Caries
Kasey Ha, DMD
PGY-2
Boston Children’s Hospital, Boston, MA
Boston Children's Hospital and Harvard School of Dental Medicine
Boston, Massachusetts, United States
Joseph Montesano, BS
Harvard School of Dental Medicine
Jason Juang, BA
Harvard School of Dental Medicine
Sunita P. Ho, MS, PhD
UCSF School of Dentistry
Clifford Beall, MS, PhD
The Ohio State University
Ann Griffen, DDS, MS
The Ohio State University
Rosalyn Sulyanto, DMD, MS
Postdoctoral Research Director
Boston Children's Hospital
Boston, Massachusetts, United States
Rosalyn Sulyanto, DMD, MS
Postdoctoral Research Director
Boston Children's Hospital
Boston, Massachusetts, United States
Isabelle Chase, DDS, FRCD(C)
Program Director
Boston Children's Hospital
Harvard School of Dental Medicine
Boston, Massachusetts, United States
Purpose: Although fungi have been isolated from saliva and carious plaque, few studies have explored fungal colonization of dental hard tissues. This novel study elucidates fungal and bacterial biogeography within carious dentin utilizing high throughout sequencing, histological, and microscopy techniques.
Methods: Carious (n=24) and non-carious (n=10) primary teeth were collected from 34 children. Dentin samples were obtained and analyzed using 16s rRNA gene and ITS region sequencing, specific for bacteria and fungi, respectively. Subsequently, specimens were stained with Periodic acid Schiff (PAS) and Grocott’s methanamine silver (GMS). Immunohistochemistry (IHC) and immunofluorescence (IF) were performed using antibodies against β-D-glucan, lipoteichoic acid, and endotoxin, specific for fungi, gram-positive, and gram-negative bacteria, respectively. Species-level distribution was explored for Streptococcus mutans and Candida albicans by IHC. Light and confocal microscopy were used to image histologic sections. Fungi and bacteria in carious dentinal tubules were visualized using field emission scanning electron microscopy (FESEM).
Results: Numerous bacterial species were detected in carious dentin by next-generation sequencing, while fungal species were quantitively less diverse. Fungal elements were detected in carious dentinal tubules by PAS and GMS while absent in non-carious teeth. Immunohistochemistry, IF, and FESEM revealed that individual tubules of carious dentin contain predominantly either fungi or bacteria. Fungi and bacteria seldomly co-colonized within the same tubule. Similar findings were observed on multiplex staining with S. mutans and C. albicans antibodies, whereas gram-positive and gram-negative bacteria primarily co-localized within the same tubules.
Conclusions: We demonstrated a previously unrecognized spatial distribution of fungi and bacteria within carious dentin whereby bacteria formed multispecies biofilms and fungi formed monospecies biofilms in separate dentin tubules. This suggests a novel cross-kingdom interaction in caries pathogenesis that warrants further exploration and could aid in the development of targeted anti-microbial and anti-mycobial treatments.