Metagenomic characterization of the gut microbiome, resistome, and pathogenic potential in fecal samples of Pied Harrier (Circus melanoleucos)

Migratory birds are increasingly recognized as potential carriers and disseminators of antimicrobial-resistant (AMR) bacteria and pathogens. This study characterized the gut microbiome, resistome, and virulome of the Pied Harrier ( Circus melanoleucos ), a migratory raptor occupying a high trophic level. Ten pooled fecal samples were collected during 2022–2023 at a major wintering site at Wiang Nong Lom, a wetland in Northern Thailand. The site exhibits substantial overlap with agricultural and human-modified land use, providing an opportunity to investigate exposure to AMR bacteria and antimicrobial resistance genes (ARGs) at the wildlife–human interface. Shotgun metagenomic analysis revealed a microbial community dominated by Escherichia spp., Clostridium spp., and Clostridioides spp. A total of 66 putative ARGs and 185 virulence factor genes (VFGs) were identified. More than half of the detected ARGs were associated with multidrug resistance, with fluoroquinolone resistance genes being the most abundant ARGs, followed by penicillin β-lactam and tetracycline resistance genes. The most abundant ARGs, Cper_mprF , tetA(P) , and tetB(P) , were associated with Clostridium spp., whereas Escherichia spp. harbored the greatest diversity of ARGs and VFGs. Network analyses revealed that the majority of ARGs and VFGs were associated with Escherichia spp., highlighting their potential as important carriers of antimicrobial resistance and virulence determinants within the Pied Harrier gut microbiome. Among ARGs and VFGs with confident genomic location assignments, plasmid-associated genes (38% and 43%, respectively) were more prevalent than chromosome-associated genes (6% and 15%, respectively). However, only limited associations with MGEs and limited evidence of HGT were identified, suggesting that plasmid carriage did not necessarily indicate widespread horizontal dissemination. These findings should be interpreted in light of the limitations of short-read metagenomic assemblies. These findings provide baseline insights into the microbiome and resistome of the Pied Harriers and suggest that migratory raptors may contribute to the environmental dissemination of antimicrobial resistance determinants along their migratory flyways.