16S metagenomics for diagnosis of bloodstream infections: opportunities and pitfalls

Abstract

Bacterial bloodstream infections (BSI) form a large public health threat worldwide. Current routine diagnosis is based on blood culture (BC) but this technique suffers from limited sensitivity. Molecular diagnostic tools have been developed for identification of bacteria in the blood of BSI patients. 16S metagenomics is an open-ended technique that can detect simultaneously all bacteria in a given sample based on PCR amplification of the 16S ribosomal RNA gene (rDNA) followed by sequencing of the PCR amplicons and taxonomic labeling of the sequence reads at genus or species level. Areas covered: Here we review the studies that have used 16S metagenomics for the identification of bacteria in human blood samples. We also discuss the potential added value of 16S metagenomics in the diagnosis of BSI, challenges as well as future directions for implementation in clinical settings. Expert commentary: 16S metagenomics has the potential to complement conventional BC; however, the technique currently suffers from several technical limitations jeopardizing implementation in routine clinical microbiology laboratories. Further studies are required to assess the cost-efficiency and clinical impact of 16S metagenomics in comparison to BC which remains the gold standard diagnostic method for BSI.

A 16S ribosomal RNA TaqMan real-time reverse transcription PCR for specific detection of Salmonella in blood

“…On the other hand, 16S metagenomics can simultaneously detect all bacteria in a given blood sample based on PCR ampli cation of the 16S ribosomal RNA gene followed by deep sequencing of the PCR amplicons and taxonomic labelling of the sequence reads at genus or species level. However, the technique currently presents various technical limitations such as limited taxonomic resolution and high background signals jeopardizing its implementation in routine patients' diagnosis [11]. Based on the proof-of-concept presented here, 16S rRNA amplicon deep sequencing may be more sensitive than conventional 16S metagenomics at DNA level.…”

“…Current reference methods for Salmonella detection in blood rely on conventional blood culture followed by microbiological work-up for species and serotype identi cation. Microbiological methods are labour-intensive and generally take 6 to 48 hours before positive growth [11]. Various selective media for the identi cation of Salmonella have been developed and are commercially available.…”

The heart microbiome of insectivorous bats from Central and South Eastern Europe

“…were found (Hornok et al 2019). Identification of infectious bacteria from the bloodstream, particularly in humans are usually performed using blood cultures, but different studies compared this method with the 16S metagenomics, showing that this later technique is more sensitive and specific (Decuypere et al 2016, Rutanga et al 2018, Watanabe et al 2018]. The blood tested for those studies was fresh however there are no studies that have tested the sensitivity in partly degraded heart or heart tissue.…”

The Problem of Microbial Dark Matter in Neonatal Sepsis

“…Another approach is 16S amplicon sequencing using PCR to amplify the common 16S ribosomal gene shared by all bacteria. DNA sequencing of this PCR product is then used to identify the specific bacteria ( 32 ). 16S amplicon techniques can, in theory, uncover bacterial pathogens in an unbiased manner.…”

“…However, contamination from clinical collection, laboratory reagents, or the laboratory environment can dominate the sequencing results if pathogen sequences in the patient sample are present in very low concentrations ( 31 ), which is often the case in NS. Bacterial DNA can be present in minute quantities and therefore difficult to detect within the much greater mass of contaminant bacterial DNA acquired during routine sequencing workflows ( 32 ).…”