DISCUSSION

Habitat-associated variation in gut metaproteomic profiles

NHPs are highly relevant animal models for human research. Extensive investigations of their microbial composition have provided critical insights into the factors shaping host–microbiome interactions. Metaproteomics has become a powerful tool for studying the expression and functions of the gut microbiome by analyzing the entire proteome of animal microbial communities. The present study provides novel protein level evidence that habitat-specific anthropogenic interactions shape expressed AMR functions in gut Escherichia spp. and Salmonella spp., complementing and extending metagenomic findings by identifying mechanisms that are actively translated in macaque populations from Chongkrachok Mountain, Prachuap Khiri Khan Province (location P), and Phromawat Temple, Si Racha District, Chonburi Province (location S). Long-tailed macaques naturally forage on leaves, fruits, and insects; however, individuals at the present sampling sites also relied heavily on anthropogenic food sources, including food waste and food provided by residents and tourists. Location P represents a nature-based ecotourism setting characterized by frequent human–wildlife interactions. In contrast, habitat fragmentation and expanding human activities have pushed macaques closer to urban areas, such as location S, leading to interactions that may have both beneficial and adverse effects on surrounding human communities. In this shared human–macaque landscape, we performed a comprehensive fecal gut microbiome proteomic analysis of wild macaques in Thailand.

The results showed that protein expression patterns of Escherichia spp. and Salmonella spp. differed markedly between the two locations. A study comparing the microbiomes of wild and captive long-tailed macaques revealed that captivity conditions may alter gut microbiome richness and key metabolic functions [26]. In addition, Muhammad et al. [27] found that long-tailed macaque populations receiving anthropogenic food had higher gut microbiota richness and evenness than populations inhabiting different habitats. Further studies have also reported that geographic location and environmental factors are critical determinants of the structure and function of gut microbial communities in animals [28, 29]. Wang et al. [29] investigated the role of geographic location and environmental factors in shaping the gut microbiota of Himalayan langurs and Xizang macaques. Differences in the prevalence of E. coli and MDR genes were observed among langurs from four locations with different geographic and altitudinal characteristics, suggesting that these animals may serve as reservoirs for antimicrobial-resistant Escherichia spp. under unique ecological pressures. Therefore, variation in gut microbial protein expression between macaque populations in the present study was likely driven by differences in dietary patterns, geographic location, and human–macaque interactions. Nevertheless, limited awareness of pathogen transmission among tourists, together with poorly managed human–wildlife interactions, may increase the risk of infectious disease transmission.

Functional interpretation of DEPs

The present study compared upregulated and downregulated proteins in gut Escherichia spp. and Salmonella spp. from fecal samples of macaques inhabiting locations P and S. Among the upregulated proteins of Escherichia spp., most were involved in translation and DNA-templated transcription. Meanwhile, the most significantly altered biological processes among downregulated proteins in location P were DNA-templated transcription, proteolysis, DNA replication, and response to antibiotics. These functions are closely associated with microbial proliferation. Excessive proliferation of E. coli in the intestine may disrupt the gut microbiota and exacerbate inflammation of the large intestine [30].

Among Salmonella spp., most of the upregulated proteins expressed at location P were involved in DNA repair, the SOS response, and enterobacterial common antigen biosynthesis. Proteins encoded by SOS-associated genes are involved in DNA repair and maintenance of crucial cell division proteins [31]. These shifts in proliferation- and repair-related proteins provide functional evidence of microbial adaptation to anthropogenic pressures and extend beyond taxonomic shifts reported in previous Thai macaque 16S rRNA studies. Meanwhile, downregulated DEPs were involved in cobalamin biosynthesis, methylation, and glutamine metabolism. A previous study using a metaproteomic approach to examine microbial protein expression during a controlled dietary intervention revealed significant metabolic alterations in microbial responses to different dietary patterns [32]. Thus, metaproteomics is a valuable approach for investigating microbial metabolism associated with diet and its potential influence on host health.

Antibiotic resistance-associated proteins and anthropogenic influence

Interestingly, the present study revealed that proteins associated with AMR responses in Escherichia spp. were more abundant at location S than at location P. The macaques in location S inhabited an urban-proximate temple environment with frequent human contact. Previous studies in India indicated that environmental AMR pollution resulting from human activity is a long-standing concern. Evidence from medicinal plants, hospital effluents, and untreated wastewater showed that hospital effluents contained E. coli resistant to extended-spectrum cephalosporins and fluoroquinolone antibiotics [33, 34]. In addition, a study on AMR and the epidemiology of E. coli isolated from town hospitals in Shandong Province, China, reported that colonization with extended-spectrum β-lactamase-producing E. coli increased by 62.8% among outpatients across three regions of Shandong Province. These rates were considered to reflect contact with food-producing animals in rural areas [35].

The present study examined molecular mechanisms of resistance to β-lactam antibiotics using a metaproteomic approach and demonstrated that the fecal microbiome may respond differently according to habitat-associated dietary and environmental exposures. β-lactam antibiotics, including penicillins, cephalon-sporins, carbapenems, monobactams, and penems, are among the most widely used drugs for treating bacterial infections [36]. These antibiotics primarily inhibit bacterial growth by disrupting bacterial cell wall synthesis, reducing selective permeability, causing cell death, and inactivating penicillin-binding proteins associated with cell wall synthesis [37, 38]. According to KEGG pathway analysis of upregulated antibiotic resistance-associated proteins of Escherichia spp. from long-tailed macaque fecal microbiomes, TolC from location P was upregulated in the β-lactam resistance pathway. This protein was associated with MDR through the RND efflux pump mechanism. Previous research reported that RND family transporters are widespread, particularly among Gram-negative bacteria, and actively efflux many antibiotics and chemotherapeutic agents [39]. The RND family includes several members relevant to antibiotic resistance in Escherichia spp. and typically consists of homomeric assemblies comprising an inner membrane pump, a membrane fusion protein, and an outer membrane porin [40]. A distinctive characteristic of RND efflux pump systems is that they span both the inner and outer bacterial membranes. Their function is to transport antibiotics out of the bacterial cell, preventing the drugs from reaching their intended targets. These systems also play important roles in bacterial physiology, pathogenicity, and metabolism.

Regulation of the RND efflux pump remains incompletely understood. Previous literature indicates that the RND efflux system in enteric bacteria is controlled by natural inducers encountered during infection, such as bile salts and fatty acids [41]. Previous studies have also reported that activation may occur through the inactivation of local repressors that block the expression of structural pump genes, such as AcrR, or through the activation of global transcriptional regulators, such as SoxS, RobA, or RamA [42, 43]. In addition, metal cations, which act as cofactors in several bacterial processes, may induce the expression of RND efflux pumps. In particular, studies on the metal-induced regulation of CusCBA proteins, members of the RND protein superfamily of proton-driven cation symporters and antiporters in E. coli, have shown that copper and silver ions may serve as natural inducers of CusCBA expression [44, 45].

In location S, AcrA and class D β-lactamases, including β-lactamase 1, β-lactamase OXA-7, and β-lactamase OXA-2, were upregulated and involved in multidrug efflux and β-lactamase enzyme activity. When exposed to antibiotics, bacteria often overexpress efflux pumps or accumulate mutations, especially in regulatory genes, allowing them to expel antibiotics more efficiently. Previous studies have shown that constitutively expressed efflux pumps often act in concert with other resistance mechanisms, such as β-lactamases, to confer antibiotic resistance [46–48]. Moreover, a review reported that multidrug efflux pumps are involved in several cellular processes, including biofilm development, and play a pivotal role in regulating specific genes and adaptation to environmental conditions [49]. The production of β-lactamases is the most common mechanism of resistance to β-lactam antibiotics in Gram-negative bacteria [50]. Evidence of extended-spectrum β-lactamase-producing E. coli in the gut microbiomes of wild animals indicates that dissemination in wildlife is increasing worldwide [51–53]. Therefore, β-lactam resistance in Gram-negative bacteria requires careful monitoring to support the development of effective control and mitigation strategies.

Wildlife as sentinels of environmental AMR

Previous research suggests that wild NHPs are generally not directly exposed to antibiotics but may acquire antibiotic-resistant, β-lactamase-producing E. coli through foraging and drinking from natural environments contaminated by anthropogenic sources [54]. Moreover, agriculture and livestock activities, together with lifestyle changes in free-living NHPs transitioning to captive or semi-captive conditions, may increase human–NHP interactions and enhance the spread of antibiotic-resistant bacteria [55]. As shown in the present study, expression of antibiotic resistance-associated proteins and β-lactam resistance mechanisms among Escherichia spp. from location S appeared more complex than those observed in location P. Location P, Chongkrachok Mountain, represents a nature-based ecotourism site with increasing interactions between humans and wild macaques, suggesting that tourism may intensify rather than reduce human–macaque conflict. Such interactions may reduce natural foraging success, increase reliance on anthropogenic foods, and contribute to the emergence of antibiotic-resistant E. coli strains. In contrast, location S, Phromawat Temple, represents an urban-proximate habitat where macaque populations interact frequently with human communities. Habitats near human activity have been shown to harbor greater diversity of antimicrobial-resistant genes in NHP E. coli isolates, indicating that macaques may have substantial potential to spread MDR [56]. By demonstrating elevated expression of β-lactam resistance proteins in urban-proximate macaques, the present metaproteomic data provide functional support for wildlife as sentinels of environmental AMR pollution, offering insights that gene-centric studies alone cannot provide and informing tourism management and surveillance strategies in Thailand.

One Health implications and future perspectives

Compared with other microbiome approaches, metaproteomic data on wildlife resistomes remain limited, particularly for Southeast Asian NHPs. Beyond microbiome monitoring, metaproteomics can estimate microbial abundance and molecular processes relevant to the One Health framework, supporting the development of effective gut microbiome control algorithms [57, 58]. The present findings suggest that M. fascicularis populations are influenced by anthropogenic activities that alter their foraging strategies and diets, particularly their access to human-derived foods. Human–macaque interactions were more common in urban habitats. Nevertheless, inducers of antibiotic resistance are complex and likely reflect environmental cues specific to each ecosystem. Environmental pollution associated with human activities and the use of antimicrobial compounds may significantly shape gut microbiota expression and negatively affect wildlife conservation and natural ecosystems. Therefore, the present findings provide important insights into how habitat, diet, and geographic variation affect AMR expression and resistance mechanisms, supporting the adoption of the One Health approach in AMR surveillance. Overall, this study improves understanding of the role of wild animals in AMR dissemination, supports the development of effective control measures to minimize transmission risks from antibiotic-resistant gut microbiota, and contributes to estimating the emergence and spread of zoonotic and anthroponotic diseases at the human–wildlife interface.