Improving the Diagnosis of Bacterial Infections: Evaluation of 16S rRNA Nanopore Metagenomics in Culture-Negative Samples

Abstract

While 16S rRNA PCR-Sanger sequencing has paved the way for the diagnosis of culture-negative bacterial infections, it does not provide the composition of polymicrobial infections. We aimed to evaluate the performance of the Nanopore-based 16S rRNA metagenomic approach, using both partial and full-length amplification of the gene, and to explore its feasibility and suitability as a routine diagnostic tool for bacterial infections in a clinical laboratory. Thirty-one culture-negative clinical samples from mono- and polymicrobial infections based on Sanger-sequencing results were sequenced on MinION using both the in-house partial amplification and the Nanopore dedicated kit for the full-length amplification of the 16S rRNA gene. Contamination, background noise definition, bacterial identification, and time-effectiveness issues were addressed. Cost optimization was also investigated with the miniaturized version of the flow cell (Flongle). The partial 16S approach had a greater sensitivity compared to the full-length kit that detected bacterial DNA in only 24/31 (77.4%) samples. Setting a threshold of 1% of total reads overcame the background noise issue and eased the interpretation of clinical samples. Results were obtained within 1 day, discriminated polymicrobial samples, and gave accurate bacterial identifications compared to Sanger-based results. We also found that multiplexing and using Flongle flow cells was a cost-effective option. The results confirm that Nanopore technology is user-friendly as well as cost- and time-effective. They also indicate that 16S rRNA targeted metagenomics is a suitable approach to be implemented for the routine diagnosis of culture-negative samples in clinical laboratories.

Utility of Metagenomic Next-Generation Sequencing in Infective Endocarditis: A Systematic Review

“…Three retrospective cohort studies on plasma cell-free DNA-based pathogen identification had a few patients with IE among other infectious syndromes [ 22 , 23 , 24 ]. Million M et al and Bouchiat et al proposed two different steps to enhance the results of mNGS, microdissection combined with human DNA depletion [ 25 ] and a nanopore-based 16S rRNA metagenomic approach [ 26 ].…”

“…Bouchiat et al evaluated a nanopore-based 16S rRNA metagenomic approach—a technology that enables rapid and user-friendly library preparation, real-time data acquisition and analysis through an online platform with dedicated pipelines—and showed that setting a threshold of 1% of total reads overcame background noise and eased the interpretation of clinical samples. However, only two samples were cardiac valves; thus, it is difficult to draw conclusions regarding the applicability of this technique in IE [ 26 ].…”

Advances in Multiplexed Microfluidics for Infectious Disease Detection

“…However, low sensitivity often occurs because of differences in primer amplification efficiency. NA amplification using single primer sets targeting the phylogenetically conserved sequences allows for a wide range of pathogen detection without compromising sensitivity 64 . In addition, broad-range PCR can identify unknown, mutated, or emerging causes of disease infection 65 .…”

“…NA amplification using single primer sets targeting the phylogenetically conserved sequences allows for a wide range of pathogen detection without compromising sensitivity. 64 In addition, broad-range PCR can identify unknown, mutated, or emerging causes of disease infection. 65 Microfluidic chip sequencing assays and high-resolution melt analysis have been used to detect pathogens without amplicons, providing a better choice for large-scale pathogen identification.…”

Metagenomics reveals microbial communities and functional differences during chili pepper (Capsicum frutescens L.) natural fermentation: Effects of brines and containers

“…Commonly used culture‐independent methods for studying the microbial composition of fermented chili peppers were 16S rDNA and ITS rDNA sequencing. However, these methods have limitations in their discriminatory power, particularly at the genus and species levels for certain microbial clades (Bouchiat et al., 2022). In contrast, metagenomic sequencing provides a comprehensive approach to identify the diverse microorganisms present in a sample and explore the functional diversity of microbial communities (Bouchiat et al., 2022; Chen, 2022).…”