Whole-genome analysis of NDM-producing associated with recurrent bacteraemia with rapid development of aztreonam-avibactam resistance.

A 60-year-old male with recurrent bacteremia associated with necrotizing pancreatitis was followed over 17 months, during which six clinical isolates of were obtained. The index isolate (R1) was susceptible to aztreonam-avibactam (MIC 4 mg/L), but resistance (MIC >64 mg/L) emerged with isolate B1, collected one month later, and persisted in subsequent isolates. To investigate the genomic and phenotypic evolution of six clinical isolates, focusing on mechanisms underlying the emergence of aztreonam-avibactam resistance. Whole-genome sequencing was performed to determine species identity, phylogenetic relationships, and resistance determinants. Average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) were used to confirm species classification. Comparative genomic analysis with R1 as a reference identified mutations associated with resistance. Structural modelling assessed the functional impact of key mutations. All isolates were sequence type ST44. Phylogenomic analysis revealed the isolates were more closely related to than , supported by ANI (97%) and DDH (76%). All isolates harboured , correlating with carbapenem resistance. Resistant isolates displayed a glycine insertion at position 420 in PBP3 (p.Gly420dup), which structural modelling indicated disrupted aztreonam-avibactam binding. A Gly151Asp substitution in OmpC was also identified, potentially affecting drug permeability. No mutations were observed in other porins (OmpA, OmpD, or OmpW). This study identifies a glycine insertion in PBP3 as a novel mechanism driving aztreonam-avibactam resistance in , supported by structural modelling and additional mutations in OmpC. This study provides evidence of a novel resistance mechanism to aztreonam-avibactam in , driven by a glycine insertion in PBP3 and supported by alterations in OmpC.