Effect of LongZhang Gargle on Biofilm Formation and Acidogenicity ofStreptococcus mutans In Vitro

Streptococcus mutans, with the ability of high-rate acid production and strong biofilm formation, is considered the predominant bacterial species in the pathogenesis of human dental caries. Natural products which may be bioactive againstS. mutanshave become a hot spot to researches to control dental caries. LongZhang Gargle, completely made from Chinese herbs, was investigated for its effects on acid production and biofilm formation byS. mutansin this study. The results showed an antimicrobial activity of LongZhang Gargle againstS. mutansplanktonic growth at the minimum inhibitory concentration (MIC) of 16% and minimum bactericidal concentration (MBC) of 32%. Acid production was significantly inhibited at sub-MIC concentrations. Biofilm formation was also significantly disrupted, and 8% was the minimum concentration that resulted in at least 50% inhibition of biofilm formation (MBIC50). A scanning electron microscopy (SEM) showed an effective disruption of LongZhang Gargle onS. mutansbiofilm integrity. In addition, a confocal laser scanning microscopy (CLSM) suggested that the extracellular polysaccharides (EPS) synthesis could be inhibited by LongZhang Gargle at a relatively low concentration. These findings suggest that LongZhang Gargle may be a promising natural anticariogenic agent in that it suppresses planktonic growth, acid production, and biofilm formation againstS. mutans.

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The vicK gene of Streptococcus mutans mediates its cariogenicity via exopolysaccharides metabolism

International Journal of Oral Science ◽

10.1038/s41368-021-00149-x ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Yalan Deng ◽

Yingming Yang ◽

Bin Zhang ◽

Hong Chen ◽

Yangyu Lu ◽

...

Keyword(s):

Dental Caries ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

Laser Scanning ◽

Single Species ◽

Laser Scanning Microscopy ◽

Gas Chromatography Mass Spectrometry ◽

Extracellular Polysaccharides ◽

Confocal Laser

AbstractStreptococcus mutans (S. mutans) is generally regarded as a major contributor to dental caries because of its ability to synthesize extracellular polysaccharides (EPS) that aid in the formation of plaque biofilm. The VicRKX system of S. mutans plays an important role in biofilm formation. The aim of this study was to investigate the effects of vicK gene on specific characteristics of EPS in S. mutans biofilm. We constructed single-species biofilms formed by different mutants of vicK gene. Production and distribution of EPS were detected through atomic force microscopy, scanning electron microscopy and confocal laser scanning microscopy. Microcosmic structures of EPS were analyzed by gel permeation chromatography and gas chromatography-mass spectrometry. Cariogenicity of the vicK mutant was assessed in a specific pathogen-free rat model. Transcriptional levels of cariogenicity-associated genes were confirmed by quantitative real-time polymerase chain reaction. The results showed that deletion of vicK gene suppressed biofilm formation as well as EPS production, and EPS were synthesized mostly around the cells. Molecular weight and monosaccharide components underwent evident alterations. Biofilms formed in vivo were sparse and contributed a decreased degree of caries. Moreover, expressional levels of genes related to EPS synthesis were down-regulated, except for gtfB. Our report demonstrates that vicK gene enhances biofilm formation and subsequent caries development. And this may due to its regulations on EPS metabolism, like synthesis or microcosmic features of EPS. This study suggests that vicK gene and EPS can be considered as promising targets to modulate dental caries.

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Effects of brief sodium fluoride treatments on the growth of early and mature cariogenic biofilms

Scientific Reports ◽

10.1038/s41598-021-97905-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ye Han

Keyword(s):

Dental Caries ◽

Sodium Fluoride ◽

Biofilm Formation ◽

Water Soluble ◽

Laser Scanning Microscopy ◽

Extracellular Polysaccharides ◽

Antibiofilm Activity ◽

Fluoride Treatment ◽

Biofilm Development ◽

Cariogenic Biofilm

AbstractAlthough fluoride has been widely used as a preventive agent for dental caries, the effects of fluoride on the activities of biofilms in different stages of cariogenic biofilm formation are less studied. This study was designed to investigate the antibiofilm activity of sodium fluoride during the early and mature stages of Streptococcus mutans (S. mutans) biofilm formation. S. mutans biofilms were formed on saliva-coated hydroxyapatite disks. In the early (0–46 h) and mature (46–94 h) biofilm stages, the biofilms were treated with different concentrations of fluoride (250, 500, 1000, 2000 ppm; 5 times in total, 1 min/treatment). Acidogenicity, dry weight, colony-forming units (CFUs), water-soluble/insoluble extracellular polysaccharides (EPSs), and intracellular polysaccharides were analysed, and confocal laser scanning microscopy images were obtained of the two stages of biofilms to determine antibiofilm activities of fluoride at varying concentrations during the formation of early and mature biofilms. In the early stages of cariogenic biofilm formation, test groups with all fluoride concentrations significantly inhibited the growth of S. mutans biofilms. The antibiofilm and anti-EPS formation activities of the brief fluoride treatments increased with a concentration-dependent pattern. At the mature biofilm stage, only the 2000 ppm fluoride treatment group significantly inhibited biofilm accumulation, activity, and intracellular/extracellular polysaccharide content compared with those of the control and other fluoride treatment groups. The antimicrobial effect of fluoride treatment on the growth of S. mutans biofilms was linked with the stage of cariogenic biofilm formation. The inhibition of S. mutans biofilm growth by fluoride treatment was easier in the early formation stage than in the mature stage. Fluoride treatment in the early stage of cariogenic biofilm formation may be an effective approach to controlling cariogenic biofilm development and preventing dental caries.

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Starvation Survival and Biofilm Formation under Subminimum Inhibitory Concentration of QAMs

BioMed Research International ◽

10.1155/2021/8461245 ◽

2021 ◽

Vol 2021 ◽

pp. 1-10

Author(s):

Sanjay Kumar Tiwari ◽

Suping Wang ◽

Yannan Huang ◽

Xuedong Zhou ◽

Hockin H. K. Xu ◽

...

Keyword(s):

Lactic Acid ◽

Biofilm Formation ◽

Acid Production ◽

Inhibitory Concentration ◽

Lactic Acid Production ◽

Oral Bacteria ◽

Laser Scanning Microscopy ◽

Prolonged Starvation ◽

Survival And Development ◽

Development Of Tolerance

Quaternary ammonium methacrylates (QAMs) are useful antimicrobial compounds against oral bacteria. Here, we investigated the effects of two QAMs, dimethylaminododecyl methacrylate (DMADDM) and dimethylaminohexadecyl methacrylate (DMAHDM), on biofilm formation, survival and development of tolerance by biofilm, and survival and development of tolerance against QAMs after prolonged starvation. Enterococcus faecalis (E. faecalis), Streptococcus gordonii (S. gordonii), Lactobacillus acidophilus (L. acidophilus), and Actinomyces naeslundii (A. naeslundii) were used. Minimum inhibitory concentration (MIC) of QAMs against multispecies biofilm was determined. Biofilm formed under sub-MIC was observed by crystal violet staining and confocal laser scanning microscopy (CLSM). Metabolic activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactic acid production measurement. Development of tolerance was determined by MIC values before and after exposure to QAMs or after prolonged starvation. It was found that E. faecalis and S. gordonii could survive and form biofilm under sub-MIC of QAMs. Lactic acid production from biofilms formed under sub-MIC was significantly higher than control specimens ( p < 0.05 ). The exposure to sub-MIC of QAMs promoted biofilm formation, and prolonged starvation or prolonged contact with sub-MIC helped bacteria develop tolerance against killing by QAMs.

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Baicalein Inhibits Streptococcus mutans Biofilms and Dental Caries-Related Virulence Phenotypes

Antibiotics ◽

10.3390/antibiotics10020215 ◽

2021 ◽

Vol 10 (2) ◽

pp. 215

Author(s):

Aparna Vijayakumar ◽

Hema Bhagavathi Sarveswari ◽

Sahana Vasudevan ◽

Karthi Shanmugam ◽

Adline Princy Solomon ◽

...

Keyword(s):

Dental Caries ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

Acid Production ◽

Cell Surface Hydrophobicity ◽

Surface Hydrophobicity ◽

Oral Disease ◽

Public Healthcare ◽

Surface Attachment ◽

First Time

Dental caries, the most common oral disease, is a major public healthcare burden and affects more than three billion people worldwide. The contemporary understanding of the need for a healthy microbiome and the emergence of antimicrobial resistance has resulted in an urgent need to identify compounds that curb the virulence of pathobionts without microbial killing. Through this study, we have demonstrated for the first time that 5,6,7-trihydroxyflavone (Baicalein) significantly downregulates crucial caries-related virulence phenotypes in Streptococcus mutans. Baicalein significantly inhibited biofilm formation by Streptococcus mutans UA159 (MBIC50 = 200 μM), without significant growth inhibition. Notably, these concentrations of baicalein did not affect the commensal S. gordonii. Strikingly, baicalein significantly reduced cell surface hydrophobicity, autoaggregation and acid production by S. mutans. Mechanistic studies (qRT-PCR) showed downregulation of various genes regulating biofilm formation, surface attachment, quorum sensing, acid production and competence. Finally, we demonstrate the potential translational value of baicalein by reporting synergistic interaction with fluoride against S. mutans biofilms.

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Antimicrobial and Antibiofilm Activity of Citral and its Derivative against Microflora from Dental Plaque

Asian Journal of Organic & Medicinal Chemistry ◽

10.14233/ajomc.2020.ajomc-p259 ◽

2020 ◽

Vol 5 (2) ◽

pp. 120-126

Author(s):

S.V. Ambade ◽

N.M. Deshpande ◽

S.S. Kale ◽

V.N. Ambade

Keyword(s):

Dental Caries ◽

Essential Oil ◽

Dental Plaque ◽

Biofilm Formation ◽

Periodontal Diseases ◽

Bacterial Species ◽

Antibiofilm Activity ◽

Oral Microflora ◽

Lemon Grass ◽

First Time

Dental biofilms inhabit the oral cavity in form of dental plaque which then causes dental caries and periodontal diseases worldwide. Lemon grass essential oil (LGEO) has been reported to exhibit antimicrobial and antibiofilm activity against. This study represents the potential of citral and derivatives as antimicrobial and antibiofilm agent against dental microflora. Three bacterial species chiefly responsible for biofilm formation, and five prime colonizer of dental plaque were selected to represent dental microflora. Citral and its derivative viz. citral semicarbazone, exhibited antimicrobial and antibiofilm activity against the selected organisms. For the first time, any citral derivative has ever demonstrated to exhibit antimicrobial and antibiofilm activity against the oral microflora. However, study could not established citral or its derivatives as more effective, powerful and better herbal material as compared to LGEO to control the oral microflora associated with dental plaque.

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Bacterial colonization of enamel in situ investigated using fluorescence in situ hybridization

Journal of Medical Microbiology ◽

10.1099/jmm.0.011213-0 ◽

2009 ◽

Vol 58 (10) ◽

pp. 1359-1366 ◽

Cited By ~ 48

Author(s):

Ali Al-Ahmad ◽

Marie Follo ◽

Ann-Carina Selzer ◽

Elmar Hellwig ◽

Matthias Hannig ◽

...

Keyword(s):

Biofilm Formation ◽

Laser Scanning ◽

Bacterial Colonization ◽

Bacterial Species ◽

Fusobacterium Nucleatum ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Actinomyces Naeslundii ◽

Oral Biofilms

Oral biofilms are one of the greatest challenges in dental research. The present study aimed to investigate initial bacterial colonization of enamel surfaces in situ using fluorescence in situ hybridization (FISH) over a 12 h period. For this purpose, bovine enamel slabs were fixed on buccal sites of individual splints worn by six subjects for 2, 6 and 12 h to allow biofilm formation. Specimens were processed for FISH and evaluated with confocal laser-scanning microscopy, using probes for eubacteria, Streptococcus species, Veillonella species, Fusobacterium nucleatum and Actinomyces naeslundii. The number of adherent bacteria increased with time and all tested bacterial species were detected in the biofilm formed in situ. The general percentage composition of the eubacteria did not change over the investigated period, but the number of streptococci, the most frequently detected species, increased significantly with time (2 h: 17.7±13.8 %; 6 h: 20.0±16.6 %; 12 h: 24.7±16.1 %). However, ≤1 % of the surface was covered with bacteria after 12 h of biofilm formation in situ. In conclusion, FISH is an appropriate method for quantifying initial biofilm formation in situ, and the proportion of streptococci increases during the first 12 h of bacterial adherence.

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Effects of brief sodium fluoride treatment on the growth of early and mature cariogenic biofilms

10.21203/rs.3.rs-586115/v1 ◽

2021 ◽

Author(s):

Ye Han

Keyword(s):

Sodium Fluoride ◽

Biofilm Formation ◽

Water Soluble ◽

Laser Scanning Microscopy ◽

Extracellular Polysaccharides ◽

Antibiofilm Activity ◽

Fluoride Treatment ◽

Formation Stage ◽

Early Formation ◽

Cariogenic Biofilm

Abstract Although fluoride has been widely used in the prevention of dental caries, the effect of fluoride on the activity of biofilm in different stages of cariogenic biofilm formation is less studied. This study aimed to investigate the antibiofilm activity of sodium fluoride during early and mature Streptococcus mutans (S. mutans) biofilms formation. S. mutans biofilms were formed on saliva-coated hydroxyapatite disks. In the early (0 ~ 46 h) and mature (46 ~ 94 h) biofilm stages, the biofilm was treated with different concentrations of fluoride (250, 500, 1000, 2000 ppm; 5 times in total, 1 min/treatment). Acidogenicity, dry weight, colony-forming units, water-soluble/insoluble extracellular polysaccharides (EPS), and intracellular polysaccharides were analyzed and confocal laser scanning microscopy images were obtained of the two stages of biofilms (early and mature biofilms). To determine the antibiofilm activity of sodium fluoride during the formation of early and mature biofilms, and to evaluate the relationship between different concentrations of sodium fluoride and antibiofilm activity. In the early cariogenic biofilm formation stage, all fluoride concentration test groups (250, 500, 1000, 2000 ppm) significantly inhibited the growth of S. mutans biofilm. The antibiofilm and anti-EPS formation activities of the brief fluoride treatment increased in a concentration-dependent pattern. At the mature biofilm stage, only the 2000 ppm fluoride treatment group significantly inhibited biofilm accumulation, activity, and intracellular/extracellular polysaccharide content compared with the control and other fluoride treatment groups. The antimicrobial activity of fluoride is related to the formation stage of cariogenic biofilm. The early formation stage of cariogenic biofilm is more susceptible to the inhibition of fluorine than the mature stage. The fluoride treatment in the early formation stage of cariogenic biofilm may be an effective means to control the development of cariogenic biofilm and prevent caries.

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Effects of Norspermidine on Dual‐Species Biofilms Composed of Streptococcus mutans and Streptococcus sanguinis

BioMed Research International ◽

10.1155/2019/1950790 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9

Author(s):

Yan Sun ◽

Yihuai Pan ◽

Yu Sun ◽

Mingyun Li ◽

Shengbin Huang ◽

...

Keyword(s):

Streptococcus Mutans ◽

Metabolic Activity ◽

Biofilm Formation ◽

Acid Production ◽

Single Species ◽

Extracellular Polysaccharides ◽

Streptococcus Sanguinis ◽

Crystal Violet Assay ◽

Sem Imaging

The present study aimed at investigating the influence of norspermidine on the formation of dual-species biofilms composed of Streptococcus mutans (S. mutans) and Streptococcus sanguinis (S. sanguinis). Crystal violet assay was conducted to assess the formation of single-species biofilms of S. mutans and S. sanguinis, and the growth curve was carefully observed to monitor the growth of these two species of bacteria. Fluorescence in situ hybridization (FISH) and MTT array were used to analyze the composition and metabolic activity of the dual-species biofilms, respectively. Extracellular polysaccharides (EPS)/bacteria staining, anthrone method, and scanning electron microscopy (SEM) imaging were conducted to study the synthesis of EPS by dual-species biofilms. Lactic acid assay and pH were measured to detect dual-species biofilm acid production. We found that norspermidine had different effects on S. mutans and S. sanguinis including their growth and biofilm formation. Norspermidine regulated the composition of the dual-species biofilms, decreased the ratio of S. mutans in dual-species biofilms, and reduced the metabolic activity, EPS synthesis, and acid production of dual-species biofilms. Norspermidine regulated dual-species biofilms in an ecological way, suggesting that it may be a potent reagent for controlling dental biofilms and managing dental caries.

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Tooth mousse containing casein phosphopeptide-amorphous calcium phosphate prevents biofilm formation of Streptococcus mutans

BMC Oral Health ◽

10.1186/s12903-021-01502-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Ronit Vogt Sionov ◽

Danae Tsavdaridou ◽

Muna Aqawi ◽

Batya Zaks ◽

Doron Steinberg ◽

...

Keyword(s):

Calcium Phosphate ◽

Streptococcus Mutans ◽

Amorphous Calcium Phosphate ◽

Biofilm Formation ◽

Laser Scanning Microscopy ◽

Planktonic Growth ◽

Casein Phosphopeptide ◽

Cariogenic Bacterium ◽

Biofilm Development ◽

Tooth Mousse

Abstract Background Streptococcus mutans is a common cariogenic bacterium in the oral cavity involved in plaque formation. Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) has been introduced into tooth mousse to encourage remineralization of dental enamel. The aim of this research was to study the effect of tooth mousse containing CPP-ACP (GC Tooth Mousse®) or CPP-ACP with 0.2% fluoride (CPP-ACPF; GC Tooth Mousse Plus®; GCP) on S. mutans planktonic growth and biofilm formation. Methods S. mutans was cultivated in the presence of different dilutions of the tooth mousse containing CPP-ACP or CPP-ACPF, and the planktonic growth was determined by ATP viability assay and counting colony-forming units (CFUs). The resulting biofilms were examined by crystal violet staining, MTT metabolic assay, confocal laser scanning microscopy (CLSM), and scanning electron microscope (SEM). Results The CPP-ACP tooth mousse (GC) at a dilution of 5–50 mg/ml (0.5–5%) did not inhibit planktonic growth, and even increased the ATP content and the number of viable bacteria after a 24 h incubation. The same was observed for the CPP-ACPF tooth mousse (GCP), except for the higher concentrations (25 and 50 mg/ml) that led to a drop in the bacterial count. Importantly, both compounds significantly decreased S. mutans biofilm formation at dilutions as low as 1.5–3 mg/ml. 12.5 mg/ml GC and 6.25 mg/ml GCP inhibited biofilm formation by 90% after 4 h. After 24 h, the MBIC90 was 6.25 mg/ml for both. CLSM images confirmed the strong inhibitory effect GC and GCP had on biofilm formation when using 5 mg/ml tooth mousse. SEM images of those bacteria that managed to form biofilm in the presence of 5 mg/ml tooth mousse, showed alterations in the bacterial morphology, where the streptococci appear 25–30% shorter on the average than the control bacteria. Conclusion Our data show that the tooth mousse containing CPP-ACP reduces biofilm formation of the cariogenic bacterium S. mutans without killing the bacteria. The use of natural substances which inhibit biofilm development without killing the bacteria, has therapeutic benefits, especially in orthodontic pediatric patients.

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Important Contribution of the Novel LocuscomEBto Extracellular DNA-Dependent Staphylococcus lugdunensis Biofilm Formation

Infection and Immunity ◽

10.1128/iai.00775-15 ◽

2015 ◽

Vol 83 (12) ◽

pp. 4682-4692 ◽

Cited By ~ 12

Author(s):

Nithya Babu Rajendran ◽

Julian Eikmeier ◽

Karsten Becker ◽

Muzaffar Hussain ◽

Georg Peters ◽

...

Keyword(s):

Biofilm Formation ◽

Bacterial Species ◽

Laser Scanning Microscopy ◽

Extracellular Dna ◽

Staphylococcus Lugdunensis ◽

Content Type ◽

Competence Gene ◽

Independent Mechanism ◽

Dna Release ◽

The Impact

The coagulase-negative speciesStaphylococcus lugdunensisis an emerging cause of serious and potentially life-threatening infections, such as infective endocarditis. The pathogenesis of these infections is characterized by the ability ofS. lugdunensisto form biofilms on either biotic or abiotic surfaces. To elucidate the genetic basis of biofilm formation inS. lugdunensis, we performed transposon (Tn917) mutagenesis. One mutant had a significantly reduced biofilm-forming capacity and carried a Tn917insertion within the competence genecomEB. Site-directed mutagenesis and subsequent complementation with a functional copy ofcomEBverified the importance ofcomEBin biofilm formation. In several bacterial species, natural competence stimulates DNA release via lysis-dependent or -independent mechanisms. Extracellular DNA (eDNA) has been demonstrated to be an important structural component of many bacterial biofilms. Therefore, we quantified the eDNA in the biofilms and found diminished eDNA amounts in thecomEBmutant biofilm. High-resolution images and three-dimensional data obtained via confocal laser scanning microscopy (CSLM) visualized the impact of thecomEBmutation on biofilm integrity. ThecomEBmutant did not show reduced expression of autolysin genes, decreased autolytic activities, or increased cell viability, suggesting a cell lysis-independent mechanism of DNA release. Furthermore, reduced amounts of eDNA in thecomEBmutant biofilms did not result from elevated levels or activity of theS. lugdunensisthermonuclease NucI. In conclusion, we defined here, for the first time, a role for the competence genecomEBin staphylococcal biofilm formation. Our findings indicate thatcomEBstimulates biofilm formation via a lysis-independent mechanism of DNA release.

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