Characterization of Integrative and Conjugative Elements Carrying bla_NDM-1 and bla_KPC-2 in an Environmental...

Urban wastewater is increasingly recognized as a major reservoir of antimicrobial resistance and horizontal gene transfer. From urban wastewater in Hanoi, Vietnam, we isolated a multidrug-resistant Pseudomonas guariconensis strain, KNHN1, resistant to most antimicrobials, including carbapenems and cephalosporins, but susceptible to cefiderocol; and intermediate to colistin. Whole-genome sequencing revealed two chromosomally integrated integrative and conjugative elements (ICEs): ICE_{PgKNHN1_KPC} (131 kb) carrying bla_KPC-2 and ICE_{PgKNHN1_NDM} (108 kb), carrying bla_NDM-1, both flanked by conserved 18-bp att sites in the tRNA^Gly loci and encoding MOB_H-type relaxases. Polymerase chain reaction and subsequent sequencing confirmed ICE excision from the chromosome and formation of circular intermediates. Conjugation to Pseudomonas putida KT2440 occurred at ~10⁻² frequency, producing transconjugants with ICE_{PgKNHN1_NDM} (~85%), ICE_{PgKNHN1_KPC} (~10%), or both, all showing broad range β-lactam resistance. Comparative analysis indicated that ICE_{PgKNHN1_NDM} has a highly conserved backbone across multiple species and often co-carries bla_PME-1 and other resistance genes. To our knowledge, this is the first report of chromosomally integrated bla_NDM‑1 and bla_KPC‑2 in P. guariconensis mediated by functional ICEs. These findings underscore the pivotal role of environmental bacteria as reservoirs of clinically significant resistance genes, and highlight ICEs as key drivers in the dissemination of carbapenem resistance.