Al-Hebshi, Nezar2025-09-302025-09-302025-08https://scholarshare.temple.edu/handle/20.500.12613/11259Endodontic infections develop when bacteria from the oral cavity infiltrate the pulp through breaches in enamel and dentin, leading to inflammation, necrosis, and apical periodontitis. Primary infections are dominated by anaerobic bacteria including Fusobacterium, Porphyromonas, and Prevotella while secondary infections often involve gram-positive facultative anaerobes like Enterococcus faecalis. Fungi, particularly Candida albicans, have also been implicated. Next-generation sequencing (NGS) has transformed microbial analysis, enabling detailed profiling of bacterial and fungal communities through 16S rRNA and ITS sequencing, respectively However, most studies have focused primarily on bacterial populations, with only a few that characterized the mycobiome or assessed both domains simultaneously. The potential interkingdom interactions between bacterial and fungal taxa whether synergistic or antagonistic remain poorly understood. This study addresses these gaps, this study sought to characterize both the bacteriome and mycobiome in primary and secondary infections, while exploring the potential interkingdom interactions in both types of infection. Methods Sixty-three patients were recruited: 30 with primary endodontic infections (infections occurring in teeth with necrotic pulps and no prior endodontic treatment), 30 with secondary/persistent infections (infections in previously treated teeth where the root canal system has been reinfected or the initial infection was not completely eliminated), and 3 negative controls undergoing extraction for orthodontic reasons. Root canal samples were collected using files and paper points under sterile conditions. DNA extracted from the samples was subjected to dual amplicon sequencing targeting the 16S rRNA (V1–V3) and ITS2 regions to profile bacterial and fungal communities, respectively. Taxonomic assignment and diversity analyses were performed using QIIME2, Phyloseq, and Microbiome Packages in R. Differential abundance analysis was performed using MaAsLin2. Results Beta diversity analysis revealed significant compositional differences in bacterial communities between primary and secondary infections (PERMANOVA, p = 0.001), whereas fungal community structure was more conserved across groups. Alpha diversity metrics, including Shannon and Chao1 indices, did not significantly differ between infection types for either domain. Both primary and secondary endodontic infections were dominated by strict anaerobes, including Propionibacterium, Fusobacterium, Prevotella, Parvimonas, Porphyromonas, Pyrimadobacter, Fretibacterium and Olsenela. However, secondary infections still showed enrichment of gram-positive facultative anaerobes, such as Streptococcus mitis, Streptococcus vestibularis, Rothia mucilaginosa, Lactobacillus gasseri, and Corynebacterium accolens compared to primary infections. Fungal communities in both primary and secondary infections were primarily composed of Candida, Malassezia and Cladosporium species but secondary infections showed significantly higher relative abundance of Malassezia globosa (novel variants 94.5%, 96.6%) and Candida dubliniensis Interkingdom correlations were identified for primary but not secondary infections including positive associations between fungi and strict anaerobes including Prevotella oris, and Parvimonas micra, suggesting potential synergistic interactions. Conversely, negative correlations were observed between fungi and several facultative species such as Streptococcus infantis and Lactobacillus gasseri, suggesting competitive exclusion or antagonism in secondary infections. Conclusions This study provides the first simultaneous characterization of the bacteriome and mycobiome in primary and secondary endodontic infections using next-generation sequencing. The identification of distinct bacterial profiles anaerobe-dominated communities in primary infections versus gram-positive facultative anaerobes in secondary infections alongside conserved yet compositionally varied fungal communities, underscores the ecological divergence between infection types. Notably, the detection of significant bacterial-fungal correlations, particularly involving Cladosporium and strict anaerobes, suggests that specific fungal taxa may participate in synergistic interactions that contribute to the persistence and biofilm resilience of endodontic infections.36 pagesengIN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available.http://rightsstatements.org/vocab/InC/1.0/DentistryOral biologyText2025-09-02Lee_temple_0225M_16247.pdf