Emergence of a KPC Variant Conferring Resistance to Ceftazidime-Avibactam in a Widespread ST11 Carbapenem-Resistant Klebsiella pneumoniae Clone in China

Abstract

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection poses a great threat to public health worldwide, and KPC-2-producing strains are the main factors responsible for resistance to carbapenems in China. Ceftazidime/avibactam (CZA) is a novel β-lactam/β-lactamase inhibitor combination with good activity against KPC-2 carbapenemase and is becoming the most important option for treating KPC-producing CRKP infection. Here, we report the emergence of a novel KPC-2 variant, designated KPC-74, produced by K. pneumoniae strain KP55, that conferred CZA resistance in a patient after CZA exposure. The novel blaKPC–74 variant showed a deletion of 6 nucleotides at positions 712–717 compared with blaKPC–2, and this deletion resulted in the consequent deletion of glycine and valine at positions 239 and 240. Antimicrobial susceptibility testing showed that KP55 presents multidrug resistance, including resistance to CZA and ertapenem, but is susceptible to imipenem, meropenem, and colistin. The blaKPC–74 gene was located on a plasmid, as determined by S1-nuclease pulsed-field gel electrophoresis followed by southern blotting, and confirmed to be 133,766 bp in length by whole-genome sequencing on both the Illumina and MinION platforms. The CZA resistance phenotype of the novel KPC variant was confirmed by both transformation of the blaKPC–74-harboring plasmid and a blaKPC–74 gene cloning assay, showing a 64-fold higher CZA minimum inhibitory concentration (MIC) than the recipient strains. The G239_V240del observed in KPC-74 was outside the omega-loop region but was still close to the active site Ser70 and omega-loop in the protein tertiary structure. The enzyme kinetic parameters and IC50 values further indicated that the hydrolytic activity of the KPC-74 enzyme against ceftazidime was potentiated twofold and that the affinity between KPC-74 and avibactam was alleviated 17-fold compared with that of the KPC-2 allele. This CZA resistance mediated by KPC-74 could be selected after CZA therapy and evolved to be more diverse and heterogeneous. Surveillance of CZA resistance is urgently needed in clinical settings.

Molecular Mechanisms Driving the In Vivo Development of KPC-71-Mediated Resistance to Ceftazidime-Avibactam during Treatment of Carbapenem-Resistant Klebsiella pneumoniae Infections

“…S1 in the supplemental material) and might fail to combine the KPC-2 protein antibody. Similar results were also observed in our previous study ( 15 ). However, the precise role of this 182S insertion outside the omega loop remains unclear and will need further studies.…”

“…In addition, the KPC-71 enzyme displayed a lower hydrolysis activity of nitrocefin (∼4,850-fold) than wild-type KPC-2 ( Table 2 ). Notably, the KPC-71 enzyme has almost no detectable hydrolysis activity of carbapenems (ertapenem, imipenem, and meropenem) under our conditions ( Table 2 ) ( 15 ), indicating that reduced hydrolysis activity caused by KPC-71 leads to susceptibility to carbapenems.…”

“…In this plasmid, several antimicrobial resistance genes were also identified, such as bla TEM-1 , bla SHV-12 , bla CTX-M-65 , and rmtb . Further sequence alignments revealed that the plasmid sequence showed almost identical nucleotide sequences (100% coverage and 99.99% identity) with the plasmids pKP55_2 (accession number CP055296 ) ( 15 ), pB (accession number CP069172 ), pKP37-KPC (accession number CP082755 ), and p3-L39 (accession number CP033956 ), indicating that bla KPC-71 gene mutation was derived from the bla KPC-2 gene based on the similarity of plasmid sequences.…”

“…Cloning, expression, and purification of β-lactamase were performed as previously described ( 15 ). Protein concentration was determined by absorbance at 280 nm using an extinction coefficient of 39,545 M −1 cm −1 .…”

Antibiotic Heteroresistance in Klebsiella pneumoniae

“…The main K. pneumoniae carbapenemase (KPC) is the most common class A serine β-lactamase, encoded by the bla KPC gene carried on plasmids and transported in the Tn4401 transposon, which facilitates its efficient transfer between bacterial isolates [ 75 ]. The increasing number of new KPC variants encoded on mobile genetic elements and their dissemination in Gramm-negative bacteria has been recently reported [ 76 , 77 ]. In addition to KPC, K. pneumoniae may produce different metallo-β-lactamases, including New Delhi metallo-β-lactamase (NDM), Verona integron-encoded metallo-b-lactamase (VIM), imipenemase (IMP), and OXY-48-like carbapenemases [ 3 , 75 ].…”