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.
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.
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.
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.