A novel KPC-166 in ceftazidime/avibactam resistant ST307 Klebsiella pneumoniae causing an outbreak in intensive care COVID Unit, Italy

Abstract

Introduction: Aim of the study was the molecular characterization of 21 ceftazidime/ avibactam resistant (CZA-R) Klebsiella pneumoniae strains, collected in the period October 2021eMarch 2022 from an Intensive Care COVID Unit in a Northern Italian Hospital. Methods: After growth on selective/chromogenic culture media and susceptibility tests assessment, resistance genes content was ascertained for all the isolates by the HybriSpot 12 multiplexing, PCR and Whole-Genome Sequencing (WGS). Clonality was assessed by PFGE and MLST according to the Pasteur scheme. A SNPs-based phylogenetic tree was obtained comparing representative isolates and global genomes. The blaKPC gene horizontal transmission was evaluated by conjugation experiments. blaKPC-166 was cloned in a pCR2.1 vector and transformed in chemically competent TOP10 cells. Results: Sixteen inpatients resulted positive for colonization and/or infection by KPCproducing K. pneumoniae (KPC-Kp) strains. The 21 CZA-R KPC-Kp isolates obtained showed MDR phenotype; susceptibility to meropenem was always retained. All the CZA-R KPC-Kp presented a novel blaKPC variant, named blaKPC-166, showing a single nucleotide substitution (T811C) compared to the blaKPC-94; but related to blaKPC-2. Two different pulsotypes were detected: A in 18/21 and B in 1/21 cases, two strains from the same patient being untypable by PFGE. Interestingly, the outbreak was sustained by the highrisk clone ST307, although the ST22, ST6342, ST6418 and ST6811 have also been identified and associated to KPC-166. Worryingly, blaKPC-166 could be transferred horizontally and, after cloning, it conferred resistance to CZA. Discussion: This novel variant confers CZAeresistance and carbapenems susceptibility restoration. As KPC-166 was found expressed by multiple Kp clones, greater efforts should be made to prevent the further dissemination of such strains in Italian clinical settings.