Genomic characterization of ceftazidime/avibactam-resistant KPC-producing Klebsiella pneumoniae in bloodstream infections indicates OmpK35 and OmpK36 as pivotal key players in resistance mechanisms

Background: has been severely challenged by the emergence of ceftazidime/avibactam (CAZ/AVI) resistance. The circulation of CAZ/AVI-resistant KPC-Kp remains a public health critical concern, warranting the need of strict epidemiological surveillance and genomic analysis to elucidate the underlying molecular mechanisms. Methods: We retrospectively investigated 16 BSIs caused by KPC-Kp isolates resistant to CAZ/AVI collected from January 2022 to July 2025. Antimicrobial susceptibility testing (AST) was performed to assess resistance profiles against last-line agents. Whole Genome Sequencing (WGS) and bioinformatic analyses were employed to identify molecular drivers of resistance and evaluate genetic relatedness. Patients' data were collected to integrate clinical outcomes with the microbiological features of the isolated strains. Results: The BSIs analyzed were characterized by a high degree of severity, with 9/16 patients presenting a Pitt bacteremia score ≥ 4 and 7/16 (44%) a 30-days mortality rate. Intestinal colonization by KPC-Kp was identified in 14/16 patients, and prior CAZ/AVI-based therapy had been administered to 12/16, suggesting the resistance development under selective antimicrobial pressure. AST revealed resistance to last-line agents: imipenem/relebactam (4/16), meropenem/vaborbactam (2/16) and colistin (3/16), alongside reduced susceptibility to cefiderocol (11/16), thereby limiting effective treatment strategies. Besides the predominance of high-risk clones, such as ST101 (8/16) and ST512 (3/16), phylogenetic analysis highlighted a cluster within ST101 group, suggesting a hospital outbreak. Furthermore, two novel sequence types, ST9507 and ST8640 were identified. Multiple KPC variants were detected, including the novel KPC-293 and KPC-296, which were not previously described. All isolates exhibited a truncated OmpK35 in combination or not with a highly mutated OmpK36, with insertion in loop3 being the most prevalent alteration. Given the heterogeneity of the KPC variants found in the isolates, and the consistent loss of porin function, the OmpK35/36 impairment may represent the pivotal driver of the MDR phenotype in these strains. Conclusion: Our findings indicate that the synergy between multiple KPC variants and porin structural alterations may represent a critical determinant underlying the clinical challenge posed by CAZ/AVI resistance in invasive infections.