Prevalent and diverse new plasmid-encoded heavy metal and antimicrobial resistance in Klebsiella strains isolated from hospital wastewater

The rise of antibiotic-resistant <i>Klebsiella pneumoniae</i> poses a significant global health threat. Plasmids, as mobile genetic elements, play a critical role in bacterial adaptation by facilitating the spread of resistance genes. To analyze plasmid-mediated antibiotic and heavy metal resistance in clinical <i>Klebsiella</i> strains, 33 <i>Klebsiella</i> strains isolated from wastewater were subjected to third-generation nanopore sequencing to obtain high-quality whole-genome assemblies. The presence and diversity of plasmids associated with antibiotic and heavy metal resistance were analyzed, and phenotypic assays were conducted to confirm metal resistance. A total of 81 plasmids were identified across 24 strains, including 28 (34.6%) novel plasmids. Among them, 22 plasmids carried antibiotic resistance genes (ARGs), with 12 containing integrons, four of which were complex Class I integrons and two unconventional integrons. Notably, a novel conjugative plasmid, pKP228-1, was discovered carrying a complex Class I integron with a unique gene cassette array encoding 12 ARGs, and harboring <i>bla</i> _NDM-1 in the adjacent IS<i>CR1</i>-associated region. Another plasmid, pKP174-2, harbored <i>mcr-8.1</i> and <i>tporJ1-tmexCD1</i>. Additionally, 24 plasmids encoded resistance to eight heavy metals/metalloids, and 12 plasmids co-harbored both ARGs and metal resistance genes, indicating potential co-selection mechanisms. This study highlights the extensive diversity and novel structures of plasmids carrying both antibiotic and heavy metal resistance in clinical <i>Klebsiella</i> isolates. The observed co-occurrence of the two resistance types highlights the need for comprehensive genomic surveillance to monitor the spread of multi-resistance determinants.