The prevalence and diversity of carbapenem-hydrolyzing bacteria and β-lactamases in the aquatic environment of the Han River, South Korea

• Diverse carbapenem-hydrolyzing bacteria and β-lactamases were prevalent in the river. • A considerable number of novel carbapenemase gene variants were identified. • Two clinically important OXA family carbapenemases were functionally expressed. • Bla OXA-652 was flanked by transposase genes, suggesting potential transmission. • The Han River serves as a reservoir of diverse carbapenemases and their producers. Antimicrobial resistance poses a critical global health threat, and carbapenem-resistant bacteria (CRB) are of a particular concern. Aquatic environments are increasingly recognized as reservoirs for CRB and associated resistance determinants. This study investigated the diversity, distribution, and functional properties of carbapenem-hydrolyzing β-lactamases (CHβLs) in environmental isolates from the Han River, South Korea. A multi-step screening of approximately 3,200 environmental isolates identified 64 carbapenem-hydrolyzing bacteria, predominantly from the genera Chryseobacterium, Stenotrophomonas , and Aeromonas . Using 41 carbapenemase-specific primer pairs and multiplex PCR, 62 distinct CHβL sequences were detected, with Ambler class B metallo-β-lactamases—particularly subclasses B1 (CGB and IND families) and B3 (L1 and GOB families)—being most prevalent. Notably, 46 sequences represented novel variants, each differing from previously reported CHβLs by at least one amino acid substitution. Two clinically relevant class D β-lactamase genes, bla OXA-334 and bla OXA-652 , were identified in Acinetobacter johnsonii isolates, and their heterologous expression confirmed their functionality by conferring resistance against carbapenems. Genomic analysis further revealed that bla OXA-652 gene was flanked by transposases associated with the insertion sequence (IS) 200/605, IS4, and IS1 families, suggesting potential for transmission via horizontal gene transfer. These results demonstrate the substantial diversity of carbapenemases and their producers of environmental origin in the Han River during the investigation period, suggesting the riverine environment as a reservoir of various CHβLs, including clinically important enzymes. Our findings underscore the potential role of aquatic ecosystems in the dissemination of clinically relevant carbapenemase genes and highlight the necessity for continuous surveillance of the environmental resistome.