Aztreonam-Avibactam Resistance Due to Synergistic Interplay of ampC-Related Mutations in Carbapenem-Resistant...

Aztreonam-avibactam (ATM-AVI) is an important antimicrobial option for metallo-β-lactamases (MBLs)-producing Enterobacterales. The ATM-AVI-resistant mechanisms remain elusive.

Through induction experiments, ATM-AVI-resistant mutants were obtained from an MBL-producing Enterobacter xiangfangensis clinical strain. Mutants were genome sequenced and analyzed. We investigated a complex combination involving mutations causing structural alteration of AmpC and enhanced expression of its encoding ampC gene leading to resistance to various degrees of ATM-AVI resistance. The expression of ampC was measured by quantitative polymerase chain reaction (PCR).

We obtained 4 ATM-AVI-resistant mutants. All mutants harbored ampC mutations encoding an amino acid substitution at position 150 (Tyr150Cys or Tyr150Ser) and mutations of ampD, leading to either an amino acid substitution (Cys108Tyr) or premature truncation (Glu106X and Trp95X). Additionally, 2 mutants had mutations in the ampC attenuator region. The minimum inhibitory concentration (MIC; ATM/AVI mg/L) changes associated with the 4 mutants were: (1) AmpC (Tyr150Ser) plus AmpD (Glu106X) MIC = 32/4; (2) AmpC (Tyr150Cys) plus AmpD (Cys108Tyr) MIC = 64/4; (3) AmpC (Tyr150Cys) plus AmpD (Cys108Tyr) plus ampC attenuator region (+13G > T) MIC = 256/4; and (4) AmpC (Tyr150Cys) plus AmpD (Trp95X) plus ampC attenuator region (+7T > C) MIC = 256/4. Either AmpC Tyr150Cys/Ser substitution or knockout of ampD alone reduced susceptibility to ATM-AVI. However, AmpC Tyr150Cys/Ser substitution and ampD mutation in combination conferred ATM-AVI resistance. Furthermore, attenuator mutations upregulated ampC expression by 2- to 4-fold and led to elevated resistance to ATM-AVI in the presence of AmpC Tyr150Cys/Ser substitution and ampD mutation.

The synergy of regulatory gene (ampD) mutations, ampC attenuator region mutations, and AmpC structural variations drive high-level ATM-AVI resistance in Enterobacter.