Department of Dentistry, Boston Children's Hospital, Boston, Massachusetts, United States of America.
Department of Developmental Biology, Harvard School of Dental Medicine, Boston, Massachusetts, United States of America.
PLoS Pathog. 2024 May 28;20(5):e1011865. doi: 10.1371/journal.ppat.1011865. eCollection 2024 May.
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.
细菌在龋齿发病机制中的作用早已确立，而真菌的作用只是最近才受到更多关注。在进展性龋齿中，牙本质的微生物入侵尤其值得进一步研究。我们评估了牙本质龋中真菌和细菌群落的组成和空间分布。采用扩增子 16S rRNA 基因测序结合定量 PCR 技术，对无龋儿童（n=43）和严重婴幼儿龋（n=32）龋进展的 4 个阶段的细菌和真菌物种进行了分析。此外，还对健康（n=10）和患龋（n=10）的乳牙进行脱钙、切片，并分别用 Grocott 的美蓝、过碘酸希夫（PAS）和钙荧光白（CW）染色观察真菌，还使用针对真菌β-D-葡聚糖、革兰阳性菌脂磷壁酸、革兰阴性菌内毒素、变形链球菌和白色念珠菌的抗体进行免疫定位。我们还进行了场发射扫描电子显微镜（FESEM）观察，以观察牙本质小管内的真菌和细菌。观察到的细菌群落包括高丰度的变形链球菌和韦荣球菌属，这是意料之中的。与病变程度较轻的牙齿相比，病变累及牙本质的牙齿中真菌与细菌的比例更高，而且频繁出现白色念珠菌、都柏林念珠菌，在一个病例中还出现了热带念珠菌。Grocott 的银染、PAS、CW 和免疫组化（IHC）显示真菌存在于牙本质小管内。多重免疫组化显示，真菌、革兰阴性菌和革兰阳性菌主要占据单独的牙本质小管，很少有共同定殖的情况。使用抗变形链球菌和抗白色念珠菌抗体的多重免疫荧光也观察到了类似的结果。电子显微镜显示，在不同的牙本质小管内存在单形性细菌和真菌生物膜。我们证明了在牙本质龋中真菌和细菌占据不同的空间生态位且很少共同定殖的这一先前未被认识到的现象。这种现象在龋病进展中的潜在意义值得进一步探索。
