Klebsiella oxytoca is an opportunistic pathogen associated with hospital-acquired infections. The clinical prevalence of multidrug-resistant (MDR) K. oxytoca poses a significant threat to human health due to limited treatment options.
In this study, K. oxytoca 611 was isolated from hospital wastewater and sequenced using both Illumina (short-read) and Oxford nanopore (long-read) sequencing. Analysis of complete sequences revealed 59 antimicrobial resistance genes (ARGs), of which 28 were encoded by a large (473,763 bp,) hybrid plasmid (pKO611) belonging to both IncA/C and IncH2 groups. Resistance gene analysis revealed a gene cassette that contained a new variant tetracycline efflux pump tet(E) and a novel AmpC β-lactamase. This gene cassette was also identified in Aeromonas spp, Klebsiella pneumonia, and Raoultella ornithinolytica and was potentially mobilized by the IS30 transposon. Tet(E) and AmpC were cloned and expressed in E. coli BL21 (DE3) where it conferred resistance to tetracyclines and beta-lactam antibiotics except carbapenems. Additionally, a prophage region identified in pKO611 was also found in Enterobacter adelaidei, a new species we isolated from Adelaide hospital wastewater recently. Finally, an antimicrobial sensitive E. coli strain was conjugated with K. oxytoca 611 and the susceptibility results showed that pKO611 conferred resistance to a broad range of antibiotics including beta-lactams, monobactam, tetracyclines, aminoglycosides, and chloramphenicol. However, curing of pKO611 does not reverse the resistance in the original K. oxytoca host presumably due to the presence of multiple other chromosome-encoded resistance mechanisms.
Here we identified a novel hybrid plasmid harbouring various ARGs from MDR K. oxytoca conferring high-level resistance to E. coli via conjugation. Sequencing analysis showed evidence that this plasmid has potentially spread to both environmental and human pathogens. The ease with which this plasmid transferred to E. coli where it conferred resistance to multiple antibiotics suggests that pKO611 may serve as a reservoir of ARGs for other pathogens.