in Clinical Specimens by Broad-Range 16S rRNA Gene Enrichment

Abstract

The broad-range detection and identification of bacterial DNA from clinical specimens are a foundational approach in the practice of molecular microbiology. However, there are circumstances under which conventional testing may yield false-negative or otherwise uninterpretable results, including the presence of multiple bacterial templates or degraded nucleic acids. Here, we describe an alternative, next-generation sequencing approach for the broad range detection of bacterial DNA using broad-range 16S rRNA gene hybrid capture ("16S Capture"). The method is able to deconvolute multiple bacterial species present in a specimen, is compatible with highly fragmented templates, and can be readily implemented when the overwhelming majority of nucleic acids in a specimen derive from the human host. We find that this approach is sensitive to detecting as few as 17 Staphylococcus aureus genomes from a background of 100 ng of human DNA, providing 19- to 189-fold greater sensitivity for identifying bacterial sequences than standard shotgun metagenomic sequencing, and is able to successfully recover organisms from across the eubacterial tree of life. Application of 16S Capture to a proof-of-principle case series demonstrated its ability to identify bacterial species that were consistent with histological evidence of infection, even when diagnosis could not be established using conventional broad range bacterial detection assays. 16S Capture provides a novel means for the efficient and sensitive detection of bacteria embedded in human tissues and for specimens containing highly fragmented template DNA.

Keywords: 16S rRNA; broad range; enrichment; hybridization capture; metagenomics; molecular diagnosis; next-generation sequencing; sequencing.

FIG 1

Schematic of 16S Capture probe design. All panels are drawn to scale, as represented by the common scale bar at bottom. (A) Schematic of the domains of a representative 16S rRNA gene (derived from E. coli), displayed 5′ to 3′, indicating the relative size of constant and variable regions. Variable regions (V1 to V9) are depicted as thick lines, and conserved domains are depicted as thin lines. (B) Relative lengths of 16S features (variable and conserved regions, according to E. coli numbering) as they have been expanded through a gapped multiple alignment across all bacterial species and subsequently used as the substrate for probe design. (C) Location of probe-design windows, represented as rectangular boxes, is shown relative to features in the multiple alignment. The count of probes designed within each windowed region is indicated by the height of shading.

FIG 2

Recovery of bacterial sequences from human background using mNGS and 16S Capture. Three biological replicates for various S. aureus genome equivalents spiked into a fixed background of 100 ng of human DNA were detected to the species level using standard mNGS (A) or 16S Capture (B). Points indicate data from individual replicates, error bars indicate standard errors of the mean for replicates within a condition, and horizontal lines denote mean value across those replicates. Asterisks indicate significance of P ≤ 0.05 (Wald test) relative to the negative control (0 genome equivalents). Note the difference in the y-axis scale between panels.

FIG 3

Affinity of 16S Capture probes across disparate species. (A and B) Enrichment of genera (A) or species (B) from a 20-organism mock community by 16S Capture. The number of 16S templates per taxon and the average length of the template fragments are indicated below each plot, with replicates performed for each condition. Matched controls prepared from specimens lacking bacterial DNA are shown for comparison for each condition. A heatmap indicates the relative abundance of each classification in a specimen. Black “X” overlays indicate classifications for which a replicate exhibited ≤10-fold relative abundance of microbial DNA compared with the paired, nontemplate control reaction.

FIG 4

Clinical case series. (A) Histology of case 1 biopsy specimen showing diplococci by Gram stain. Original magnification, ×100. (B) Case 1 histology. Original magnification, ×400. (C) Relative abundance of top 10 identifications established by 16S Capture of case 1, shown relative to patient matched negative control material. (D) Histology of case 2 biopsy specimen showing Gram-variable bacilli by Gram stain. Original magnification, ×100. (E) Case 2 histology. Original magnification, ×400. (F) Relative abundance of top 10 identifications established by 16S Capture of case 2, shown relative to patient-matched negative-control material.

FIG 5

Organism-specific PCR confirmation of 16S Capture diagnoses of patient material. (A) L. iners-specific PCR for defined control specimens and patient autopsy material. (B) P. oris-specific PCR for defined control specimens and patient autopsy material. Arrowheads in both panels indicate the expected amplicon size.