A Framework for Gut Microbiome and Resistome Research in Indigenous Orang Asli Communities of Peninsular Malaysia

Indigenous groups across the world have been underrepresented in the ongoing efforts to map human microbiome diversity. This study investigates the faecal microbiome and resistome diversities of Indigenous Orang Asli communities in Malaysia with different lifestyles and level of urbanisation. Healthy adults (>18 years) from three Indigenous communities: Temuan (urban), Temiar (semi-urban), and Jahai (rural hunter-gatherer), as well as urban Malays, were recruited, and their stools samples were subjected to shotgun metagenomic sequencing. Microbial alpha diversity (Shannon) decreased but antibiotic resistance genes (ARGs) diversity increased as the degree of urbanisation in the groups increased (P < 0.05). The groups contributed to 13% of the variation observed in the microbial composition (PERMANOVA, P = 0.001), and 14.5% in resistome composition (PERMANOVA, P = 0.001). Romboutsia timonensis was significantly depleted in Jahai compared to Malay (FDR = 0.04). Shared ARGs conferring resistance to beta-lactams (cfxA), tetracyclines (tet), and macrolides (erm) were observed across all groups, irrespective of geographical location, ethnicity and lifestyle. These results establish a baseline framework for urbanisation-linked microbial shifts in the under-studied Malaysian gut microbiome and provide foundational evidence of antimicrobial resistance patterns and underscores the need for broader inclusion of underrepresented populations in national surveillance and stewardship efforts.Indigenous groups across the world have been underrepresented in the ongoing efforts to map human microbiome diversity. This study investigates the faecal microbiome and resistome diversities of Indigenous Orang Asli communities in Malaysia with different lifestyles and level of urbanisation. Healthy adults (>18 years) from three Indigenous communities: Temuan (urban), Temiar (semi-urban), and Jahai (rural hunter-gatherer), as well as urban Malays, were recruited, and their stools samples were subjected to shotgun metagenomic sequencing. Microbial alpha diversity (Shannon) decreased but antibiotic resistance genes (ARGs) diversity increased as the degree of urbanisation in the groups increased (P < 0.05). The groups contributed to 13% of the variation observed in the microbial composition (PERMANOVA, P = 0.001), and 14.5% in resistome composition (PERMANOVA, P = 0.001). Romboutsia timonensis was significantly depleted in Jahai compared to Malay (FDR = 0.04). Shared ARGs conferring resistance to beta-lactams (cfxA), tetracyclines (tet), and macrolides (erm) were observed across all groups, irrespective of geographical location, ethnicity and lifestyle. These results establish a baseline framework for urbanisation-linked microbial shifts in the under-studied Malaysian gut microbiome and provide foundational evidence of antimicrobial resistance patterns and underscores the need for broader inclusion of underrepresented populations in national surveillance and stewardship efforts.