Abstract
To investigate the effects of ficin on biofilm formation of conditionally cariogenic Streptococcus mutans (S. mutans). Biomass and metabolic activity of biofilm were assessed using crystal violet assay, colony-forming unit (CFU) counting, and MTT assay. Extracellular polysaccharide (EPS) synthesis was displayed by SEM imaging, bacteria/EPS staining, and anthrone method while acid production was revealed by lactic acid assay. Growth curve and live/dead bacterial staining were conducted to monitor bacterial growth state in both planktonic and biofilm form. Total protein and extracellular proteins of S. mutans biofilm were analyzed by protein/bacterial staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), severally. qRT-PCR was conducted to detect acid production, acid tolerance, and biofilm formation associated genes. Crystal violet assay, CFU counting, and MTT assay showed that the suppression effect of ficin on S. mutans biofilm formation was concentration dependent. 4 mg/mL ficin had significant inhibitory effect on S. mutans biofilm formation including biomass, metabolic activity, EPS synthesis, and lactic acid production (p < 0.05). The growth curves from 0 mg/mL to 4 mg/mL ficin were aligned with each other. There was no significant difference among different ficin groups in terms of live/dead bacterial staining result (p > 0.05). Protein/bacterial staining outcome indicated that ficin inhibit both total protein and biofilm formation during the biofilm development. There were more relatively small molecular weight protein bands in extracellular proteins of 4 mg/mL ficin group when compared with the control. Generally, ficin could inhibit biofilm formation and reduce cariogenic virulence of S. mutans effectively in vitro; thus, it could be a potential anticaries agent.
Highlights
Dental caries, a biofilm relevant disease, is caused by destruction of mineralized tooth tissue due to acidic catabolites from the bacterial fermentation of sugars [1]
Extracellular polymeric substances produced by S. mutans that is composed of extracellular polysaccharides (EPS), extracellular DNA, proteins, and lipoteichoic acids provide crucial scaffold for biofilm and are essential for expression of cariogenic virulence [5]
The total culture volume was 200 μL, twofold serial dilutions of ficin range from 0 mg/mL to 16 mg/mL were prepared in 96-well plate and overnight culture of S. mutans was diluted to final concentration of 106 colony-forming unit (CFU)/mL. 0.12% chlorhexidine (CHX) was used as a positive control group
Summary
A biofilm relevant disease, is caused by destruction of mineralized tooth tissue due to acidic catabolites from the bacterial fermentation of sugars [1]. Streptococcus mutans (S. mutans), a major extracellular polymeric substances producer, is the most well-known biofilm forming bacteria in oral cavity and was reported as most associated microbe in the transition of oral flora from a healthy one to a cariogenic one [3] Extracellular polymeric substances produced by S. mutans that is composed of extracellular polysaccharides (EPS), extracellular DNA (eDNA), proteins, and lipoteichoic acids provide crucial scaffold for biofilm and are essential for expression of cariogenic virulence [5]. Biofilm showed more resistant to antimicrobial agents than that of planktonic microorganism, which could be up to 1000 times [7]
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