Oral Microbiota of Children Undergoing Comprehensive Treatment for Severe Early Childhood Caries

Abstract

Severe early childhood caries (S-ECC) is one of the leading oral health problems that affect the growth, development and quality of life of many preschool children. Currently, the management of S-ECC includes preventive and restorative approaches but it is clear that the children remain at high risk for dental caries despite these approaches. Dental caries is a microbiologically related disease and the oral microbiota is composed of diverse groups of bacterial species, each one possessing its own specific nutritional and physico-chemical requirements. Further understanding of the composition and ecological events that drive changes in the structure, from health to disease, of oral microbial communities is an important step in the development of improved preventive strategies to promote oral health. The significance of the research presented in this thesis is that for the first time in New Zealand, the oral microbiota in children with S-ECC has been profiled before and at two time points after comprehensive restorative dental and preventive treatment under general anaesthesia. This information will inform further research to target specific bacteria that contribute to the risk of dental caries. The aim of this research was to investigate the oral microbiome before and after comprehensive restorative and preventive dental treatment for S-ECC in preschool-aged children using a metagenomic approach with next-generation semiconductor (Ion Torrent™) DNA sequencing technology to perform bacterial profiling on dental plaque samples. Two groups of participants were recruited: (i) S-ECC group of thirty children aged two to six years, and (ii) age-matched control group of thirty children with no clinically detectable dental caries. The S-ECC participants were monitored with respect to dietary and oral hygiene habits at baseline (i.e., pre-treatment), two weeks and at three months after comprehensive restorative and preventive dental treatment (post-treatment). Their oral bacterial profiles were characterized at those time points also. Supra- and sub-gingival plaque samples were collected from both study groups: at baseline for the control group and at baseline/pre-treatment, two weeks and three months post-treatment for the S-ECC group. Total genomic DNA purified from each plaque sample and bacterial 16S rRNA genes were specifically amplified by the polymerase chain reaction (PCR). Each participant oral sample was assigned individual barcodes and all PCR amplicons were sequenced using the Life Technologies Ion Torrent™ Personal Genome Machine. The sequence data obtained were analysed using the Quantitative Insights Into Microbial Ecology (QIIME) bioinformatics software package which sorted the barcoded sequences and delineated the identities of bacteria to phylum and genus levels. Further analyses using the BLASTN homology search algorithm determined the bacterial profiles to species level. Furthermore, a limited analysis using traditional culture-based microbiological techniques followed by 16S rRNA gene sequencing of the putative cariogenic bacteria in these children was undertaken. Firmicutes and Fusobacteria were the predominant phyla identified followed by Actinobacteria, Proteobacteria and Bacteroidetes. Firmicutes were the most dominant in the S-ECC group. The most prevalent species in the S-ECC group were Streptococcus mitis, Veillonella dispar, Streptococcus mutans, Streptococcus oralis, Neisseria flavescens, Neisseria bacilliformis, Prevotella denticola and Treponema socranskii, and statistically significant differences were observed with respect to these species between the two study groups. Conversely, species including as Actinomyces naeslundii and Rothia aeria were identified at higher levels in the control group. Comparison of the microbiota in the S-ECC group at the three time points showed no statistically significant differences between the time points. Despite the lack of statistical significance, there was a reduction in the proportions of some bacterial species (S. mutans, S.mitis, V. dispar, N. flavescens, N. bacilliformis and P. denticola) after comprehensive restorative and preventive dental treatment. The microbiological analyses of plaque samples from the S-ECC group revealed the presence of the expected cariogenic species such as S. mutans, Streptococcus sobrinus, Lactobacillus casei and Lactobacillus rhamnosus, and also other streptococcal species (S. mitis, Streptococcus anginosus). Traditional culture-based microbiological analysis did show a reduction in the number of detectable species observed in the pre-treatment and post-treatment samples. Both culture-based and culture-independent next-generation DNA sequencing analyses confirmed the relationship of S. mutans with dental caries. Species in addition to S. mutans, such as Veillonella spp., Neisseria spp. and Prevotella spp. also may play an important role in caries development and progression. On the other hand, some genera such as Actinomyces and Rothia may be associated with low caries risk. This study indicates the need for further research involving larger sample sizes, at the depth of next generation sequencing, to understand oral microbiome ecology and to investigate potential intervention to reduce the effect of bacteria that contribute to caries risk.