Dental Caries Etiopathogenesis: Microbial Composition, Functional Activity and Host Recognition

For decades, the sugar-fermenting, acidogenic species Streptococcus mutans has been considered the main causative agent of dental caries and most diagnostic and therapeutic strategies have been targeted toward this microorganism. However, the DNA- and RNA-based studies from carious lesions reported in this thesis, have uncovered an extraordinarily diverse ecosystem where S. mutans accounts only for a tiny fraction of the bacterial community. This supports the concept that consortia formed by multiple microorganisms act collectively, probably synergistically, to initiate and expand the carious lesion. The data also show that these microbial consortia are different between individuals, between the affected tissue, and even between different lesions from the same individual. Thus, antimicrobial therapies are not expected to be effective in the treatment of caries and other polymicrobial diseases that do not follow Koch's postulates, and that I propose cannot be considered infectious diseases in classical terms. In addition, the data also indicate a prominent role for the immune system in caries risk, suggesting that therapies directed towards stimulating immunological competence should be explored. Based on the results from this Thesis, I propose that dental caries is a dysbiotic polymicrobial disease caused by pathobionts

Revealing microbial recognition by specific antibodies

Background: Recognition of microorganisms by antibodies is a vital component of the human immune response. However, there is currently very limited understanding of immune recognition of 50 % of the human microbiome which is made up of as yet un-culturable bacteria. We have combined the use of flow cytometry and pyrosequencing to describe the microbial composition of human samples, and its interaction with the immune system. Results: We show the power of the technique in human faecal, saliva, oral biofilm and breast milk samples, labeled with fluorescent anti-IgG or anti-IgA antibodies. Using Fluorescence-Activated Cell Sorting (FACS), bacterial cells were separated depending on whether they are coated with IgA or IgG antibodies. Each bacterial population was PCR-amplified and pyrosequenced, characterizing the microorganisms which evade the immune system and those which were recognized by each immunoglobulin. Conclusions: The application of the technique to healthy and diseased individuals may unravel the contribution of the immune response to microbial infections and polymicrobial diseases

Impact of the repurposed drug thonzonium bromide on host oral-gut microbiomes

Drug repurposing is a feasible strategy for the development of novel therapeutic applications. However, its potential use for oral treatments and impact on host microbiota remain underexplored. Here, we assessed the influences of topical oral applications of a repurposed FDA-approved drug, thonzonium bromide, on gastrointestinal microbiomes and host tissues in a rat model of dental caries designed to reduce cross-contamination associated with coprophagy. Using this model, we recapitulated the body site microbiota that mirrored the human microbiome profile. Oral microbiota was perturbed by the treatments with specific disruption of Rothia and Veillonella without affecting the global composition of the fecal microbiome. However, disturbances in the oral-gut microbial interactions were identified using nestedness and machine learning, showing increased sharing of oral taxon Sutterella in the gut microbiota. Host-tissue analyses revealed caries reduction on teeth by thonzonium bromide without cytotoxic effects, indicating bioactivity and biocompatibility when used orally. Altogether, we demonstrate how an oral treatment using a repurposed drug causes localized microbial disturbances and therapeutic effects while promoting turnover of specific oral species in the lower gut in vivo

Problem Solving Skills Development in Mathematics Teaching

Diplomdarbā „Problēmrisināšanas prasmju attīstīšana matemātikas mācību procesā” tiek noskaidrota skolēnu pieredze problēmu risināšanā, un, domājot par mācību procesa kvalitātes celšanu, tiek izstrādāts skolotāju atbalsta materiāls skolēnu problēmrisināšanas prasmju attīstīšanai matemātikas stundās pamatskolas 7. – 9. klasēs. Darbs sastāv no trīs nodaļām: 1. nodaļā tie apzināti mācību mērķi matemātikā pamatskolā; 2. nodaļā tiek noskaidrota skolēnu pieredze problēmu risināšanā; 3. nodaļā tiek piedāvāti ieteikumi problēmrisināšanas prasmju attīstīšanai. Atslēgvārdi: problēmrisināšanas prasmes, problēmrisināšanas stratēģijas, pētnieciskā mācīšanās, aktīvās mācību metodesThe Diploma Paper “Problem Solving Skills Development in Mathematics Teaching” outlines students' experience in solving of problems. Furthermore, it presents supplementary teachers' material to encourage development of students' problem solving skills via teaching of mathematics. This material is aimed at improving the quality of the teaching process and is intended for Grades 7 – 9 of elementary schools. The Paper consists of three chapters: Chapter I identifies learning objectives in mathematics in elementary schools; Chapter II deals with students' experience in solving problems, while Chapter III contains recommendations for developing of problem solving skills. Keywords: problem solving skills, problem solving strategies, investigative learning, active learning method

Precision targeting of bacterial pathogen via bi-functional nanozyme activated by biofilm microenvironment

Human dental caries is an intractable biofilm-associated disease caused by microbial interactions and dietary sugars on the host's teeth. Commensal bacteria help control opportunistic pathogens via bioactive products such as hydrogen peroxide (H2O2). However, high-sugar consumption disrupts homeostasis and promotes pathogen accumulation in acidic biofilms that cause tooth-decay. Here, we exploit the pathological (sugar-rich/acidic) conditions using a nanohybrid system to increase intrinsic H2O2 production and trigger pH-dependent reactive oxygen species (ROS) generation for efficient biofilm virulence targeting. The nanohybrid contains glucose-oxidase that catalyzes glucose present in biofilms to increase intrinsic H2O2, which is converted by iron oxide nanoparticles with peroxidase-like activity into ROS in acidic pH. Notably, it selectively kills Streptococcus mutans (pathogen) without affecting Streptococcus oralis (commensal) via preferential pathogen-binding and in situ ROS generation. Furthermore, nanohybrid treatments potently reduced dental caries in a rodent model. Compared to chlorhexidine (positive-control), which disrupted oral microbiota diversity, the nanohybrid had significant higher efficacy without affecting soft-tissues and the oral-gastrointestinal microbiomes, while modulating dental health-associated microbial activity in vivo. The data reveal therapeutic precision of a bi-functional hybrid nanozyme against a biofilm-related disease in a controlled-manner activated by pathological conditions.National Institutes of Health/ National Institute of Dental and Craniofacial Researc

Evaluation of possible biomarkers for caries risk in children 6 to 12 years of age

Background: Electrolytes, proteins, and other salivary molecules play an important role in tooth integrity and can serve as biomarkers associated with caries. Objective: To determine the concentration of potential biomarkers in children without caries (CF) and children with caries (CA). Methods: Unstimulated saliva was collected, and the biomarkers quantified in duplicate, using commercial Enzyme Linked Immunosorbent Assay (ELISA) kits to determine IgA, fibronectin, cathelicidin LL-37, and statherin levels, as well as colorimetric tests to detect formate and phosphate. Results: Significantly higher concentrations of statherin was detected in the CF group (Median: 94,734.6; IQR: 92,934.6-95,113.7) compared to the CA2 group (90,875.0; IQR: 83,580.2-94,633.4) (p = 0.03). Slightly higher median IgA (48,250.0; IQR: 31,461.9-67,418.8) and LL-37 levels (56.1; IQR 43.6-116.2) and a lower concentration of formate were detected in the CF group (0.02; IQR 0.0034-0.15) compared to the group with caries (IgA: 37,776.42; IQR: 33,383.9-44,128.5; LL-37: 46.3; IQR: 40.1011-67.7; formate: 0.10; IQR: 0.01-0.18), but these differences were not statistically significant. Conclusion: The fact that these compounds have been identified as good markers for caries among European adults highlights the difficulty of identifying universal biomarkers that are applicable to all ages or to different populations

In situ substrate-formed biofilms using IDODS mimic supragingival tooth-formed biofilms

This study aimed to compare the bacterial viability and diversity of a substrate-formed biofilm (SF-biofilm) in situ to a supragingival tooth-formed biofilm (TF-biofilm) in the same group of individuals. The impact of the device/disc position and toothbrushing during the formation of SF-biofilm was also assessed. Two tests were run. In test 1, 15 volunteers wore two hemi-splints carrying six discs of human enamel, glass, and hydroxyapatite for 2 days, and were instructed to not perform any oral hygiene measure. Biofilm samples were collected from the substrates and the contralateral tooth and were analysed using CLSM. In five volunteers, half of the biofilm present on the discs and their contralateral teeth were scraped and analysed using 16S  pyrosequencing. In test 2, the microscopic analysis was repeated only on the SF-biofilm samples, and the volunteers were allowed to brush their teeth. Multivariate analyses revealed that the donors had a significant effect on the composition of the biofilm, confirming its subject-dependent character. The bacterial composition of the SF-biofilm was similar to the TF-biofilm, with significant differential abundance detected in very few taxa of low abundance. The toothbrushing during the formation of SF-biofilm was the only factor that conditioned the thickness or bacterial viability