Understanding the development of the oral microbiota in healthy children is of great importance to oral and general health. However, limited data exist on a healthy maturation of the oral microbial ecosystem in children. Moreover, the data are biased by mislabeling “caries-free” populations. Therefore, we aimed to characterize the healthy salivary and dental plaque microbiome in young children. Caries-free (ICDAS [International Caries Detection and Assessment System] score 0) children (n = 119) and their primary caregivers were followed from 1 until 4 y of child age. Salivary and dental plaque samples were collected from the children at 3 time points (T1, ~1 y old; T2, ~2.5 y old; and T3, ~4 y old). Only saliva samples were collected from the caregivers. Bacterial V4 16S ribosomal DNA amplicons were sequenced using Illumina MiSeq. The reads were denoised and mapped to the zero-radius operational taxonomic units (zOTUs). Taxonomy was assigned using HOMD. The microbial profiles of children showed significant differences (P = 0.0001) over time. Various taxa increased, including Fusobacterium, Actinomyces, and Corynebacterium, while others showed significant decreases (e.g., Alloprevotella and Capnocytophaga) in their relative abundances over time. Microbial diversity and child-caregiver similarity increased most between 1 and 2.5 y of age while still not reaching the complexity of the caregivers at 4 y of age. The microbiome at 1 y of age differed the most from those at later time points. A single zOTU (Streptococcus) was present in all samples (n = 925) of the study. A large variation in the proportion of shared zOTUs was observed within an individual child over time (2% to 42% of zOTUs in saliva; 2.5% to 38% in dental plaque). These findings indicate that the oral ecosystem of caries-free toddlers is highly heterogeneous and dynamic with substantial changes in microbial composition over time and only few taxa persisting across the 3 y of the study. The salivary microbiome of 4-y-old children is still distinct from that of their caregivers.

Dental caries lesions are a clinical manifestation of disease, preceded by microbial dysbiosis, which is poorly characterized and thought to be associated with saccharolytic taxa. Here, we assessed the associations between the oral microbiome of children and various caries risk factors such as demographics and behavioral and clinical data across early childhood and characterized over time the salivary and dental plaque microbiome of children before clinical diagnosis of caries lesions. Children ( N = 266) were examined clinically at ~1, 2.5, 4, and 6.5 y of age. The microbiome samples were collected at 1, 2.5, and 4 y. Caries groups consisted of children who remained caries free (International Caries Detection and Assessment System [ICDAS] = 0) at all time points (CFAT) ( n = 50); children diagnosed with caries (ICDAS ≥ 1) at 6.5 y (C6.5), 4 y (C4), or 2.5 y of age (C2.5); and children with early caries or advanced caries lesions at specific time points. Microbial community analyses were performed on zero-radius operational taxonomic units (zOTUs) obtained from V4 of 16S ribosomal RNA gene amplicon sequences. The oral microbiome of the children was affected by various factors, including antibiotic use, demographics, and dietary habits of the children and their caregivers. At all time points, various risk factors explained more of the variation in the dental plaque microbiome than in saliva. At 1 y, composition of saliva of the C4 group differed from that of the CFAT group, while at 2.5 y, this difference was observed only in plaque. At 4 y, multiple salivary and plaque zOTUs of genera Prevotella and Leptotrichia were significantly higher in samples of the C6.5 group than those of the CFAT group. In conclusion, up to 3 y prior to clinical caries detection, the oral microbial communities were already in a state of dysbiosis that was dominated by proteolytic taxa. Plaque discriminated dysbiotic oral ecosystems from healthy ones better than saliva.

Caries remains a major oral health burden among young children. While the impact of diet and oral hygiene is well established, the relationship between the oral microbiota, including both bacteria and fungi, and oral health has not been comprehensively explored. This thesis investigated the development of oral bacterial and fungal communities in children aged 1 to 4 years and their primary caregivers, focusing on microbial shifts associated with Early Childhood Caries (ECC). Through a longitudinal cohort design and amplicon sequencing of the 16S rRNA (V4) region of oral samples, we gained insight into the microbial maturation, bacterial dysbiosis, bacterio-fungal interkingdom associations, and the behavioral and environmental influences on the oral microbiota of children with and without ECC. First, we examined the bacterial component of the oral microbiota and found that the oral ecosystem of caries-free toddlers is highly heterogeneous, with significant maturation of oral bacterial diversity and composition, and that a few bacterial taxa persist across 1 and 4 years of age (Chapter 3). Second, we demonstrated that ecological dysbiosis preceding caries can occur up to 3 years before its clinical manifestation, emphasizing the potential for microbiota-based early caries risk assessments (Chapter 4). Specifically, proteolytic and anaerobic taxa, such as Prevotella, and saccharolytic and anaerobic taxa, such as Leptotrichia, were associated with dysbiosis (Chapter 4). This finding contradicts the ecological plaque hypothesis of caries, which proposes an enrichment of aciduric and acidogenic, facultatively anaerobic taxa, like certain streptococci (Marsh 2003). Our assessment of various caries risk factors revealed that demographics, dietary habits, and exposure to antibiotics explained significant variation in the oral bacterial composition of the children (Chapter 4). Next, we studied another presumably equally important component: the fungal communities, or the mycobiota. Considering the methodological challenges of processing of samples with low fungal biomass (Chapter 5), our study revealed that the oral fungal composition of children is niche-dependent (saliva vs. supragingival plaque), dynamic over time, and associated with caries development (Chapter 6). Specifically, Candida albicans was predominant in dental plaque samples and was linked to caries (Chapter 6). A crucial connection across these studies is the interplay between bacteria and fungi. Our results showed that a high fungal load was correlated with a higher prevalence of caries-associated bacteria, particularly Streptococcus mutans and Actinomyces spp. (Chapter 6). These findings support the hypothesis that fungi and bacteria engage in interkingdom interactions that influence biofilm stability and caries progression (Koo et al. 2014). In conclusion: • The oral bacterial composition in children is highly dynamic and influenced by microbial and environmental factors from an early age. • Bacterial dysbiosis can precede clinical caries by years, with complex communities displacing early commensals. • Though less abundant, fungi may significantly contribute to caries through synergy with bacteria, especially in dental plaque biofilms. • Caregiver behaviors and bacterial composition strongly shape the child’s oral ecosystem, reinforcing the importance of family-centered preventive strategies.

Worldwide, there are significant differences in caries prevalence among groups differing in socio-economic position. Understanding these differences is crucial to improving accessibility to oral healthcare for all population groups. Our study assessed caries prevalence and severity in a random sample of children aged 1 to 6.5 years from high and low socio-economic positions. At 4 years of age, 39% versus 57% of children of high and low socio-economic positions, respectively, had dental caries. This was 43% versus 72% at 6.5 years of age. The number of surfaces affected by caries and the percentage of carious teeth increased significantly over time among children with caries experience from low socio-economic positions. From the ages of 4 and 6.5 years, children with low socio-economic positions had significantly more carious surfaces and a higher percentage of carious teeth compared to those in the high socio-economic positions group. This emphasizes an inequality in caries experience among groups differing in socio-economic position.