High-level ceftazidime-avibactam resistance by in-host evolution of blaKPC genes: Emergence of a novel blaKPC-102 variant and increase of blaKPC-33 copy number in Klebsiella pneumoniae strains from a lung transplantation recipient.

OBJECTIVE

KPC-producing Klebsiella pneumoniae (Kp) infections have become one of the major threats to public health in China. Recently, the emergence of ceftazidime-avibactam (CZA) resistance due to the blaKPC mutations has been increasingly reported.

METHODS

Two CZA-susceptible Kp strains (Kp36854 carrying blaKPC-2 and Kp37523 without blaKPC) and two CZA-resistant Kp strains (Kp38935 carrying double copies of blaKPC-33 and Kp38097 carrying a newly identified blaKPC-102 gene) were isolated from a lung-transplanted patient during CZA treatment. This study analyzed the within-host evolutionary dynamics of blaKPC in KPC-Kp strains.

RESULTS

Compared with KPC-2, KPC-33 possessed only a D179Y substitution while KPC-102 harbored both D179Y and Y241D substitutions. We constructed KPC-δ33 with only a Y241D substitution, which means the correction of the first mutation that is also present in KPC-33 (D179Y). Cloning and expression experiments showed that CZA-MIC value of E. coli DH5α/pKPC102 was 512 mg/L, while CZA-MIC values of both E. coli DH5α/pKPC-33 and E. coli DH5α/pKPC-δ33 were 32/4 mg/L. Enzymatic kinetic analysis revealed that KPC-2 demonstrated the highest catalytic efficiency to nitrocefin and meropenem, whereas KPC-33 displayed higher catalytic efficiency against ceftazidime compared to other tested KPC variants. Regarding avibactam, KPC-2 showed the highest sensitivity, while KPC-δ33 and KPC-102 were less sensitive.

CONCLUSION

High-level CZA resistance was mediated by the novel blaKPC-102 and double copies of blaKPC-33 gene, respectively, in two porin-deficient Kp strains from one patient. The CZA resistance resulting from blaKPC mutation could be selected and evolved to be more diverse and heterogeneous within the host after CZA therapy. It is very essential to perform the surveillance of CZA-resistance for clinicians during treatment.