scholarly journals Effects of pH on the Properties of Membrane Vesicles Including Glucosyltransferase in Streptococcus mutans

2021 ◽  
Vol 9 (11) ◽  
pp. 2308
Author(s):  
Yusuke Iwabuchi ◽  
Tomoyo Nakamura ◽  
Yasuka Kusumoto ◽  
Ryoma Nakao ◽  
Tsutomu Iwamoto ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Membrane Vesicles ◽  
Extracellular Dna ◽  
Tooth Surface ◽  
Initial Ph ◽  
Effects Of Ph ◽  
Low Levels ◽  
Quantitative Changes ◽  
Biofilm Development

Streptococcus mutans releases membrane vesicles (MVs) and induces MV-dependent biofilm formation. Glucosyltransferases (Gtfs) are bound to MVs and contribute to the adhesion and glucans-dependent biofilm formation of early adherent bacteria on the tooth surface. The biofilm formation of S. mutans may be controlled depending on whether the initial pH tends to be acidic or alkaline. In this study, the characteristics and effects of MVs extracted from various conditions {(initial pH 6.0 and 8.0 media prepared with lactic acid (LA) and acetic acid (AA), and with NaOH (NO), respectively)} on the biofilm formation of S. mutans and early adherent bacteria were investigated. The quantitative changes in glucans between primary pH 6.0 and 8.0 conditions were observed, associated with different activities affecting MV-dependent biofilm formation. The decreased amount of Gtfs on MVs under the initial pH 6.0 conditions strongly guided low levels of MV-dependent biofilm formation. However, in the initial pH 6.0 and 8.0 solutions prepared with AA and NO, the MVs in the biofilm appeared to be formed by the expression of glucans and/or extracellular DNA. These results suggest that the environmental pH conditions established by acid and alkaline factors determine the differences in the local pathogenic activities of biofilm development in the oral cavity.

scholarly journals Effects of Complex DNA and MVs with GTF Extracted from Streptococcus mutans on the Oral Biofilm

Molecules ◽  
2019 ◽  
Vol 24 (17) ◽  
pp. 3131 ◽  
Author(s):  
Hidenobu Senpuku ◽  
Tomoyo Nakamura ◽  
Yusuke Iwabuchi ◽  
Satoru Hirayama ◽  
Ryoma Nakao ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Complex Form ◽  
Membrane Vesicles ◽  
Microtiter Plate ◽  
Extracellular Dna ◽  
Proteinase K ◽  
Human Tooth ◽  
Tooth Surface ◽  
Oral Biofilm

Streptococcus mutans is one of the principal pathogens for the development of dental caries. Oral biofilms formed by S. mutans are constructed of insoluble glucan formation induced by the principal enzymes, GTF-I and GTF-SI, in sucrose-containing conditions. However, as another means of biofilm formation, extracellular DNA (eDNA) and membrane vesicles (MVs) are also contributors. To explore the roles of eDNA and MVs for biofilm formation, short and whole size pure DNAs, two types of sub-purified DNAs and MVs were extracted from S. mutans by beads destruction, treatment of proteinase K, and ultracentrifugation of culture supernatant, and applied into the biofilm formation assay using the S. mutans UA159 gtfBC mutant, which lost GTF-I and GTF-SI, on a human saliva-coated 96 well microtiter plate in sucrose-containing conditions. Sub-purified DNAs after cell lysis by beads destruction for total 90 and 180 s showed a complex form of short-size DNA with various proteins and MVs associated with GTF-I and GTF-SI, and induced significantly higher biofilm formation of the S. mutans UA159.gtfBC mutant than no sample (p < 0.05). Short-size pure DNA without proteins induced biofilm formation but whole-size pure DNA did not. Moreover, the complex form of MV associated with GTFs and short-size DNA showed significantly higher biofilm formation of initial colonizers on the human tooth surface such as Streptococcus mitis than no sample (p < 0.05). The short-size DNAs associated with MVs and GTFs are important contributors to the biofilm formation and may be one of additional targets for the prevention of oral biofilm-associated diseases.

scholarly journals Essential Roles of thesppRAFructose-Phosphate Phosphohydrolase Operon in Carbohydrate Metabolism and Virulence Expression byStreptococcus mutans

2018 ◽  
Vol 201 (2) ◽  
Author(s):  
Lin Zeng ◽  
Robert A. Burne
Keyword(s):  
Dental Caries ◽  
Organic Acids ◽  
Streptococcus Mutans ◽  
Biochemical Characterization ◽  
Constitutive Expression ◽  
Extracellular Dna ◽  
Tooth Surface ◽  
Oral Diseases ◽  
Sugar Phosphate ◽  
Content Type

ABSTRACTThe dental caries pathogenStreptococcus mutanscan ferment a variety of sugars to produce organic acids. Exposure ofS. mutansto certain nonmetabolizable carbohydrates, such as xylitol, impairs growth and can cause cell death. Recently, the presence of a sugar-phosphate stress inS. mutanswas demonstrated using a mutant lacking 1-phosphofructokinase (FruK) that accumulates fructose-1-phosphate (F-1-P). Here, we studied an operon inS. mutans,sppRA, which was highly expressed in thefruKmutant. Biochemical characterization of a recombinant SppA protein indicated that it possessed hexose-phosphate phosphohydrolase activity, with preferences for F-1-P and, to a lesser degree, fructose-6-phosphate (F-6-P). SppA activity was stimulated by Mg2+and Mn2+but inhibited by NaF. SppR, a DeoR family regulator, repressed the expression of thesppRAoperon to minimum levels in the absence of the fructose-derived metabolite F-1-P and likely also F-6-P. The accumulation of F-1-P, as a result of growth on fructose, not only inducedsppAexpression, but it significantly altered biofilm maturation through increased cell lysis and enhanced extracellular DNA release. Constitutive expression ofsppA, via a plasmid or by deletingsppR, greatly alleviated fructose-induced stress in afruKmutant, enhanced resistance to xylitol, and reversed the effects of fructose on biofilm formation. Finally, by identifying three additional putative phosphatases that are capable of promoting sugar-phosphate tolerance, we show thatS. mutansis capable of mounting a sugar-phosphate stress response by modulating the levels of certain glycolytic intermediates, functions that are interconnected with the ability of the organism to manifest key virulence behaviors.IMPORTANCEStreptococcus mutansis a major etiologic agent for dental caries, primarily due to its ability to form biofilms on the tooth surface and to convert carbohydrates into organic acids. We have discovered a two-gene operon inS. mutansthat regulates fructose metabolism by controlling the levels of fructose-1-phosphate, a potential signaling compound that affects bacterial behaviors. With fructose becoming increasingly common and abundant in the human diet, we reveal the ways that fructose may alter bacterial development, stress tolerance, and microbial ecology in the oral cavity to promote oral diseases.

scholarly journals Suppressive Effects of Octyl Gallate on Streptococcus mutans Biofilm Formation, Acidogenicity, and Gene Expression

Molecules ◽  
2019 ◽  
Vol 24 (17) ◽  
pp. 3170 ◽  
Author(s):  
Vika Gabe ◽  
Tomas Kacergius ◽  
Saleh Abu-Lafi ◽  
Mouhammad Zeidan ◽  
Basheer Abu-Farich ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dental Caries ◽  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Colorimetric Method ◽  
Solid Surfaces ◽  
Dependent Manner ◽  
Oral Diseases ◽  
Expression Change ◽  
Biofilm Development

The accumulation of biofilm by Streptococcus mutans bacteria on hard tooth tissues leads to dental caries, which remains one of the most prevalent oral diseases. Hence, the development of new antibiofilm agents is of critical importance. The current study reports the results from testing the effectiveness of octyl gallate (C8-OG) against: (1) S. mutans biofilm formation on solid surfaces (polystyrene, glass), (2) acidogenicity, (3) and the expression of biofilm-related genes. The amount of biofilm formed by S. mutans bacteria was evaluated using the colorimetric method and optical profilometry. The pH of the biofilm growth medium was measured with microelectrode. A quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to assess the expression of genes encoding glucan binding protein B (gbpB), glucosyltransferases B, -C, -D (gtfB, -C, -D), and the F-ATPase β subunit of the F1 protein (atpD). The results show that C8-OG significantly diminished biofilm formation by exposed S. mutans on solid surfaces and suppressed acidogenicity in a dose-dependent manner, compared to unexposed bacteria (p < 0.05). The C8-OG concentration of 100.24 µM inhibited S. mutans biofilm development on solid surfaces by 100% and prevented a decrease in pH levels by 99%. In addition, the RT-qPCR data demonstrate that the biofilm-producing bacteria treated with C8-OG underwent a significant reduction in gene expression in the case of the four genes under study (gbpB, gtfC, gtfD, and atpD), and there was a slight decrease in expression of the gtfB gene. However, C8-OG treatments did not produce significant expression change compared to the control for the planktonic cells, although there was a significant increase for the atpD gene. Therefore, C8-OG might be a potent antibiofilm and/or anticaries agent for oral formulations that aim to reduce the prevalence of dental caries.

scholarly journals Role of the Streptococcus mutans irvA Gene in GbpC-Independent, Dextran-Dependent Aggregation and Biofilm Formation

2009 ◽  
Vol 75 (22) ◽  
pp. 7037-7043 ◽  
Author(s):  
Min Zhu ◽  
Dragana Ajdić ◽  
Yuan Liu ◽  
David Lynch ◽  
Justin Merritt ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Growth Conditions ◽  
Parental Background ◽  
A Cell ◽  
Major Surface ◽  
Biofilm Development ◽  
Small Aggregation

ABSTRACT Dextran-dependent aggregation (DDAG) of Streptococcus mutans is an in vitro phenomenon that is believed to represent a property of the organism that is beneficial for sucrose-dependent biofilm development. GbpC, a cell surface glucan-binding protein, is responsible for DDAG in S. mutans when cultured under defined stressful conditions. Recent reports have described a putative transcriptional regulator gene, irvA, located just upstream of gbpC, that is normally repressed by the product of an adjacent gene, irvR. When repression of irvA is relieved, there is a resulting increase in the expression of GbpC and decreases in competence and synthesis of the antibiotic mutacin I. This study examined the role of irvA in DDAG and biofilm formation by engineering strains that overexpressed irvA (IrvA+) on an extrachromosomal plasmid. The IrvA+ strain displayed large aggregation particles that did not require stressful growth conditions. A novel finding was that overexpression of irvA in a gbpC mutant background retained a measure of DDAG, albeit very small aggregation particles. Biofilms formed by the IrvA+ strain in the parental background possessed larger-than-normal microcolonies. In a gbpC mutant background, the overexpression of irvA reversed the fragile biofilm phenotype normally associated with loss of GbpC. Real-time PCR and Northern blot analyses found that expression of gbpC did not change significantly in the IrvA+ strain but expression of spaP, encoding the major surface adhesin P1, increased significantly. Inactivation of spaP eliminated the small-particle DDAG. The results suggest that IrvA promotes DDAG not only by GbpC, but also via an increase in P1.

scholarly journals Expression of biofilm-associated genes of Streptococcus mutans in response to glucose and sucrose

2007 ◽  
Vol 56 (11) ◽  
pp. 1528-1535 ◽  
Author(s):  
Moshe Shemesh ◽  
Avshalom Tam ◽  
Doron Steinberg
Keyword(s):  
Gene Expression ◽  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Fold Increase ◽  
Extracellular Polysaccharide ◽  
Tooth Surface ◽  
Differential Analysis ◽  
Nutrient Contents ◽  
Genes Encoding

Streptococcus mutans is known as a primary pathogen of dental caries, one of the most common human infectious diseases. Exopolysaccharide synthesis, adherence to tooth surface and biofilm formation are important physiological and virulence factors of S. mutans. In vitro comparative gene expression analysis was carried out to differentiate 10 selected genes known to be mostly involved in S. mutans biofilm formation by comparing the expression under biofilm and planktonic environments. Real-time RT-PCR analyses indicated that all of the genes tested were upregulated in the biofilm compared to cells grown in planktonic conditions. The influence of simple dietary carbohydrates on gene expression in S. mutans biofilm was tested also. Among the tested genes, in the biofilm phase, the greatest induction was observed for gtf and ftf, which are genes encoding the extracellular polysaccharide-producing enzymes. Biofilm formation was accompanied by a 22-fold induction in the abundance of mRNA encoding glucosyltransferase B (GTFB) and a 14.8 -fold increase in mRNA encoding GTFC. Levels of mRNA encoding fructosyltransferase were induced approximately 11.8-fold in biofilm-derived cells. Another notable finding of this study suggests that glucose affects the expression of S. mutans GS5 biofilm genes. In spite of a significant upregulation in biofilm-associated gene expression in the presence of sucrose, the presence of glucose with sucrose reduced expression of most tested genes. Differential analysis of the transcripts from S. mutans, grown in media with various nutrient contents, revealed significant shifts in the expression of the genes involved in biofilm formation. The results presented here provide new insights at the molecular level regarding gene expression in this bacterium when grown under biofilm conditions, allowing a better understanding of the mechanism of biofilm formation by S. mutans.

Extracellular DNA and Outer Membrane Vesicles contribute to P. fluorescens SBW25 air-liquid interface biofilms.

2020 ◽  
Author(s):  
Olena Moshynets ◽  
Airat Kayumov ◽  
Olga Iungin ◽  
Svitlana Rymar ◽  
Ianina Pokholenko ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Extracellular Dna ◽  
Exogenous Dna ◽  
Rhizosphere Bacterium ◽  
Cellulose Matrix ◽  
Dnase Treatment ◽  
Biofilm Development ◽  
Biofilm Matrix

&lt;p&gt;Outer membrane vesicles (OMVs) and extracellular DNA (eDNA) are important for biofilm formation for many bacteria. OMVs are a perfect transport system to deliver biofilm-related components including eDNA beyond the boundaries of cells, and eDNA itself is an important structural component of biofilms as well as enabling horizontal gene transfer and local adaptation. Both OMVs and eDNA are found in the biofilms produced by the opportunistic human pathogen P. aeruginosa and the plant pathogen P. syringae, but as yet, they have not been reported in the cellulose matrix-based biofilms produced by the related model rhizosphere bacterium Pseudomonas fluorescens SBW25.&lt;/p&gt; &lt;p&gt;In this work we have gone back to re-assess the complexity of SBW25 biofilms by looking for evidence of OMVs and eDNA associated with biofilm&amp;#8211;formation. OMVs were first imaged by SEM and LC-MC analysis used to identify 51 biofilm matrix-associated proteins of which 12 were also identified in biofilm OMVs. Interestingly, only 5 proteins were identified in both biofilm matrix and OMV samples, but not in planktonic OMVs, suggesting that these may be biofilm-specific components. &amp;#160;&lt;/p&gt; &lt;p&gt;We also observed eDNA by CLSM in both the weak and poorly-attached Viscous Mass (VM) and robust and well-attached Wrinkly Spreader (WS) air-liquid (A-L) interface biofilms produced by wild-type SBW25 and the Wrinkly Spreader mutant. The eDNA fraction could be precipitated from biofilm cell-free supernatant samples which demonstrated that WS biofilms had two-fold&amp;#8211;higher levels than VM biofilms. DNAse treatment effected the development of both types of biofilm and reduced the strength and attachment levels when added to mature VM and WS biofilms. Testing with exogenous DNA suggests that high molecular weight (HMW) DNA is involved in both strength and attachment, perhaps by surface conditioning and interactions with the primary cellulose matrix common to both biofilms. HMW eDNA could be isolated directly from biofilm supernatants whereas two different HMW size fractions could be isolated from OMVs, presumably, from the outer OMV surface because DNAse treatment led to a substantially reduced DNA signal. This suggest that eDNA persistence and degradation in SBW25 biofilms is complex and eDNA fractions may play different roles in biofilm development, protection and adaptation.&lt;/p&gt;

scholarly journals Biofilm Formation by Streptococcus pneumoniae: Role of Choline, Extracellular DNA, and Capsular Polysaccharide in Microbial Accretion

2006 ◽  
Vol 188 (22) ◽  
pp. 7785-7795 ◽  
Author(s):  
Miriam Moscoso ◽  
Ernesto García ◽  
Rubens López
Keyword(s):  
Streptococcus Pneumoniae ◽  
Biofilm Formation ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Dimensional Structure ◽  
Confocal Laser ◽  
Upper Respiratory Tract ◽  
Teichoic Acids ◽  
Abiotic Surfaces ◽  
Biofilm Development

ABSTRACTStreptococcus pneumoniaecolonizes the human upper respiratory tract, and this asymptomatic colonization is known to precede pneumococcal disease. In this report, chemically defined and semisynthetic media were used to identify the initial steps of biofilm formation by pneumococcus during growth on abiotic surfaces such as polystyrene or glass. Unencapsulated pneumococci adhered to abiotic surfaces and formed a three-dimensional structure about 25 μm deep, as observed by confocal laser scanning microscopy and low-temperature scanning electron microscopy. Choline residues of cell wall teichoic acids were found to play a fundamental role in pneumococcal biofilm development. The role in biofilm formation of choline-binding proteins, which anchor to the teichoic acids of the cell envelope, was determined using unambiguously characterized mutants. The results showed that LytA amidase, LytC lysozyme, LytB glucosaminidase, CbpA adhesin, PcpA putative adhesin, and PspA (pneumococcal surface protein A) mutants had a decreased capacity to form biofilms, whereas no such reduction was observed in Pce phosphocholinesterase or CbpD putative amidase mutants. Moreover, encapsulated, clinical pneumococcal isolates were impaired in their capacity to form biofilms. In addition, a role for extracellular DNA and proteins in the establishment ofS. pneumoniaebiofilms was demonstrated. Taken together, these observations provide information on conditions that favor the sessile mode of growth byS. pneumoniae. The experimental approach described here should facilitate the study of bacterial genes that are required for biofilm formation. Those results, in turn, may provide insight into strategies to prevent pneumococcal colonization of its human host.

scholarly journals Extracellular DNA and Type IV Pilus Expression Regulate the Structure and Kinetics of Biofilm Formation by Nontypeable Haemophilus influenzae

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Jayajit Das ◽  
Elaine Mokrzan ◽  
Vinal Lakhani ◽  
Lucia Rosas ◽  
Joseph A. Jurcisek ◽  
...  
Keyword(s):  
Middle Ear ◽  
In Silico ◽  
Biofilm Formation ◽  
Rational Design ◽  
Bacterial Biofilm ◽  
Confocal Imaging ◽  
Extracellular Dna ◽  
Fractal Structures ◽  
Biofilm Development

ABSTRACT Biofilms formed in the middle ear by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and refractive nature of otitis media (OM). However, mechanisms that underlie the emergence of specific NTHI biofilm structures are unclear. We combined computational analysis tools and in silico modeling rooted in statistical physics with confocal imaging of NTHI biofilms formed in vitro during static culture in order to identify mechanisms that give rise to distinguishing morphological features. Our analysis of confocal images of biofilms formed by NTHI strain 86-028NP using pair correlations of local bacterial densities within sequential planes parallel to the substrate showed the presence of fractal structures of short length scales (≤10 μm). The in silico modeling revealed that extracellular DNA (eDNA) and type IV pilus (Tfp) expression played important roles in giving rise to the fractal structures and allowed us to predict a substantial reduction of these structures for an isogenic mutant (ΔcomE) that was significantly compromised in its ability to release eDNA into the biofilm matrix and had impaired Tfp function. This prediction was confirmed by analysis of confocal images of in vitro ΔcomE strain biofilms. The fractal structures potentially generate niches for NTHI survival in the hostile middle ear microenvironment by dramatically increasing the contact area of the biofilm with the surrounding environment, facilitating nutrient exchange, and by generating spatial positive feedback to quorum signaling. IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies. IMPORTANCE NTHI is a major bacterial pathogen for OM, which is a common ear infection in children worldwide. Chronic OM is associated with bacterial biofilm formation in the middle ear; therefore, knowledge of the mechanisms that underlie NTHI biofilm formation is important for the development of therapeutic strategies for NTHI-associated OM. Our combined approach using confocal imaging of NTHI biofilms formed in vitro and mathematical tools for analysis of pairwise density correlations and agent-based modeling revealed that eDNA and Tfp expression were important factors in the development of fractal structures in NTHI biofilms. These structures may help NTHI survive in hostile environments, such as the middle ear. Our in silico model can be used in combination with laboratory or animal modeling studies to further define the mechanisms that underlie NTHI biofilm development during OM and thereby guide the rational design of, and optimize time and cost for, benchwork and preclinical studies.

scholarly journals Murein Hydrolase LytF of Streptococcus sanguinis and the Ecological Consequences of Competence Development

2017 ◽  
Vol 83 (24) ◽  
Author(s):  
Nyssa Cullin ◽  
Sylvio Redanz ◽  
Kirsten J. Lampi ◽  
Justin Merritt ◽  
Jens Kreth
Keyword(s):  
Dental Caries ◽  
Oral Cavity ◽  
Cell Morphology ◽  
Biofilm Formation ◽  
Oral Bacteria ◽  
Tooth Surface ◽  
Content Type ◽  
Streptococcus Sanguinis ◽  
Murein Hydrolase ◽  
Biofilm Development

ABSTRACT The overall health of the oral cavity is dependent on proper homeostasis between health-associated bacterial colonizers and bacteria known to promote dental caries. Streptococcus sanguinis is a health-associated commensal organism, a known early colonizer of the acquired tooth pellicle, and is naturally competent. We have shown that LytF, a competence-controlled murein hydrolase, is capable of inducing the release of extracellular DNA (eDNA) from oral bacteria. Precipitated LytF and purified LytF were used as treatments against planktonic cultures and biofilms. Larger amounts of eDNA were released from cultures treated with protein samples containing LytF. Additionally, LytF could affect biofilm formation and cellular morphology. Biofilm formation was significantly decreased in the lytF-complemented strain, in which increased amounts of LytF are present. The same strain also exhibited cell morphology defects in both planktonic cultures and biofilms. Furthermore, the LytF cell morphology phenotype was reproducible in wild-type cells using purified LytF protein. In sum, our findings demonstrate that LytF can induce the release of eDNA from oral bacteria, and they suggest that, without proper regulation of LytF, cells display morphological abnormalities that contribute to biofilm malformation. In the context of the oral biofilm, LytF may play important roles as part of the competence and biofilm development programs, as well as increasing the availability of eDNA. IMPORTANCE Streptococcus sanguinis, a commensal organism in the oral cavity and one of the pioneer colonizers of the tooth surface, is associated with the overall health of the oral environment. Our laboratory showed previously that, under aerobic conditions, S. sanguinis can produce H2O2 to inhibit the growth of bacterial species that promote dental caries. This production of H2O2 by S. sanguinis also induces the release of eDNA, which is essential for proper biofilm formation. Under anaerobic conditions, S. sanguinis does not produce H2O2 but DNA is still released. Determining how S. sanguinis releases DNA is thus essential to understand biofilm formation in the oral cavity.

Streptococcus mutans biofilm formation is dependent on extracellular DNA in primary low pH conditions

2016 ◽  
Vol 58 (2) ◽  
pp. 55-61 ◽  
Author(s):  
Taketo Kawarai ◽  
Naoki Narisawa ◽  
Yusuke Suzuki ◽  
Ryo Nagasawa ◽  
Hidenobu Senpuku
Keyword(s):  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Low Ph ◽  
Extracellular Dna ◽  
Ph Conditions
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