MATERIALS AND METHODS

Ethical approval

This study was reviewed and approved by the Animal Care and Use Ethics Committee (ACUC), Faculty of Veterinary Medicine, Rajamangala University of Technology Tawan-ok, Thailand, under approval reference number RMUTTO-ACUC-2-2023-010.

The study was conducted in accordance with the institutional guidelines for the ethical use of animals in research and complied with internationally accepted principles for wildlife research and animal welfare. Free-living long-tailed macaques (Macaca fascicularis) were not captured, restrained, sedated, anesthetized, marked, or experimentally manipulated at any stage of the investigation. Fecal samples were collected exclusively through non-invasive sampling immediately after natural defecation to avoid disturbance, stress, or injury to the animals. Collection was performed at the center of freshly voided feces using sterile sampling equipment, while minimizing environmental contamination and avoiding repeated sampling from the same individual, as observed in the field.

All field activities were conducted with minimal interference to the animals' normal behavior and habitat. No food was provided, and no procedures were undertaken that could alter animal movement, social interactions, or foraging behavior. Sample transportation, storage, and laboratory processing were conducted in accordance with established biosafety and biosecurity procedures to ensure personnel safety and the integrity of biological specimens.

This non-invasive study involved only observational fieldwork and fecal sample collection from naturally occurring wildlife populations; therefore, no invasive procedures, euthanasia, or experimental interventions were performed. All efforts were made to ensure animal welfare and minimize potential impacts on free-living macaque populations and their natural environment.

Study period and location

The study was conducted from January to February 2025 at two geographically distinct locations in Thailand, selected for differences in habitat characteristics, food availability, and the degree of human–macaque interaction. The first study site was Chongkrachok Mountain, Mueang Prachuap Khiri Khan District, Prachuap Khiri Khan Province (11°48'55.77"N, 99°47'54.00"E), representing a natural habitat with ecotourism activities and supplementary feeding by visitors (location P). The second study site was Phromawat Temple in Si Racha District, Chonburi Province (13°10'32.03"N, 100°57'08.16"E), representing an urban-proximate habitat characterized by frequent human–macaque interactions, tourism, and religious gatherings (location S).

As shown in Figure 1, the study sites were selected to represent contrasting ecological conditions and varying levels of anthropogenic influence on free-living M. fascicularis populations. These habitats are typical environments inhabited by long-tailed macaques in Thailand, including temples, community forests, public parks, and nature-based ecotourism areas.

Study design

A cross-sectional comparative metaproteomic study was conducted to investigate habitat-associated differences in the gut resistome of free-living long-tailed macaques. A total of 54 fecal samples were collected and categorized according to sampling location. Group P consisted of samples collected from Chongkrachok Mountain (n = 16), whereas Group S consisted of samples collected from Phromawat Temple (n = 38). Comparative analyses were subsequently performed to evaluate differences in protein expression profiles, AMR-associated proteins, and functional pathways between the two populations.

Fecal sample collection

Fresh fecal samples were non-invasively collected from free-living long-tailed macaque colonies inhabiting natural environments adjacent to human communities and tourist attractions. Sample collection was performed immediately after defecation to minimize the possibility of repeated sampling from the same individual. Fecal material was collected from the center of each stool mass using sterile scoops and transferred into sterile fecal collection tubes to minimize contamination from soil, substrate, and other environmental sources.

The sex and age of individual macaques could not be accurately determined because samples were collected after direct observation of defecation without animal capture or identification. Based on field observations, the sampled animals likely included adult males and females. During transportation, samples were stored in insulated containers containing ice packs and subsequently preserved at −80°C until laboratory analysis.

Figure 1: Map of the sampling locations of free-living long-tailed macaques (Macaca fascicularis) from two distinct habitats in Thailand: Chongkrachok Mountain, Mueang Prachuap Khiri Khan District, Prachuap Khiri Khan Province (11°48'55.77"N, 99°47'54.00"E) and Phromawat Temple, Si Racha District, Chonburi Province (13°10'32.03"N, 100°57'08.16"E). The map was generated using OSM data and QGIS version 4.0.1.

Protein extraction and quantification

Frozen fecal samples were processed by adding phosphate-buffered saline (pH 7.2), then vortexed and centrifuged at 2,000 × g for 20 min at 4°C. The resulting supernatants were collected and stored at −80°C until further analysis.

Fecal supernatants were mixed with acetone at a ratio of 2:1 (v/v) and centrifuged at 10,000 × _g _for 10 min. The resulting pellets were suspended in lysis buffer containing 0.25% (w/v) sodium dodecyl sulfate and 50 mM Tris-HCl (pH 9.0). Protein concentrations were determined using the Lowry method [22] with bovine serum albumin as the reference standard. Pooled samples for each location group were prepared by combining equal amounts of protein from individual samples. All analyses were performed in triplicate.

For protein digestion, 5 μg of protein from each sample was reduced using dithiothreitol and subsequently alkylated with iodoacetamide. Samples were then digested with sequencing-grade trypsin (Promega, Mannheim, Germany) at a ratio of 1:20 and incubated overnight at 37°C.

Liquid chromatography–tandem mass spectrometry (LC-MS/MS)

Protein expression profiles were determined using LC-MS/MS. Digested peptide samples were dried, reconstituted in 0.1% formic acid, and injected into an Ultimate 3000 Nano/Capillary LC system (Thermo Scientific, Loughborough, UK) coupled to a Hybrid Quadrupole Time-of-Flight Impact II mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a CaptiveSpray ion source.

One microliter of each peptide digest was loaded onto a μ-Precolumn (300 μm internal diameter × 5 mm, C18 PepMap 100, 5 μm, 100 Å; Thermo Fisher Scientific) for online desalting and preconcentration. Peptide separation was performed using an analytical column (75 μm internal diameter × 15 cm, Acclaim PepMap C18, 2 μm, 100 Å; Thermo Fisher Scientific) maintained at 60°C.

The mobile phases consisted of 0.1% formic acid in water (Solvent A) and 0.1% formic acid in 80% acetonitrile (Solvent B). Peptides were eluted using a linear gradient of 5%–55% Solvent B over 30 min at a flow rate of 0.30 μL/min.

Electrospray ionization was performed using the CaptiveSpray source at 1.6 kV. Nitrogen was used as the drying gas at approximately 50 L/h and as the collision gas. Mass spectra and tandem mass spectra were acquired in positive-ion mode at a frequency of 2 Hz over an m/z range of 150–2,200. Collision energy was optimized to 10 eV for each m/z value. All samples were analyzed in triplicate to ensure reproducibility.

Protein identification and bioinformatics analysis

A label-free shotgun metaproteomic workflow integrating differential expression analysis and functional annotation was employed to characterize active AMR-associated proteins in Escherichia spp. and Salmonella spp. Protein identification and label-free quantification were performed using MaxQuant software version 2.5.0.0 (Max Planck Institute of Biochemistry, Martinsried, Germany) with the Andromeda search engine [23].

Mass spectra were searched against the UniProt databases for Escherichia and Salmonella. MaxQuant parameters were applied, including trypsin specificity with a maximum of two missed cleavages, carbamido-methylation of cysteine as a fixed modification, and oxidation of methionine and N-terminal acetylation as variable modifications. Proteins were identified based on peptides containing at least seven amino acids, with a minimum requirement of two peptides per protein, including at least one unique peptide. Peptide and protein false discovery rates (FDR) were controlled at 1% using a reversed decoy database strategy.

The ProteinGroups.txt output generated by MaxQuant was imported into Perseus software version 2.0.11.0 (Max Planck Institute of Biochemistry, Martinsried, Germany) for downstream analyses [23]. Contaminant proteins were removed before statistical analyses. Protein intensities were log2-transformed, and missing values were imputed using a constant value of zero. Pairwise comparisons between experimental groups were performed using Student's t-test.

Functional annotation of proteins was performed using Gene ontology (GO) terms retrieved from the UniProt database. Statistical analyses, including heatmaps, volcano plots, principal component analysis (PCA), and partial least squares-discriminant analysis (PLS-DA), were performed using MetaboAnalyst 6.0 [24]. Differentially expressed proteins were identified using an FDR-adjusted p < 0.05 and fold change >2 as significance thresholds.

Venn diagrams were generated using Jvenn software (https://jvenn.toulouse.inrae.fr/app/example.html) to compare differentially expressed proteins among study groups [25]. Functional pathway enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Mapper reconstruction tool based on gene names and the abundance of upregulated and downregulated proteins.