CONCLUSION

This study provides the first shotgun metaproteomic characterization of habitat-associated gut resistomes in free-living M. fascicularis from Thailand. Distinct protein expression profiles were observed between macaque populations inhabiting natural ecotourism and urban-proximate environments, demonstrating that habitat characteristics and anthropogenic influences can shape the functional expression of AMR-associated proteins within the gut microbiome. A total of 1,299 DEPs were identified in Escherichia spp. and 231 DEPs in Salmonella spp., highlighting substantial habitat-related differences in microbial activity. Functional analyses revealed that proteins associated with microbial proliferation predominated in Escherichia spp., whereas DNA repair and stress-response functions were more prominent in Salmonella spp. from location P. Notably, Escherichia spp. from location S exhibited a greater abundance and diversity of antibiotic resistance-associated proteins, including multidrug efflux components and β-lactamase-related mechanisms.

From a practical perspective, these findings suggest that free-living macaques inhabiting environments with greater human influence may serve as important reservoirs and sentinels for the dissemination of environmental AMR. The identification of habitat-specific resistance mechanisms provides valuable information for wildlife health monitoring, environmental surveillance programs, and One Health-based AMR management strategies. Furthermore, the results emphasize the importance of managing human–wildlife interactions, anthropogenic food provisioning, and environmental contamination to reduce the potential spread of antimicrobial-resistant microorganisms at the human–animal–environment interface.

A major strength of this study is the application of shotgun metaproteomics, which enabled direct assessment of actively expressed resistance-associated proteins rather than merely detecting the presence of resistance genes. This approach provides a more biologically relevant understanding of microbial functional responses to environmental pressures. However, the study was limited by its relatively small sample size, restricted geographic coverage, pooled-sample design, and the lack of complementary metagenomic, metabo-lomic, and environmental exposure data. Consequently, causal relationships between habitat characteristics, dietary factors, and AMR expression could not be fully established.

Future studies should incorporate larger multicenter sampling schemes, longitudinal monitoring, dietary profiling, environmental contaminant assessments, and integrated multi-omics approaches to elucidate the ecological drivers of AMR expression in wildlife populations. Comparative investigations involving additional primate species and habitats would further enhance understanding of wildlife-associated resistome dynamics.

Overall, the present findings demonstrate that habitat-associated anthropogenic pressures influence the functional expression of AMR-associated proteins in the gut microbiota of free-living long-tailed macaques. These results advance current knowledge of wildlife resistome ecology and reinforce the importance of incorporating wildlife populations into One Health surveillance frameworks aimed at mitigating the emergence and spread of AMR.