Intrinsic class C extended-spectrum β-lactamases mediate resistance to oxyimino-cephalosporins in Antarctic Pseudomonas fluorescens complex bacteria

ABSTRACT From a One Health perspective, identifying environmental reservoirs of antibiotic resistance determinants with potential clinical relevance is increasingly important. Among resistance mechanisms, β-lactamases are of particular concern because they compromise the efficacy of β-lactam antibiotics, the most widely used class in clinical practice. Antarctic soils harbor thousands of putative β-lactamase genes, yet the functional properties and resistance contributions of these enzymes remain largely unexplored. Here, we investigated the distribution, diversity, and functional impact of intrinsic class C β-lactamases (AmpC-type) in Antarctic members of the Pseudomonas fluorescens species complex, a lineage widely distributed in Antarctic soils and increasingly recognized as an opportunistic pathogen of humans, animals, and plants. Genome-scale analyses revealed that class C β-lactamases are intrinsic, widespread, and highly diverse within this lineage. Phenotypic assays demonstrated that Antarctic isolates exhibit elevated resistance to β-lactam antibiotics, particularly oxyimino-cephalosporins such as cefotaxime and ceftazidime, and that this phenotype is largely attenuated by β-lactamase inhibition. Heterologous expression of selected Antarctic AmpC variants in a susceptible Escherichia coli host confirmed their ability to increase minimum inhibitory concentrations to oxyimino-cephalosporins. Notably, several Antarctic β-lactamases harbor amino acid substitutions previously associated with extended-spectrum AmpC (ESAC) variants, including M174L and N346I, while others display broader substrate profiles despite lacking known ESAC-associated signatures. Comparative analyses further showed that the sequence diversity of Antarctic class C β-lactamases exceeds that reported for clinical PDC variants from Pseudomonas aeruginosa . Together, these findings provide functional evidence that pristine Antarctic environments constitute reservoirs of naturally occurring class C β-lactamases with extended-spectrum potential. Our results highlight the evolutionary depth and functional diversity of environmental β-lactamases and underscore the importance of incorporating remote ecosystems into One Health–oriented antimicrobial resistance surveillance frameworks.