Abstract

Post-treatment endodontic infections remain a major clinical challenge driven by microbial persistence. The influence of host comorbidities and gender on the endodontic microbiome, resistome, and virulence repertoire remains poorly understood. This study profiled microbial ecology, antimicrobial resistance (AMR), and virulence factors across infection type, comorbidity, and gender using shotgun metagenomics. Fourteen samples were analyzed, including primary infections (n=7) and post-treatment infections (n=8). Total DNA was sequenced on an Illumina NovaSeq X Plus platform, followed by taxonomic profiling, AMR gene detection (MEGARes), virulence factor identification (VFDB), and statistical analyses. Although alpha and beta diversity did not differ significantly, pronounced compositional shifts were observed. Failed root canal treatments (RCTs) were enriched with periodontal pathogens (Porphyromonas gingivalis, Tannerella forsythia) and environmental opportunists (Burkholderia cepacia complex), alongside a ribosomal mutation–dominated AMR profile (MLS23S 49.47%; O23S 24.28%). Comorbidities strongly shaped microbial structure: diabetic patients exhibited enrichment of periodontal pathobionts, whereas a hypertension-only case showed extreme Lactobacillus dominance with a broad 28-gene resistome. Gender-associated patterns were also evident, with females enriched in Lactobacillus spp. and fluoroquinolone resistance genes, and males enriched in periodontal pathogens and mutation-based resistance. Network analysis revealed antagonism between a probiotic-associated Lactobacillus cluster and key pathogens (Parvimonas micra, B. cepacia). Virulence profiles were infection-specific, with acute abscesses displaying broader immune evasion genes and chronic lesions showing restricted, niche-adapted traits. Overall, failed RCTs represent a specialized, treatment-resistant pathobiome rather than increased diversity, highlighting the importance of host factors and suggesting probiotic-based therapeutic potential.

Key words: Antibiotic resistance, Endodontic failure, Metagenomics, Microbial dysbiosis, Personalized dentistry