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SEM analysis of biofilms by S. mutans strains. Biofilms of S. mutans strains UA159 (panels 1 and 3) and TW14 (panels 2 and 4) were allowed to form for 24 h on hydroxylapatite disks that were deposited in 24-well cell culture clusters in semidefined BM medium with 20 mM glucose (panels 1 and 2) or sucrose (panels 3 and 4) as the supplemental carbohydrate sources. Data presented here are representative of three independent experiments.
Source publication
Streptococcus mutans, the primary etiological agent of human dental caries, has developed multiple mechanisms to colonize and form biofilms on
the tooth surface. The brpA gene codes for a predicted surface-associated protein with apparent roles in biofilm formation, autolysis, and cell division.
In this study, we used two models to further characte...
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Citations
... For biofilm grown in 96-well plate, the sessile populations were stained with 0.1% crystal violet and measured in absorbance at wavelength of 575 nm with a spectrophotometer. Biofilms grown in HA discs were analyzed by using a laser scanning confocal microscope (Olympus Fluoview BX61, Center Valley, PA, USA) similarly as described previously [17,30]. For confocal microscopic analysis, HA discs were briefly washed in phosphate-buffered saline (PBS), 20 mM pH 7.0 to remove loosely attached bacterial cells, and then stained using Live/Dead bacterial staining kit (Invitrogen, Waltham, MA, USA) for 30 min which confers live cell with green fluorescence and membrane-compromised and dead cells with red fluorescence. ...
... Biofilms were then dissected using a confocal laser scanning microscope with a 60× water immersion objective lens. Post-acquisition analysis was carried out using SLIDEBOOK 5.0 (Olympus) and COMSTAT 2.0 [31], and the average thickness, biovolume, and surface area of the biofilms were calculated and compared as detailed previously [30,31]. ...
Locus SMU.243 in Streptococcus mutans was annotated as a member of the DUF2207 family proteins highly conserved in all bacteria but with unknown function. To investigate its role in S. mutans physiology, a SMU.243-deficient mutant was constructed using allelic exchange mutagenesis, and the impacts of SMU.243 deletion on bacterial growth, stress tolerance response, and biofilm formation were analyzed. Compared to the wild-type UA159, S. mutans lacking SMU.243 displayed a reduced growth rate and a reduced overnight culture density (p < 0.01) when grown at low pH and in the presence of methyl viologen. Relative to the parent strain, the deficient mutant also had a reduced survival rate following incubation in a buffer of pH 2.8 (p < 0.01) and in a buffer containing hydrogen peroxide at 58 mM after 60 min (p < 0.001) and had a reduced capacity in biofilm formation especially in the presence of sucrose (p < 0.01). To study any ensuing functional/phenotypical links between SMU.243 and uppP, which is located immediately downstream of SMU.243 and encodes an undecaprenyl pyrophosphate phosphatase involved in recycling of carrier lipid undecaprenyl phosphate, a uppP deficient mutant was generated using allelic exchange mutagenesis. Unlike the SMU.243 mutant, deletion of uppP affected cell envelope biogenesis and caused major increases in susceptibility to bacitracin. In addition, two variant morphological mutants, one forming rough colonies and the other forming mucoid, smooth colonies, also emerged following the deletion of uppP. The results suggest that the SMU.243-encoded protein of the DUF2207 family in S. mutans plays an important role in stress tolerance response and biofilm formation, but unlike the downstream uppP, does not seem to be involved in cell envelope biogenesis, although the exact roles in S. mutans’ physiology awaits further investigation.
... Although not investigated here, our results suggest that inhibition of ATR development in common oral bacteria in the presence of L. reuteri ATCC PTA5289 could contribute to this effect. To shed light on possible mechanisms by which L. reuteri PTA5289 exerted an inhibitory effect on ATR development, we investigated the expression of three key genes known to be involved in acid tolerance in S. mutans (luxS, a global regulator of cellular function [26], brpA [39], and ldh [27]) during ATR induction in the presence and absence of L. reuteri ATCC PTA5289. The results showed a significant down-regulation of all three genes compared to when an ATR was induced in S. mutans alone. ...
Probiotic bacteria show promising results in prevention of the biofilm-mediated disease caries, but the mechanisms are not fully understood. The acid tolerance response (ATR) allows biofilm bacteria to survive and metabolize at low pH resulting from microbial carbohydrate fermentation. We have studied the effect of probiotic strains: Limosilactobacillus reuteri and Lacticaseibacillus rhamnosus on ATR induction in common oral bacteria. Communities of L. reuteri ATCC PTA5289 and Streptoccus gordonii, Streptococcus oralis, Streptococcus mutans or Actinomyces naeslundii in the initial stages of biofilm formation were exposed to pH 5.5 to allow ATR induction, followed by a low pH challenge. Acid tolerance was evaluated as viable cells after staining with LIVE/DEAD®BacLight™. The presence of L. reuteri ATCC PTA5289 caused a significant reduction in acid tolerance in all strains except S. oralis. When S. mutans was used as a model organism to study the effects of additional probiotic strains (L. reuteri SD2112, L. reuteri DSM17938 or L. rhamnosus GG) as well as L. reuteri ATCC PTA5289 supernatant on ATR development, neither the other probiotic strains nor supernatants showed any effect. The presence of L. reuteri ATCC PTA5289 during ATR induction led to down-regulation of three key genes involved in tolerance of acid stress (luxS, brpA and ldh) in Streptococci. These data suggest that live cells of probiotic L. reuteri ATCC PTA5289 can interfere with ATR development in common oral bacteria and specific strains of L. reuteri may thus have a role in caries prevention by inhibiting development of an acid-tolerant biofilm microbiota.
... The observed reduction in the expression of these genes may result in suppression of biofilm formation, cell adhesion and integrity of the cell wall, although the underlying mechanism is unknown. Similarly, brpA, which is involved in oxidative stress, acid tolerance and biofilm formation, has shown a 0.16-fold reduction in its expression [32,59] and the stringent response gene relA, which is involved in the accumulation of the bacterial alarmones [60], showed 0.030-fold downregulation, which may reduce stress tolerance and induce defective biofilm formation. ...
Introduction. Dextransucrase produced by Streptococcus mutans plays a vital role in the formation of dental caries by synthesizing exopolysaccharides from sucrose, which helps in the attachment of microbes to the tooth surface, causing caries. Exploring antibody production against S. mutans antigens could be an effective method to protect against dental caries.
Hypothesis. Dextransucrase antibodies may help in the prevention of caries formation by inhibiting essential cariogenic factors.
Aims. The aim of this study was to investigate the effects of dextransucrase antibodies on biofilm formation and certain associated cariogenic factors of S. mutans .
Methodology. Dextransucrase was purified from culture of S. mutans . The antisera against the enzyme were raised in rabbits. The effect of dextransucrase antibodies on biofilm formation was studied using scanning electron microscopy, fluorescence microscopy and quantitative real-time polymerase chain reaction. The effects of the antibodies on associated cariogenic factors were examined using established methods. The cross-reactivity of antibodies with human lung, liver, heart, thyroid and kidney tissues was evaluated by immunohistochemistry.
Results. Our findings showed impaired biofilm formation in S. mutans in the presence of dextransucrase antibodies. Genes associated with biofilm formation such as gtfB, gtfC, brpA, relA, Smu.630 and vicK were downregulated (50–97 %) by dextransucrase antibodies in S. mutans . The adherence of S. mutans to glass surface was reduced by 58 % and hydrophobicity was reduced by 55.2 % in the presence of the antibodies compared to the controls. Immunohistochemistry studies revealed no cross-reactivity of human tissues with dextransucrase antibodies.
Conclusions. These findings suggest that antibodies raised against dextransucrase exhibit a profound inhibitory effect on biofilm formation and vital cariogenic factors of S. mutans , which supports the contention that dextransucrase could be a promising antigen to study for its anticariogenic potential.
... ComD and ComE are involved in a twocomponent signal transduction system, a quorumsensing cascade of S. mutans [1]. The gene brpA encodes a predicted surface-associated protein which is known to regulate biofilm formation [53]. S. mutans can produce organic acid and endure acid in the enclosed biofilm environment, which results in demineralization [54]. ...
Streptococcus mutans is known as a contributor to dental caries. In this work, Lactobacillus pentosus MJM60383 was selected for its strong antagonistic activity against S. mutans and was characterized by good oral probiotic properties including lysozyme tolerance, adhesive ability to oral cells, good aggregation (auto-aggregation, co-aggregation) ability, hydrogen peroxide production and inhibition of biofilm formation of S. mutans. L. pentosus MJM60383 also exhibited safety as a probiotic characterized by no hemolytic activity, no D-lactate production, no biogenic amine production, and susceptibility to antibiotics. Furthermore, the biofilm formation of S. mutans was also significantly inhibited by the supernatant of L. pentosus MJM60383. An anti-biofilm mechanism study revealed that sucrose decomposition and the production of water-insoluble exopolysaccharides by S. mutans were inhibited by the treatment with L. pentosus MJM60383 supernatant. Real-time PCR analysis indicated that the supernatant of L. pentosus MJM60383 significantly inhibited the mRNA expression of S. mutans glycosyltransferases, which synthesize glucan to construct biofilm architecture and mediate bacterial adherence. Our study demonstrated L. pentosus MJM60383 as a potential oral probiotic and revealed its anti-biofilm mechanism.
... Dalia and Weiser described that an increase in the S. pneumoniae chain length in a mutant strain correlated with an increase in cell death through C3-mediated neutrophil opsonophagocytosis (20). Alternatively, other studies show that longer-chain phenotypes in mutants are associated with decreased biofilm formation and adherence and colonization abilities, like what is seen in our studies with BvaP (21,22). ...
Neonatal GBS disease is a major cause of morbidity and mortality, and maternal vaginal colonization is the leading risk factor for the disease. Colonization prevention would greatly impact the rates of disease transmission, but vaccine development has stalled as capsular polysaccharide vaccines have low immunogenicity in vivo. While these vaccines are still in development, the addition of a protein conjugate may prove fruitful in increasing immunogenicity and strain coverage across GBS serotypes.
... In acidic dental biofilm, which had the highest %PMS in our patients, spaP might not be predominantly expressed compared with gtfB and gbpB. The expression of brpA in S. mutans has been proposed to be associated with acid tolerance and biofilm development [15,17]. The increased brpA expression found in the present study might play an important role in dental biofilm cariogenicity after fixed orthodontic appliance insertion. ...
Background:
Dental caries commonly occurs during orthodontic treatment because fixed appliances can impede effective oral hygiene practices. This study investigated the effects of fixed orthodontic treatment on dental biofilm maturity and virulence gene (gtfB, ldh, brpA, spaP, luxS, and gbpB) expression.
Methods:
Dental biofilms and virulence gene expression were determined in 24 orthodontic patients before and after treatment of ≥6 months. A three-tone disclosing gel was used to stain dental biofilm and assess its maturity by its color change-pink (new dental biofilm), purple (mature dental biofilm), and light blue (cariogenic dental biofilm). Gene expression levels were determined using real-time PCR.
Results:
After fixed orthodontic appliance insertion, the percentage of new dental biofilm decreased, whereas that of cariogenic dental biofilm significantly increased (p < 0.05). There was no significant difference in the percentage of mature dental biofilm (p > 0.05). Fixed orthodontic appliances increased gtfB, ldh, brpA, and gbpB gene expression above 1.5-fold in dental biofilm. In contrast, there was no change in spaP or luxS gene expression after treatment.
Conclusions:
Fixed orthodontic appliance insertion induced ecological changes and cariogenic virulence gene expression in dental biofilm.
... Its activity might be affected by SCH-79797 since it is located at the cell membrane resulting in upregulation of atpD. Analogously, upregulation of brpA might arise as a response to SCH-79797 targeting the cell envelope [37] since this gene is related to the integrity of the cell envelope [38,39]. In this study, SCH-79797 did not show thorough germicidal action but prevented S. mutans biofilm formation, which made it having potential to be a brand-new anti-caries agent without affecting the oral microecological environment [40]. ...
Background
SCH-79797 was recently shown to be a broad-spectrum antibacterial agent with a dual-bactericidal mechanism. However, its anti-biofilm effect remains unknown.
Purpose
To investigate the effect of SCH-79797 on the biofilm formation of the cariogenic Streptococcus mutans
Methods and Results
Crystal violet staining, colony forming units count and MTT assays (for cell metabolic activity) revealed that S. mutans biofilm formation was significantly suppressed. In addition, virulence factors, including extracellular polysaccharides (investigated by bacterial/exopolysaccharide staining and the anthrone method) and acid production (investigated by lactic acid and supernatant pH detection) were also inhibited significantly. Moreover, the biofilm inhibitory effect of SCH-79797 was mediated through its repression of bacterial growth and not by a bactericidal effect, which was verified by growth curve and bacterial live/ dead staining, respectively. Quantitative real-time PCR results disclosed that SCH-79797 affected bacterial acid production and tolerance, polysaccharide synthesis and remodeling, biofilm formation and quorum sensing-related gene expression. In addition, SCH-79797 showed good biocompatibility as determined by cytotoxicity assays.
Conclusion
SCH-79797 had an anti-biofilm effect and showed application prospects in the control of dental caries.
... As far as molecular mechanism was concerned, we analyzed the transcription level of several amyloid related genes quantitatively. A large number of data suggest that bacterial cells interact or respond to a variety of genes when they are influenced with external stimulus (Wen et al. 2006). Amyloid formation was firmly related to srtA gene and pacR gene. ...
Amyloid fibrils are important scaffold in bacterial biofilms. Streptococcus mutans is an established cariogenic bacteria dwelling within biofilms, and C123 segment of P1 protein is known to form amyloid fibrils in S. mutans biofilms, among which C3 segment could serve as a promising anti-amyloid target due to its critical role in C123-P1 interactions. Recently, small molecules have been found to successfully inhibit biofilms by targeting amyloid fibrils. Thus, our study aimed to screen small molecules targeting C3 segment with the capacity to influence amyloid fibrils and S. mutans biofilms. In silico screening was utilized to discover promising small molecules, which were evaluated for their effects on bacterial cells and amyloid fibrils. We selected 99 small molecules and enrolled 55 small molecules named D1–D55 for crystal violet staining. Notably, D25 selectively inhibit S. mutans biofilms but had no significant influence on biofilms formed by Streptococcus gordonii and Streptococcus sanguinis , and D25 showed no bactericidal effects and low cytotoxicity. In addition, amyloid fibrils in free-floating bacteria, biofilms and purified C123 were quantified with ThT assays, and the differences were not statistically significant in the presence or absence of D25. Morphological changes of amyloid fibrils were visualized with TEM images, where amorphous aggregates were obvious coupled with long and atypical amyloid fibrils. Moreover, amyloid-related genes were upregulated in response to D25. In conclusion, D25 is a promising antimicrobial agent with the capacity to influence amyloid fibrils and inhibit S. mutans biofilms.
... Using RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/ rnahybrid) [44], target mRNAs were predicted to encode factors involved in acid tolerance, including the glutamate transporter components GlnQ and GlnM [45], Ffh, homologous to the signal recognition particle of E. coli [46], the surface-associated protein BrpA, homologous to LytR-CpsA-Psr family proteins [47], and RelA responsible for synthesis and hydrolysis of guanosine tetraphosphate/pentaphosphate [48] (glnQ, glnM, brpA, and relA for srn884837; ffh, brpA, and relA for srn133480). The expression levels of the two sRNAs were decreased with lowered pH (pH 6.5, 5.5, 4.5), while those of the corresponding putative target genes were increased, implying that these sRNAs have negative effects on expression of acid tolerance related genes. ...
Small regulatory RNAs (sRNAs) belong to a family of non-coding RNAs, and many of which regulate expression of genes via interaction with mRNA. The recent popularity of high-throughput next generation sequencers have presented abundant sRNA-related data, including sRNAs of several different oral bacterial species. Some sRNA candidates have been validated in terms of their expression and interaction with target mRNAs. Since the oral cavity is an environment constantly exposed to various stimuli, such as fluctuations in temperature and pH, and osmotic pressure, as well as changes in nutrient availability, oral bacteria require rapid control of gene expression for adaptation to such diverse conditions, while regulation via interactions of sRNAs with mRNA provides advantages for rapid adaptation. This review summarizes methods effective for identification and validation of sRNAs, as well as sRNAs identified to be associated with oral bacterial species, including cariogenic and periodontal pathogens, together with their confirmed and putative target genes.
... For example, the genes SMU.629 and SMU.1591 encode superoxide dismutase and catabolite control protein A, which are involved in the oxidative stress tolerance and the expression of biofilm-associated genes in Streptococcus mutans, respectively. SMU.629 and SMU.1591 were impressively increased in biofilm [4,5]. ...
Pathogenic oral biofilms are now recognized as a key virulence factor in many microorganisms that cause the heavy burden of oral infectious diseases. Recently, new investigations in the nanotechnology field have propelled the development of novel biomaterials and approaches to control bacterial biofilms, either independently or in combination with other substances such as drugs, bioactive molecules, and photosensitizers used in antimicrobial photodynamic therapy (aPDT) to target different cells. Moreover, nanoparticles (NPs) showed some interesting capacity to reverse microbial dysbiosis, which is a major problem in oral biofilm formation. This review provides a perspective on oral bacterial biofilms targeted with NP-mediated treatment approaches. The first section aims to investigate the effect of NPs targeting oral bacterial biofilms. The second part of this review focuses on the application of NPs in aPDT and drug delivery systems.
