Multiple mechanisms mediate aztreonam-avibactam resistance in Klebsiella pneumoniae: Driven by KPC-2 and OmpK36 mutations.

Aztreonam-avibactam (ATM-AVI) is a promising β-lactam/β-lactamase inhibitor combination with an antimicrobial spectrum covering serine carbapenemase- or metallo-β-lactamase-producing Enterobacterales. Although ATM-AVI has not been widely used in clinical practice, resistance to it in Escherichia coli has been widely reported. In this study, we investigated an ATM-AVI-resistant Klebsiella pneumoniae strain, designated as 1705R, derived from K. pneumoniae ATCC BAA-1705 by induction, with a minimal inhibitory concentration of 128 µg/mL. The 1705R strain contained two copies of the bla variant, which encodes for a K. pneumoniae carbapenemase (KPC) variant with a Ser109Pro substitution, as well as a premature termination in OmpK36 and OmpK35 porins. This KPC variant decreased susceptibility to ATM-AVI by four-fold and demonstrated a reduced affinity for ATM and AVI in molecular docking analysis. In porin-deficient strains harbouring this KPC variant, ATM-AVI susceptibility was further diminished, exhibiting a 32-fold reduction. Whole-genome sequencing revealed that the transposition of Tn4401 carrying bla from the IncFIB/FIIK plasmid into the ColRNAI plasmid produced a second copy of bla. Quantitative polymerase chain reaction revealed that the copy number of bla and its carrier plasmid increased, which significantly up-regulated their mRNA expression. Overexpression of the AcrAB-TolC efflux pump may also be associated with high levels of ATM-AVI resistance. Furthermore, collateral susceptibility and costs of growth and biofilm formation developed after the acquisition of ATM-AVI resistance. This study demonstrates that multiple molecular mechanisms collectively contribute to ATM-AVI resistance in K. pneumoniae 1705R strain, which may represent a mode of resistance to ATM-AVI.