Tandem amplification of the umpA allele contributes to ceftazidime-avibactam heteroresistance in clinical carbapenem-resistant Klebsiella pneumoniae isolates

Ceftazidime-avibactam (CAZ/AVI, CZA) combination has shown promising efficacy against carbapenem-resistant Klebsiella pneumoniae (CRKP); however, several recent studies have reported resistance to CZA in CRKP. Hence, the present study aimed to elucidate the potential mechanisms of CZA resistance and heteroresistance in clinical carbapenemase (KPC)-producing K. pneumoniae. A total of 25 KPC-producing K. pneumoniae were collected and identified 19 CZA-resistant and 3 CZA-heteroresistant isolates using broth microdilution and population analysis profile methods, respectively. Whole-genome sequencing and comparative genomic analysis of the heteroresistant parental strain and its resistant subpopulation showed mutations in blaKPC-2 (KPC-71 variant) and tandem amplification of the lipoprotein diacylglyceryl transferase umpA allele in the resistant subpopulation. Reverse transcription-quantitative PCR (qRT-PCR) results demonstrated that the copy number of umpA gene is associated with resistance to CZA. umpA overexpression was induced in K. pneumoniae strains involved in CZA heteroresistance and failed CZA therapy in infected mice. Flow cytometry and parallel reaction monitoring (PRM) assays were performed to investigate the heteroresistance mechanisms in umpA overexpression strains. Flow cytometry analysis showed that overexpression of the umpA gene decreased propidium iodide incorporation in the cells of this strain. PRM assays showed that the peptidoglycan-associated lipoprotein Pal and the outer membrane (OM) protein OmpA were significantly upregulated in the umpA-overexpressing K. pneumoniae strain. In conclusion, we demonstrated a new mechanism for CZA heteroresistance wherein tandem umpA amplification generated population heterogeneity and failed CZA therapy in vivo experimentally infected mice. Lipoprotein biosynthetic pathways are considered a potential target for CZA resistance.IMPORTANCEThe novel synthetic β-lactamase inhibitor combination ceftazidime-avibactam has been available clinically for a few years. However, evidence that resistance and heteroresistance to ceftazidime-avibactam are responsible for treatment failure in clinical settings is increasing, and the underlying molecular mechanisms involved in developing heteroresistance have not been elucidated in detail. This study highlights that the lipoprotein diacylglyceryl transferase umpA gene amplification conferred heteroresistance to K. pneumoniae strains. Moreover, we found that umpA gene amplification-driven ceftazidime-avibactam heteroresistance might have resulted in vivo treatment failure during K. pneumoniae infection in mice. We also observed that the umpA gene amplification upregulated the expression levels of lipoproteins and outer membrane proteins and decreased outer membrane permeability in K. pneumoniae strains. These findings suggest that ceftazidime-avibactam heteroresistance is a major clinical concern in eradicating bacterial infections. Furthermore, lipoprotein biosynthetic pathways are considered a promising target for overcoming ceftazidime-avibactam (CZA) resistance.