Efficacy of the GentleWave System in the removal of biofilm from the mesial roots of mandibular molars before and after minimal instrumentation: An ex vivo study.

AIM: To compare the efficacy of Enterococcus faecalis biofilm removal using the GentleWave System (GWS) (Sonendo Inc, CA) on non-instrumented versus minimally instrumented root canal systems. METHODOLOGY: Thirty-four mandibular molars were autoclaved and allocated to four groups: Negative control (n = 5); positive control (n = 5); Group 1: non-instrumentation + GWS (NI + GWS) (n = 12); and Group 2: minimal instrumentation + GWS (MI + GWS) (n = 12). Of 34 samples, 24 samples with Vertucci type 2 configuration within the mesial root of each sample were allocated to Groups 1 and 2 and then matched based on the working length and root canal configuration. After inoculation of samples with E. faecalis for 3 weeks, the GWS was used on Group 1 without any instrumentation and Group 2 after instrumentation of mesial canals until size 20/06v. CFU and SEM analysis were used. RESULTS: Log10 (CFU/mL) from the positive control, and Group 1 and 2 were 7.41 ± 0.53, 3.41 ± 1.54, and 3.21 ± 1.54, respectively. Both groups showed a statistically significant difference in the reduction of viable E. faecalis cells compared to the positive control (Group 1 [p = .0001] and Group 2 [p < .0001]), whilst showing no significant difference between the two tested groups (p < .05). CONCLUSION: The use of GWS on the non-instrumented root canal system could be an effective disinfection protocol in removing the biofilm without dentin debris formation.

Genetic Analysis of Mutacin B-Ny266, a Lantibiotic Active against Caries Pathogens.

Bacteriocins are ribosomally synthesized proteinaceous antibacterial peptides. They selectively interfere with the growth of other bacteria. The production and secretion of bacteriocins confer a distinct ecological advantage to the producer in competing against other bacteria that are present in the same ecological niche. Streptococcus mutans, a significant contributor to the development of dental caries, is one of the most prolific producers of bacteriocins, known as mutacins in S. mutans In this study, we characterized the locus encoding mutacin B-Ny266, a lantibiotic with a broad spectrum of activity. The chromosomal locus is composed of six predicted operon structures encoding proteins involved in regulation, antimicrobial activity, biosynthesis, modification, transport, and immunity. Mutacin B-Ny266 was purified from semisolid cultures, and two inhibitory peptides, LanA and LanA', were detected. Both peptides were highly modified. Such modifications include dehydration of serine and threonine and the formation of a C-terminal aminovinyl-cysteine (AviCys) ring. While LanA peptide alone is absolutely required for antimicrobial activity, the presence of LanA' enhanced the activity of LanA, suggesting that B-Ny266 may function as a two-peptide lantibiotic. The activation of lanAA' expression is most likely controlled by the conserved two-component system NsrRS, which is activated by LanA peptide but not by LanA'. The chromosomal locus encoding mutacin B-Ny266 was not universally conserved in all sequenced S. mutans genomes. Intriguingly, the genes encoding LanAA' peptides were restricted to the most invasive serotypes of S. mutansIMPORTANCE Although dental caries is largely preventable, it remains the most common and costly infectious disease worldwide. Caries is initiated by the presence of dental plaque biofilm that contains Streptococcus mutans, a species extensively characterized by its role in caries development and formation. S. mutans deploys an arsenal of strategies to establish itself within the oral cavity. One of them is the production of bacteriocins that confer a competitive advantage by targeting and killing closely related competitors. In this work, we found that mutacin B-Ny266 is a potent lantibiotic that is effective at killing a wide array of oral streptococci, including nearly all S. mutans strains tested. Lantibiotics produced by oral bacteria could represent a promising strategy to target caries pathogens embedded in dental plaque biofilm.

Trans-cinnamaldehyde potently kills Enterococcus faecalis biofilm cells and prevents biofilm recovery.

Enterococcus faecalis is a biofilm-forming, nosocomial pathogen that is frequently isolated from failed root canal treatments. Contemporary root canal disinfectants are ineffective in eliminating these biofilms and preventing reinfection. As a result, there is a pressing need to identify novel and safe antibiofilm molecules. The effect of short-term (5 and 15 min) and long-term (24 h) treatments of trans-cinnamaldehyde (TC) on the viability of E. faecalis biofilms was compared with currently used root canal disinfectants. Treatment for 15 min with TC reduced biofilm metabolic activity as effective as 1% sodium hypochlorite and 2% chlorhexidine. Treatment with TC for 24 h was significantly more effective than 2% chlorhexidine in reducing the viable cell counts of biofilms. This serendipitous effect of TC was sustained for 10 days under growth-favoring conditions. For the first time, our study highlights the strong antibacterial activity of TC against E. faecalis biofilms, and notably, its ability to prevent biofilm recovery after treatment.

Association of Streptococcus mutans collagen binding genes with severe childhood caries.

OBJECTIVE: An important factor in the assessment of caries risk is the presence of specific oral microflora, especially Streptococcus mutans. Some S. mutans strains possess proteins capable of binding collagen, such as the Cnm and Cbm proteins. The aim is to determine the presence of S. mutans strains carrying collagen binding proteins in a group of subjects with severe early childhood caries (S-ECC). MATERIALS AND METHODS: S. mutans strains isolated from 15 S-ECC children were analyzed for collagen binding domains (cbd) of the cnm (cbd/cnm) and cbm (cbd/cbm) genes and their ability to bind to collagen. RESULTS: S. mutans strains positive for cbd/cnm or cbd/cbm were only found in 3 subjects with the most severe caries profile, with one subject having both cbd/cnm and cbd/cbm, and the other two with one of each. cnm/cbm-positive S. mutans strains bound to collagen substrate more avidly compared with negative S. mutans strains from each of the three groups. CONCLUSIONS: Our findings of an association between the presence of the collagen binding domains of the cnm/cbm genes in plaque S. mutans and the most aggressive form of caries profile in children offer a potential strategy to identify an individual's risk for caries progression. Our study should be replicated in other settings and communities in longitudinal and longer-term studies. CLINICAL RELEVANCE: Our data offer a potential tool in the caries risk management and assessment in children.

Short-term effects of fixed orthodontic appliance on concentrations of mutans streptococci and persister cells in adolescents.

INTRODUCTION: Orthodontic patients are at an increased risk for developing caries. Dental caries is a biofilm-mediated disease, with mutans streptococci (MS) as the primary etiologic bacterial group. It has been suggested that persister cells (PCs), a subset of cells within the biofilm, contribute to the chronic infectious nature of dental caries. PC formation can be induced by environmental stressors such as orthodontic treatment. The aim of this study was to quantify MS, aerobic and facultative anaerobe bacterial PC proportions from plaque samples during the initial stage of orthodontic treatment. This study is the first to analyze the role of PCs in a population of patients highly susceptible to caries, that is, patients undergoing orthodontic treatment. METHODS: Plaque samples were collected from 17 participants (11 males and 6 females; age range: 11-18 years) before and 1 month after insertion of fixed orthodontic appliances. Percentages of MS and PCs were determined with selective media and a classical persister microbial assay, respectively. RESULTS: There was a statistically significant decrease in %MS (P = 0.039) but no statistically significant difference in %PCs (P = 0.939) after 1 month of orthodontic appliance placement. CONCLUSION: Our study illustrated the technical feasibility of analysis of PCs in plaque samples of patients during orthodontic treatment and revealed that PC formation during orthodontic treatment is highly variable across individuals.

The formation of Streptococcus mutans persisters induced by the quorum-sensing peptide pheromone is affected by the LexA regulator.

The presence of multidrug-tolerant persister cells within microbial populations has been implicated in the resiliency of bacterial survival against antibiotic treatments and is a major contributing factor in chronic infections. The mechanisms by which these phenotypic variants are formed have been linked to stress response pathways in various bacterial species, but many of these mechanisms remain unclear. We have previously shown that in the cariogenic organism Streptococcus mutans, the quorum-sensing peptide CSP (competence-stimulating peptide) pheromone was a stress-inducible alarmone that triggered an increased formation of multidrug-tolerant persisters. In this study, we characterized SMU.2027, a CSP-inducible gene encoding a LexA ortholog. We showed that in addition to exogenous CSP exposure, stressors, including heat shock, oxidative stress, and ofloxacin antibiotic, were capable of triggering expression of lexA in an autoregulatory manner akin to that of LexA-like transcriptional regulators. We demonstrated the role of LexA and its importance in regulating tolerance toward DNA damage in a noncanonical SOS mechanism. We showed its involvement and regulatory role in the formation of persisters induced by the CSP-ComDE quorum-sensing regulatory system. We further identified key genes involved in sugar and amino acid metabolism, the clustered regularly interspaced short palindromic repeat (CRISPR) system, and autolysin from transcriptomic analyses that contribute to the formation of quorum-sensing-induced persister cells.

The copYAZ Operon Functions in Copper Efflux, Biofilm Formation, Genetic Transformation, and Stress Tolerance in Streptococcus mutans.

UNLABELLED: In bacteria, copper homeostasis is closely monitored to ensure proper cellular functions while avoiding cell damage. Most Gram-positive bacteria utilize the copYABZ operon for copper homeostasis, where copA and copB encode copper-transporting P-type ATPases, whereas copY and copZ regulate the expression of the cop operon. Streptococcus mutans is a biofilm-forming oral pathogen that harbors a putative copper-transporting copYAZ operon. Here, we characterized the role of copYAZ operon in the physiology of S. mutans and delineated the mechanisms of copper-induced toxicity in this bacterium. We observed that copper induced toxicity in S. mutans cells by generating oxidative stress and disrupting their membrane potential. Deletion of the copYAZ operon in S. mutans strain UA159 resulted in reduced cell viability under copper, acid, and oxidative stress relative to the viability of the wild type under these conditions. Furthermore, the ability of S. mutans to form biofilms and develop genetic competence was impaired under copper stress. Briefly, copper stress significantly reduced cell adherence and total biofilm biomass, concomitantly repressing the transcription of the gtfB, gtfC, gtfD, gbpB, and gbpC genes, whose products have roles in maintaining the structural and/or functional integrity of the S. mutans biofilm. Furthermore, supplementation with copper or loss of copYAZ resulted in significant reductions in transformability and in the transcription of competence-associated genes. Copper transport assays revealed that the ΔcopYAZ strain accrued significantly large amounts of intracellular copper compared with the amount of copper accumulation in the wild-type strain, thereby demonstrating a role for CopYAZ in the copper efflux of S. mutans. The complementation of the CopYAZ system restored copper expulsion, membrane potential, and stress tolerance in the copYAZ-null mutant. Taking these results collectively, we have established the function of the S. mutans CopYAZ system in copper export and have further expanded knowledge on the importance of copper homeostasis and the CopYAZ system in modulating streptococcal physiology, including stress tolerance, membrane potential, genetic competence, and biofilm formation. IMPORTANCE: S. mutans is best known for its role in the initiation and progression of human dental caries, one of the most common chronic diseases worldwide. S. mutans is also implicated in bacterial endocarditis, a life-threatening inflammation of the heart valve. The core virulence factors of S. mutans include its ability to produce and sustain acidic conditions and to form a polysaccharide-encased biofilm that provides protection against environmental insults. Here, we demonstrate that the addition of copper and/or deletion of copYAZ (the copper homeostasis system) have serious implications in modulating biofilm formation, stress tolerance, and genetic transformation in S. mutans. Manipulating the pathways affected by copper and the copYAZ system may help to develop potential therapeutics to prevent S. mutans infection in and beyond the oral cavity.

Cell death of Streptococcus mutans induced by a quorum-sensing peptide occurs via a conserved streptococcal autolysin.

Streptococcus mutans, a member of the human indigenous oral microbiome, produces a quorum-sensing peptide called the competence-stimulating peptide (CSP) pheromone. We previously demonstrated that S. mutans expresses its CSP pheromone under specific stresses and responds to high levels of CSP by inducing cell death in a fraction of the bacterial population. Streptococci lack the classical SOS response, and the induction of the SigX regulon has been proposed to act as a general stress response in Gram-positive bacteria. We show here that inactivation of SigX abolished the CSP-induced cell death phenotype. Among SigX-regulated genes, SMU.836 (now named lytF(Sm)), encoding a conserved streptococcal protein, is a functional peptidoglycan hydrolase involved in CSP-induced cell lysis. We also demonstrated that LytF(Sm) is most likely a self-acting autolysin, since LytF(Sm) produced by attacker cells cannot trigger CSP-induced lysis of LytF(Sm)-deficient target cells present in the same environment. Electron microscopy revealed important morphological changes accompanying autolysis of CSP-induced wild-type cultures that were absent in the LytF(Sm)-deficient mutant. The LytF(Sm) promoter was activated in the physiological context of elevated concentrations of the CSP pheromone under stress conditions, such as exposure to heat, hydrogen peroxide, and acid. In a long-term survival assay, the viability of a mutant deficient in LytF(Sm) autolysin was significantly lower than that observed for the wild-type strain. The results of this study suggest that cell death of S. mutans induced by its quorum-sensing CSP pheromone may represent a kind of altruistic act that provides a way for the species to survive environmental stresses at the expense of some of its cells.

Microencapsulation of Probiotic Streptococcus salivarius LAB813.

Probiotics are living microorganisms that confer a health benefit on the host when administered in adequate amounts. Streptococcus salivarius, a commensal bacterium found in the oral cavity, has been shown to secrete antimicrobial peptides and can be used as probiotics. This study aimed to develop a delivery system for the probiotic LAB813, a novel S. salivarius strain first identified in the laboratory. Probiotics can be delivered and protected through the encapsulation of biomaterials such as polysaccharides. Their biocompatibility, biodegradability, user-friendliness, and ease of access make polysaccharides useful for encapsulating probiotics. Alginate (Alg) and chitosan (Ch) are naturally obtained polysaccharides and, hence, tested for LAB813 encapsulation. An extrusion method of encapsulation was performed to form Alg microcapsules (Alg-LAB813), some of which were coated with Ch (Alg-LAB813-Ch) to provide dual-layered protection. Inhibitory assays of the Alg-LAB813 and Alg-LAB813-Ch microcapsules were assayed against an indicator strain. Alg-LAB813-Ch microcapsules showed superior antibacterial properties compared to Alg-LAB813 microcapsules over 24 h and when subject to temperatures ranging from 4 to 68 °C. In addition, Alg-LAB813-Ch microcapsules retained antibacterial activity for up to 28 days of storage at 4 °C. The strong and sustained inhibitory activities of Ch-coated Alg encapsulated LAB813 signify the potential for their use to improve oral health.

Transcriptome Analysis of Streptococcus mutans Quorum Sensing-Mediated Persisters Reveals an Enrichment in Genes Related to Stress Defense Mechanisms.

Persisters are a small fraction of growth-arrested phenotypic variants that can survive lethal concentrations of antibiotics but are able to resume growth once antibiotics are stopped. Their formation can be a stochastic process or one triggered by environmental cues. In the human pathogen Streptococcus mutans, the canonical peptide-based quorum-sensing system is an inducible DNA repair system that is pivotal for bacterial survival. Previous work has shown that the CSP-signaling peptide is a stress-signaling alarmone that promotes the formation of stress-induced persisters. In this study, we exposed S. mutans to the CSP pheromone to mimic DNA damage conditions and isolated the antibiotic persisters by treating the cultures with ofloxacin. A transcriptome analysis was then performed to evaluate the differential gene expression between the normal stationary-phase cells and the persisters. RNA sequencing revealed that triggered persistence was associated with the upregulation of genes related to several stress defense mechanisms, notably, multidrug efflux pumps, the arginine deaminase pathway, and the Opu/Opc system. In addition, we showed that inactivation of the VicK kinase of the YycFG essential two-component regulatory system abolished the formation of triggered persisters via the CSP pheromone. These data contribute to the understanding of the triggered persistence phenotype and may suggest new therapeutic strategies for treating persistent streptococcal infections.

Antimicrobial activity of probiotic Streptococcus salivarius LAB813 on in vitro cariogenic biofilms.

OBJECTIVE: To investigate the antimicrobial activity of a novel commensal strain of Streptococcus salivarius, LAB813, against Streptococcus mutans biofilms. METHODS: The inhibitory activity of LAB813 towards S. mutans was tested using mono-, dual-, and multi-species cariogenic biofilms formed on three types of orthodontic appliances (metal, ceramic, aligner). The activity of the commercially available probiotic, BLIS M18™ was used as control. RESULTS: LAB813 significantly inhibited S. mutans biofilms with cell killing approximating 99% for all materials. LAB813 showed effectiveness at inhibiting S. mutans in more complex multi-species biofilms with cell killing approximating 90% for all three materials. When comparing the killing kinetics of the probiotics, LAB813 had a faster rate of killing biofilms than M18. Experiments conducted with cell-free culture supernatant confirmed the presence of an inhibitory substance of proteinaceous nature. The addition of xylitol, a common sugar substitute used for human consumption, potentiated the inhibitory effects of LAB813 against S. mutans embedded in a more complex fungal-bacterial biofilm. CONCLUSIONS: LAB813 possesses strong antimicrobial activity, potent anti-biofilm properties, and enhanced antimicrobial activity in the presence of xylitol. The identification and characterization of strain LAB813 exhibiting antimicrobial activity towards S. mutans hold exciting promise for this novel strain to be developed as an oral probiotic for use in the prevention of dental caries.

A stress-inducible quorum-sensing peptide mediates the formation of persister cells with noninherited multidrug tolerance.

Within a given microbial population, a small subpopulation known as dormant persister cells exists. This persistence property ensures the survival of the population as a whole in the presence of lethal factors. Although persisters are highly important in antibiotic therapy, the mechanism for persistence is still not thoroughly understood. We show here that the cariogenic organism Streptococcus mutans forms persister cells showing noninherited multidrug tolerance. We demonstrated that the ectopic expression of the type II toxin-antitoxin systems, MazEF and RelBE, caused an increase in the number of persisters. In a search for additional persistence genes, an expression library was constructed, and several clones exhibiting a significant difference in persister formation after prolonged antibiotic treatment were selected. The candidate persister genes include genes involved in transcription/replication, sugar metabolism, cell wall synthesis, and energy metabolism, clearly pointing to redundant pathways for persister formation. We have previously reported that the S. mutans quorum-sensing peptide, CSP pheromone, was a stress-inducible alarmone capable of conveying sophisticated messages in the bacterial population. In this study, we demonstrate the involvement of the intraspecies quorum-sensing system during the formation of stress-induced multidrug-tolerant persisters. To the best of our knowledge, this is the first study reporting the induction of bacterial persistence using a quorum-sensing regulatory system.

Identification of a novel adhesin involved in acid-induced adhesion of enterohaemorrhagic Escherichia coli O157 : H7.

Enterohaemorrhagic Escherichia coli (EHEC) survives exposure to acute acid stress during gastric passage and progresses to colonize the large intestine. We previously reported that acid stress significantly increases host adhesion of EHEC O157 : H7 and is associated with a coincident upregulation of the expression of a putative adhesin gene, yadK. Further gene expression analysis now confirms that yadK is minimally transcribed under unstressed conditions and is significantly upregulated under acid stress. Immunoblotting with an anti-YadK polyclonal antiserum demonstrates that YadK protein is also upregulated after acid stress. Disruption of yadK results in loss of the acid-induced adhesion increase seen for wild-type EHEC to human epithelial cells in vitro and complementation in trans fully restores the acid-induced adhesion phenotype to the wild-type level. Significantly, no difference is observed in adhesion of the unstressed yadK mutant relative to wild-type, indicating that YadK does not play a role in adhesion of unstressed EHEC. Anti-YadK antiserum inhibits the acid-induced adhesion enhancement of EHEC but has no effect on adhesion of unstressed EHEC. There is no significant difference in the viability of either the unstressed or the acid-stressed yadK mutant relative to the similarly treated wild-type, suggesting that yadK is not involved in acid tolerance. These results provide persuasive evidence that YadK plays a significant role in the adhesion of acid-stressed EHEC to epithelial cells, and support a role for acid stress as a factor which may regulate bacteria-host attachment and lead to increased EHEC colonization and virulence.

Chromosomal bacterial type II toxin-antitoxin systems.

Most prokaryotic chromosomes contain a number of toxin-antitoxin (TA) modules consisting of a pair of genes that encode 2 components, a stable toxin and its cognate labile antitoxin. TA systems are also known as addiction modules, since the cells become "addicted" to the short-lived antitoxin product (the unstable antitoxin is degraded faster than the more stable toxin) because its de novo synthesis is essential for their survival. While toxins are always proteins, antitoxins are either RNAs (type I, type III) or proteins (type II). Type II TA systems are widely distributed throughout the chromosomes of almost all free-living bacteria and archaea. The vast majority of type II toxins are mRNA-specific endonucleases arresting cell growth through the mechanism of RNA cleavage, thus preventing the translation process. The physiological role of chromosomal type II TA systems still remains the subject of debate. This review describes the currently known type II toxins and their characteristics. The different hypotheses that have been proposed to explain their role in bacterial physiology are also discussed.

Fsr quorum sensing system modulates the temporal development of Enterococcus faecalis biofilm matrix.

Quorum sensing (QS) is a cell-to-cell communication process that regulates major pathogenic attributes in bacteria including biofilm formation, secretion of virulence factors, and antimicrobial resistance. The two-component Fsr-QS system of the nosocomial pathogen Enterococcus faecalis controls the production of extracellular gelatinase that contributes to biofilm development by enhancing the release of nucleic acids into the biofilm matrix. However, the contribution of this system to the deposition of other biofilm matrix components such as polysaccharides and proteins remains unknown. Using wild type and mutant strains, we discovered that biofilm formation was attenuated by inactivation of the Fsr system or its downstream gelatinase production. Inactivation of the Fsr system caused a modest, yet significant reduction in biofilm metabolic activity without affecting cell counts. Inactivation of the QS-signal sensor FsrC and response regulator FsrA resulted in decreased extracellular polysaccharides and proteins in biofilms in a temporal manner. Irrespective of biofilm age, eDNA levels were reduced in the gelatinase mutant strain. Our results collectively suggest that the Fsr system contributes to the temporal deposition of polysaccharides and proteins into the extracellular polymeric matrix (EPS) of E. faecalis biofilm, without affecting bacterial viability. This understanding of the role of the Fsr-QS system in biofilm development may reveal a novel target to develop effective antibiofilm agents to tackle E. faecalis-mediated infections such as in dental root canals, heart valves, and surgical sites.

A DNA-Damage Inducible Gene Promotes the Formation of Antibiotic Persisters in Response to the Quorum Sensing Signaling Peptide in Streptococcus mutans.

Bacteria use quorum sensing (QS) to communicate with each other via secreted small autoinducers produced by individuals. QS allows bacteria to display a unified response that benefits the species during adaptation to environment, colonization, and defense against competitors. In oral streptococci, the CSP-ComDE QS is an inducible DNA damage repair system that is pivotal for bacterial survival. In the oral pathogen Streptococcus mutans, the QS system positively influences the formation of antibiotic persisters, cells that can survive antibiotic attack by entering a non-proliferative state. We recently identified a novel gene, pep299, that is activated in the persister cell fraction induced by QS. In this study, we focused our investigation on the role of pep299, a gene encoding a bacteriocin-like peptide, in the formation of antibiotic persisters. Mutant Δ299, unable to produce Pep299, showed a dramatic reduction in the number of stress-induced persisters. Using a co-culture assay, we showed that cells overproducing pep299 induced the formation of persisters in the mutant, suggesting that Pep299 was actively secreted and detected by neighboring cells. Cells exposed to DNA damage conditions activated the gene expression of pep299. Interestingly, our results suggested that the pep299 gene was also involved in the regulation of a QS-inducible toxin−antitoxin system. Our study suggests that the pep299 gene is at the core of the triggered persistence phenotype in S. mutans, allowing cells to transition into a state of reduced metabolic activity and antibiotic tolerance.

Interfacial Biomaterial-Dentin Bacterial Biofilm Proliferation and Viability Is Affected by the Material, Aging Media and Period.

Biomaterial−dentin interfaces undergo degradation over time, allowing salivary, tissue fluid, and bacterial movement between the root filling or restoration and dentin. This study aims to investigate the effect of aging in simulated human salivary/bacterial/blood esterases (SHSE) on proliferation and viability of Enterococcus faecalis biofilm within the dentin interface with four materials used to fill/restore the endodontic space. Root canals of human anterior teeth were prepared and filled with gutta-percha and one of the following: self-cured resin composite (BisfilTM 2B, Bisco, Schaumburg, IL, USA) with either self-etch (SE) (EasyBond) or total-etch (TE) (ScotchbondTM, 3M, Saint Paul, MN, USA) methacrylate-based adhesives, epoxy-resin sealer (AH Plus®, Dentsply Sirona, York, PA, USA), or bioceramic sealer (EndoSequence® BC Sealer™, Brasseler USA, Savannah, GA, USA). Specimens were aged in SHSE or phosphate-buffered saline (PBS) for up to 360 days, followed by cultivation of steady-state E. faecalis biofilm. Depth and viability of interfacial bacterial biofilm proliferation were assessed by confocal laser scanning microscopy and live/dead staining. Data were analyzed using three-way ANOVA and Scheffe's post hoc analyses. Initial depths of biofilm proliferation were similar among material groups (p > 0.05). All groups showed significantly deeper biofilm proliferation with increased aging period (p < 0.05). SHSE aging increased interfacial biofilm depth for TE, SE and BC (p < 0.05) but not AH. For unaged interfaces, BC exhibited the lowest ratio of live bacteria, followed by AH, TE, and SE (p < 0.05). Interfacial bacterial biofilm proliferation and viability were dependent on the biomaterial, aging media, and period.

Effectiveness of the Lorodent Probiotic Lozenge in Reducing Plaque and Streptococcus mutans Levels in Orthodontic Patients: A Double-Blind Randomized Control Trial.

Orthodontic patients are at a significant risk for oral diseases due to increased plaque accumulation and oral bacterial dysbiosis. We aimed to determine the efficacy of the commercially available Lorodent Probiotic Complex at reducing plaque accumulation and Streptococcus mutans bacterial levels in adolescent orthodontic patients. Sixty adolescents undergoing fixed orthodontic treatment for a minimum of 6 months were recruited in a randomized, double-blind, parallel-group, placebo-controlled trial. They received either Lorodent probiotic lozenge (intervention, n = 30) or placebo lozenge (control, n = 30) orally every day for a 28-day administration period. Participants were assessed at four appointments (T1-T4) over a total of 56 days. Compliance and lozenge satisfaction were monitored. Saliva samples and supragingival plaques were collected for evaluation of S. mutans levels. Clinical assessment using a Plaque Index (PI) was used. Compliance with lozenge intake of all participants was over 90%. There was no significant change in the PI and composite PI scores in both placebo and probiotic groups at each time frame (all p > 0.05) or the relative S. mutans DNA levels in the saliva and plaque between the probiotic and placebo groups. The findings of high compliance and satisfaction with the probiotic lozenges combined with the study's rigorous design offer a baseline for subsequent testing of further potential probiotics (of varying formulations, concentrations), especially in adolescents.

Regulation of the competence pathway as a novel role associated with a streptococcal bacteriocin.

The oral biofilm organism Streptococcus mutans must face numerous environmental stresses to survive in its natural habitat. Under specific stresses, S. mutans expresses the competence-stimulating peptide (CSP) pheromone known to induce autolysis and facilitate the uptake and incorporation of exogenous DNA, a process called DNA transformation. We have previously demonstrated that the CSP-induced CipB bacteriocin (mutacin V) is a major factor involved in both cellular processes. Our objective in this work was to characterize the role of CipB bacteriocin during DNA transformation. Although other bacteriocin mutants were impaired in their ability to acquire DNA under CSP-induced conditions, the ΔcipB mutant was the only mutant showing a sharp decrease in transformation efficiency. The autolysis function of CipB bacteriocin does not participate in the DNA transformation process, as factors released via lysis of a subpopulation of cells did not contribute to the development of genetic competence in the surviving population. Moreover, CipB does not seem to participate in membrane depolarization to assist passage of DNA. Microarray-based expression profiling showed that under CSP-induced conditions, CipB regulated ∼130 genes, among which are the comDE locus and comR and comX genes, encoding critical factors that influence competency development in S. mutans. We also discovered that the CipI protein conferring immunity to CipB-induced autolysis also prevented the transcriptional regulatory activity of CipB. Our data suggest that besides its role in cell lysis, the S. mutans CipB bacteriocin also functions as a peptide regulator for the transcriptional control of the competence regulon.

The chromosomal mazEF locus of Streptococcus mutans encodes a functional type II toxin-antitoxin addiction system.

Type II chromosomal toxin-antitoxin (TA) modules consist of a pair of genes that encode two components: a stable toxin and a labile antitoxin interfering with the lethal action of the toxin through protein complex formation. Bioinformatic analysis of Streptococcus mutans UA159 genome identified a pair of linked genes encoding a MazEF-like TA. Our results show that S. mutans mazEF genes form a bicistronic operon that is cotranscribed from a σ70-like promoter. Overproduction of S. mutans MazF toxin had a toxic effect on S. mutans which can be neutralized by coexpression of its cognate antitoxin, S. mutans MazE. Although mazF expression inhibited cell growth, no cell lysis of S. mutans cultures was observed under the conditions tested. The MazEF TA is also functional in E. coli, where S. mutans MazF did not kill the cells but rather caused reversible cell growth arrest. Recombinant S. mutans MazE and MazF proteins were purified and were shown to interact with each other in vivo, confirming the nature of this TA as a type II addiction system. Our data indicate that MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a complex. Our results suggest that the MazEF TA module might represent a cell growth modulator facilitating the persistence of S. mutans under the harsh conditions of the oral cavity.