Genomic epidemiology and aztreonam–avibactam resistance mechanisms of Proteus mirabilis in china: an eight-year retrospective study

Proteus mirabilis has emerged as an important multidrug-resistant opportunistic pathogen, with the production of metallo-β-lactamases (MBLs) being a major contributor to its broad-spectrum resistance. Although the aztreonam-avibactam (ATM-AVI) combination represents a key therapeutic option against MBL-producing Enterobacteriaceae, the mechanisms underlying ATM-AVI resistance in P. mirabilis has not yet been reported. A total of 176 multidrug-resistant P. mirabilis isolates were collected from a tertiary hospital in China (2017-2024). Antimicrobial susceptibility testing identified ATM-AVI-resistant isolates (MIC ≥ 8/4 µg/mL). Whole-genome sequencing, gene cloning, RT-qPCR, and copy number analyses were used to determine resistance mechanisms. Growth rate assays evaluated fitness costs, and global phylogenetic analysis elucidated evolutionary and dissemination patterns. Twelve isolates (6.8%, 12/176) were resistant to ATM-AVI, all carrying the bla PER-4 gene. Cloning experiments confirmed that bla PER-4 conferred significantly higher ATM-AVI resistance than bla PER-1 . Increased resistance correlated with bla PER-4 overexpression and gene copy number amplification. Whole-genome analysis showed that bla PER-4 was embedded in IS CR1 -associated class 1 integrons located on both plasmids and chromosomes, with a strain carrying eight tandem chromosomal copies. These structures likely mediated gene amplification via rolling-circle replication and homologous recombination. Phylogenetic analysis revealed that bla PER-4 -positive isolates were mainly associated with the ST135 lineage, suggesting transmission event within hospitals. Global data demonstrated that bla PER-4 -carrying P. mirabilis strains were predominantly found in China (80%, 12/15), while bla PER-1 strains were more common in the United States. The bla PER-4 -carrying P. mirabilis , particularly the ST135 clone, represents a high-risk lineage associated with high-level ATM-AVI resistance mediated by gene overexpression and copy number amplification. This finding highlights a novel mechanism of ATM-AVI resistance and underscores the need for continuous genomic surveillance and rational antimicrobial stewardship to prevent its further dissemination.