AUTHOR=Bouchiat Coralie , Ginevra Christophe , Benito Yvonne , Gaillard Tiphaine , Salord Hélène , Dauwalder Olivier , Laurent Frédéric , Vandenesch François 

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

JOURNAL=Frontiers in Microbiology

VOLUME=Volume 13 - 2022

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.943441

DOI=10.3389/fmicb.2022.943441

ISSN=1664-302X

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 full-length amplification of the 16S rRNA gene. Contamination, background noise definition, bacterial identification, and time-effectiveness issues were addressed. Cost optimisation was also investigated with the miniaturized version of 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 at 1% of total reads overcame the background noise issue and eased interpretation of clinical samples. Results were obtained within one 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 routine diagnosis of culture-negative samples in clinical laboratories.