New resistance threat in difficult-to-treat resistance Pseudomonas aeruginosa co-producing AFM and KPC carbapenemases: plasmid dynamic transfer and global phylogeography perspective.

Metallo-β-lactamase (MBL) production is one of the primary carbapenem resistance mechanisms in carbapenem-resistant Pseudomonas aeruginosa (CRPA). The emergence of the novel MBL gene blaAFM poses a significant threat to global public health. Concerningly, we have identified clinical CRPA strains co-producing AFM and the widely-disseminated carbapenemase KPC-2. Here, we describe AFM-producing, KPC-2-producing, and AFM/KPC-2 co-producing clinical CRPA isolates that were collected from three patients in two different hospital buildings in China. Comparative genomics suggested horizontal transfer of a blaAFM-2-harboring plasmid may have contributed to the spread of the AFM carbapenemase between different hospital areas, and to the emergence of dual carbapenemase-producing CRPA. Further epidemiological source tracing revealed the likely involvement of cross-patient nursing care and cross-area patient transfer in carbapenemase transmission. Experimental data confirmed the transfer ability of clinical blaAFM-2-bearing plasmids into P. aeruginosa PAO1. As the global epidemiology of blaAFM has not been systematically evaluated, we further examined 30,800 publicly available P. aeruginosa genome sequences. Including those generated in this study, blaAFM genes were detected in 36 isolates in total, which were derived from China (35/36) or Australia (1/36). AFM-containing genomes were sourced from six Chinese provinces, with 63.9% (23/36) isolated in Zhejiang between 2020 and 2024. The most prominent AFM-associated P. aeruginosa clone was ST463 (17/36 genomes). Our study highlights the concerning challenge presented by blaAFM-harboring CRPA in clinical settings. Horizontal transfer of blaAFM-bearing plasmids can contribute to difficult-to-treat resistance (DTR) phenotypes. Surveillance should be strengthened to prevent the further spread of these plasmids, particularly into and within ICUs.