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).ResultsThe 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.ConclusionOverall, MinION sequencing rapidly detected the XDR/PDR K. pneumoniae resistome, and direct RNA sequencing provided accurate estimation of expression levels of these genes.
Highlights
Klebsiella pneumoniae is one of the leading causes of nosocomial infections, with reports of mortality rates as high as 50% [1,2,3,4,5]
MinION DNA sequencing for all isolates was run for ≥20 hours, which generated 1.19 gigabase pairs (Gb) (215×) for 1 GR 13, 0.39 Gb (67×) for 2 GR 12, 0.56 Gb (101×) for 16 GR 13, and 0.64 Gb (115×) for 20 GR 12 (Supplementary Table S2)
Across the differing assembly tools, the chromosome sequence commonly circularized as a 5.0–5.4 megabase pairs (Mb) contig including plasmids ranging between 13 and 193 kb with the exception of 2 GR 12
Summary
Klebsiella pneumoniae is one of the leading causes of nosocomial infections, with reports of mortality rates as high as 50% [1,2,3,4,5]. The long reads coupled with the ability to multiplex samples have immensely aided with the assembly of bacterial genomes [16,17,18] This capability allows for the rapid determination of whether resistance is residing on the chromosome or plasmid(s). Klebsiella pneumoniae frequently harbours multidrug resistance, and current diagnostics struggle to rapidly identify appropriate antibiotics to treat these bacterial infections. 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 quinolones, rifampicin, fosfomycin, and phenicol in some isolates. 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
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
