Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2018

Evaluating the Extensively Drug-Resistant Klebsiella pneumoniae Resistome via MinION Sequencing (#350)

Miranda E Pitt 1 , Son Hoang Nguyen 1 , Tânia PS Duarte 1 , Mark AT Blaskovich 1 , Matthew A Cooper 1 , Lachlan JM Coin 1
  1. Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia

Introduction

Klebsiella pneumoniae is one of the leading causes of nosocomial infections, frequently possesses multidrug resistance and subsequently results in high mortality1. Utilising 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.

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 an mRNA enrichment, poly(A) ligation and direct RNA sequencing on MinION R9.4 flowcells. The real-time emulation was conducted as previously described4.

Results

DNA sequencing identified the majority of acquired resistance (≥75%) resided on up to 5 plasmids in these isolates. The real-time emulation detected ≥70% of resistance genes in 2 hours for all isolates. Direct RNA sequencing successfully revealed aminoglycoside, beta-lactam, trimethoprim and sulphonamide resistance within 6 hours. In several instances, quinolone, rifampicin and phenicol resistance was apparent, although dependent on the level of transcription. Fosfomycin, macrolide and tetracycline resistance was absent, however, these genes were validated to have low expression via qRT-PCR.

Conclusion

MinION sequencing was capable of detecting antibiotic resistance in these XDR K. pneumoniae isolates within hours and differential expression of these genes was successfully validated via direct 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, Elliott AG, Cao MD, Ganesamoorthy D, Karaiskos I, Giamarellou H, Abboud CS, Blaskovich MAT, Cooper MA, Coin LJM. 2018. Microb Genom. doi: 10.1099/mgen.0.000158.
  4. Cao MD, Ganesamoorthy D, Elliott AG, Zhang H, Cooper MA, Coin LJ. 2016. Gigascience. 5:32.