Molecular analysis of clinical isolates of ceftazidime-avibactam-resistant Klebsiella pneumoniae

Abstract Objectives To analyse the strains collected during a one-year survey of ceftazidime-avibactam (CZA)-resistant KPC-producing Klebsiella pneumoniae (KPC-Kp), in order to investigate the molecular mechanisms potentially responsible for their resistant phenotype. Methods Clinical KPC-Kp isolates were collected from 31 patients in 6 different hospitals in Rome. For 8/31 patients, an additional strain grown before the start of treatment was also available, bringing the total of isolates studied to 39. Antimicrobial susceptibility was determined by automated system, broth microdiluition and E-test as appropriate. In silico analysis of acquired resistance genes was achieved by whole-genome sequencing, while MLST and core genome multi locus sequence typing (cgMLST) were employed for molecular typing. Mutations associated with CZA resistance were identified by Sanger sequencing of the blaKPC gene. Possible mutations in OmpK35 and OmpK36 outer membrane proteins were also investigated. Results Molecular analyses highlighted the circulation of the ST512, 101 and 307 high-risk clones; 26/31 carried a mutated KPC variant, 5/31 a wild-type KPC-3. Among the KPC variants detected, 11 were different mutations within the blaKPC-3 gene, 4 of which were novel mutational changes. Conclusions Different mutations including single amino-acid substitutions, insertions or deletions within the blaKPC gene were found in 26/31 CZA -resistant KPC-Kp strains belonging to high-risk clones circulating in Italy. Of note, in 14/31 cases the isolates displayed resistance to both CZA and carbapenems, raising the alarm for the possible selection of a multi-drug-resistant phenotype.