{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,8,15]],"date-time":"2025-08-15T02:06:28Z","timestamp":1755223588588,"version":"3.43.0"},"reference-count":51,"publisher":"Wiley","issue":"9","license":[{"start":{"date-parts":[[2025,6,15]],"date-time":"2025-06-15T00:00:00Z","timestamp":1749945600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Int Endodontic J"],"published-print":{"date-parts":[[2025,9]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Aim<\/jats:title><jats:p>This case report explored the application of next\u2010generation sequencing (NGS) and fluorescence in\u00a0situ hybridization (FISH) to visualize and characterize microbial populations in a refractory endodontic infection with apical calculus\u2010like deposits, a rarely reported phenomenon.<\/jats:p><\/jats:sec><jats:sec><jats:title>Summary<\/jats:title><jats:p>Histopathologic analysis revealed bacterial biofilms and calcifications on the root surface, with Gram\u2010positive bacteria predominant in both hard and soft tissues. Microbial sequencing showed <jats:italic>Pseudomonadota<\/jats:italic> dominated hard tissues, whereas <jats:italic>Bacillota<\/jats:italic> were prevalent in soft tissues, with distinct genera like <jats:italic>Lactibacterium<\/jats:italic> and <jats:italic>Streptococcus<\/jats:italic> identified. FISH imaging confirmed spatially distributed bacterial taxa, including <jats:italic>Actinomycetota<\/jats:italic> and <jats:italic>Chloroflexota<\/jats:italic>, within the biofilm, aligning with NGS findings. Notably, <jats:italic>Bacteroidota<\/jats:italic> was exclusive to soft tissues, whereas <jats:italic>Chloroflexota<\/jats:italic> was detected only in hard tissues. The presence of extensive calculus\u2010like deposits on the root surface provided new insights into the microbial complexity of persistent endodontic infections and their management.<\/jats:p><\/jats:sec><jats:sec><jats:title>Key learning points<\/jats:title><jats:p>\n<jats:list>\n\n<jats:list-item><jats:p>The combination of NGS and FISH provided unprecedented insights into the microbial composition of refractory endodontic infections, revealing a diverse and spatially organized ecosystem.<\/jats:p><\/jats:list-item>\n\n<jats:list-item><jats:p>Distinct microbial compositions in hard and soft tissues emphasize the importance of targeted therapeutic strategies for endodontic infections.<\/jats:p><\/jats:list-item>\n\n<jats:list-item><jats:p>The presence of unique bacterial taxa and biofilms in calculus\u2010like deposits offers new avenues for research into the pathogenesis and persistence of endodontic 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