Shotgun metaproteomics reveals habitat-specific antimicrobial resistance-associated proteins of Escherichia spp. and Salmonella spp. in the gut resistome of free-living long-tailed macaques in Thailand.

Abstract

Background and aim: Antimicrobial resistance (AMR) is a growing global health concern affecting humans, animals, and the environment within the One Health framework. Free-living non-human primates may act as reservoirs and disseminators of antimicrobial-resistant microorganisms through close interactions with humans and shared environments. This study aimed to characterize habitat-specific AMR-associated proteins of Escherichia spp. and Salmonella spp. in the gut resistome of free-living long-tailed macaques (Macaca fascicularis) inhabiting distinct ecological settings in Thailand using a shotgun metaproteomics approach.

Materials and methods: A total of 54 fecal samples were non-invasively collected from free-living long-tailed macaques at two locations in Thailand between January and February 2025: Chongkrachok Mountain, Prachuap Khiri Khan Province (natural ecotourism habitat; n = 16) and Phromawat Temple, Chonburi Province (urban-proximate habitat; n = 38). Proteins were extracted and analyzed using liquid chromatography-tandem mass spectrometry. Label-free shotgun metaproteomics was performed using MaxQuant and Perseus platforms. Differential protein expression, functional annotation, and pathway analyses were conducted using MetaboAnalyst 6.0, Gene Ontology classification, and Kyoto Encyclopedia of Genes and Genomes pathway mapping.

Results: Distinct protein expression profiles of Escherichia spp. and Salmonella spp. were observed between habitats. In Escherichia spp., 1,299 proteins were differentially expressed, whereas 231 differentially expressed proteins were identified in Salmonella spp. Functional enrichment analysis indicated that proteins associated with microbial proliferation predominated in Escherichia spp., while DNA repair and stress-response proteins were enriched in Salmonella spp. from the ecotourism habitat. Notably, AMR-associated proteins were substantially more abundant in Escherichia spp. from the urban-proximate habitat, where 25 resistance-related proteins were identified compared with six in the ecotourism habitat. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed enhanced β-lactam resistance mechanisms involving multidrug efflux systems and β-lactamases in the urban-proximate population, whereas TolC-mediated efflux activity predominated in the ecotourism population.

Conclusion: This shotgun metaproteomic analysis provides the first direct evidence of differentially expressed AMR-associated proteins varying by habitat conditions, revealing active functional mechanisms under anthropogenic pressure and geographic location. Our findings suggest that environmental shifts and dietary habits could directly affect AMR-related functions of the gut microbiota of animals. This finding contributes to understanding the dynamics of AMR under the One Health framework and may inform strategies to reduce zoonotic and environmental transmission risks.