Influence of Streptococcus mutans on matrix construction during the development of microcosm biofilms exposed to nutritional challenge

Author(s)

Elkin Jahir Florez Salamanca

• Document type: Doctoral Thesis
• Press: Araraquara. 2021-07-13.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Odontologia. Araraquara
• Defense date: 2021-03-30
• Advisor: Marlise Inêz Klein Furlan

Abstract

Dental caries is the most prevalent chronic disease in the world. Its pathophysiology is associated with factors such as biofilms (microorganisms and extracellular matrix), carbohydrate-rich diet, dental tissues, and interaction time. Although many laboratory models have simplified the study of caries by focusing only on understanding specific microorganisms, mimicking the oral cavity to control the variables that influence caries is still one of the greatest challenges. The study aimed to evaluate longitudinally the importance of Streptococcus mutans constructing the extracellular matrix and structuring polymicrobial biofilms associated with dental caries. Microcosm biofilms were developed to assess the dynamics of these communities. For this purpose, the human salivary microbiota was inoculated on bovine enamel discs in four biofilm models in vitro. The following were assessed: pH culture media; microhardness and roughness of enamel discs; total biomass and population dynamics (via CFU mL-1) of biofilms. Among the models studied, model 3 (M3) - 'Three meals a day' presented characteristics closest to the oral cavity since the pH values remained close to neutral, subtle and progressive enamel demineralization, did not favor specific microbial groups, and allowed the accumulation of biofilm, which was evidenced by the increase in biomass and total microbiota. M3 was used to test two single (UNI) and multiple (MULTI) inoculation strategies, the second simulating the secondary colonization of microorganisms. The following were assessed: pH of the culture media; biofilms (architecture; microbial population dynamics via CFU mL-1, DNA and qPCR sequencing; insoluble biomass and matrix components); and enamel demineralization (via microhardness). The fluctuations observed in the pH were similar to those reported in the oral cavity; there was acidification after exposure to carbohydrates, and the buffering action of the saliva was simulated by returning biofilms to a saliva culture broth with a neutral pH. The spatial configuration was similar to that found in oral biofilms associated with caries, represented by hedgehogs and corn cobs architectures. The representativeness of streptococci from the mutans group was ≤ 2% of the total cultivable microbiota. The genus Streptococcus was the most abundant; however, S. mutans was not detected in any sample via sequencing. In the MULTI strategy, a greater microbial diversity was detected. When quantifying the number of bacterial cells via qPCR, it was observed that S. mutans represents <0.01% of saliva and <0.005% in biofilms. As the biofilm aged, there was an increase in biomass, exopolysaccharides, and extracellular DNA in the extracellular matrix in both inoculation strategies. The accumulation of these components is associated with a cohesive structure that can favor the formation of acidic niches. The maturation of the biofilm and enamel demineralization occurred independently of the low proportion of S. mutans and the inoculation strategy, reducing the role of specific species in the multifactorial disease. (AU)

• FAPESP's process: 17/26623-5 - Influence of Streptococcus mutans on the matrix assembly during the development of microcosm biofilms exposed to nutritional challenge
• Grantee: Elkin Jahir Florez Salamanca
• Support Opportunities: Scholarships in Brazil - Doctorate