Hashmi, S. B. | bioRxiv Science

bioRxiv2023

Fungi and Bacteria Occupy Distinct Spatial Niches within Carious Dentin

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 Grocotts methenamine silver, periodic acid Schiff (PAS) and calcofluor white (CW) for fungi. Immunolocalization was also performed using antibodies against fungal {beta}-D-glucan, gram-positive bacterial lipoteichoic acid, 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. Grocotts silver, PAS, CW and immunohistochemistry (IHC) demonstrated the presence of fungi within carious dentinal tubules. Multiplex IHC revealed that fungi 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.

microbiology↗

bioRxiv2020

Acquisition of Oral Microbiota is Driven by Environment, Not Host Genetics

The human oral microbiota is acquired early in an organized pattern, but the factors driving this acquisition are not well understood. Microbial "heritability" could have far-reaching consequences for health, yet no studies have specifically examined the fidelity with which the oral microbiota are passed from parents to offspring. Some previous studies comparing monozygotic (MZ) and dizygotic (DZ) twins had suggested that host genetics has a role in shaping oral microbial communities, and also identified so called "heritable" taxa. However, these findings are likely to be confounded by shared environmental factors resulting from the well-established greater behavioral similarity among MZ twins. In addition, MZ and DZ twins share an equal portion of their parents genome, and so this model is not informative for studying direct parent to offspring transmission. To specifically examine the contribution of genetics to the fidelity of transmission of bacteria from parents to offspring, we used a novel study design comparing fraction of shared species and strains between our genetically related group consisting of children and their biological mothers, with that of children and their adoptive mothers, constituting our genetically unrelated group. Fifty-five biological and 50 adoptive mother-child pairs were recruited along with 23 biological fathers and 16 siblings. Subjects were carefully selected to ensure the two groups were matched on childs age. Three distinct habitats within the oral cavity: the saliva/soft tissue surface, supragingival biofilm, and subgingival biofilm, were sampled to comprehensively profile the oral microbiome. Our recently developed strategy for subspecies level characterization of bacterial communities by targeted sequencing of the ribosomal 16-23S intergenic spacer region (ISR) was utilized in the present study to track strain sharing between subjects, in addition to 16S rRNA gene sequencing for species analysis. Results showed that oral bacterial community profiles of adoptive and biological mother-child pairs were equally similar, indicating no effect of host genetics on the fidelity of transmission. This was consistent at both species and strain level resolutions, and across all three habitats sampled. We also found that all children more closely resembled their own mother as compared to unrelated women, suggesting that contact and shared environment were the major factors shaping the oral microbiota. Individual analysis of the most abundant species also did not detect any effect of host genetics on strain sharing between mother and child. Mother-child strain similarity increased with the age of the child, ruling out early effects that are lost over time. No effect on the fidelity of mother-child strain sharing from vaginal birth or breast feeding was seen. Analysis of extended families showed that fathers and mothers were equally similar to their children. Cohabitating couples showed even greater strain similarity than mother-child pairs, further supporting the role of age, contact and shared environment as determinants of microbial similarity. Based on these findings we suggest that the genetic effects on oral microbial acquisition observed in twin studies are more likely the result of confounding environmental factors based on greater behavioral similarity among MZ twins. Our findings suggest that these host mechanisms are universal to humans, since no effect of genetic relatedness on fidelity of microbial transmission could be detected. Instead, our findings point toward contact and shared environment being the driving factors of microbial transmission, with a unique combination of these factors ultimately shaping a highly personalized human oral microbiome.

microbiology↗