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 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 A Concise Review of Silver Diamine Fluoride on Oral Biofilm

2021 ◽  
Vol 11 (7) ◽  
pp. 3232
Author(s):  
Jingyang Zhang ◽  
Sofiya-Roksolana Got ◽  
Iris Xiaoxue Yin ◽  
Edward Chin-Man Lo ◽  
Chun-Hung Chu
Keyword(s):  
Clinical Study ◽  
Streptococcus Mutans ◽  
Bacterial Adhesion ◽  
Clinical Studies ◽  
Antibacterial Properties ◽  
Tooth Surface ◽  
Original Research ◽  
Laboratory Studies ◽  
Oral Biofilm ◽  
Silver Diamine Fluoride

Studies have shown that silver diamine fluoride (SDF) is an effective agent to arrest and prevent dental caries due to its mineralizing and antibacterial properties. While plenty of studies have investigated the mineralizing properties, there are few papers that have examined its antibacterial effect on oral biofilm. The objective of this study was to identify the effect of silver diamine fluoride on oral biofilm. Method: The keywords used were (silver diamine fluoride OR silver diammine fluoride OR SDF OR silver fluoride OR AgF AND biofilm OR plaque). Two reviewers screened the titles and abstracts and then retrieved the full text of the potentially eligible publications. Publications of original research investigating the effect of SDF on oral biofilm were selected for this review. Results: This review included 15 laboratory studies and six clinical studies among the 540 papers identified. The laboratory studies found that SDF could prevent bacterial adhesion to the tooth surface. SDF also inhibited the growth of cariogenic bacteria, including Streptococcus mutans, Lactobacillus acidophilus, Streptococcus sobrinus, Lactobacillus rhamnosus, Actinomyces naeslundii, and Enterococcus faecalis, thus contributing to its success in caries arrest. One clinical study reported a decrease in Streptococcus mutans and Lactobacillus sp. in arrested caries after SDF treatment, and another clinical study found that SDF inhibited the growth of periodontitis microbiota, including Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia/nigrescens. However, three clinical studies reported no significant change in the microbial diversity of the plaque on the tooth after SDF treatment. Moreover, one laboratory study and one clinical research study reported that SDF inhibited the growth of Candida albicans. Conclusion: Not many research studies have investigated the effects of SDF on oral biofilm, although SDF has been used as a caries-arresting agent with antibacterial properties. However, a few publications have reported that SDF prevented bacterial adhesion to the teeth, inhibited the growth of cariogenic and periodontal bacteria, and possessed antifungal properties.

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 Strain variation in Bacillus cereus biofilms and their susceptibility to extracellular matrix-degrading enzymes

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0245708
Author(s):  
Eun Seob Lim ◽  
Seung-Youb Baek ◽  
Taeyoung Oh ◽  
Minseon Koo ◽  
Joo Young Lee ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Bacillus Cereus ◽  
Biofilm Formation ◽  
Dnase I ◽  
Extracellular Dna ◽  
Proteinase K ◽  
Strain Variation ◽  
Protein Ratio ◽  
Degrading Enzymes ◽  
Matrix Degrading Enzymes

Bacillus cereus is a foodborne pathogen and can form biofilms on food contact surfaces, which causes food hygiene problems. While it is necessary to understand strain-dependent variation to effectively control these biofilms, strain-to-strain variation in the structure of B. cereus biofilms is poorly understood. In this study, B. cereus strains from tatsoi (BC4, BC10, and BC72) and the ATCC 10987 reference strain were incubated at 30°C to form biofilms in the presence of the extracellular matrix-degrading enzymes DNase I, proteinase K, dispase II, cellulase, amyloglucosidase, and α-amylase to assess the susceptibility to these enzymes. The four strains exhibited four different patterns in terms of biofilm susceptibility to the enzymes as well as morphology of surface-attached biofilms or suspended cell aggregates. DNase I inhibited the biofilm formation of strains ATCC 10987 and BC4 but not of strains BC10 and BC72. This result suggests that some strains may not have extracellular DNA, or their extracellular DNA may be protected in their biofilms. In addition, the strains exhibited different patterns of susceptibility to protein- and carbohydrate-degrading enzymes. While other strains were resistant, strains ATCC 10987 and BC4 were susceptible to cellulase, suggesting that cellulose or its similar polysaccharides may exist and play an essential role in their biofilm formation. Our compositional and imaging analyses of strains ATCC 10987 and BC4 suggested that the physicochemical properties of their biofilms are distinct, as calculated by the carbohydrate to protein ratio. Taken together, our study suggests that the extracellular matrix of B. cereus biofilms may be highly diverse and provides insight into the diverse mechanisms of biofilm formation among B. cereus strains.

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.

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

scholarly journals Strain variation in Bacillus cereus biofilms and their susceptibility to extracellular matrix-degrading enzymes

2021 ◽  
Author(s):  
Eun Seob Lim ◽  
Seung-Youb Baek ◽  
Taeyoung Oh ◽  
Minseon Koo ◽  
Joo Young Lee ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Bacillus Cereus ◽  
Biofilm Formation ◽  
Dnase I ◽  
Extracellular Dna ◽  
Proteinase K ◽  
Strain Variation ◽  
Protein Ratio ◽  
Degrading Enzymes ◽  
Matrix Degrading Enzymes

Bacillus cereus is a foodborne pathogen and can form biofilms on food contact surfaces, which causes food hygiene problems. While it is necessary to understand strain-dependent variation to effectively control these biofilms, strain-to-strain variation in the structure of B. cereus biofilms is poorly understood. In this study, B. cereus strains from tatsoi and the ATCC 10987 reference strain were incubated at 30? to form biofilms in the presence of the extracellular matrix-degrading enzymes DNase I, proteinase K, dispase II, cellulase, amyloglucosidase, and α-amylase to assess the susceptibility to these enzymes. The four strains exhibited four different patterns in terms of biofilm susceptibility to the enzymes as well as morphology of surface-attached biofilms or suspended cell aggregates. DNase I inhibited the biofilm formation of strains ATCC 10987 and BC4 but not of strains BC10 and BC72. This result suggests that some strains may not have extracellular DNA, or their extracellular DNA may be protected in their biofilms. In addition, the strains exhibited different patterns of susceptibility to protein- and carbohydrate-degrading enzymes. While other strains were resistant, strains ATCC 10987 and BC4 were susceptible to cellulase, suggesting that cellulose or its similar polysaccharides may exist and play an essential role in their biofilm formation. Our compositional analysis of strains ATCC 10987 and BC4 suggested that the physicochemical properties of their biofilms are distinct, as calculated by the carbohydrate to protein ratio. Taken together, our study suggests that the extracellular matrix of B. cereus biofilms may be highly diverse and provides insight into the diverse mechanisms of biofilm formation among B. cereus strains.

scholarly journals Characterization of eDNA from the Clinical StrainAcinetobacter baumanniiAIIMS 7 and Its Role in Biofilm Formation

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Praveen K. Sahu ◽  
Pavithra S. Iyer ◽  
Amrita M. Oak ◽  
Karishma R. Pardesi ◽  
Balu A. Chopade
Keyword(s):  
Biofilm Formation ◽  
Membrane Vesicles ◽  
Restriction Enzymes ◽  
Dnase I ◽  
Extracellular Dna ◽  
Clinical Strain ◽  
Biofilm Inhibition ◽  
Whole Cell Lysate

Release of extracellular DNA (eDNA) was observed duringin vitrogrowth of a clinical strain ofAcinetobacter baumannii. Membrane vesicles (MV) of varying diameter (20–200 nm) containing DNA were found to be released by transmission electron microscopy (TEM) and atomic force microscopy (AFM). An assessment of the characteristics of the eDNA with respect to size, digestion pattern by DNase I/restriction enzymes, and PCR-sequencing, indicates a high similarity with genomic DNA. Role of eDNA in static biofilm formed on polystyrene surface was evaluated by biofilm augmentation assay using eDNA available in different preparations, for example, whole cell lysate, cell-free supernatant, MV suspension, and purified eDNA. Biofilm augmentation was seen up to 224.64%, whereas biofilm inhibition was 59.41% after DNase I treatment: confirming that eDNA facilitates biofilm formation inA. baumannii. This is the first paper elucidating the characteristics and role of eDNA inA. baumanniibiofilm, which may provide new insights into its pathogenesis.

scholarly journals Methicillin-ResistantStaphylococcus aureusBiofilms and Their Influence on Bacterial Adhesion and Cohesion

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Khulood Hamid Dakheel ◽  
Raha Abdul Rahim ◽  
Vasantha Kumari Neela ◽  
Jameel R. Al-Obaidi ◽  
Tan Geok Hun ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Protein A ◽  
Extracellular Dna ◽  
Proteinase K ◽  
Staphylococcal Protein A ◽  
Sodium Metaperiodate ◽  
Predominant Genotype ◽  
Dna And Rna ◽  
Proteinase K Treatment ◽  
Main Components

Twenty-five methicillin-resistantStaphylococcus aureus(MRSA) isolates were characterized by staphylococcal protein A gene typing and the ability to form biofilms. The presence of exopolysaccharides, proteins, and extracellular DNA and RNA in biofilms was assessed by a dispersal assay. In addition, cell adhesion to surfaces and cell cohesion were evaluated using the packed-bead method and mechanical disruption, respectively. The predominant genotype wasspatype t127 (22 out of 25 isolates); the majority of isolates were categorized as moderate biofilm producers. Twelve isolates displayed PIA-independent biofilm formation, while the remaining 13 isolates were PIA-dependent. Both groups showed strong dispersal in response to RNase and DNase digestion followed by proteinase K treatment. PIA-dependent biofilms showed variable dispersal after sodium metaperiodate treatment, whereas PIA-independent biofilms showed enhanced biofilm formation. There was no correlation between the extent of biofilm formation or biofilm components and the adhesion or cohesion abilities of the bacteria, but the efficiency of adherence to glass beads increased after biofilm depletion. In conclusion, nucleic acids and proteins formed the main components of the MRSA clone t127 biofilm matrix, and there seems to be an association between adhesion and cohesion in the biofilms tested.

scholarly journals Detection and Quantification of eDNA-Associated Bacterial Membrane Vesicles by Flow Cytometry

2019 ◽  
Vol 20 (21) ◽  
pp. 5307 ◽  
Author(s):  
Valentina Puca ◽  
Eva Ercolino ◽  
Christian Celia ◽  
Giuseppina Bologna ◽  
Luisa Di Marzio ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Microscopy ◽  
Biofilm Formation ◽  
Lactobacillus Reuteri ◽  
Bacterial Species ◽  
Cell Communication ◽  
Membrane Vesicles ◽  
Extracellular Dna ◽  
Clinical Samples ◽  
H Pylori

Bacteria generate membrane vesicles, which are structures known as extracellular vesicles (EVs), reported to be involved in different pathogenic mechanisms, as it has been demonstrated that EVs participate in biofilm formation, cell-to-cell communication, bacteria–host interactions, and nutrients supply. EVs deliver nucleic acids, proteins, and polysaccharides. It has been reported that Helicobacter pylori (H. pylori) and Lactobacillus reuteri (L. reuteri), of both planktonic and biofilm phenotypes, produce EVs carrying extracellular DNA (eDNA). Here, we used polychromatic flow cytometry (PFC) to identify, enumerate, and characterize EVs as well as the eDNA-delivering EV compartment in the biofilm and planktonic phenotypes of H.pylori ATCC 43629 and L. reuteri DSM 17938. Biofilm formation was demonstrated and analyzed by fluorescence microscopy, using a classical live/dead staining protocol. The enumeration of EVs and the detection of eDNA-associated EVs were performed by PFC, analyzing both whole samples (cells plus vesicles) and EVs isolated by ultracentrifugation confirm EVs isolated by ultracentrifugation. PFC analysis was performed relying on a known-size beaded system and a mix of three different fluorescent tracers. In detail, the whole EV compartment was stained by a lipophilic cationic dye (LCD), which was combined to PKH26 and PicoGreen that selectively stain lipids and DNA, respectively. Fluorescence microscopy results displayed that both H. pylori and L. reuteri produced well-structured biofilms. PFC data highlighted that, in both detected bacterial species, biofilms produced higher EVs counts when paralleled to the related planktonic phenotypes. Furthermore, the staining with PicoGreen showed that most of the generated vesicles were associated with eDNA. These data suggest that the use of PFC, set according to the parameters here described, allows for the study of the production of eDNA-associated EVs in different microbial species in the same or several phases of growth, thus opening new perspectives in the study of microbial derived EVs in clinical samples.

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