Supporting data for "Evaluating the Genome and Resistome of Extensively Drug-Resistant Klebsiella pneumoniae using Native DNA and RNA Nanopore Sequencing"

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 four extensively drug-resistant (XDR) K. pneumoniae clinical isolates, however, further antimicrobial susceptibility testing identified three isolates as pandrug-resistant (PDR). The majority of acquired resistance (≥75%) resided on plasmids including several megaplasmids (≥100 kbp). 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 approximately 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 qRT-PCR across eleven resistance genes. Overall, MinION sequencing rapidly detected the XDR/ PDR K. pneumoniae resistome and direct RNA sequencing provided accurate estimation of expression levels of these genes.