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Aim: This case report explored the application of next-generation sequencing (NGS) and fluorescence in situ hybridization (FISH) to visualize and characterize microbial populations in a refractory endodontic infection with apical calculus-like deposits, a rarely reported phenomenon.

Summary: Histopathologic analysis revealed bacterial biofilms and calcifications on the root surface, with Gram-positive bacteria predominant in both hard and soft tissues. Microbial sequencing showed Pseudomonadota dominated hard tissues, whereas Bacillota were prevalent in soft tissues, with distinct genera like Lactibacterium and Streptococcus identified. FISH imaging confirmed spatially distributed bacterial taxa, including Actinomycetota and Chloroflexota, within the biofilm, aligning with NGS findings. Notably, Bacteroidota was exclusive to soft tissues, whereas Chloroflexota was detected only in hard tissues. The presence of extensive calculus-like deposits on the root surface provided new insights into the microbial complexity of persistent endodontic infections and their management.

Key learning points:

1. The combination of NGS and FISH provided unprecedented insights into the microbial composition of refractory endodontic infections, revealing a diverse and spatially organized ecosystem.
2. Distinct microbial compositions in hard and soft tissues emphasize the importance of targeted therapeutic strategies for endodontic infections.
3. The presence of unique bacterial taxa and biofilms in calculus-like deposits offers new avenues for research into the pathogenesis and persistence of endodontic infections.