Poster Presentation 41st Lorne Genome Conference 2020

Evaluating the "-omes" of Extensively Drug-Resistant Klebsiella pneumoniae using Native DNA and RNA Nanopore Sequencing (#226)

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

Background: Klebsiella pneumoniae (K. pneumoniae) is one of the leading causes of nosocomial infections, frequently harbours multidrug resistance and subsequently results in high mortality1. 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 expression of resistance genes or pathways and ascertain the time required for detection of antimicrobial resistance.

Methods: 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 a rRNA depletion, poly(A) tailing and direct RNA sequencing was performed on MinION R9.4 flowcells. The real-time simulation analysis was conducted as previously described4.

Results: Long-read DNA sequencing, in combination with prior Illumina sequencing3, enabled the completion of these genomes and the majority of acquired resistance (≥75%) was identified to reside on at least 3 to 4 plasmids. A real-time emulation analysis 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. Resistance gene co-expression, regulated via operons, was also evident. Tetracycline and fosfomycin resistance was absent, however, these genes were validated to have low expression via qRT-PCR. Heightened expression of phoPQ and pmrHFIJKLM were indicative of colistin resistance.

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

  1. Martin RM, Bachman MA. 2018. Front Cell Infect Microbiol. 8:4.
  2. Gardy JL, Loman NJ. 2018. Nat Rev Genet. 19:9-20.
  3. Pitt ME, et al. 2018. Microb Genom. 4:e000158.
  4. Cao MD, et al. 2016. Gigascience. 5:32.