Whole-genome shotgun metagenomics reveals environmental perturbations in the gut microbiome and resistome of wild raccoon dogs rescued at a wildlife center.

Raccoon dogs are potential vectors for the transmission of zoonotic pathogens and antimicrobial resistance, with their gut microbiome carrying genetic determinants of virulence and resistance. However, previous studies have primarily relied on traditional culture-based approaches, limiting our understanding of the entire dynamics and genetic potential of the gut microbiome in raccoon dogs. In this study, we employed both culture-dependent approach and whole-genome shotgun sequencing in raccoon dogs undergoing rehabilitation for eight weeks. We integratively assessed how rehabilitation-related environmental shifts influence the gut microbiome and resistome. Rehabilitation induced shifts in gut microbiome composition. Notably, within-group diversity decreased at week eight, indicating increased compositional similarity. Throughout rehabilitation, a total of 18 genera (e.g., Paeniclostridium, Psychrobacter) significantly increased, and 40 genera (e.g., Intestinimonas, Erysipelatoclostridium) decreased in abundance. Rehabilitation also induced shifts in the gut resistome, with reduced within-group diversity at weeks four and eight, indicating increased compositional similarity over time. Overall, 253 antimicrobial resistance genes (ARGs) present at week 0 disappeared, whereas 273 ARGs were acquired during rehabilitation. Additionally, the abundance of 15 ARGs significantly decreased and that of 33 increased, with beta-lactamases being the most common among the latter. Culture-dependent methods revealed a marked increase in bacteria resistant to third-generation cephalosporins, monobactams, and sulfonamides. Changes in microbiome composition during rehabilitation indirectly influenced the resistome and virulome through mobile genetic elements. Our findings suggest that rehabilitation-induced perturbations in the gut microbiome and resistome of raccoon dogs are likely driven by environmental shifts such as diet and habitat. These changes may affect their post-release fitness and raise concerns owing to the potential dissemination of zoonotic pathogens and antimicrobial resistance.