| Summary: | Carbapenem-resistant Enterobacterales (CRE) is a critical public health problem in South America, where the prevalence of NDM metallo-betalactamases has increased substantially in recent years. In this study, we used whole genome sequencing to characterize a multidrug-resistant (MDR) <i>Klebsiella pneumoniae</i> (UCO-361 strain) clinical isolate from a teaching hospital in Chile. Using long-read (Nanopore) and short-read (Illumina) sequence data, we identified a novel un-typeable megaplasmid (314,976 kb, pNDM-1_UCO-361) carrying the <i>bla</i><sub>NDM-1</sub> carbapenem resistance gene within a Tn<i>3000</i> transposon. Strikingly, conjugal transfer of pNDM-1_UCO-361 plasmid only occurs at low temperatures with a high frequency of 4.3 × 10<sup>−6</sup> transconjugants/receptors at 27 °C. UCO-361 belonged to the ST1588 clone, previously identified in Latin America, and harbored aminoglycoside, extended-spectrum β-lactamases (ESBLs), carbapenem, and quinolone-resistance determinants. These findings suggest that <i>bla</i><sub>NDM-1</sub>-bearing megaplasmids can be adapted to carriage by some <i>K. pneumoniae</i> lineages, whereas its conjugation at low temperatures could contribute to rapid dissemination at the human–environmental interface.
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