Hanseob Shin, Min Ki Jeon, Hor‐Gil Hur
ABSTRACT Metagenomic analyses rely heavily on contig assembly and reference databases, which can introduce substantial bias when predicting the hosts of antibiotic resistance genes (ARGs) in complex environmental microbiomes. Reference‐based metagenomic pipelines assign ARGs mostly to clinically important pathogens because publicly available genomic repositories are dominated by clinically relevant isolates. Motivated by this limitation, we investigated whether metagenomic inferences accurately reflect the true bacterial hosts of ARGs in a wastewater treatment plant, also integrating culture‐based validation. Metagenomic screening suggested that ARGs ( bla OXA ) were primarily associated with clinical taxa. In contrast, culture‐based screening identified a wider host distribution of bla OXA genes. Our results imply that environmental bacteria, rather than clinically important taxa, are also hosts of bla OXA genes. Phenotypic testing showed elevated cephalosporin minimal but no carbapenem resistance, consistent with the nature of carbapenem‐hydrolysing class D β‐lactamases. Our findings reveal that reliance on reference‐based metagenomic host prediction can underestimate the diversity of environmental ARG reservoirs. This integrated approach highlights the need for cautious interpretation of metagenomic host assignments and the importance of coupling metagenomic pipelines with culture‐dependent validation when assessing ARG ecology in the natural environments.