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Purpose: Antibacterial dental implants and related prosthetic components could help to reduce infection and prevent peri-implantitis. The purpose of this study was to determine the effect of ultraviolet (UV) light treatment of titanium on biofilm formation of human oral bacteria., Materials and Methods: Machine-prepared commercially pure titanium disks were treated with UV light for 12 minutes. Human oral bacteria were seeded onto untreated and UV-treated disks. Early bacterial attachment to titanium was assessed at 12 hours. Surface topography of initial biofilms was evaluated by 3D scanning electron microscopy at 24 hours. The quantity and morphology of subsequent colony development and biofilm formation were examined by confocal laser scanning microscopy for up to 7 days., Results: Throughout the time course, significantly fewer bacterial cells attached to UV-treated titanium surfaces compared to untreated ones. While biofilm developed rapidly to a final thickness of about 16 μm by day 3 on untreated titanium, on UV-treated surfaces it remained below 8 μm, even at day 7. Similarly, UV treatment resulted in 70% less exopolysaccharides (EPS) volume than on untreated surfaces at day 7. This is consistent with the finding that EPS production per cell was significantly lower on UV-treated surfaces. Untreated titanium surfaces covered with biofilm were 5-fold rougher than the original machined surface, while UV-treated surfaces remained 2-fold rougher due to a significantly less biofilm formation., Conclusion: UV treatment of titanium surfaces significantly reduces attachment of human oral bacteria and subsequent biofilm formation as well as EPS production for at least 7 days. UV treatment prevented the escalation of surface colonization, mitigating an unfavorable bacteriophilic cascade and environmental trigger for biofilm formation.

The human oral cavity is home to a large number of bacteria and bacteriophages (phages). However, the biology of oral phages as members of the human microbiome is not well understood. Recently, we isolated Actinomyces odontolyticus subsp. actinosynbacter strain XH001 from the human oral cavity, and genomic analysis revealed the presence of an intact prophage named xhp1. Here, we demonstrated that xhp1 is a linear plasmid-like prophage, which is a newly identified phage of A. odontolyticus The prophage xhp1 genome is a 35-kb linear double-stranded DNA with 10-bp single-stranded, 3' cohesive ends. xhp1 exists extrachromosomally, with an estimated copy number of 5. Annotation of xhp1 revealed 54 open reading frames, while phylogenetic analysis suggests that it has limited similarity with other phages. xhp1 phage particles can be induced by mitomycin C and belong to the Siphoviridae family, according to transmission electron microscopic examination. The released xhp1 particles can reinfect the xhp1-cured XH001 strain and result in tiny blurry plaques. Moreover, xhp1 promotes XH001 biofilm formation through spontaneous induction and the release of host extracellular DNA (eDNA). In conclusion, we identified a linear plasmid-like prophage of A. odontolyticus , which enhances bacterial host biofilm assembly and could be beneficial to the host for its persistence in the oral cavity. IMPORTANCE The biology of phages as members of the human oral microbiome is understudied. Here, we report the characterization of xhp1, a novel linear plasmid-like prophage identified from a human oral isolate, Actinomyces odontolyticus subsp. actinosynbacter strain XH001. xhp1 can be induced and reinfect xhp1-cured XH001. The spontaneous induction of xhp1 leads to the lysis of a subpopulation of bacterial hosts and the release of eDNA that promotes biofilm assembly, thus potentially contributing to the persistence of A. odontolyticus within the oral cavity., (Copyright © 2018 American Society for Microbiology.)

Statement of Problem: Peri-implantitis is considered the most important biological complication responsible for late implant failure. The physical chemical properties intrinsic to each material can affect the first step to biofilm development and is an important precursor to the adaptive behavior of pathogenic bacteria species., Purpose: The purpose of this in vitro study was to evaluate the effect of 2 commercially available implant abutment materials on the adhesion phase and biofilm formation., Material and Methods: Disks (8 mm in diameter, 2 mm thick) of machined pure titanium (Ti) and yttrium-stabilized zirconia (ZrO 2 ) materials were used to mimic implant abutments. The physical chemical surface properties were investigated using different approaches. Initial adherent bacteria and biofilm formation were evaluated after 16 and 48 hours by incubating the disks in a rich medium containing representative saliva-derived oral microbial community. Unpaired t test, 2 tailed, was used to compare the groups., Results: Ti presented lower hydrophobicity and surface free energy values than the ZrO 2 , and 6.1-fold fewer bacteria adhered to the Ti. After 48 hours, detailed quantitative analysis showed that biofilm biomass and biofilm density were lower on the Ti disks than on ZrO 2 . The quantity of phylotypes on the Ti and ZrO 2 surfaces was relatively similar during the attachment and early biofilm formation periods., Conclusions: Although no difference in the bacteria profile was observed between both materials independent of the time point, the highest level of colonization was on ZrO 2 ., (Copyright © 2017 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.)

Myxococcus xanthus is a Gram-negative soil bacterium capable of performing sophisticated cellular behaviors and growing one of the most intricate bacterial single-species biofilms in nature. During the process of biofilm formation, social behaviors of M. xanthus cells dominate key steps of the biofilm establishment, e.g., cellular motility on solid surface, predatory behavior by the grouped cells, kin recognition in the community, fruiting body development, myxospore differentiation, and programmed cell death. This review introduces the recent research progress about the M. xanthus biofilms.

To successfully colonize the oral cavity, bacteria must directly or indirectly adhere to available oral surfaces. Fusobacterium nucleatum plays an important role in oral biofilm community development due to its broad adherence abilities, serving as a bridge between members of the oral biofilm that cannot directly bind to each other. In our efforts to characterize the molecular mechanisms utilized by F. nucleatum to physically bind to key members of the oral community, we investigated the involvement of F. nucleatum outer membrane proteins in its ability to bind to the pioneer biofilm colonizer, Streptococcus gordonii. Here, we present evidence that in addition to the previously characterized fusobacterial adhesin RadD, the interaction between F. nucleatum ATCC 23726 and S. gordonii V288 involves a second outer membrane protein, which we named coaggregation mediating protein A (CmpA). We also characterized the role of CmpA in dual-species biofilm formation with S. gordonii V288, evaluated growth-phase-dependent as well as biofilm expression profiles of radD and cmpA, and confirmed an important role for CmpA, especially under biofilm growth conditions. Our findings underscore the complex set of specific interactions involved in physical binding and thus community integration of interacting bacterial species. This complex set of interactions could have critical implications for the formation and maturation of the oral biofilms in vivo, and could provide clues to the mechanism behind the distribution of organisms inside the human oral cavity., (© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Surface attachment is the first step in biofilm formation, and the ability of bacteria to adhere to surfaces and develop a biofilm is directly influenced by electrostatic interactions between the bacteria and the chemical composition of material surfaces. Here, we investigated the influence of physical and chemical characteristics of titanium (Ti) and zirconia (ZrO2) as implant abutment surfaces on the bacterial adhesion phase and compared the results to bovine enamel (BE) simulating a human tooth. To achieve this goal, we used 2 common pathogens of the oral cavity, Streptococcus mutans UA140 and Porphyromonas gingivalis 33277. To investigate the influence of material surfaces on bacterial adhesion, we studied the surface free energy as well as the topography by atomic force microscopy, and the chemical elements composition by scanning electron microscopy equipped with an energy dispersive X-ray spectroscope. Our results indicated a hydrophobic characteristic for all of the materials; however, the presence of polar and nonpolar components could aid in understanding why greater numbers of bacteria had adhered to BE compared to the other surfaces. Our confocal microscopy data support the proposition that electrostatic interactions, indeed, affected the initial adhesion phase. Within the limitations of a laboratory study, the results revealed bacterial adhered on BE and no bacteria could be observed by confocal images on Ti and ZrO2 implant abutment surfaces.

Bacterial biofilm infections remain prevalent reasons for implant failure. Dental implant placement occurs in the oral environment, which harbors a plethora of biofilm-forming bacteria. Due to its trans-mucosal placement, part of the implant structure is exposed to oral cavity and there is no effective measure to prevent bacterial attachment to implant materials. Here, we demonstrated that UV treatment of titanium immediately prior to use (photofunctionalization) affects the ability of human polymicrobial oral biofilm communities to colonize in the presence of salivary and blood components. UV-treatment of machined titanium transformed the surface from hydrophobic to superhydrophilic. UV-treated surfaces exhibited a significant reduction in bacterial attachment as well as subsequent biofilm formation compared to untreated ones, even though overall bacterial viability was not affected. The function of reducing bacterial colonization was maintained on UV-treated titanium that had been stored in a liquid environment before use. Denaturing gradient gel-electrophoresis (DGGE) and DNA sequencing analyses revealed that while bacterial community profiles appeared different between UV-treated and untreated titanium in the initial attachment phase, this difference vanished as biofilm formation progressed. Our findings confirm that UV-photofunctionalization of titanium has a strong potential to improve outcome of implant placement by creating and maintaining antimicrobial surfaces., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The oral opportunistic pathogen Fusobacterium nucleatum is known to interact with a large number of different bacterial species residing in the oral cavity. It adheres to a variety of Gram-positive bacteria, including oral streptococci via the arginine-inhibitable adhesin RadD. In this study, we describe a novel protein encoded by the predicted open reading frame FN1253 that appears to play a role in interspecies interactions of F. nucleatum, particularly with oral streptococci and related Gram-positive species. We designated FN1253 as aid1 (Adherence Inducing Determinant 1). Expression analyses demonstrated that this gene was induced in F. nucleatum single species biofilms, while the presence of representative members of the oral microbiota known to adhere to F. nucleatum triggered its suppression. Inactivation as well as overexpression of aid1 affected the ability of F. nucleatum to coaggregate with oral streptococci and the closely related Enterococcus faecalis, but not other Gram-positive oral species tested. Furthermore, overexpression of aid1 led to a drastic change in the structure of dual species biofilms of F. nucleatum with oral streptococci. Aid1 function was abolished in the presence of arginine and found to be dependent on RadD. Interestingly, differential expression of aid1 did not affect messenger RNA and protein levels of RadD. These findings indicate that RadD-mediated adhesion to oral streptococci involves more complex cellular processes than the simple interaction of adhesins on the surface of partner strains. Aid1 could potentially play an important role in facilitating RadD-mediated interaction with oral streptococci by increasing binding specificity of F. nucleatum to other microbial species.

A major aetiological factor of dental caries is the pathology of the dental plaque biofilms. The amino acid L-arginine (Arg) is found naturally in saliva as a free molecule or as a part of salivary peptides and proteins. Plaque bacteria metabolize Arg to produce alkali and neutralize glycolytic acids, promoting a less cariogenous oral microbiome. Here, we explored an alternative and complementary mechanism of action of Arg using atomic force microscopy. The nanomechanical properties of Streptococcus mutans biofilm extracellular matrix were characterized under physiological buffer conditions. We report the effect of Arg on the adhesive behaviour and structural properties of extracellular polysaccharides in S. mutans biofilms. High-resolution imaging of biofilm surfaces can reveal additional structural information on bacterial cells embedded within the surrounding extracellular matrix. A dense extracellular matrix was observed in biofilms without Arg compared to those grown in the presence of Arg. S. mutans biofilms grown in the presence of Arg could influence the production and/or composition of extracellular membrane glucans and thereby affect their adhesion properties. Our results suggest that the presence of Arg in the oral cavity could influence the adhesion properties of S. mutans to the tooth surface., (© 2014 The Authors.)

Purpose: Dentures are often colonized with a variety of microorganisms, including Candida albicans, that contribute to denture stomatitis. Several in vitro models have been previously established to study denture-related microbial colonization and evaluate treatment efficacy of denture cleansers; however, those models typically fail to appreciate the complex topology and heterogeneity of denture surfaces and lack effective ways to accurately measure microbial colonization. The purpose of this study was to study microbial colonization with a new model system based on real dentures, to more realistically mimic in vivo conditions., Materials and Methods: Scanning electron microscopy was used to observe topological structures among surfaces from different parts of the denture. Employing C. albicans as a model microorganism, we established microbial colonization on different denture surfaces. Moreover, we applied a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) colorimetric assay to quantify C. albicans colonization on dentures without the necessity of biofilm removal and to evaluate treatment efficacy of denture cleansers., Results: There were significant variations in topological structures among surfaces from different parts of the denture, with the unpolished side having the highest amounts of indentations and pores. The distinct denture surfaces support microbial colonization differently, with the unpolished side containing the highest level of microbial colonization and biofilm formation. Furthermore, the modified MTT colorimetric assay proved to be an accurate assay to measure biofilm formation on dentures and evaluate treatment efficacy of denture cleansers., Conclusion: This new denture model system in conjunction with the MTT colorimetric assay is a valuable tool to study denture-related microbiology and treatment approaches., (© 2013 by the American College of Prosthodontists.)

One intriguing discovery in modern microbiology is the extensive presence of extracellular DNA (eDNA) within biofilms of various bacterial species. Although several biological functions have been suggested for eDNA, including involvement in biofilm formation, the detailed mechanism of eDNA integration into biofilm architecture is still poorly understood. In the biofilms formed by Myxococcus xanthus, a Gram-negative soil bacterium with complex morphogenesis and social behaviors, DNA was found within both extracted and native extracellular matrices (ECM). Further examination revealed that these eDNA molecules formed well organized structures that were similar in appearance to the organization of exopolysaccharides (EPS) in ECM. Biochemical and image analyses confirmed that eDNA bound to and colocalized with EPS within the ECM of starvation biofilms and fruiting bodies. In addition, ECM containing eDNA exhibited greater physical strength and biological stress resistance compared to DNase I treated ECM. Taken together, these findings demonstrate that DNA interacts with EPS and strengthens biofilm structures in M. xanthus.

Objective: The objective of this study was to compare the antimicrobial activity of commercially available antiseptic mouthrinses against saliva-derived plaque biofilms in static and flow-through biofilm systems in vitro., Methods: Nine mouthrinses were tested in a recirculating flow-through biofilm model (RFTB) with viability assessment by ATP bioluminescence. In addition, five mouthrinses were evaluated in a batch chamber slide biofilm (BCSB) model, using live/dead staining and confocal laser scanning microscopy., Results: In the RFTB model, essential oil (EO) and chlorhexidine (CHX)-containing rinses showed equivalent antimicrobial activity and were more effective than a range of cetyl pyridinium chloride (CPC1) formulations. In the BCSB model, twice-daily mouthrinse exposure demonstrated that the EO rinse was significantly more effective than rinses containing amine and stannous fluorides (AFSF), a combination of CPC/CHX, and another CPC formulation (CPC2). EO showed biofilm kill comparable to the CHX rinse., Conclusions: The present studies have shown that mouthrinses vary significantly in their capability to kill plaque biofilm bacteria in BCSB and RFTB models. The EO mouthrinse demonstrated superior antiplaque biofilm activity to AFSF, CPC/CHX, and CPC rinses and comparable activity to CHX. The methods tested may be of value for the in-vitro screening of antiseptic rinses with different modes of antimicrobial action., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Despite the fact that most bacteria grow in biofilms in natural and pathogenic ecosystems, very little is known about the ultrastructure of their component cells or about the details of their community architecture. We used high-pressure freezing and freeze-substitution to minimize the artifacts of chemical fixation, sample aggregation, and sample extraction. As a further innovation we have, for the first time in biofilm research, used electron tomography and three-dimensional (3D) visualization to better resolve the macromolecular 3D ultrastructure of a biofilm. This combination of superb specimen preparation and greatly improved resolution in the z axis has opened a window in studies of Myxococcus xanthus cell ultrastructure and biofilm community architecture. New structural information on the chromatin body, cytoplasmic organization, membrane apposition between adjacent cells, and structure and distribution of pili and vesicles in the biofilm matrix is presented.

A defining characteristic of the suspected periodontal pathogen Fusobacterium nucleatum is its ability to adhere to a plethora of oral bacteria. This distinguishing feature is suggested to play an important role in oral biofilm formation and pathogenesis, with fusobacteria proposed to serve as central 'bridging organisms' in the architecture of the oral biofilm bringing together species which would not interact otherwise. Previous studies indicate that these bacterial interactions are mediated by galactose- or arginine-inhibitable adhesins although genetic evidence for the role and nature of these proposed adhesins remains elusive. To characterize these adhesins at the molecular level, the genetically transformable F. nucleatum strain ATCC 23726 was screened for adherence properties, and arginine-inhibitable adhesion was evident, while galactose-inhibitable adhesion was not detected. Six potential arginine-binding proteins were isolated from the membrane fraction of F. nucleatum ATCC 23726 and identified via mass spectroscopy as members of the outer membrane family of proteins in F. nucleatum. Inactivation of the genes encoding these six candidates for arginine-inhibitable adhesion and two additional homologues revealed that only a mutant derivative carrying an insertion in Fn1526 (now designated as radD) demonstrated significantly decreased co-aggregation with representatives of the gram-positive 'early oral colonizers'. Lack of the 350 kDa outer membrane protein encoded by radD resulted in the failure to form the extensive structured biofilm observed with the parent strain when grown in the presence of Streptococcus sanguinis ATCC 10556. These findings indicate that radD is responsible for arginine-inhibitable adherence of F. nucleatum and provides definitive molecular evidence that F. nucleatum adhesins play a vital role in inter-species adherence and multispecies biofilm formation.

While reductionism has greatly advanced microbiology in the past 400 years, assembly of smaller pieces just could not explain the whole! Modern microbiologists are learning "system thinking" and "holism." Such an approach is changing our understanding of microbial physiology and our ability to diagnose/treat microbial infections. This review uses oral microbial communities as a focal point to describe this new trend. With the common name "dental plaque," oral microbial communities are some of the most complex microbial floras in the human body, consisting of more than 700 different bacterial species. For a very long time, oral microbiologists endeavored to use reductionism to identify the key genes or key pathogens responsible for oral microbial pathogenesis. The limitations of reductionism forced scientists to begin adopting new strategies using emerging concepts such as interspecies interaction, microbial community, biofilms, polymicrobial disease, etc. These new research directions indicate that the whole is much more than the simple sum of its parts, since the interactions between different parts resulted in many new physiological functions which cannot be observed with individual components. This review describes some of these interesting interspecies-interaction scenarios.

Oral biofilms are multispecies communities, and in their nascent stages of development, numerous bacterial species engage in interspecies interactions. Better insight into the spatial relationship between different species and how species diversity increases over time can guide our understanding of the role of interspecies interactions in the development of the biofilms. Quantum dots (QD) are semiconductor nanocrystals and have emerged as a promising tool for labeling and detection of bacteria. We sought to apply QD-based primary immunofluorescence for labeling of bacterial cells with in vitro and in vivo biofilms and to compare this approach with the fluorophore-based primary immunofluorescence approach we have used previously. To investigate QD-based primary immunofluorescence as the means to detect distinct targets with single-cell resolution, we conjugated polyclonal and monoclonal antibodies to the QD surface. We also conducted simultaneous QD conjugate-based and fluorophore conjugate-based immunofluorescence and showed that these conjugates were complementary tools in immunofluorescence applications. Planktonic and biofilm cells were labeled effectively by considering two factors: the final nanomolar concentration of QD conjugate and the amount of antibody conjugated to the QD, which we define as the degree of labeling. These advances in the application of QD-based immunofluorescence for the study of biofilms in vitro and in vivo will help to define bacterial community architecture and to facilitate investigations of interactions between bacterial species in these communities.

Streptococcus oligofermentans is a newly characterized species belonging to the mitis group of oral streptococci. So far no correlation has been demonstrated between S. oligofermentans and dental caries. Furthermore, a reverse correlation has been observed between the number of S. oligofermentans and the number of Streptococcus mutans, a major cariogenic pathogen, in the oral cavity. These properties suggest that S. oligofermentans may have a potential to be used as a 'probiotics' for caries prevention. In this study, we aim to establish a genetic system in S. oligofermentans to further study the biology of this new species. Using homologous regions of the comCDE locus in other streptococci, the comC gene was isolated and sequenced. A synthetic competence-stimulating peptide (CSP) was synthesized and shown to be able to effectively induce competence in S. oligofermentans. This CSP-induced transformation system in S. oligofermentans was used to construct green fluorescent protein (gfp) and luciferase (luc) reporter systems, both of which are driven by the lactate dehydrogenase (ldh) promoter. These reporter systems were further shown to be highly expressed in planktonic and biofilm cells, suggesting that these reporter systems can be used in future ecological studies of S. oligofermentans.

The human mucosal surface is colonized by the indigenous microflora, which normally maintains an ecological balance among different species. Certain environmental or biological factors, however, may trigger disruption of this balance, leading to microbial diseases. In this study, we used two oral bacterial species, Streptococcus mutans and Streptococcus sanguinis (formerly S. sanguis), as a model to probe the possible mechanisms of competition/coexistence between different species which occupy the same ecological niche. We show that the two species engage in a multitude of antagonistic interactions temporally and spatially; occupation of a niche by one species precludes colonization by the other, while simultaneous colonization by both species results in coexistence. Environmental conditions, such as cell density, nutritional availability, and pH, play important roles in determining the outcome of these interactions. Genetic and biochemical analyses reveal that these interspecies interactions are possibly mediated through a well-regulated production of chemicals, such as bacteriocins (produced by S. mutans) and hydrogen peroxide (produced by S. sanguinis). Consistent with the phenotypic characteristics, production of bacteriocins and H2O2 are regulated by environmental conditions, as well as by juxtaposition of the two species. These sophisticated interspecies interactions could play an essential part in balancing competition/coexistence within multispecies microbial communities.

Noninvasive in situ detection of suspected cariogenic bacterial species within dental biofilms could facilitate monitoring of the dynamic change of oral microbial flora and assist in the assessment of the treatment efficacy of therapeutic agents. In this study, we explore the possibility to use three well-characterized monoclonal antibodies (MAbs) against Streptococcus mutans, Actinomyces naeslundii, and Lactobacillus casei to identify these three important members of the oral microbial community in the complex environment of oral biofilms. These MAbs, which were conjugated to different fluorescent labels and visualized with confocal laser scanning microscopy (CLSM), proved to be an useful tool to identify the three species of interest (S. mutans, A. naeslundii, and L. casei) under various experimental conditions including in vitro and in vivo derived oral biofilms. Manifold addition of the MAbs on consecutive days did not alter the biofilm structure thus allowing monitoring of the same biofilm over extended time periods. Using this MAb-based method the effect of sucrose challenge on the biofilm composition and the distribution of S. mutans, A. naeslundii, and L. casei were examined. S. mutans was found to be the predominant species under the various biofilm conditions tested. These studies indicate that MAbs based bacterial detection with CLSM is a versatile tool which permits new insights into the ecology of oral biofilm development.

Streptococcus mutans is one of a small number of recognized pathogens that lives among the hundreds of other bacterial species which comprise the oral flora. The virulence of this organism is intimately associated with its ability to live as an attached biofilm community on the tooth surface, and consequently, there is a great interest in its biofilm lifestyle. Currently, there are no established protocols that facilitate drug screening against this organism while it is entrenched in the biofilm. Furthermore, greater complications arise when attempting to perform these experiments in a multi-species setting. In an effort to circumvent these problems, we developed a quick, real-time, and non-disruptive method to probe the metabolic status of S. mutans growing as either a planktonic culture or a biofilm community. This assay takes advantage of the proven utility of luciferase measurements for drug screening. We placed the luciferase gene under the control of the S. mutans lactate dehydrogenase promoter (ldh) and integrated the construct onto its native position on the chromosome. We found this construct to be both highly expressed (<10000 cells easily detectable) and insensitive to many different growth parameters. When testing this reporter in both planktonic and biofilm cultures receiving either bacteriostatic or bactericidal antibiotics, we found the ldh-luc reporter to be a very accurate measurement of cell viability. Furthermore, we also demonstrated that this assay can generate useful information about the characteristics of intoxication caused by antibiotic activity. In addition, we modified the biofilm assay into the 96-well format and demonstrated the feasibility of high throughput drug screening of biofilm embedded S. mutans.

Many clinical isolates of Streptococcus mutans produce peptide antibiotics called mutacins. Mutacin production may play an important role in the ecology of S. mutans in dental plaque. In this study, inactivation of a histidine kinase gene, ciaH, abolished mutacin production. Surprisingly, the same mutation also diminished competence development, stress tolerance, and sucrose-dependent biofilm formation.

Quorum sensing is a bacterial mechanism for regulating gene expression in response to changes in population density. Many bacteria are capable of acyl-homoserine lactone-based or peptide-based intraspecies quorum sensing and luxS-dependent interspecies quorum sensing. While there is good evidence about the involvement of intraspecies quorum sensing in bacterial biofilm, little is known about the role of luxS in biofilm formation. In this study, we report for the first time that luxS-dependent quorum sensing is involved in biofilm formation of Streptococcus mutans. S. mutans is a major cariogenic bacterium in the multispecies bacterial biofilm commonly known as dental plaque. An ortholog of luxS for S. mutans was identified using the data available in the S. mutans genome project (http://www.genome.ou.edu/smutans.html). Using an assay developed for the detection of the LuxS-associated quorum sensing signal autoinducer 2 (AI-2), it was demonstrated that this ortholog was able to complement the luxS negative phenotype of Escherichia coli DH5alpha. It was also shown that AI-2 is indeed produced by S. mutans. AI-2 production is maximal during mid- to late-log growth in batch culture. Mutant strains devoid of the luxS gene were constructed and found to be defective in producing the AI-2 signal. There are also marked phenotypic differences between the wild type and the luxS mutants. Microscopic analysis of in vitro-grown biofilm structure revealed that the luxS mutant biofilms adopted a much more granular appearance, rather than the relatively smooth, confluent layer normally seen in the wild type. These results suggest that LuxS-dependent signal may play an important role in biofilm formation of S. mutans.

Adhesin-mediated bacterial interspecies interactions are important elements in oral biofilm formation. They often occur on a species-specific level, which could determine health or disease association of a biofilm community. Among the key players involved in these processes are the ubiquitous fusobacteria that have been recognized for their ability to interact with numerous different binding partners. Fusobacterial interactions with Streptococcus mutans, an important oral cariogenic pathogen, have previously been described but most studies focused on binding to non-mutans streptococci and specific cognate adhesin pairs remain to be identified. Here, we demonstrated differential binding of oral fusobacteria to S. mutans. Screening of existing mutant derivatives indicated SpaP as the major S. mutans adhesin specific for binding to Fusobacterium nucleatum ssp. polymorphum but none of the other oral fusobacteria tested. We inactivated RadD, a known adhesin of F. nucleatum ssp. nucleatum for interaction with a number of gram-positive species, in F. nucleatum ssp. polymorphum and used a Lactococcus lactis heterologous SpaP expression system to demonstrate SpaP interaction with RadD of F. nucleatum ssp. polymorphum. This is a novel function for SpaP, which has mainly been characterized as an adhesin for binding to host proteins including salivary glycoproteins. In conclusion, we describe an additional role for SpaP as adhesin in interspecies adherence with RadD-SpaP as the interacting adhesin pair for binding between S. mutans and F. nucleatum ssp. polymorphum. Furthermore, S. mutans attachment to oral fusobacteria appears to involve species- and subspecies-dependent adhesin interactions.

Bacterial biofilm infections remain prevalent reasons for implant failure. Dental implant placement occurs in the oral environment, which harbors a plethora of biofilm-forming bacteria. Due to its trans-mucosal placement, part of the implant structure is exposed to oral cavity and there is no effective measure to prevent bacterial attachment to implant materials. Here, we demonstrated that UV treatment of titanium immediately prior to use (photofunctionalization) affects the ability of human polymicrobial oral biofilm communities to colonize in the presence of salivary and blood components. UV-treatment of machined titanium transformed the surface from hydrophobic to superhydrophilic. UV-treated surfaces exhibited a significant reduction in bacterial attachment as well as subsequent biofilm formation compared to untreated ones, even though overall bacterial viability was not affected. The function of reducing bacterial colonization was maintained on UV-treated titanium that had been stored in a liquid environment before use. Denaturing gradient gel-electrophoresis (DGGE) and DNA sequencing analyses revealed that while bacterial community profiles appeared different between UV-treated and untreated titanium in the initial attachment phase, this difference vanished as biofilm formation progressed. Our findings confirm that UV-photofunctionalization of titanium has a strong potential to improve outcome of implant placement by creating and maintaining antimicrobial surfaces.

One major challenge to studying human microbiome and its associated diseases is the lack of effective tools to achieve targeted modulation of individual species and study its ecological function within multispecies communities. Here, we show that C16G2, a specifically targeted antimicrobial peptide, was able to selectively kill cariogenic pathogen Streptococcus mutans with high efficacy within a human saliva-derived in vitro oral multispecies community. Importantly, a significant shift in the overall microbial structure of the C16G2-treated community was revealed after a 24-h recovery period: several bacterial species with metabolic dependency or physical interactions with S. mutans suffered drastic reduction in their abundance, whereas S. mutans' natural competitors, including health-associated Streptococci, became dominant. This study demonstrates the use of targeted antimicrobials to modulate the microbiome structure allowing insights into the key community role of specific bacterial species and also indicates the therapeutic potential of C16G2 to achieve a healthy oral microbiome.

ObjectiveSince dentures can serve as a reservoir for halitosis-causing oral bacteria, halitosis development is a concern for denture wearers. In this study, we surveyed the prevalence of four selected halitosis-related species (Fusobacterium nucleatum, Tannerella forsythia, Veillonella atypica and Klebsiella pneumoniae) in clinical denture plaque samples, and developed denture biofilm models for these species in vitro to facilitate assessment of antimicrobial treatment efficacy. Design : Denture plaque from ten healthy and ten denture stomatitis patients was screened for the presence of aforementioned four species by PCR. Biofilm formation by these halitosis-associated species on the surfaces of denture base resin (DBR) discs was evaluated by crystal violet staining and confocal laser scanning microscopy. The efficacy of denture cleanser treatment on these mono-species biofilms was evaluated by colony counting. Results : 80% of the subjects in the denture stomatitis group and 60% in the healthy group contained at least one of the targeted halitosis-related species in their denture plaque. All halitosis species tested were able to form biofilms on DBR disc surfaces to varying degrees. These in vitro mono-species resin biofilm models were used to evaluate the efficacy of denture cleansers, which exhibited differential efficacies. When forming biofilms on resin surfaces, the halitosis-related species displayed enhanced resistance to denture cleansers compared with their planktonic counterparts. Conclusion : The four selected halitosis-related bacterial species examined in this study are present on the majority of dentures. The mono-species biofilm models established on DBR discs for these species are an efficient screening tool for dental product evaluation.

Acidogenicity and aciduricity are the main virulence factors of the cavity-causing bacterium Streptococcus mutans. Monitoring at the individual cell level the temporal and spatial distribution of acid produced by this important oral pathogen is central for our understanding of these key virulence factors especially when S. mutans resides in multi-species microbial communities. In this study, we explored the application of pH-sensitive green fluorescent proteins (pHluorins) to investigate these important features. Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP. The resulting strain (O87) was used to monitor temporal and spatial pH changes in the microenvironment of S. mutans cells under both planktonic and biofilm conditions. Using strain O87, we revealed a rapid pH drop in the microenviroment of S. mutans microcolonies prior to the decrease in the macro-environment pH following sucrose fermentation. Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity. Furthermore, strain O87 was successfully used to monitor the S. mutans acid production profiles within dual- and multispecies oral biofilms. Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.

Streptococcus mutans is a primary pathogen associated with dental caries. Its bacteriocin (mutacin) production ability is thought to play an important role in maintaining competitiveness in the multispecies oral biofilm. Previous studies have demonstrated that the production of the lantibiotic, mutacin I, is responsive to multiple input signals and that a putative inducible repressor, irvA, seems to be involved in the luxS-mediated mutacin I gene regulation pathway. In this study, we demonstrate that these multiple inputs can be divided into two pathways: irvA-dependent and irvA-independent. Similar to luxS, signals mediated through vicK, pttB and hk03 exert their effect possibly through modulating irvA transcription, whereas signals mediated through ciaH, hrcA, adhE, and Smu1281 exert their effect through an unknown mechanism independent of irvA. [ABSTRACT FROM AUTHOR]

Abstract: Streptococcus mutans is considered one of the main causative agents of human dental caries. Cell–cell communication through two-component signal transduction systems (TCSTS) plays an important role in the pathogenesis of S. mutans. One of the S. mutans TCSTS, ComDE, controls both competence development and biofilm formation. In this study, we showed that addition of exogenous competence-stimulating peptide (CSP) beyond the levels necessary for competence inhibited the growth of S. mutans in a ComDE-dependent manner. We also demonstrated that further increases of CSP stopped S. mutans cell division leading to cell death. Use of CSP as a possible therapeutic agent is discussed. [Copyright &y& Elsevier]

The authors wish to make the following corrections to this paper [[1]]: In the Materials and Methods Section 2.2.1, part of the method was incorrect and instead of "The plasmid DNA of the recombinants were isolated with the Qiagen Mini prep kit (Qiagen, Hilden, Germany) and the presence of construct was confirmed by restriction digestion and sequencing. The method should be: "The plasmid DNA of the recombinants were isolated with the Qiagen Mini prep kit (Qiagen, Hilden, Germany) and the presence of the construct was confirmed by restriction digestion and sequencing. Further, purified plasmids were electroporated into the fusobacterial strains used in this study to generate the respective derivatives lacking target genes according to previously described protocols (Supplementary Figure S1) [29]". [Extracted from the article]