Evaluating the genome and resistome of extensively drug-resistant Klebsiella pneumoniae using native DNA and RNA Nanopore sequencing

Abstract

Background Klebsiella pneumoniae frequently harbours multidrug resistance, and current diagnostics struggle to rapidly identify appropriate antibiotics to treat these bacterial infections. The MinION device can sequence native DNA and RNA in real time, providing an opportunity to compare the utility of DNA and RNA for prediction of antibiotic susceptibility. However, the effectiveness of bacterial direct RNA sequencing and base-calling has not previously been investigated. This study interrogated the genome and transcriptome of 4 extensively drug-resistant (XDR) K. pneumoniae clinical isolates; however, further antimicrobial susceptibility testing identified 3 isolates as pandrug-resistant (PDR). Results The majority of acquired resistance (≥75%) resided on plasmids including several megaplasmids (≥100 kb). DNA sequencing detected most resistance genes (≥70%) within 2 hours of sequencing. Neural network–based base-calling of direct RNA achieved up to 86% identity rate, although ≤23% of reads could be aligned. Direct RNA sequencing (with ∼6 times slower pore translocation) was able to identify (within 10 hours) ≥35% of resistance genes, including those associated with resistance to aminoglycosides, β-lactams, trimethoprim, and sulphonamide and also quinolones, rifampicin, fosfomycin, and phenicol in some isolates. Direct RNA sequencing also identified the presence of operons containing up to 3 resistance genes. Polymyxin-resistant isolates showed a heightened transcription of phoPQ (≥2-fold) and the pmrHFIJKLM operon (≥8-fold). Expression levels estimated from direct RNA sequencing displayed strong correlation (Pearson: 0.86) compared to quantitative real-time PCR across 11 resistance genes. Conclusion Overall, MinION sequencing rapidly detected the XDR/PDR K. pneumoniae resistome, and direct RNA sequencing provided accurate estimation of expression levels of these genes.

Nanopore sequencing technology, bioinformatics and applications

“…In both cases, only the RNA strand passes through the nanopore, and therefore direct sequencing of RNA molecules does not generate a consensus sequence (for example, 2D or 1D 2 ). Compared to DNA sequencing, direct RNA sequencing is typically of lower average accuracy, around 83-86%, as reported by independent research 53,54 .…”

Mapping the complex transcriptional landscape of the phytopathogenic bacterium Dickeya dadantii

“…This method allows native RNAs to be sequenced directly as near full-length transcripts from the 3' to 5' direction, with a weaker depth than the previous datasets. Only a few transcriptomes were analysed by this technique, mostly from viral and eukaryotic organisms (33,34,35,36), and, to our knowledge, a single prokaryotic one (37). This dataset provides a direct isoform-level validation of the TUs, and an accurate definition of transcription termination sites.…”

Finishing the Job - Utility of Long-Read Sequencing Using the Minion for Bacterial Genomics

“…However, TGS platforms such as ONT's MinION can directly measure RNA in its native molecules without any preparatory shearing and qPCR steps and, even more, there is no limit to the length of the sequenced molecules (Smith et al, 2019). Thus far, ONT's MinION has been used to evaluate the resistome of four extensively drugresistant Klebsiella pneumoniae clinical isolates (Pitt et al, 2020), estimate 3' poly(A) tail length (Krause et al, 2019;Workman et al, 2019), produce an accurate profile of the transcriptome (Bolisetty et al, 2015;Sessegolo et al, 2019), and identify novel isoforms (Byrne et al, 2017;Križanović et al, 2018) and RNA modifications (extensively referenced in Cozzuto et al, 2020).…”