The oral cavity is one of the most important habitats of the human body and one of the richest environments in microorganisms, consisting of bacteria, fungi, viruses, and archaea (1–3). It is formed by an ecological community of commensal, symbiotic, and pathogenic microorganisms (4) with a significant impact on both oral and general health and is a potential diagnostic indicator for various diseases such as cardiovascular disease, rheumatoid arthritis, cancer, or diabetes, among others (5–7). The oral environment constantly transforms with age, and, consequently, the microbiota also changes (8). At birth, most children do not have a colonized microbiome, and during the first two months of life, bacteria colonize the mucosal surfaces, and later, with the eruption of primary teeth, these surfaces are also colonized (8). During childhood, primary dentition exfoliates, leading to mixed dentition and finally to permanent dentition (2), with observed changes in bacterial colonization at different dentition stages (6, 9), resulting in known differences between the microbiota of children and adults (10, 11). In this regard, Ling et al. (12) observed that Actinobacteria (syn. Actinomycetota), Bacteroidetes (syn. Bacteroidota), and Fusobacteria (syn. Fusobacteriota) were overrepresented in children, whereas Firmicutes (syn. Bacillota) and Proteobacteria (syn. Pseudomonadota) were more abundant in adults.

Although oral microbiota is resistant to minor ecological changes, factors such as diet, environment, inadequate oral hygiene (13), the use of medications or prolonged use of antibiotics (8) can also induce structural changes in the microbiota (14) leading to dysbiosis of the resident microbiota (4), which can result in caries (1).

Untreated dental caries is the most prevalent condition worldwide and is estimated to affect 2.5 billion people of which 530 million are children (15). Although Streptoccocus is the main genera related to dental caries, other microorganisms have been identified in relation to the presence of caries lesions (i.e., Fusobacterium, Prevotella, Leptotrichia, Veillonella, Bifidobacterium, and Capnocytophaga species) (4). However, several important limitations in the existing studies on the oral microbiota and its association with dental caries have to be considered. Many of these investigations rely on non-omics-based techniques, which restrict the detection and characterization of non-culturable bacteria, thus providing an incomplete picture of the microbial diversity involved. Furthermore, most studies have been conducted in Asian populations, with limited data available from Spanish or broader European cohorts, making it difficult to generalize findings across different populations. In addition, there is a lack of studies that simultaneously analyse both children and adults, despite known age-related differences in oral microbiota composition. In this sense, confounding factors such as sex and age are often not adequately controlled or considered in the analysis, potentially biasing the results and limiting the reliability of observed associations.

Another aspect to consider is the site of sample collection. In this regard, saliva is a biological fluid that bathes the oral cavity, and it is essential for maintaining both oral and general health (14). It is estimated that over 700 bacterial species coexist in the oral cavity (16), among them, only two-thirds belong to in vitro cultivable species (17). The rich microbiota provides a composite representation of microorganisms from diverse niches within the oral cavity (18) and reflects local alterations of the supragingival and subgingival microbiota (4). Salivary collection is non-invasive, simple, and painless collection method, which makes it particularly suitable for studies involving children (16). Moreover, it has been reported that saliva composition parameters, such as total protein content and antioxidant capacity, are associated with the composition of the salivary microbiota (19) which may in turn influence susceptibility to oral conditions such as caries. These biochemical properties could potentially modulate the oral environment and shape microbial communities, highlighting the importance of integrating salivary biomarkers into microbiome research. These gaps underscore the need for more comprehensive, omics-based, and demographically diverse research that considers developmental stages, relevant confounding variables, and optimized sampling strategies to better understand the complex interactions between the oral microbiome and dental caries.

This study aimed to investigate the relationship between salivary microbiota composition, based on 16S rRNA gene V3–V4 amplicon sequencing, and the presence of active dentin caries (ADC), in Spanish children and adults. Additionally, the relation between salivary parameters, including pH, flow rate, total protein content, and antioxidant activity, and caries status was evaluated in both age groups.

A case-control, cross-sectional, observational, and analytical study was conducted, with two parallel and matched arms. The study design was approved by the Bioethics Committee of the San Carlos Clinical Hospital (code 22/334-E, 31st May 2022) complying with current Spanish and European regulations on personal data protection and the principles of the Declaration of Helsinki for research involving human subjects. Additionally, the STROBE guidelines for cross-sectional studies were followed as reporting guidelines (20) (Supplementary Table S1).

The main objective of this study was to evaluate the relationship between the level of ADC with salivary biochemical parameters and salivary microbiota composition in both, children and adults from Spain. In line with most of the previous work, the present study employed a cross-sectional design, which, although limited in its ability to infer causality, remains a practical and commonly used approach in oral microbiome research. Our sample size (n = 80), comprising 40 children and 40 adults evenly distributed by ADC status, is comparable to similar investigations, which have included between 30 and 50 paediatric participants or 40–50 adult subjects. In the present study, we adhered to WHO criteria to define the presence of caries and further classified them into enamel and dentin lesions according to ICDAS, allowing a more precise stratification of disease severity. To minimize confounding variables related to age, participants were selected within a narrow age range, with mean ages of approximately 8 years for children and 30 years for adults. As in many metagenomic studies, 16S rRNA gene sequencing was used to characterize the microbial communities present in saliva. All participants exhibited salivary values within normal ranges for flow rate, pH, TPC, TAC, and microbial composition.

Regarding salivary metagenomic studies, most of them have been conducted in Asian countries such as China (1, 13, 24–26, 28, 35–43) or Korea (44, 45), although they have also been carried out in European countries (9, 10, 12, 14, 16, 17, 27, 46–49), Turkey (50, 51), USA (2, 5, 23, 52, 53) and Australia (54), while only one study conducted in Spain to date (55). It is important to consider the dietary, hygienic and social differences between different races and cultures, which underlines the need to carry out population studies that describe salivary bacterial composition, helping to detect intercultural differences (56). In relation to the design of the published studies, most are cross-sectional, similar to the present study (2, 9, 14, 16, 17, 23–25, 28, 35–43, 46–50, 52, 57), although longitudinal studies have also been conducted (5, 10, 12, 13, 26, 27, 45, 51). Regarding the population characteristics, variables studied, and sample size, we find that the sample size is very heterogeneus, with some studies including only two participants (52), up to studies with 293 subjects (47). In our case, we studied a total of 80 subjects (40 children and 40 adults, evenly divided according to ADC), similar to previous studies, with 30–50 children (13, 24–27) or 46 adults (28), respectively. Regarding the age of the studied population, there is great heterogeneity, with studies analyzing ages ranging from 17 to 63 years in adults (10, 28, 35, 38, 39, 41–44, 46–49, 51) and from 3 months-18 years in children (1, 5, 9, 12, 13, 16, 24–27, 40, 42, 45, 50, 54, 57). Similar to our study, with a mean age of 30.15 ± 5.6 and 31 ± 6.3 years old for adults (non-ADC and ADC respectively) and 7.85 ± 1.18 and 8.4 ± 1.23 years old for children (non-ADC and ADC respectively), with a narrow range to avoid biases associated with age changes. Regarding the variables of interest evaluated in relation to salivary metagenomics, studies in adults have focused on caries (10, 28, 38, 39, 42, 48, 51), periodontal disease (14, 35, 44, 46, 49) or both (41, 43, 47, 52). In contrast, in children, the studies conducted evaluated caries disease (1, 5, 9, 12, 13, 16, 24–27, 40, 42, 45, 54, 57). To determine the status of oral health or disease, different diagnostic criteria and reference indices were used in published studies. In previous research the WHO criteria were employed as the diagnostic method for caries (12, 23, 24, 45), while in other studies used ICDAS method (1, 16, 27, 51).

In relation to the association between salivary biochemical parameters (salivary flow rate, pH, TPC, and TAC) and ADC in both children and adults, our analysis did not reveal any significant correlations in either age group. These findings were somewhat unexpected, given prior evidence suggesting otherwise. For instance, Pandey et al. (58) reported higher salivary pH levels in caries-free children and observed an increase in antioxidant capacity in children with active caries, irrespective of age. Consistent with our results, however, they found no significant differences in the salivary flow rate between caries-active and caries-free individuals. In contrast, Pyati et al. (59) observed significant differences in salivary flow rate, pH, and total protein levels between children with active caries and healthy controls. Similarly, a recent systematic review focusing on paediatric populations (60) supported these findings, indicating that salivary parameters such as flow rate, pH, and buffering capacity tend to decrease in children with caries, regardless of age or sex. However, the authors reported no significant changes in total protein concentration among children aged 6–12 years, which aligns with our results, especially considering the narrow age range employed in our study to minimize age-related variability.

One possible explanation for the lack of significant associations in our data could be the multifactorial nature of dental caries, which involves complex interactions between host factors, diet, oral hygiene practices, and microbiota composition. Biochemical parameters such as pH and antioxidant capacity may fluctuate over short periods and be influenced by recent food intake or stress, making them less reliable as isolated markers of caries activity. Additionally, the narrow range of biochemical values observed in our participants, most of whom exhibited values within normal physiological limits, might have limited our ability to detect statistically significant differences. It is also possible that microbial composition and ecological shifts in the biofilm play a more critical role in caries development than the salivary biochemistry alone.

Thus, we explored the relationship between salivary microbiota composition with ADC in both age groups. In the present study, we did not observe significant correlations between ADC and the relative abundance of bacterial phyla in either children or adults. These findings suggest that shifts in the salivary microbiota related to caries may occur more prominently at lower taxonomic levels, such as genus or species, rather than at the broader phylum level. This observation aligns with previous studies indicating that caries-associated microbial changes are often subtle and localized to specific taxa rather than being characterized by global shifts in major bacterial phyla (32, 61). In relation to periodontal status, although in children no statistical correlation was found, a significant and negative relationship was found between CPI and Bacillota and Actinobacteriota, according to previous research, stating that Fusobacteria and Saccharibacteria (TM7) are the most abundant phyla associated with gingivitis, while Actinobacteriota and Bacteroidetes are less frequently associated with gingivitis (35).

At the genus level, several taxa showed significant positive correlations with ADC in adults, including Corynebacterium, Porphyromonas, Tanerella, Catonella, Oribacterium, Mogibacterium, Filifactor, Parvimonas, Dialister, or Leptotrichia. Many of these genera have been associated with periodontal and endodontic infections, and their presence may reflect a microbial dysbiosis that supports both caries progression and other oral pathologies (62, 63). For example, Porphyromonas have been implicated in pulp and periapical infections and have been increasingly associated with the progression of deep carious lesions (62–67). Similarly, Filifactor and Parvimonas are anaerobic genera linked to endodontic and periodontal diseases, supporting the notion that late-stage dentin caries may share microbial signatures with these conditions. These results suggest that the adult oral microbiota harbours a more diverse array of genera associated with caries, many of which have been previously implicated in dysbiotic biofilms and periodontal or endodontic infections. The presence of multiple anaerobic and proteolytic taxa (e.g., Filifactor, Mogibacterium) in this group may reflect the complex microbial consortia contributing to caries progression beyond classical acidogenic bacteria. However, it should be noted that in our study, adults with higher ADC level also presented higher level of CPI. Thus, these findings should be confirmed in later studies. Conversely, Haemophilus showed a significant negative correlation with ADC in adults, and thus, its abundance could potentially protect from caries development. This genus is commonly associated with health-associated oral microbiota and may exert protective effects via competitive exclusion or production of neutral or alkali metabolic byproducts.

At the species level, in adult saliva, the presence of ADC was positively correlated with P. gingivalis, P. endodontalis, P. stomatis, L. buccalis, P. oris, and C. matruchotii. Interestingly, P. gingivalis, traditionally linked to periodontitis, and P. stomatis, typically associated with endodontic infections, showed positive correlations with caries in adults, suggesting potential ecological shifts in the salivary microbiome under cariogenic conditions. C. matruchotii, though traditionally considered a commensal, may contribute structurally to the development of cariogenic biofilms through its characteristic filamentous morphology and central positioning in the “hedgehog” structure of supragingival plaque (68). A positive correlation between P. gingivalis and CPI was found, according tio Kim et al. (44), who reported an increase in the group of patients with periodontitis, along with abundance of T. denticola and T. forsythia. In children, by contrast, the associations between microbiota and caries were more limited. Only the genera Scardovia, particularly known for its role in early childhood caries and acidogenic potential, was significantly and positively correlated with ADC. Scardovia wiggsiae, in particular, has been well documented as a caries-associated species in paediatric populations and has been proposed as an early colonizer in the development of severe early childhood caries (69). At the species level, P. stomatis was significantly and negatively associated with caries, suggesting a potentially protective role in the paediatric population. Interestingly, P. stomatis showed opposing correlations with caries in adults and children, positive in the former and negative in the latter. This discrepancy may reflect age-related differences in the oral microbiome's composition, maturity, and ecological dynamics. In adults, P. stomatis may participate in dysbiotic consortia involving anaerobic and proteolytic taxa that contribute to caries progression. In contrast, in children whose microbiome is less mature and more dominated by acidogenic species, P. stomatis may play a less pathogenic or even modulatory role. These divergent associations may also be influenced by differences in salivary parameters, immune responses, or microbial interactions across age groups. Further studies are needed to clarify the functional role of P. stomatis in distinct developmental contexts.

This study presents several limitations that should be considered when interpreting the results. First, the sequencing of the V3–V4 regions of the 16S rRNA gene, although widely used in microbiome studies, does not always provide sufficient resolution to reliably distinguish between closely related bacterial species. As a result, a substantial proportion of sequences could not be taxonomically classified at the species level, potentially limiting the precision of our microbial profiling. Second, the classification of participants was based solely on the presence or absence of ADC. While none of the individuals in the non-ADC group presented clinical signs of periodontitis, the ADC group included participants with varying periodontal health status, ranging from high periodontal indices to no clinical signs. This heterogeneity may function as a confounding factor, particularly considering the overlapping microbial profiles between caries and periodontal diseases. Future studies should aim to better control for periodontal status to isolate the specific microbial signatures associated with ADC. Third, the cross-sectional nature of this study prevents the establishment of causal relationships between microbiota composition, salivary biochemical parameters, and caries status. Longitudinal studies are needed to elucidate temporal dynamics and causality. Additionally, the sample size, although comparable to previous studies in this field, was relatively limited, which may reduce statistical power. Besides, due to the theoretical calculation of sample size the generalization could be reduced. Therefore, correlation analyses were employed as an exploratory approach to detect potential associations, recognizing the inherent limitations in generalizability and robustness. The statistical analysis of beta diversity lacks model adjusting of covariables as sex or age, due to the low sample size. Although that adjusted analysis would improve the quality of the interpretation and generability in a wide sample size, in our sample the sample size divided by sex and age would rise the biases (i.e., low data precision, atypical data, high confidence intervals, data distortion) of the data analysis. Besides, sampling was paired by sex in order to prevent sex influence in results. Furthermore, some aspects of the CPI index constitute limitations, as it is a population-based epidemiological index, but it is an index that measures the need for treatment associated with pathology. According to the WHO and the American Society of Pediatric Dentistry, its use is recommended for assessments in both adults and child populations. Besides, it has been used in previous research (30, 49, 70, 71).

Despite these limitations, this study has several notable strengths. Most existing salivary metagenomic studies have been conducted in Asian populations, where cultural practices, dietary habits, and oral hygiene behaviours differ significantly from those in other regions. Our study contributes novel data from a Western European population, specifically from Spain, where only limited microbiome data is currently available. This adds valuable intercultural insight into the salivary microbiota and its association with oral health. Moreover, the study was carefully designed to minimize confounding variables. Thus, adults and children, with and without ADC were balanced for sex and body mass index (BMI), and in both age groups, participants were matched by age to reduce potential age-related microbiome variability. Saliva collection followed standardized and validated protocols, ensuring consistency in sample handling and processing. Additionally, rigorous inclusion and exclusion criteria were applied, helping to control for variables such as recent antibiotic use, systemic diseases, and other factors known to influence the oral microbiome. Finally, the integration of microbiota data with salivary biochemical markers, such as TAC and total TPC, adds depth to the findings and provides a more holistic view of the oral ecosystem. The balanced study design, along with the novelty of exploring both microbiota and oxidative markers in relation to caries in adults and children, enhances the relevance and scientific value of this work.

Finally, interpreting the data obtained, significant differences were found in salivary bacterial composition in the presence of ADC, as well as periodontal disease, the most common diseases in dentistry. Furthermore, data suggest that salivary parameters do not play as important a role as salivary microbiological composition. However, the aforementioned limitations do not allow generalization to broad populations, making it necessary to conduct broad-spectrum, multicentre, and controlled studies that allow for firm conclusions and allow for extrapolation of data.

This study explored the relationship between salivary microbiota composition, salivary biochemical parameters, and ADC in both children and adults. Although no significant associations were observed between phylum-level microbial composition and ADC status, specific genera and species showed positive correlations with caries activity, particularly in adults. These included Corynebacterium, Porphyromonas, Filifactor, Olsenella, Dialister or Catonella, among other microorganisms previously associated with oral dysbiosis and disease. In children, the genus Scardovia was positively related to ADC. This supports the idea that distinct microbial profiles may underlie caries development in different age groups, which may be influenced by factors such as diet, oral hygiene habits, maturity of the oral microbiome, and host immune responses. In this sense, the specie P. stomatis showed opposing correlations with caries in adults and children, positive in the former and negative in the latter.

Despite initial hypotheses, salivary biochemical parameters, including flow rate, pH, total protein concentration, and total antioxidant capacity, did not show significant associations with ADC presence in either age group.

Taken together, these findings suggest that microbial factors may play a more significant role than salivary biochemical markers in distinguishing caries activity and suggest age-specific microbial risk markers, particularly in adults. Future studies with larger sample sizes and longitudinal designs are needed to confirm these associations and explore potential mechanistic pathways.