Modified Lactoperoxidase System as a Promising Anticaries Agent: In Vitro Studies on Streptococcus mutans Biofilms.

The lactoperoxidase (LPO) system shows promise in the prevention of dental caries, a common chronic disease. This system has antimicrobial properties and is part of the non-specific antimicrobial immune system. Understanding the efficacy of the LPO system in the fight against biofilms could provide information on alternative strategies for the prevention and treatment of caries. In this study, the enzymatic system was modified using four different (pseudo)halide substrates (thiocyanate, thiocyanate-iodide mixture, selenocyanate, and iodide). The study evaluated the metabolic effects of applying such modifications to Streptococcus mutans; in particular: (1) biofilm formation, (2) synthesis of insoluble polysaccharides, (3) lactate synthesis, (4) glucose and sucrose consumption, (5) intracellular NAD+ and NADH concentrations, and (6) transmembrane glucose transport efficiency (PTS activity). The results showed that the LPO-iodide system had the strongest inhibitory effect on biofilm growth and lactate synthesis (complete inhibition). This was associated with an increase in the NAD+/NADH ratio and an inhibition of glucose PTS activity. The LPO-selenocyanate system showed a moderate inhibitory effect on biofilm biomass growth and lactate synthesis. The other systems showed relatively small inhibition of lactate synthesis and glucose PTS but no effect on the growth of biofilm biomass. This study provides a basis for further research on the use of alternative substrates with the LPO system, particularly the LPO-iodide system, in the prevention and control of biofilm-related diseases.

Safety Assessment of the Modified Lactoperoxidase System-In Vitro Studies on Human Gingival Fibroblasts.

One strategy in caries prevention is to inhibit the formation of cariogenic biofilms. Attempts are being made to develop oral hygiene products enriched with various antimicrobial agents. One of them is lactoperoxidase-an enzyme that can oxidise (pseudo)halide ions to reactive products with antimicrobial activity. Currently, commercially available products utilise thiocyanate as a substrate; however, several alternatives that are oxidised to products with greater antimicrobial potential have been found. In this study, toxicity against human gingival fibroblasts of the lactoperoxidase system was evaluated using four different (pseudo)halide substrate systems-thiocyanate, iodide, selenocyanate, and a mixture of thiocyanate and iodide. For this purpose, cells were treated with the systems and then apoptosis, cell cycle, intracellular glutathione concentration, and mitochondrial superoxide production were assessed. The results showed that each system, after generating 250 µM of the product, inhibited cell divisions, increased apoptosis, and increased the percentage of dead cells. It was concluded that the mechanism of the observed phenomena was not related to increased superoxide production or the depletion of glutathione concentration. These findings emphasised the need for the further in vitro and in vivo toxicity investigation of the modified lactoperoxidase system to assess its safety and the possibility of use in oral hygiene products.

Effect of a Short-Term Intervention with Lactobacillus salivarius Probiotic on Early Childhood Caries-An Open Label Randomized Controlled Trial.

ECC is a significant therapeutic and social problem and a global burden on public health. The aim of this clinical trial was to test whether a 2-week daily consumption of chewing tablets containing thermally inactivated L. salivarius reduces the 12-month caries increment compared to the control group. The investigation was a single-center, randomized, controlled open-label, blinded end-point evaluation trial in two parallel groups. At baseline, 140 generally healthy children between 3 and 6 years of age with or without ECC were randomly assigned to the probiotic test group (n = 70) or to the treatment as the usual control group (n = 70). The primary outcome measure was the 1-year increment in caries incidence and prevalence. Secondary endpoints assessed were the initial, cavitated and obvious dentinal caries increment as well as the measurement of dental plaque accumulation, as an indicator of the ECC risk. Data were collected through the clinical assessment of the children's caries (dmft and ICDAS II) and oral hygiene status (DI-S of OHI-S index). Caries incidence and prevalence were statistically significantly lower in the probiotic group versus the control group (p < 0.001 and p = 0.0075). The initial and final mean OHI-S scores in the probiotic group did not show any significant differences. In conclusion, the regular short-term intake of probiotics may reduce caries development. Our findings suggest that self-administered probiotic therapy may provide a good complement to increase the effectiveness of individual preventive home care in preschool children. This is the first clinical study evaluating the effect of a short-term probiotic intervention on reducing early childhood caries with 12 months of follow-up.

Differences in Sweet Taste Perception and Its Association with the Streptococcus mutans Cariogenic Profile in Preschool Children with Caries.

The aim of the study was to verify the hypothesis about differences in sweet taste perception in the group of preschool children with and without caries, and to determine its relationship with cariogenic microbiota and the frequency of sweets consumption in children. The study group included of 63 children aged 2-6 years: 32 with caries and 31 without caries. The study consisted of collecting questionnaire data and assessment of dental status using the decayed, missing, filled in primary teeth index (dmft) and the International Caries Detection and Assessment System (ICDAS II). The evaluation of sweet taste perception was carried out using a specific method that simultaneously assessed the level of taste preferences and the sensitivity threshold for a given taste. The microbiological analysis consisted of the assessment of the quantitative and qualitative compositions of the oral microbiota of the examined children. The sweet taste perception of children with caries was characterized by a lower susceptibility to sucrose (the preferred sucrose solution concentration was >4 g/L) compared to children without caries (in the range ≤ 4 g/L, p = 0.0015, chi-square test). A similar relationship was also observed for frequent snacking between meals (p = 0.0038, chi-square test). The analysis of studied variables showed the existence of a strong positive correlation between the perception of sweet taste and the occurrence and intensity of the cariogenic process (p = 0.007 for dmft; and p = 0.012 for ICDAS II), as well as the frequency of consuming sweets (p ≤ 0.001 for frequent and repeated consumption of sweets during the day, Spearman test) in children with caries. Additionally, children with an elevated sucrose taste threshold were more than 10-times more likely to develop S. mutans presence (OR = 10.21; 95% CI 3.11-33.44). The results of this study suggest the future use of taste preferences in children as a diagnostic tool for the early detection of increased susceptibility to caries through microbial dysbiosis towards specific species of microorganisms.

Effect of Polyols and Selected Dental Materials on the Ability to Create a Cariogenic Biofilm-On Children Caries-Associated Streptococcus Mutans Isolates.

Secondary caries is a disease associated with the formation of biofilm on the border of the tooth and dental filling. Its development is strongly influenced by the dietary sweet foods and the type of dental material. The aim of the study was to assess the effect of sweeteners on the ability of clinical Streptococcus mutans strains to form biofilm on dental materials. Strains were isolated from plaque samples from 40 pediatric patients from the 3-6 ICADS II group. The ability to form biofilm was tested on composite and glass ionomer dental materials used for milk teeth filling in the presence of sucrose, xylitol, sorbitol, and erythritol. The bacterial film mass after 12, 24, 48, and 72 h and the number of bacterial colonies significantly decreased (p < 0.01) compared to the initial value for 5% erythritol and sorbitol on examined materials. A greater inhibitory effect was noted for glass ionomers compared to composites. Sucrose and xylitol supported biofilm formation, while erythritol had the best inhibitory effect. The use of fluoride-releasing glass ionomers exerted an effect synergistic to erythritol, i.e., inhibited plaque formation and the amount of cariogenic S. mutans. Selection of proper type of dental material together with replacing sucrose with polyols can significantly decrease risk of secondary caries development. Erithritol in combination with glass ionomer seems to be the most effective in secondary caries prevention.

The Relationship between the Concentration of Salivary Tyrosine and Antioxidants in Patients with Oral Lichen Planus.

The diagnosis of oral lichen planus (OLP) is based on clinical examination and histopathological criteria. Noninvasive diagnostics of saliva may be considered as a confirmation of OLP diagnosis and a potential alternative to an invasive method. The objective of the present study was to evaluate the relationship between the level of tyrosine (Tyr) as well as antioxidants like uric acid (UA) and glutathione peroxidase (GPx) activity in the saliva of patients with OLP in comparison with the control group (healthy subjects without any oral changes). A total of 40 patients with OLP and 40 healthy volunteers were selected for the study based on the modified WHO diagnostic (clinical and histopathological) criteria. High-performance liquid chromatography (HPLC) was performed for Tyr concentration, while GPx activity and uric acid levels were determined by a colorimetric method. The concentrations of Tyr, UA, and GPx activity were statistically lowered in OLP patients compared to the control group. All examined parameters correlated strongly and positively with each other. Mean values of salivary UA concentrations differed between the groups of OLP patients (reticular and erosive forms) and controls (206.66 vs. 196.54 vs. 218.49 µmol/L, respectively, p = 0.001). A similar trend was demonstrated in salivary Tyr concentration which differed statistically between the study and control groups (0.08 vs. 0.07 vs. 0.13 µmol/L, respectively, p = 0.001). Determining of a relationship between the concentrations of Tyr, UA, and GPx activity may be useful in the prognosis of OLP. The HPLC method may be employed, as an additional noninvasive diagnostic procedure to screen OLP patients, during the routine diagnostics of salivary biochemical parameters such as aromatic amino acids.

Biomarkers of inflammatory external root resorption as a result of traumatic dental injury to permanent teeth in children.

OBJECTIVE: External inflammatory root resorption (EIRR) is a common complication of traumatic dental injury (TDI) that can be detected radiologically. During EIRR, various proteins are released into gingival sulcus fluid (GCF). The aim of the study was to monitor the levels of selected proteins in GCF in children (8-16 years of age) in order to assess their utility in the early diagnosis of EIRR. DESIGN: Twenty five children who experienced TDI to permanent incisors with ended root development were enrolled. GCF was collected from injured and control teeth with paper strips within seven days after TDI and on three visits during six-month follow-up. Concentrations of IL-1α, IL-1ß, IL-6, IL-8, TNFα, RANKL and MMP-9 in GCF were measured using enzyme-linked immunosorbent assays. EIRR was confirmed by radiological imaging techniques. RESULTS: Of all analyzed proteins, only the levels of IL-1α, Il-1ß and TNFα in GCF from the injured teeth with resorption were higher than in GCF from control teeth on the visit during which the EIRR was diagnosed. In univariate logistic regression model, the concentration of IL-1α in GCF was found as the strongest risk factor for the occurrence of EIRR. CONCLUSIONS: The composition of GCF may be indicative of EIRR after TDI. The monitoring of selected biomarkers in GCF may help to detect EIRR at its early stage and might be useful in reducing radiological exposure in children after TDI. IL-1α can be considered as a potential marker of the EIRR in children after TDI to the permanent teeth.

Effect of a Lactobacillus Salivarius Probiotic on a Double-Species Streptococcus Mutans and Candida Albicans Caries Biofilm.

The aim of the study was to evaluate the anti-cariogenic effects of Lactobacillus salivarius by reducing pathogenic species and biofilm mass in a double-species biofilm model. Coexistence of S. mutans with C. albicans can cause dental caries progression or recurrence of the disease in the future. Fifty-nine children with diagnosed early childhood caries (ECC) were recruited onto the study. The condition of the children's dentition was defined according to the World Health Organization guidelines. The participants were divided into children with initial enamel demineralization and children showing dentin damage. The study was performed on the S. mutans and C. albicans clinical strains, isolated from dental plaque of patients with ECC. The effect of a probiotic containing Lactobacillus salivarius on the ability of S. mutans and C. albicans to produce a double-species biofilm was investigated in an in vitro model. The biomass of the formed/non-degraded biofilm was analyzed on the basis of its crystal violet staining. The number of colonies of S. mutans and C. albicans (CFU/mL, colony forming units/mL) forming the biofilm was determined. Microorganism morphology in the biofilm was evaluated using a scanning electron microscope (SEM). In vitro analysis demonstrated that the presence of S. mutans increased the number of C. albicans colonies (CFU/mL); the double-species biofilm mass and hyphal forms produced in it by the yeast. L. salivarius inhibited the cariogenic biofilm formation of C. albicans and S. mutans. Under the influence of the probiotic; the biofilm mass and the number of S. mutans; C. albicans and S. mutans with C. albicans colonies in the biofilm was decreased. Moreover; it can be noted that after the addition of the probiotic; fungi did not form hyphae or germ tubes of pathogenic potential. These results suggest that L. salivarius can secrete intermediates capable of inhibiting the formation of cariogenic S. mutans and C. albicans biofilm; and may inhibit fungal morphological transformation and thereby reduce the pathogenicity of C. albicans; weakening its pathogenic potential. Further research is required to prove or disprove the long-term effects of the preparation and to achieve preventive methods.

Methods of Biotyping of Streptococcus mutans Species with the Routine Test as a Prognostic Value in Early Childhood Caries.

PURPOSE: In order to investigate the suitability of Streptococcus mutans species biotyping by measuring the activity of selected enzymes from a commercial test, criteria were established for biotyping clinical strains from children with dental caries. In addition, the relationships between the selected biotypes, sensitivity to commonly used antibiotics, and early childhood caries were determined. METHODS: A total of 142 S. mutans isolates from dental plaque of children with caries were divided into different biotypes. Patients were divided into two groups: noncavitated (1-2 in ICDAS) and cavitated (5-6 in ICDAS) lesions. Biotyping criteria were determined based on both the arbitrary method and the clusterization method. The susceptibility of the strains to amoxicillin, cefazolin, erythromycin, and teicoplanin was studied by diluting a solid medium. RESULTS: Biotype I was the most common. Mean MIC values showed that the strains belonging to biotypes II and IV were the most sensitive to amoxicillin. For predetermined biotypes, observed differences were dependent on the severity of dental caries. CONCLUSIONS: The proposed method of S. mutans strains biotyping is relatively quick and simple to use, provided the application of suitable biotyping criteria, and may contribute to the effective prevention of dental caries induced by S. mutans.

Relationship between Pyruvate Kinase Activity and Cariogenic Biofilm Formation in Streptococcus mutans Biotypes in Caries Patients.

Streptococcus mutans (MS) and its biotype I are the strains most frequently found in dental plaque of young children. Our results indicate that in children pyruvate kinase (PK) activity increases significantly in dental plaque, and this corresponds with caries progression. The MS strains isolated in this study or their main glycolytic metabolism connected with PK enzymes might be useful risk factors for studying the pathogenesis and target points of novel therapies for dental caries. The relationship between PK activity, cariogenic biofilm formation and selected biotypes occurrence was studied. S. mutans dental plaque samples were collected from supragingival plaque of individual deciduous molars in 143 subjects. PK activity was measured at different time points during biofilm formation. Patients were divided into two groups: initial stage decay, and extensive decay. Non-parametric analysis of variance and analysis of covariance were used to determine the connections between S. mutans levels, PK activity and dental caries biotypes. A total of 143 strains were derived from subjects with caries. Biotyping data showed that 62, 23, 50, and 8 strains were classified as biotypes I, II, III, IV, respectively. PK activity in biotypes I, II, and IV was significantly higher in comparison to that in biotype III. The correlation between the level of S. mutans in dental plaque and PK activity was both statistically significant (p < 0.05) and positive. The greater the level of S. mutans in the biofilm (colony count and total biomass), the higher the PK activity; similarly, a low bacterial count correlated with low PK activity.

A study on ß-defensin-2 and histatin-5 as a diagnostic marker of early childhood caries progression.

BACKGROUND: Recently, a continuous growth of interest has been observed in antimicrobial peptides (AMPs) in the light of an alarming increase in resistance of bacteria and fungi against antibiotics. AMPs are used as biomarkers in diagnosis and monitoring of oral cavity pathologies. Therefore, the determination of specific protein profiles in children diagnosed with early childhood caries (ECC) might be a basis for effective screening tests and specialized examinations which may enable progression of disease. METHODS: The objective of the studies was to determine the role of histatin-5 and ß-defensing-2 as a diagnostic marker of early childhood caries progression. In this work, results of concentration determination of two salivary proteins (histatin-5 and ß-defensin-2) were presented. In addition, bacterial profiles from dental plaque in various stages of ECC and control were marked. The assessment of alteration in the concentration of these two proteins in a study group of children with various stages of ECC and a control group consisting of children with no symptoms was performed by enzyme-linked immunosorbent assays. RESULTS: The statistical analysis showed a significant increase in the concentration of histatin-5 and ß-defensin-2 in the study group compared to the control group and correlated with the progression of the disease. CONCLUSIONS: The confirmation of concentration changes in these proteins during the progression of dental caries may discover valuable disease progression biomarkers.

Effect of histatin-5 and lysozyme on the ability of Streptococcus mutans to form biofilms in in vitro conditions.

INTRODUCTION: The mechanisms of adhesion to solid surfaces enable S. mutans to colonize oral cavities and form biofilms, which play an important role in caries development. Additional properties enabling the survival of S. mutans in the oral cavity include its ability to survive in acidic environments and specific interactions with other microorganisms inhabiting this ecosystem. AIM OF THE STUDY: The aim of this study was to determine the antibacterial activity of saliva histatin-5 (peptide) and lysozyme (protein) against S. mutans and L. rhamnosus, as representatives of physiological flora. MATERIALS AND METHODS: The study involved strains of physiological (L. rhamnosus) and cariogenic (S. mutans) flora isolated from one patient with diagnosed early caries of the deciduous teeth. RESULTS: It was proved that the presence of probiotic L. rhamnosus bacteria in the environment had a negative impact on the ability of S. mutans to produce biofilm. Moreover, the antibacterial activity of histatin-5 was confirmed, and it inhibited S. mutans growth at concentrations of 27.2 µg/ml and 54.4 µg/ml, both individually and in a mixture with lysozyme (in a total concentration of 54.4 µg/ml). CONCLUSIONS: The data obtained constitute a promising result due to their potential future application in the prevention and early diagnosis of caries.

A study on ß-defensin-2 and histatin-5 as a diagnostic marker of early childhood caries progression

BACKGROUND: Recently, a continuous growth of interest has been observed in antimicrobial peptides (AMPs) in the light of an alarming increase in resistance of bacteria and fungi against antibiotics. AMPs are used as biomarkers in diagnosis and monitoring of oral cavity pathologies. Therefore, the determination of specific protein profiles in children diagnosed with early childhood caries (ECC) might be a basis for effective screening tests and specialized examinations which may enable progression of disease. METHODS: The objective of the studies was to determine the role of histatin-5 and ß-defensing-2 as a diagnostic marker of early childhood caries progression. In this work, results of concentration determination of two salivary proteins (histatin-5 and ß-defensin-2) were presented. In addition, bacterial profiles from dental plaque in various stages of ECC and control were marked. The assessment of alteration in the concentration of these two proteins in a study group of children with various stages of ECC and a control group consisting of children with no symptoms was performed by enzyme-linked immunosorbent assays. RESULTS: The statistical analysis showed a significant increase in the concentration of histatin-5 and ß-defensin-2 in the study group compared to the control group and correlated with the progression of the disease. CONCLUSIONS: The confirmation of concentration changes in these proteins during the progression of dental caries may discover valuable disease progression biomarkers.

The usefulness of biotyping in the determination of selected pathogenicity determinants in Streptococcus mutans.

BACKGROUND: Streptococcus mutans is known to be a primary etiological factor of dental caries, a widespread and growing disease in Polish children. Recognition of novel features determining the pathogenicity of this pathogen may contribute to understanding the mechanisms of bacterial infections.The goal of the study was to determine the activity of prephenate dehydrogenase (PHD) and to illuminate the role of the enzyme in S. mutans pathogenicity. The strains were biotyped based on STREPTOtest 24 biochemical identification tests and the usefulness of biotyping in the determination of S. mutans pathogenicity determinants was examined. RESULTS: Out of ninety strains isolated from children with deciduous teeth fifty three were classified as S. mutans species. PDH activity was higher (21.69 U/mg on average) in the experimental group compared to the control group (5.74 U/mg on average) (P <0.001). Moreover, it was demonstrated that biotype I, established basing on the biochemical characterization of the strain, was predominant (58.5%) in oral cavity streptococcosis. Its dominance was determined by higher PDH activity compared to biotypes II and III (P = 0.0019). CONCLUSIONS: The usefulness of biotyping in the determination of Streptococcus mutans pathogenicity determinants was demonstrated. The obtained results allow for better differentiation of S. mutans species and thus may contribute to recognition of pathogenic bacteria transmission mechanisms and facilitate treatment.

Salivary proteins in health and disease.

Besides their structural catalytic and diverse regulatory functions, proteins are also precursors of many important biological compounds essential for normal functioning of humans. Many of these compounds may be used as markers for identification of specific pathological states. A comprehensive knowledge about the metabolism of salivary proteins and the mechanisms of action of their metabolites allowed the development of effective treatment for many disorders. However, it should not be forgotten that in some pathological conditions, these compounds not only could be involved in the pathogenesis but also could be used as tool in the prediction of many diseases. This paper is a review of the published literature on selected salivary proteins in the context of the physiological processes of the human body and chosen chronic disorders, such as diabetes, diabetic nephropathy, mucositis, oral mycoses and caries.