Fungi and bacteria occupy distinct spatial niches within carious dentin

• Published in: PLoS pathogens 2024-05, Vol.20 (5), p.e1011865
• Main Authors: Sulyanto, Rosalyn M, Beall, Clifford J, Ha, Kasey, Montesano, Joseph, Juang, Jason, Dickson, John R, Hashmi, Shahr B, Bradbury, Seth, Leys, Eugene J, Edgerton, Mira, Ho, Sunita P, Griffen, Ann L
• Format: Article
• Language: English
• Subjects: Acids; Analysis; Antibacterial agents; Antibodies; Antifungal agents; Bacteria; Bacteria - genetics; Biofilms; Biology and Life Sciences; Care and treatment; Child; Child, Preschool; Children; Community composition; Dental caries; Dental Caries - microbiology; Dental Caries - pathology; Dental enamel; Dentin; Dentin - microbiology; Development and progression; Diagnosis; Dosage and administration; Electron microscopy; Endotoxins; Female; Field emission microscopy; Fungi; Gene sequencing; Glucan; Gram-negative bacteria; Gram-positive bacteria; Humans; Immunofluorescence; Immunohistochemistry; Lesions; Lipoteichoic acid; Male; Medicine and Health Sciences; Microorganisms; Microscopy; Multiplexing; Niches; Oral hygiene; Pathophysiology; Research and Analysis Methods; RNA; RNA, Ribosomal, 16S; rRNA 16S; Scanning electron microscopy; Spatial distribution; Teeth; Tubules; Viral antibodies
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1011865

The role of bacteria in the etiology of dental caries is long established, while the role of fungi has only recently gained more attention. The microbial invasion of dentin in advanced caries especially merits additional research. We evaluated the fungal and bacterial community composition and spatial distribution within carious dentin. Amplicon 16S rRNA gene sequencing together with quantitative PCR was used to profile bacterial and fungal species in caries-free children (n = 43) and 4 stages of caries progression from children with severe early childhood caries (n = 32). Additionally, healthy (n = 10) and carious (n = 10) primary teeth were decalcified, sectioned, and stained with Grocott's methenamine silver, periodic acid Schiff (PAS) and calcofluor white (CW) for fungi. Immunolocalization was also performed using antibodies against fungal β-D-glucan, gram-positive bacterial lipoteichoic acid, gram-negative endotoxin, Streptococcus mutans, and Candida albicans. We also performed field emission scanning electron microscopy (FESEM) to visualize fungi and bacteria within carious dentinal tubules. Bacterial communities observed included a high abundance of S. mutans and the Veillonella parvula group, as expected. There was a higher ratio of fungi to bacteria in dentin-involved lesions compared to less severe lesions with frequent preponderance of C. albicans, C. dubliniensis, and in one case C. tropicalis. Grocott's silver, PAS, CW and immunohistochemistry (IHC) demonstrated the presence of fungi within carious dentinal tubules. Multiplex IHC revealed that fungi, gram-negative, and gram-positive bacteria primarily occupied separate dentinal tubules, with rare instances of colocalization. Similar findings were observed with multiplex immunofluorescence using anti-S. mutans and anti-C. albicans antibodies. Electron microscopy showed monomorphic bacterial and fungal biofilms within distinct dentin tubules. We demonstrate a previously unrecognized phenomenon in which fungi and bacteria occupy distinct spatial niches within carious dentin and seldom co-colonize. The potential significance of this phenomenon in caries progression warrants further exploration.