Target enrichment improves culture-independent detection of Neisseria gonorrhoeae and antimicrobial resistance determinants direct from clinical samples with Nanopore sequencing

• Published in: Microbial genomics Vol. 10; no. 3
• Main Authors: Street, Teresa L, Sanderson, Nicholas D, Barker, Leanne, Kavanagh, James, Cole, Kevin, The GonFast Investigators Group, Llewelyn, Martin, Eyre, David W
• Format: Journal Article
• Language: English
• Published: England 01.03.2024
• Subjects: Anti-Bacterial Agents - pharmacology; Anti-Infective Agents; DNA; Drug Resistance, Bacterial - genetics; Gonorrhea - diagnosis; Humans; Nanopore Sequencing; Neisseria gonorrhoeae - genetics; Phylogeny; nanopore sequencing; Neisseria gonorrhoeae; SureSelect; culture-independent; target enrichment; AMR
• ISSN: 2057-5858; 2057-5858
• DOI: 10.1099/mgen.0.001208

Multi-drug-resistant infection is a significant public health risk. Rapidly detecting and antimicrobial-resistant (AMR) determinants by metagenomic sequencing of urine is possible, although high levels of host DNA and overgrowth of contaminating species hamper sequencing and limit genome coverage. We performed Nanopore sequencing of nucleic acid amplification test-positive urine samples and culture-positive urethral swabs with and without probe-based target enrichment, using a custom SureSelect panel, to investigate whether selective enrichment of DNA improves detection of both species and AMR determinants. Probes were designed to cover the entire genome, with tenfold enrichment of probes covering selected AMR determinants. Multiplexing was tested in a subset of samples. The proportion of sequence bases classified as increased in all samples after enrichment, from a median (IQR) of 0.05 % (0.01-0.1 %) to 76 % (42-82 %), giving a corresponding median improvement in fold genome coverage of 365 times (112-720). Over 20-fold coverage, required for robust AMR determinant detection, was achieved in 13/15(87 %) samples, compared to 2/15(13 %) without enrichment. The four samples multiplexed together also achieved >20-fold genome coverage. Coverage of AMR determinants was sufficient to predict resistance conferred by changes in chromosomal genes, where present, and genome coverage also enabled phylogenetic relationships to be reconstructed. Probe-based target enrichment can improve genome coverage when sequencing DNA extracts directly from urine or urethral swabs, allowing for detection of AMR determinants. Additionally, multiplexing prior to enrichment provided enough genome coverage for AMR detection and reduces the costs associated with this method.