Cross-sectional comparative shotgun metagenomic analysis of the subgingival resistome in healthy subjects and patients with periodontitis from four countries

Background: The oral cavity is a known reservoir of antibiotic resistance genes (ARGs), but little is known about their subgingival distribution across health states and regions. Objective: This study aimed to characterize and compare the subgingival resistome and mobile genetic elements (MGEs) in healthy subjects (HS) and periodontitis patients (PP) from Belgium, Chile, Peru and Spain. Design: Subgingival samples pooled from the deepest site of each quadrant of 40 HS and 40 PP were analyzed via shotgun metagenomic sequencing. After human DNA depletion, the microbial composition was assessed with MetaPhlAn 4.0; ARGs were identified using MEGAHIT and AMRFinderPlus; and MGEs with MGEfinder. Results: ARG richness was significantly higher in PP (mean 3.98) than in HS (2.15). PP from Peru showed more ARGs than HS from Chile and Spain. In total, 28 ARGs were found, conferring resistance to eight antibiotic classes. β-lactam, tetracycline and aminoglycoside resistance were more abundant in PP. Macrolide resistance was lower in Chilean samples than in Peruvian and Spanish ones. Additionally, 99 MGE-associated genes were detected, with 16 differing by diagnosis and 78 by country. Conclusions: Subgingival resistome profiles vary significantly by periodontal status and geography, underscoring the influence of clinical and regional factors on antimicrobial resistance in the oral microbiome.