Unlabelled: The emergence of carbapenemase-producing Enterobacterales represents a significant clinical challenge. Resistance mechanisms involve carbapenemase production, porin and efflux pump alterations, penicillin-binding protein (PBP) modifications, and biofilm formation. This study characterizes a KPC-producing Klebsiella pneumoniae and a concurrent Escherichia coli isolate harboring the same resistance genes, with E. coli also exhibiting PBP3 mutations. Whole-genome sequencing and plasmid analysis identified an IncFII(K) plasmid carrying the blaKPC-3 gene. Both strains shared two resistance genes (blaKPC-3, blaOXA-9). K. pneumoniae contained a single copy of blaKPC-3 on Tn4401a, whereas E. coli carried two copies on separate Tn4401a transposons. Plasmid reconstruction revealed high homology (99.85%) with E. coli plasmid pECAZ147_KPC and K. pneumoniae plasmid pKPC. This suggests that transposon-mediated blaKPC-3 transfer between the two strains may have occurred via the same plasmid. Moreover, E. coli harbored PBP3 mutations (A233T, I332V), which have been previously linked to increased ceftazidime MICs and two missense mutations in marR. Despite carrying two copies of the blaKPC-3 gene, along with marR and PBP3 mutations, our E. coli isolate did not exhibit a significant increase in ceftazidime/avibactam MIC. This phenomenon could be explained by avibactam’s ability to bind to E. coli PBP2, potentially compensating for the reduced binding of ceftazidime to the mutated PBP3. Supplementary information: The online version contains supplementary material available at 10.1038/s41598-025-07624-z.
