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EX VIVO MODELLING OF THE MICROBIOME OF DENTAL CARIES
Gandikota , Kumari Akshara
Gandikota , Kumari Akshara
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Thesis/Dissertation
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2025-05
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Oral Biology
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https://doi.org/10.34944/tn3h-xc90
Abstract
The complexity of the microbiome associated with dental caries necessitates the need for dental-caries-specific in vitro/ex vivo microbial models to enable a better understanding of the microbial interactions, drivers of dysbiosis, and response to various treatments. Several such models have been described but none include a reference healthy (normobiotic) microbiome along with the dental-caries associated (dysbiotic) microbiome. There is also a gap in the literature with respect to the optimal medium and growth conditions required to maximize similarity to the in vivo microbiomes. The aim of this study is to (i) develop an ex vivo model that recaptures health- and dental caries-associated microbiomes in parallel, and (ii) directly compare nutrient-rich and nutrient-limited media to assess their ability to support the growth of microbiomes similar to the in vivo microbiomes. Saliva samples were collected from 5 caries-free individuals and 4 individuals with dental caries. The levels of Streptococcus mutans were quantified by qPCR in each sample to exclude caries-free samples with high S. mutans and caries samples with low S. mutans. Subsequently, samples from caries-free and caries subjects were separately pooled to form healthy and caries inocula. An additional inoculum was prepared by spiking the healthy inoculum with S. mutans. Ex vivo microbiomes were grown from each inoculum type in MBEC devices using two types of media: saliva with 0.1% sucrose and SHI with 0.1% sucrose. The microbiomes were harvested after 24 hours, 48 hours without changing the media, or 48 hours with a media change. The generated biofilms were subjected to biomass quantification (DNA yield) and microbiome analysis using 16S rRNA gene sequencing.
Biomass significantly increased as a function of growth time, medium change, and medium type (SHI vs. saliva). However, SHI, but not saliva, was associated with a significant drop in viability after 48 h. Beta diversity analysis revealed separate clustering of health- and caries-derived microbiomes with their respective inocula regardless of medium type (PERMANOVA, p: 0.002). Spiking with S. mutans didn’t result in significant microbial shifts. Caries-derived microbiomes were enriched in Prevotella and Selenomonas spp., while health-derived microbiomes were enriched in Streptococcus and Veillonella spp. No significant differences in alpha diversity were found among microbiomes grown in the different conditions. Based on a similarity index, microbiomes grown in SHI for 24 h had the highest similarity to their respective inocula, followed by those grown in saliva for 24 h. However, changes in similarity were not statistically significant.
This study successfully developed an ex vivo model that replicates differences between health- and dental caries-associated microbiomes. While SHI maximized the similarity to the clinical inocula, the use of saliva is recommended where the generated microbiomes are used for further experiments that require biofilm viability.
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