Evaluation of the relationship between salivary levels of Streptococcus mutans, statherin, and cathelicidin LL-37 and early childhood caries.

Evaluation of the relationship between salivary levels of Streptococcus mutans, statherin, and cathelicidin LL-37 and early childhood caries - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Search Log in Primary site navigation Search Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice BMC Oral Health . 2026 Feb 9;26:954. doi: 10.1186/s12903-026-07819-4 Evaluation of the relationship between salivary levels of Streptococcus mutans, statherin, and cathelicidin LL-37 and early childhood caries Ceren Pamuk 1 , Nazan Ersin 1 , Gülnur Emingil 2 , Güven Ozdemir 3 , Caner Vural 4 , Cansu Vural 3 , Aytül Gül 5 , Handan Çelik 6, ✉ , Yüksel Gezgin 5 Author information Article notes Copyright and License information 1 Department of Pediatric Dentistry, Faculty of Dentistry, Ege University, Izmir, 35100 Turkey 2 Department of Periodontology, Faculty of Dentistry, İstinye University, Istanbul, 34408 Turkey 3 Department of Biology, Faculty of Science, Ege University, Izmir, 35100 Turkey 4 Department of Biology, Faculty of Science, Pamukkale University, Denizli, 20160 Turkey 5 Department of Bioengineering, Faculty of Engineering, Ege University, Izmir, 35100 Turkey 6 Department of Pediatric Dentistry, Faculty of Dentistry, Izmir Democracy University, Izmir, 35100 Turkey ✉ Corresponding author. Received 2024 Nov 21; Accepted 2026 Jan 28; Collection date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13235084  PMID: 41656215 Abstract Background Although caries is known to be influenced by diet and oral hygiene factors, susceptibility to the disease may be reduced by host factors, such as saliva antimicrobial peptides (AMPs). However, the relationship between salivary AMPs and early childhood caries (ECC) remains limited. Among the AMPs, cathelicidin LL-37 is mentioned in the literature for its bactericidal activity against oral pathogens, while statherin is recognized for its mechanism of reducing hydroxyapatite demineralization on the tooth surface. Cathelicidin LL-37 is a significant class of proteins within the immune system, and statherin prevents the precipitation of calcium phosphate in saliva, maintaining high levels of calcium for enamel remineralization and high phosphate levels for buffering. Aim This study aimed to compare the salivary levels of statherin, cathelicidin LL-37, and Streptococcus mutans (S. mutans), as well as the oral hygiene and dietary habits, of children with and without early childhood caries (ECC). Design A total of 24 children with ECC and 34 children without ECC were selected to participate in this study. The levels of S. mutans in unstimulated saliva collected from the children were measured by real-time polymerase chain reaction (PCR), and the levels of statherin and cathelicidin LL-37 in saliva were measured using an enzyme-linked immunosorbent assay (ELISA). Results There was no statistically significant difference in salivary statherin levels between the two groups, but salivary cathelicidin LL-37 levels were significantly higher in the ECC group ( p = 0.024). The ECC group had significantly elevated S. mutans levels compared to the non-ECC group ( p < 0.05). The frequency of sugar-containing foods and/or beverages consumed more than three times a day between meals was statistically significantly higher in the ECC group compared to the non-ECC group ( p = 0.021). The statistical analysis also revealed a positive correlation between the dmfs score and age ( p = 0.022). Supplementary Information The online version contains supplementary material available at 10.1186/s12903-026-07819-4. Keywords: Childhood caries, Antimicrobial peptides, Streptococcus mutans, COVID-19 pandemic. Introduction Childhood caries are also known as “labial caries,” “rampant caries,” “baby bottle caries,” “infantile melanodontia,” and “breastfeeding caries” due to their etiological factors and clinical appearance [ 1 , 2 ]. Because childhood caries have a multifactorial etiology that cannot be adequately described by these terms, the American Academy of Pediatric Dentistry (AAPD) has currently classified this condition as early childhood caries (ECC) and severe early childhood caries (S-ECC). ECC is defined as the presence of ≥ 1 cavitated or non-cavitated caries lesion, tooth loss due to caries, or the presence of any filled primary tooth surface in children below the age of 71 months. The oral cavity hosts a complex microbial community that includes cariogenic microorganisms, like Streptococcus mutans colonization, which usually leads to an extremely high caries occurrence as a primary etiological agent in the development of dental caries during early childhood [ 3 ]. Host-associated defense systems, especially antimicrobial proteins, play an important role in both preventing caries and maintaining oral cavity health [ 4 ]. Caries-sensitive tooth surfaces, low socioeconomic status, the education level of the family, and lack of oral hygiene care in childhood are also associated with ECC [ 5 ]. The change in children’s dietary habits, such as increased sweet consumption, on increased sweet consumption and snacking habits before bedtime rather than a decrease in oral hygiene, could lead to an increase in the prevalence of caries disease during the COVID-19 pandemic in the Italian pediatric population [ 6 ]. Salivary antimicrobial peptides, including the cathelicidin LL-37, are considered important biological factors involved in innate immunity against dental caries. Variations in the salivary levels of these peptides may influence individuals’ susceptibility to dental caries by affecting the oral microbial balance and immune response [ 7 ]. Specifically, analysis of the concentration–response relationship demonstrated that LL-37 induces a reduction in Streptococcus mutans viability. LL-37 has been detected in gingival crevicular fluid at concentrations exceeding 1 mM, and a pronounced decrease in bacterial viability was observed within a relatively narrow concentration range of 1–8 mM [ 7 ]. Among these peptides, cathelicidin LL-37 has been reported to exhibit bactericidal activity against cariogenic microorganisms. However, the specific effects of LL-37 on S. mutans remain unclear, as the cited reference does not directly mention this species, and existing studies show conflicting results regarding its role in the immunopathogenesis of dental caries [ 8 – 10 ]. According to studies, the LL-37 peptide is one of the AMPs that appears to be vital for maintaining eubiotics in the oral cavity and thus serves as a potential agent for pharmaceutical prophylaxis or the treatment of various dysbiotic conditions, for example by direct application, the application of its synthetic derivatives, or modulation of its expression [ 11 ]. Statherin, another antimicrobial peptide, binds calcium in the saliva. Thus, it maintains the salivary saturation of calcium and phosphate by preventing their precipitation; this increases the remineralization of the enamel and protects the integrity of the teeth [ 12 ]. There is a limited number of studies on the relationship between statherin and ECC [ 13 – 18 ]. Our study hypothesized that salivary antimicrobial peptide levels may be associated with caries potential in children with and without ECC. Thus, we intended to examine the salivary levels of statherin, cathelicidin LL-37, and S. mutans in 4- to 6-year-old children with and without active caries during the COVID-19 pandemic. In this study, the relationship between the children’s salivary levels and their socio-demographic characteristics, oral hygiene, and (dietary) nutritional habits was also examined. Materials and methods Sample size Using the Mann–Whitney U test for two independent groups (ECC vs. non-ECC), a difference in S. mutans, statherin, and cathelicidin LL-37 rankings that can affect large classes (large class effect size, d = 0.8) was detected. A minimum of 54 participants was found to be sufficient to achieve 80% power at the 0.05 significance level. It was calculated that 27 participants per group (27 + 27) would be sufficient. Subjects The study group included 24 children with ECC, while 34 children without caries (no lesions, including precavitated ones) were chosen as the control group. All participants were aged 4 to 6 years. However, children with systemic diseases, those undergoing regular medication such as antibiotics within 1 month prior to the dental examination, those with gingivitis or mucosal lesions, and children who could not communicate verbally were excluded from the study. All children included in the study successfully completed the saliva collection procedure. Ethical approval for the study was obtained from the medical research ethics committee (Decision number: 19-8.1T/46, date of approval: 21 August 2019). All the children and their parents received verbal explanations and were informed about the procedures required for the implementation of the study. The participants’ parents signed written informed consent forms. A single dentist performed intraoral examinations for all participants under natural daylight using a mirror and probe in a classroom environment. Caries were detected in all caries-active children using the DMFS index established by the World Health Organization [ 19 ]. Saliva samples Unstimulated whole saliva samples were collected from each subject. The children and teachers were informed in advance to ensure that the subjects refrained from eating, drinking, or chewing gum for 2 h before sample collection, thereby preventing contamination with non-salivary components. To minimize variations due to the circadian rhythm of salivary secretion, all samples were collected between 9:00 and 11:00 in the morning. During this procedure, the children were asked to sit comfortably, tilt their heads slightly forward, and minimize mouth movements. They were then instructed to spit the saliva accumulating in their mouths into sterile tubes over a 5–10-minute period. A total of approximately 4–5 mL of saliva was collected [ 8 ]. Gloves were used during all procedures to avoid contamination. The collected saliva samples were transported on ice within 1 h to the Bioengineering Department, Faculty of Engineering, Ege University. Supernatants were obtained by centrifuging the saliva samples at 3000 rpm at 4 °C for 5 min. Aliquots of 500 µL from each sample were stored at − 80 °C until analysis for statherin and cathelicidin LL-37 levels [ 20 ]. Antimicrobial peptide level measurements The concentrations of cathelicidin LL-37 and statherin in the saliva samples were identified using ELISA kits specific to each protein (Sunred Biological Technology, Shanghai, China; catalog no. for cathelicidin LL-37: 201-12−2198; catalog no. for statherin: 201-12−2557), in accordance with the manufacturer’s instructions. For the analysis of salivary cathelicidin LL-37 and statherin levels, each standard dilution was added to the standard wells in duplicate, along with the standard dilution and streptavidin-HRP-conjugate agent. Saliva samples, biotin-LL-37 antibody or biotin-STATH antibody, and streptavidin-HRP-conjugate agent were added to the test wells, respectively. Only washing solution was added to the blank wells. After preparing the standard, test, and blank wells, the plates were covered with sealing membrane and incubated. The liquid in the wells was then removed, and the washing process was performed. Chromogen solutions were added to the wells in the dark and incubated. Subsequently, stop solution was added in the dark, and absorbance values were measured using a spectrophotometer at a wavelength of 450 nm. Determination of S. mutans level in saliva DNA isolation Saliva samples (2 mL) in microcentrifuge tubes were centrifuged at 10,000 rpm for 5 min. After centrifugation, the supernatants were discarded. The resulting pellets were homogenized by brief vortexing after the addition of 200 µL phosphate-buffered saline. DNA isolation from the saliva was carried out using a High Pure PCR Template Preparation Kit (Roche), according to the manufacturer’s instructions. Real-time PCR In this study, forward (5′-CTACACTTTCGGGTGGCTG-3′) and reverse (5′-GAAGCTTTTCACCATTAGAAGCTG-3′) primers targeting the S. mutans glucosyltransferase I gene were used. Real-time PCR analyses were conducted using the LightCycler 96 instrument (Roche Instruments) and the LightCycler 480 SYBR Green I Master Kit. The PCR protocol consisted of one pre-incubation cycle at 95 °C for 5 min; 40 amplification cycles at 95 °C for 10 s, 58 °C for 10 s, and 72 °C for 11 s; one melting cycle at 95 °C for 5 s, 65 °C for 60 s, and heating up to 97 °C; followed by a final cooling cycle at 40 °C for 10 s. Following the 40 amplification cycles, the melting temperatures (Tm) of the PCR products were analyzed to confirm the specificity of amplification. PCR products were purified using the NucleoSpin ® Gel and PCR Clean-up Kit (Macherey-Nagel GmbH & Co., Germany) and quantified by UV absorbance at 260 nm using a Nanodrop (2000c, Thermo Scientific, USA). Standard curves were constructed over a concentration range of 10² to 10¹⁰ amplicons of target DNA per µL. The cycle threshold (Ct) was determined during the exponential phase of amplification. DNA dilutions were processed in the real-time PCR device, and a standard curve graph was generated. Tm values of PCR products amplified with the selected primer set were calculated, and the generated standard curve plots were used to evaluate the PCR reaction yield. Obtaining survey data In this study, to evaluate children’s oral hygiene habits, nutritional habits, and access to professional dental care, the parents of both caries-active and caries-free groups were administered a questionnaire based on the Caries Risk Assessment Tool published by the AAPD in 2020 [ 21 ]. In addition, they were asked to provide a list detailing their child’s weekday diet. This list included data on all meals and snacks, along with the time of their consumption. All sweets consumed during the day were categorized as liquid, solid and sticky, or slow-dissolving. A frequency graph was generated for each sugar-containing food item eaten at the end of a meal or with at least 20-minute intervals between meals. In each group, the consumption frequency of each sweet category was multiplied by a weighting factor: 5 for liquids, 10 for solid and sticky foods, and 15 for slowly dissolving foods. The results were then summed to obtain the total daily sugar consumption score, following the protocol outlined by Nizel and Papas [ 22 ]. Statistical analysis Statistical analyses were conducted using IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp., Released 2017, Armonk, NY, USA). Data distribution was evaluated using the Shapiro–Wilk test. The Chi-Square or Fisher’s exact test was applied to compare categorical variables between the two groups. The Mann–Whitney U test was used for comparing numerical variables. Spearman’s rho correlation analysis was employed to assess linear relationships between numerical variables. Univariable and multivariable logistic regression analyses were used to examine the relationships among salivary components, frequency of sugar-containing food and beverage consumption, and caries presence. The calibration of the multivariable model was assessed with the Hosmer–Lemeshow test. All statistical analyses were conducted at a significance level of 0.05. Results The demographic characteristics, dietary habits, and oral hygiene behaviors of the study groups are presented in Table 1 . The mean age of the children, oral hygiene practices (tooth brushing frequency, brushing since the eruption of the first tooth, brushing with parental assistance), and feeding behaviors (sleeping with a baby bottle during infancy, nighttime breastfeeding, and total daily sugar consumption score) were found to be similar between children with and without ECC ( p > 0.05; Table 1 ; Fig. 1 ). Table 1. Characterization of subjects studied as regards age, diet, and oral hygiene habits Variable ECC No ( n = 34) ECC Yes ( n = 34) p -value Age 0.244 4 13 7 5 14 16 6 7 11 Age (mean ± SD) 4.82 ± 0.75 5.11 ± 0.72 Toothbrushing frequency 0.442 2–3 times a day 10 13 < 1 time a day 24 21 Brushing since the first tooth erupted 0.804 Yes 21 20 No 13 14 Tooth brushing with parental assistance 0.209 Yes 19 24 No 15 10 Frequency of sugar-containing foods/beverages consumption 0.021* > 3 times 7 16 < 3 times 27 18 Sleeping with a baby bottle during infancy 0.787 Yes 9 10 No 25 24 Pacifier use during infancy 0.465 Yes 14 17 No 20 17 Breastfeeding at night 0.709 Yes 31 29 No 3 5 Open in a new tab Fig. 1. Open in a new tab The levels of total daily sugar consumption score in the groups without and with ECC ( p = 0.678). Whiskers show minimum and maximum values; boxes represent 25–75% of the data ranges; horizontal lines within boxes are medians However, the frequency of consuming sugar-containing foods and/or beverages more than three times a day between meals was statistically significantly higher in the ECC group compared to the group without ECC ( p < 0.05, Table 1 ). There was no statistically significant difference in salivary statherin levels between both groups but salivary cathelicidin LL-37 levels were significantly higher in the ECC group ( p = 0.778, p = 0.024 respectively) (Table 2 ; Fig. 2 A and B). The ECC group had significantly elevated S. mutans compared to those without the ECC group ( p < 0.05, Table 2 ; Fig. 3 ). Table 2. The relationship of early childhood caries with total daily sugar consumption score and salivary parameters Variable ECC No ( n = 34) ECC Yes ( n = 24) p -value Median Min–max Median Min–max Total sugar score 15.00 0–40 15.00 0–55 0.678 S. mutans (gene copy number/1 ml) 316.00 70.0–13200.0 872.50 227.0–625000.0 < 0.001* Statherin (ng/ml) 144.69 25.93–320.62 149.44 37.77–292.46 0.778 Cathelicidin LL-37 (ng/ml) 23.90 3.42–85.83 39.97 7.40–115.31 0.024* Open in a new tab Fig. 2. Open in a new tab The levels of antimicrobial peptides. A The levels of statherin in the groups without ECC and with ECC ( p = 0.778), ( B ) The levels of cathelicidin LL-37 in the groups without and with ECC ( p = 0.024). Whiskers show minimum and maximum values; boxes represent 25–75% of the data ranges; horizontal lines within boxes are medians Fig. 3. Open in a new tab DNA copy numbers of S. mutans in the groups without and with ECC ( p < 0.05). Whiskers show minimum and maximum values; boxes represent 25–75% of the data ranges; horizontal lines within boxes are medians The results of the statistical analysis showed a positive correlation between the dmfs and age ( p = 0.022; Table 3 ). Although there was a statistically significant and positive correlation between the dmfs scores and S. mutans and cathelicidin LL-37 levels, no statistically significant relationship was observed between the dmfs scores and statherin protein levels ( p < 0.0005, p = 0.012, p = 0.834, respectively; Table 3 ). There was no statistically significant correlation between the levels of S. mutans, cathelicidin LL-37, and statherin ( p = 0.237, p = 0.829, respectively; Table 3 ). On the other hand, a statistically significant difference and positive correlation were seen between the frequency of sugar-containing food or beverage consumption > 3 times a day between meals and the levels of S. mutans ( p < 0.001; Table 4 ). Table 3. Correlation coefficients and p values (r,p) were obtained from salivary parameters, dmfs, and age analysis Parameter Statherin LL-37 dmfs Age S.Mutans 0.027, 0.829 0.145, 0.237 0.511*, <0.001 0.133, 0.281 Statherin - 0.671*, <0.001 −0.026, 0.834 −0.161, 0.191 LL-37 - 0.303*, 0.012 0.166, 0.177 dmfs - 0.277*, 0.022 Open in a new tab *p value <0.005, significant correlation Table 4. S. mutans and the frequency of the consumption of sugar-containing foods or beverages >3 times a day between meals relationship Variable Salivary S. mutans Median (min-max) p- value Frequency of the consumption of sugar-containing foods and/or beverages between meals >3 times 5440.0 (208.0–625000.0.0.0) <0.001* <3 times 419.0 (70.9–14300.0.9.0) Open in a new tab *p value < 0.001, Significant difference The univariable logistic regression analysis determined that the consumption of sugar-containing foods or beverages more than 3 times a day, increased salivary S. mutans and LL-37 levels were caries risk predictors (OR: 3.43, 1.89, 1.03, respectively; Table 5 ). The multivariable analysis demonstrated that higher salivary Streptococcus mutans levels were associated with an 83% increased risk of dental caries (OR: 1.83). In contrast, salivary LL-37 levels showed only a weak association with caries risk, whereas salivary statherin levels exhibited a slight inverse association with the presence of dental caries. Table 5. Univariable and multivariable logistic regression analysis of potential caries risk predictors VARIABLE CRUDE ADJUSTED OR 95% CI OR 95% CI Lower Upper Lower Upper Frequency of sugar-containing foods or beverages consumption >3 times 3.43 1.18 9.99 - - - Salivary S. Mutans * 1.89 1.27 2.82 1.83 1.19 2.81 Statherin 0.99 0.99 1.01 0.983 0.970 0.996 Cathelicidin LL-37 1.03 1.01 1.05 1.061 1.020 1.104 Open in a new tab OR Odds ratio, 95% CI 95% confidence interval *Ln transformation was performed because the range was too wide for salivary S. mutans Discussion The oral cavity hosts a complex microbial community with Streptococcus mutans being a primary etiological agent in the development of dental caries [ 4 ]. Therefore, controlling the levels of S. mutans is a strategic approach to mitigating the occurrence of dental caries. However, the natural protective mechanisms of biofilms and the risk of antimicrobial resistance pose a challenge to the controlling of S. mutans levels. As a result, interest in endogenous AMPs such as statherin and LL-37 has been piqued due to their potential roles in the modulation of oral microbiota. The results of the present study have demonstrated that children with caries had elevated S. mutans and cathelicidin LL-37 levels while statherin levels were similar between study groups. Moreover, the dmfs scores S. mutans , and cathelicidin LL-37 levels were significantly correlated. On the other hand, there was no significant association between the dmfs scores and statherin levels. The presence of elevated salivary LL-37 and its link with the dmfs scores and S. mutans might suggest its significant role in caries susceptibility. Some researchers did not find any statistically significant relationship between the presence of caries and age [ 23 – 26 ]. The findings of our study indicated that the incidence of caries was similar across the 4-, 5-, and 6-year-old age groups, and there was not a link between the two. However, a positive correlation was found between age and dmfs scores. Hallett and O’rourke [ 27 ] found that ECC was more common among 5-year-old children compared with 4-year-old ones and age and ECC were statistically and significantly associated [ 28 , 29 ]. Some researchers proved a strong relationship between high-frequency sugar intake and ECC in their studies [ 30 ]. Some studies showed a positive correlation between the frequency of consumption of sugar-containing foods or beverages and S. mutans levels in saliva [ 25 ]. In the present study, we observed that the frequency of consumption of sugar-containing foods or beverages > 3 times a day between meals in caries-active children was higher. Also, it was verified that S. mutans levels in the saliva of the children who consumed sugar-containing foods or beverages > 3 times a day between meals were higher than those who consumed them less frequently. When the relationship between oral hygiene habits and ECC was evaluated, according to the study conducted by Rosenblatt & Zarzar [ 29 ], caries were detected at a higher rate in children who did not brush their teeth. Fan et al. [ 23 ] found a statistically significant relationship between the frequency of tooth brushing and the presence of caries. Colombo et al. [ 31 ] did not observe a statistically significant relationship between the caries-free, ECC, and S-ECC groups in terms of daily tooth brushing frequency. In our study, no significant relationship was found between the presence of caries and the frequency of daily tooth brushing. We consider this to be a result of ineffective and insufficient tooth brushing durations. Colombo et al. [ 31 ] evaluated the initiation of tooth brushing with the eruption of the first tooth in children in the caries-free, ECC, and S-ECC groups and did not detect a significant difference between the groups. Similar results were obtained in our study. Hallet & O’Rourke [ 27 ], found a lower rate of ECC in children who started tooth brushing before 12 months of age compared with children who started tooth brushing after 12 months of age. In our study, when parents’ help with brushing was evaluated, no significant difference was detected between the groups. Oral bacteria are known to be responsible for the initiation and progression of caries. In a study, a positive correlation was found between the severity of caries and the salivary levels of S. mutans , and the relationship was found to be substantial [ 32 ]. Our study had similar results proving that salivary levels of S. mutans were considerably higher in caries-active children. At the same time, a significant positive correlation was found between salivary levels of S. mutans and dmfs values. The present findings are evidence of the role of S. mutans as a marker for caries. Control of microbial dental plaque is of primary importance in the prevention and treatment of dental caries. Controlling the levels of S. mutans is therefore a strategic approach to mitigating the occurrence of dental caries. This approach is made challenging by the natural protective mechanisms of biofilms and the risk of antimicrobial resistance. As a result, interest in endogenous antimicrobial AMPs such as statherin and LL-37 has been piqued due to their potential roles in the modulation of oral microbiota. Studies investigated the association between salivary LL37 and dental caries in children with controversial results. The role of antimicrobial peptides that make up the salivary defense system in the assessment of caries risk is currently a very interesting topic. found that salivary cathelicidin LL-37 peptide levels were lower in children with caries. Colombo et al. [ 8 ] evaluated cathelicidin LL-37 levels in the unstimulated saliva of children aged 3–5 years and detected a positive correlation between dmfs scores and cathelicidin LL-37 values. Similarly, in our study, a positive correlation was found between the dmfs scores and cathelicidin LL-37 peptide concentration. Additionally, a significantly higher LL-37 peptide amount was measured in the caries-active group. Malcolm et al. [ 33 ] found a positive correlation between increased S. mutans levels in dental plaque and increased cathelicidin LL-37 peptide levels in saliva. Colombo et al. [ 8 ] on the other hand, could not detect any correlation between salivary levels of S. mutans and cathelicidin LL-37 peptide in their study. Similar results were obtained in the present study. Although these results show that the cathelicidin LL-37 peptide may cause increased susceptibility to caries, a comparative evaluation with the contradictory results in the literature will bring forth a need for further research. In a study by Vitorino et al. [ 14 ], significant correlation was spotted between the increase in saliva statherin values and the decrease in dmfs scores. As Wang et al. [ 15 ] noted, statherin level was higher in the dental caries free group compared to both low and high caries prevalance groups. Similarly, Bakkal and Kargul [ 16 ] reported a higher rate of statherin protein bands in children without caries. In the present study, salivary levels of statherin were found similar between groups with and without caries, and no correlation was found between dmfs scores and salivary statherin levels but in logistic regression analysis, we found that statherin was protective against caries. We consider further studies necessary to clarify the role of statherin levels as a biomarker in the determination of caries risk. Shimoyotodome et al. [ 17 ] stated that statherin competitively inhibits the adsorption of salivary high molecular weight glycoprotein in acquired enamel pellicle. Thus, S. mutans reduced its adhesion to hydroxyapatite surfaces in the tooth structure. Rudney et al. [ 18 ] suggested an inverse connection between salivary levels of statherin peptide, occlusal caries, and total streptococcal levels along with an inhibition of bacterial adhesion by statherin peptide. The present study does not contain a considerable link between salivary levels of S. mutans and statherin peptide. Hence, due to the shortage of currently available studies in the literature on the relationship between statherin peptide and S. mutans in the saliva and existing contradictory results, the conduct of supplementary studies on the field is suggested. This study primarily focused on the cathelicidin LL-37 peptide. However, previous studies have demonstrated that histatins play a protective role against acid-induced damage when adsorbed onto the enamel surface. The absence of histatin level evaluation represents a limitation of the present study. Future research including histatin and other biologically relevant salivary proteins may provide a more comprehensive understanding of caries development. Another limitation of this study is the relatively small sample size. Studies with larger cohorts are expected to yield more robust results. In addition, functional characteristics of LL-37, salivary mineralization-related parameters, and behavioral factors such as the use of fluoridated versus non-fluoridated toothpaste were not assessed. Finally, considering the multifactorial nature of early childhood caries, potential recall bias related to parent-reported data should also be taken into account. Conclusions Early childhood caries (ECC) is one of the most common chronic diseases of childhood and represents a major concern for both parents and dental professionals. It is a biofilm-associated condition that affects a large proportion of the pediatric population and remains a significant public health problem in many countries. Cariogenic bacteria, diet, susceptible all together contribute to the occurrence of dental caries. Therefore, recent studies have focused on the evaluation of oral defense mechanisms for early diagnosis and prevention of ECC. The prediction of caries risk is of importance especially in childhood. Among the caries-related etiological factors analyzed in this study, consuming sugar-containing foods and beverages more than 3 times a day was associated with ECC. In addition, frequent intake of sugar via food and beverages causes an increase in S. mutans , which causes caries. Saliva is a fluid that can be used to measure and monitor caries risk due to its easy noninvasive collection. Based on our study design, we cannot yet determine whether salivary cathelicidin levels are a predictor of future caries and whether salivary statherin is a protective marker against caries, but we have shown that children with caries have higher levels of salivary cathelicidin LL-37. Last but not least, it is crucial to further a longer-term investigation for a better understand on whether these antimicrobial peptides in saliva can be used as caries risk predictors or therapeutic agent. Supplementary Information Supplementary Material 1 (2.2MB, pdf) Supplementary Material 2 (616.8KB, pdf) Acknowledgements We are thankful to Assoc. Dr. Timur Köse, Department of Biostatistics and Medical Informatics, Faculty of Medicine, Ege University, for an analyses of the data. Abbreviations AAPD The American Academy of Pediatric Dentistry DMFS Number of decayed missed and filled surfaces ECC Early childhood caries ELISA Enzyme-linked immunosorbent assay PCR Polymerase chain reaction S-ECC Severe early childhood caries Authors’ contributions CP, NE and GE designed the research study. CP, NE, GÖ, CV, Cansu V, AG, HÇ and YG performed the research. CV, Cansu V, AG, YG, HÇ analyzed the data. CP, GE, GÖ, CV, AG, YG, HÇ wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. Funding This research was funded by Ege University Scientific Research Projects Coordination Unit (Project number: 21020). Data availability All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author. Declarations Ethics approval and consent to participate This study was conducted in accordance with the ethical principles of the Declaration of Helsinki and its later amendments. Ethical approval of the study was obtained from the Medical Research Ethics Committee of the Faculty of Medicine, Ege University (Decision number: 19-8.1T/46, date of approval:21 August 2019). Written informed consent was obtained from all participants (and from parents or legal guardians when applicable). Consent for publication No identifiable personal data, images, or details of individual participants are included in this manuscript. Competing interests The authors declare no competing interests. Footnotes Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Supplementary Materials Supplementary Material 1 (2.2MB, pdf) Supplementary Material 2 (616.8KB, pdf) Data Availability Statement All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author. Articles from BMC Oral Health are provided here courtesy of BMC ACTIONS View on publisher site PDF (1.1 MB) Cite Collections Permalink RESOURCES Similar articles Cited by other articles Links to NCBI Databases Follow NCBI NCBI on X (formerly known as Twitter) NCBI on Facebook NCBI on LinkedIn NCBI on GitHub NCBI RSS feed Connect with NLM NLM on X (formerly known as Twitter) NLM on Facebook NLM on YouTube National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894 Web Policies FOIA HHS Vulnerability Disclosure Help Accessibility Careers NLM NIH HHS USA.gov Back to Top

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