Emerging reservoirs of metallo-β-lactamase genes among non-aeruginosa Pseudomonas and Stutzerimonas species in a Spanish tertiary hospital

Introduction Pseudomonas is one of the largest genera among Gram-negative bacteria, currently comprising nearly 400 valid species. Among them, non- aeruginosa Pseudomonas species, together with Stutzerimonas —a genus recently separated from Pseudomonas —are becoming important reservoirs of antimicrobial resistance genes. However, accurate species-level identification of these organisms remains challenging when relying on the methods routinely implemented in most clinical microbiology laboratories, such as MALDI-TOF mass spectrometry. Therefore, this study aimed to identify and characterize metallo-β-lactamase-producing non- aeruginosa Pseudomonas spp. and Stutzerimonas spp. isolates obtained in a Spanish tertiary hospital, focusing on their species-level identification, dissemination and role as reservoirs of carbapenemase-encoding genes. Methods Whole-genome sequencing (WGS) and antimicrobial susceptibility testing (AST) were used to identify and characterize a total of 22 carbapenemase-producing non- aeruginosa Pseudomonas and Stutzerimonas isolates collected from patient and environmental samples from March 2022 to July 2024 in a Spanish tertiary hospital. Results WGS analysis identified eight species: Pseudomonas alloputida ( n = 7), Pseudomonas mosselii ( n = 5), Pseudomonas kurunegalensis ( n = 4), Pseudomonas asiatica ( n = 2), Pseudomonas oleovorans ( n = 1), Pseudomonas soli ( n = 1), Stutzerimonas chloritidismutans ( n = 1) and Stutzerimonas stutzeri ( n = 1). Multilocus sequence typing (MLST) classified isolates of the P. putida group into nine STs, revealing three predominant clones: P. alloputida ST69, P. mosselii ST115 and P. kurunegalensis ST114. AST results showed resistance rates >55% to all tested β-lactams, aminoglycosides and fluoroquinolones, except for cefiderocol, amikacin and colistin (0–5%). The following carbapenemase combinations were detected: bla VIM−1 ( n = 16), bla VIM−1 + bla VIM−2 ( n = 4), bla VIM−1 + bla NDM−1 ( n = 1) and bla VIM−11 ( n = 1). Seven distinct bla VIM -harboring plasmids were detected in 16 isolates, whereas the remaining six carried chromosomally-integrated bla VIM . Eight different bla VIM -harboring class I integrons were detected, two of which also occurred in P. aeruginosa clinical isolates obtained in the same hospital. Conclusion This study highlights the epidemiological relevance of non- aeruginosa Pseudomonas and Stutzerimonas species as reservoirs of metallo-β-lactamase genes encoded in mobile genetic elements. These findings reinforce the need for systematic WGS-based surveillance of both clinical and environmental isolates, enabling the detection of hidden transmission networks and interspecies dissemination of antimicrobial resistance determinants.