Klebsiella pneumoniae carbapenemase (KPC) variants, predominantly KPC-2 and KPC-3, are a significant global resistance mechanism in K. pneumoniae. KPC-2 and KPC-3 confer resistance to a broad range of β-lactams, including carbapenems, while remaining susceptible to ceftazidime-avibactam (CZA). Lately, new KPC variants have emerged and developed resistance to CZA, often through mutations, insertions, or deletions in key regions such as the Omega loop, the 237–243 loop, and the 266–275 loop. In the present work we aim to understand the role of these mutations in KPC in the emergence of cross-resistance to last-resort antibiotics like cefiderocol (FDC) and cefepime/zidebactam (FPZ). Fifteen clinical isolates of KPC-producing Klebsiella spp. were analyzed, representing 15 distinct KPC variants. CZA resistance was confirmed in 12 of the 15 KPC variants tested. Cross-resistance to FDC was observed in eight isolates, with five of these exhibiting spontaneous resistant subpopulations, including one originally categorized as FDC-susceptible. Six FDC-resistant strains carried mutations in the 266–275 loop. Cross-resistance to FPZ was observed in five KPC variants, particularly those with mutations in the 266–275 loop, although many omega loop and 237–243 loop mutants remained susceptible to FPZ. WGS of FDC-resistant subpopulations revealed additional mutations in genes such as ompC, rpoC, dksA, and cirA.This study demonstrates that KPC variants show resistance to both CZA and FDC, with cross-resistance to FPZ observed to a lesser degree. The emergence of cross-resistance in strains that had not been exposed to these antibiotics raises concerns about the spread of resistance. The identification of mutations in blaKPC, cirA, and other novel genes highlights the importance of understanding the molecular mechanisms driving resistance to develop effective therapeutic strategies.

Files Description ---rawseqs_ref.tar.gz: FASTQ file of wild type strains ---annot_assemblies_ref.tar.gz: FASTA files of wild-type strains. Genome annotation files of wild-type strains ---IHC_seqs.tar.gz: FASTQ files of IHC strains --- annot_IHC.tar.gz: Genome annotation files of IHC strains --- assemblies_IHC.tar.gz: FASTA files of IHC strains.