In vitro mimicry of in vivo KPC mutations by ceftazidime-avibactam: phenotypes, mechanisms, genetic structure and kinetics of enzymatic hydrolysis

Abstract

Ceftazidime-avibactam (CZA) is employed for the treatment of infections caused by <i>Klebsiella pneumoniae</i> carbapenemase-producing <i>K. pneumoniae</i> (KPC-KP). Resistance to CZA is frequently linked to point mutations in the <i>bla</i>_KPC. We conducted <i>in vitro</i> simulations of <i>in vivo bla</i>_KPC mutations using CZA. Four pre-therapy KPC-KP isolates (K1, K2, K3, and K4) were evaluated, all initially exhibited susceptibility to CZA and produced KPC-2. The crucial distinction was that following CZA treatment, the <i>bla</i>_KPC-2 mutated in K1, K2, and K3, rendering them resistant to CZA, while K4 achieved microbiological clearance, and <i>bla</i>_KPC-2 remained unaltered. The induction assay identified various <i>bla</i>_KPC-2 variants, including <i>bla</i>_KPC-25, <i>bla</i>_KPC-127, <i>bla</i>_KPC-100, <i>bla</i>_KPC-128, <i>bla</i>_KPC-137, <i>bla</i>_KPC-138, <i>bla</i>_KPC-144 and <i>bla</i>_KPC-180. Our findings suggest that the resistance of KPC-KP to CZA primarily results from the emergence of KPC variants, complemented by increased <i>bla</i>_KPC expression. A close correlation exists between avibactam concentration and the rate of increased CZA minimum Inhibitory concentration, as well as <i>bla</i>_KPC mutation. Inadequate avibactam concentration is more likely to induce resistance in strains against CZA, there is also a higher likelihood of mutation in the <i>bla</i>_KPC-2 and the optimal avibactam ratio remains to be determined. Simultaneously, we selected a <i>bla</i>_KPC-33-producing <i>K. pneumoniae</i> strain (mutated from <i>bla</i>_KPC-2) and induced it with imipenem and meropenem, respectively. The <i>bla</i>_KPC-2 was detected during the process, indicating that the mutation is reversible. Clinical use of carbapenems to treat KPC variant strains increases the risk of infection, as the gene can mutate back to <i>bla</i>_KPC-2, rendering the strain even more cross-resistant to carbapenems and CZA.