E-Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2021

Evaluating the "-omes" of Extensively Drug-Resistant Klebsiella pneumoniae using Native DNA and RNA Nanopore Sequencing (#249)

Miranda E. Pitt 1 2 , Son H. Nguyen 1 , Tania P.S. Duarte 1 , Haotian Teng 1 , Mark A.T. Blaskovich 1 , Matthew A. Cooper 1 , Lachlan J.M. Coin 1 2
  1. Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia
  2. The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, Victoria, Australia

Klebsiella pneumoniae is one of the leading causes of nosocomial infections, frequently harbours multidrug resistance and subsequently results in high mortality[1]. Utilizing sequencing as a rapid diagnostic for bacterial infections has advanced significantly, in particular, MinION sequencing (Oxford Nanopore Technologies)[2]. This portable device is capable of real-time analysis and reading long fragments of DNA and RNA. This study sequenced four extensively drug-resistant (XDR) K. pneumoniae clinical isolates in order to assemble these genomes, discern the differential expression of resistance genes and ascertain the time required for detection of antimicrobial resistance.

Isolates were obtained from the Hygeia General Hospital (Greece)[3]. DNA and RNA were extracted from a paired inoculum and long fragment DNA was acquired using the MagAttract HMW DNA kit. RNA underwent an mRNA enrichment, poly(A) tailing and direct RNA sequencing was performed on MinION R9.4 flowcells[4].

Long-read DNA sequencing enabled the completion of these genomes and the majority of acquired resistance (≥75%) was identified to reside on at least 3 to 4 plasmids. Further antimicrobial susceptibility testing, including 35 antibiotics and combinations, determined 3 of the 4 isolates were pandrug-resistant (PDR). A real-time emulation analysis[5] of DNA detected ≥70% of resistance genes in 2 hours for all isolates. Native RNA sequencing successfully revealed aminoglycoside, beta-lactam, trimethoprim and sulphonamide resistance within 2 hours. In several instances, quinolone, rifampicin and phenicol resistance was apparent, although dependent on the level of transcription. Tetracycline and fosfomycin resistance were absent, however, these genes were validated to have low expression via qRT-PCR. Long-read RNA sequencing identified co-expression of genes, associated with operons, which included up to 3 resistance genes encoded on these transcripts. Furthermore, aminoglycoside resistance could also be detected via an RNA modification (m7G, 16S rRNA, position 1396) using native RNA sequencing.

Nanopore sequencing was capable of detecting antibiotic resistance in these XDR/ PDR K. pneumoniae isolates within hours and differential expression of these genes was successfully validated via native RNA sequencing.

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  4. Pitt ME et al. 2020. Gigascience. 9:giaa002.
  5. Cao MD et al. 2016. Gigascience. 5:32.