The acquired CTX-M-type extended-spectrum-β-lactamase (ESBL)-producing Enterobacterales are of great concern in clinical settings because they limit therapeutic options for patients infected by the pathogens. An intriguing clonality of CTX-M ESBL-producing Klebsiella pneumoniae blood isolates was observed from a national cohort study, and comparative genomics were assessed for the 115 K. pneumoniae blood isolates carrying the bla _CTX-M gene. The plasmid preference of particular clones of a sequence type (ST) was assessed by liquid mating. A quarter of the bla _CTX-M gene-carrying K. pneumoniae blood isolates harbor the gene in their chromosome, and most of those with the built-in bla _CTX-M gene belonged either to ST307 or ST48. Notably, all 16 K. pneumoniae ST48 isolates harbored two copies of the bla _CTX-M-15 gene in the chromosome. The chromosomal integration of the bla _CTX-M-15 gene was mostly derived from the ISEcp1-targeting 5-bp AT-rich locus in the chromosome. The IS26-mediated chromosomal integration occurred when the upstream ISEcp1 from the bla _CTX-M gene was truncated, targeting the anchor IS26 copy in the chromosome. Higher transfer efficiency of the bla _CTX-M-15 gene-carrying FIA:R plasmid was observed in ST17 than that of the bla _CTX-M-14 gene-carrying FIB:FII plasmid. The transfer efficiency of the plasmid differed by isolate among the ST307 members. The K. pneumoniae clones ST307 and ST48 harboring the bla _CTX-M-15 gene in the chromosome were able to disseminate stably in clinical settings regardless of the environmental pressure, and the current population of K. pneumoniae blood isolates was constructed. Further follow-up is needed for the epidemiology of this antimicrobial resistance.IMPORTANCE Dominant F-type plasmids harboring the gene have been pointed out to be responsible for the dissemination of the CTX-M extended-spectrum-β-lactamase (ESBL)-producing K. pneumoniae Recently, the emergence of K. pneumoniae isolates with the bla _CTX-M gene in their chromosomes has been reported occasionally worldwide. Such a chromosomal location of the resistance gene could be beneficial for stable propagation, as was the Acinetobacter baumannii ST191 harboring chromosomal bla _OXA-23 that is endemic to South Korea. Through the present study, particular clones were identified as having built-in resistance genes in their chromosomes, and the chromosomal integration events were tracked by assessing their genomes. The cefotaxime-resistant K. pneumoniae clones of this study were particularized as results of the fastidiousness for plasmids to acquire the bla _CTX-M gene for securing the diversity and of the chromosomal addiction of the bla _CTX-M gene for ensuring propagation.

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