Association between root canals and gingival sulci microbiota in secondary and persistent endodontic infections

Secondary/persistent endodontic infections (SPEIs) result from failed root canal therapy, causing persistent apical periodontitis. Current diagnostic methods for SPEIs predominantly rely on clinical and radiographic indicators, which often lack adequate sensitivity and specificity. Consequently, there is an urgent need to effectively detect SPEIs or monitor their progression. The aim of this study was to compare and characterize the microbiota of root canals and gingival sulci of teeth affected by SPEI to identify keystone pathogens as potential diagnostic biomarkers through advanced next-generation sequencing (NGS) techniques. Ninety samples from 30 affected teeth in 25 patients undergoing nonsurgical retreatment were analyzed. Bacterial DNA was extracted, the V3–V4 region of the 16S rRNA gene was amplified, and sequencing was performed (Illumina MiSeq). Amplicon sequence variants (ASVs) identified 16 phyla, 182 genera, and 390 species. Microbiota in root canals differed from gingival sulci, with Acinetobacter and Veillonella prevalent in canals, and Streptococcus and Actinomyces dominant in sulci. Certain species, including Shuttleworthella satelles, Olsenella uli, Dialister invisus, Massilia timonae, and Klebsiella pneumoniae were detected in both sites, suggesting microbial migration via anatomical structures. Detecting these potential keystone pathogens of SPEI as biomarkers in readily accessible sulcus fluid could facilitate diagnoses and monitoring of progression and/or resolution. These insights provide a foundation for more accurate and targeted diagnostic and therapeutic strategies for management of SPEIs.