Emergence and evolution of multidrug-resistant Klebsiella pneumoniae with both bla_KPC and bla_CTX-M integrated in the chromosome

The extended-spectrum-β-lactamase (ESBL)-and Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae represent serious and urgent threats to public health. In a retrospective study of multidrug-resistant K. pneumoniae, we identified three clinical isolates, CN1, CR14, and NY9, carrying both bla_CTX-M and bla_KPC genes. The complete genomes of these three K. pneumoniae isolates were de novo assembled by using both short-and long-read whole-genome sequencing. In CR14 and NY9, bla_CTX-M and bla_KPC were carried on two different plasmids. In contrast, CN1 had one copy of bla_KPC-2 and three copies of bla_CTX-M-15 integrated in the chromosome, for which the bla_CTX-M-15 genes were linked to an insertion sequence, ISEcp1, whereas the bla_KPC-2 gene was in the context of a Tn4401a transposition unit conjugated with a PsP3-like prophage. Intriguingly, downstream of the Tn4401a-bla_KPC-2-prophage genomic island, CN1 also carried a clustered regularly interspaced short palindromic repeat (CRISPR)-cas array with four spacers targeting a variety of K. pneumoniae plasmids harboring antimicrobial resistance genes. Comparative genomic analysis revealed that there were two subtypes of type I-E CRISPR-cas in K. pneumoniae strains and suggested that the evolving CRISPR-cas, with its acquired novel spacer, induced the mobilization of antimicrobial resistance genes from plasmids into the chromosome. The integration and dissemination of multiple copies of bla_CTX-M and bla_KPC from plasmids to chromosome depicts the complex pandemic scenario of multidrug-resistant K. pneumoniae. Additionally, the implications from this study also raise concerns for the application of a CRISPR-cas strategy against antimicrobial resistance.