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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).<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>The majority of acquired resistance (\u226575%) resided on plasmids including several megaplasmids (\u2265100 kb). DNA sequencing detected most resistance genes (\u226570%) within 2 hours of sequencing. Neural network\u2013based base-calling of direct RNA achieved up to 86% identity rate, although \u226423% of reads could be aligned. Direct RNA sequencing (with ~6 times slower pore translocation) was able to identify (within 10 hours) \u226535% of resistance genes, including those associated with resistance to aminoglycosides, \u03b2-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 (\u22652-fold) and the pmrHFIJKLM operon (\u22658-fold). Expression levels estimated from direct RNA sequencing displayed strong correlation (Pearson: 0.86) compared to quantitative real-time PCR across 11 resistance genes.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>Overall, MinION sequencing rapidly detected the XDR\/PDR K. pneumoniae resistome, and direct RNA sequencing provided accurate estimation of expression levels of these genes.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1093\/gigascience\/giaa002","type":"journal-article","created":{"date-parts":[[2020,2,12]],"date-time":"2020-02-12T21:36:19Z","timestamp":1581543379000},"source":"Crossref","is-referenced-by-count":36,"title":["Evaluating the genome and resistome of extensively drug-resistant\n                    <i>Klebsiella pneumoniae<\/i>\n                    using native DNA and RNA Nanopore sequencing"],"prefix":"10.1093","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8255-4036","authenticated-orcid":false,"given":"Miranda E","family":"Pitt","sequence":"first","affiliation":[{"name":"Institute for Molecular Bioscience, The University of Queensland , 306 Carmody Road, Brisbane, Queensland, 4072,","place":["Australia"]},{"name":"The Peter Doherty Institute for Infection and Immunity, The University of Melbourne , 792 Elizabeth Street, Melbourne, Victoria, 3000,","place":["Australia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8711-1587","authenticated-orcid":false,"given":"Son H","family":"Nguyen","sequence":"additional","affiliation":[{"name":"Institute for Molecular Bioscience, The University of Queensland , 306 Carmody Road, Brisbane, Queensland, 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