Measurement and analysis of microbial fluoride resistance in dental biofilm models.

The interactions between communities of microorganisms inhabiting the dental biofilm is a major determinant of oral health. These biofilms are periodically exposed to high concentrations of fluoride, which is present in almost all oral healthcare products. The microbes resist fluoride through the action of membrane export proteins. This chapter describes the culture, growth and harvest conditions of model three-species dental biofilm comprised of cariogenic pathogens Streptococcus mutans and Candida albicans and the commensal bacterium Streptococcus gordonii. In order to examine the role of fluoride export by S. mutans in model biofilms, procedures for generating a strain of S. mutans with a genetic knockout of the fluoride exporter are described. We present a case study examining the effects of this mutant strain on the biofilm mass, acid production and mineral dissolution under exposure to low levels of fluoride. These general approaches can be applied to study the effects of any gene of interest in physiologically realistic multispecies oral biofilms.

Spatiotemporal monitoring of a periodontal multispecies biofilm model: demonstration of prebiotic treatment responses.

Biofilms are complex polymicrobial communities which are often associated with human infections such as the oral disease periodontitis. Studying these complex communities under controlled conditions requires in vitro biofilm model systems that mimic the natural environment as close as possible. This study established a multispecies periodontal model in the drip flow biofilm reactor in order to mimic the continuous flow of nutrients at the air-liquid interface in the oral cavity. The design is engineered to enable real-time characterization. A community of five bacteria, Streptococcus gordonii-GFPmut3*, Streptococcus oralis-GFPmut3*, Streptococcus sanguinis-pVMCherry, Fusobacterium nucleatum, and Porphyromonas gingivalis-SNAP26 is visualized using two distinct fluorescent proteins and the SNAP-tag. The biofilm in the reactor develops into a heterogeneous, spatially uniform, dense, and metabolically active biofilm with relative cell abundances similar to those in a healthy individual. Metabolic activity, structural features, and bacterial composition of the biofilm remain stable from 3 to 6 days. As a proof of concept for our periodontal model, the 3 days developed biofilm is exposed to a prebiotic treatment with L-arginine. Multifaceted effects of L-arginine on the oral biofilm were validated by this model setup. L-arginine showed to inhibit growth and incorporation of the pathogenic species and to reduce biofilm thickness and volume. Additionally, L-arginine is metabolized by Streptococcus gordonii-GFPmut3* and Streptococcus sanguinis-pVMCherry, producing high levels of ornithine and ammonium in the biofilm. In conclusion, our drip flow reactor setup is promising in studying spatiotemporal behavior of a multispecies periodontal community.ImportancePeriodontitis is a multifactorial chronic inflammatory disease in the oral cavity associated with the accumulation of microorganisms in a biofilm. Not the presence of the biofilm as such, but changes in the microbiota (i.e., dysbiosis) drive the development of periodontitis, resulting in the destruction of tooth-supporting tissues. In this respect, novel treatment approaches focus on maintaining the health-associated homeostasis of the resident oral microbiota. To get insight in dynamic biofilm responses, our research presents the establishment of a periodontal biofilm model including Streptococcus gordonii, Streptococcus oralis, Streptococcus sanguinis, Fusobacterium nucleatum, and Porphyromonas gingivalis. The added value of the model setup is the combination of simulating continuously changing natural mouth conditions with spatiotemporal biofilm profiling using non-destructive characterization tools. These applications are limited for periodontal biofilm research and would contribute in understanding treatment mechanisms, short- or long-term exposure effects, the adaptation potential of the biofilm and thus treatment strategies.

Inflammatory macrophages exploited by oral streptococcus increase IL-1B release via NLRP6 inflammasome.

Chronic inflammatory periodontal disease develops in part from the infiltration of a large number of classically activated inflammatory macrophages that release inflammatory cytokines important for disease progression, including inflammasome-dependent interleukin (IL)-1ß. Streptococcus gordonii is a normally commensal oral microorganism; while not causative, recent evidence indicates that commensal oral microbes are required for the full development of periodontal disease. We have recently reported that inflammatory macrophages counterintuitively allow for the increased survival of phagocytosed S. gordonii over nonactivated or alternatively activated macrophages. This survival is dependent on increased reactive oxygen species production within the phagosome of the inflammatory macrophages, and resistance by the bacterium and can result in S. gordonii damaging the phagolysosomes. Here, we show that activated macrophages infected with live S. gordonii release more IL-1ß than non-activated macrophages infected with either live or dead S. gordonii, and that the survival of oral Streptococci are more dependent on macrophage activation than other Gram positive microbes, both classical pathogens and commensals. We also find that S. gordonii-dependent inflammatory macrophage inflammasome activation requires the cytoplasmic NLRP6. Overall, our results suggest S. gordonii is capable of evading immune destruction, increasing inflammatory mediators, and increasing inflammatory macrophage response, and that this ability is increased under conditions of inflammation. This work reveals additional mechanisms by which normally commensal oral streptococci-macrophage interactions can change, resulting in increased release of mature IL-1ß, potentially contributing to an environment that perpetuates inflammation.

Antagonistic interactions by a high H2 O2 -producing commensal streptococcus modulate caries development by Streptococcus mutans.

Dental caries (tooth-decay) is caused by biofilms harboring polymicrobial communities on teeth that leads to the onset of localized areas of enamel demineralization. Streptococcus mutans has been clinically associated with severe caries in childhood. Although commensal bacteria can combat S. mutans using self-generated antimicrobials such as hydrogen peroxide (H2 O2 ), constant sugar-rich diet consumption disrupts microbial homeostasis shifting toward cariogenic community. Recently, Streptococcus oralis subsp. tigurinus strain J22, an oral isolate, was identified as a uniquely potent H2 O2 producer. Here, we assess whether a high H2 O2 -producing commensal streptococcus can modulate the spatial organization and virulence of S. mutans within biofilms. Using an experimental biofilm model, we find that the presence of S. oralis J22 can effectively inhibit the clustering, accumulation, and spatial organization of S. mutans on ex vivo human tooth surface, resulting in significant reduction of enamel demineralization. Notably, the generation of H2 O2 via pyruvate oxidase (SpxB) from S. oralis J22 is not repressed by sugars (a common repressor in other mitis group streptococci), resulting in enhanced inhibition of S. mutans growth (vs. Streptococcus gordonii). We further investigate its impact on biofilm virulence using an in vivo rodent caries model under sugar-rich diet. Coinfection of S. mutans with S. oralis results in reduced caries development compared to either species infected alone, whereas coinfection with S. gordonii has negligible effects, suggesting that the presence of an efficient, high H2 O2 -producer can disrupt S. mutans virulence. This work demonstrates that oral isolates with unusual high H2 O2 production may be capable of modulating biofilm cariogenicity in vivo. The findings also highlight the importance of bacterial antagonistic interactions within polymicrobial communities in health and in disease-causing state.

Quantification of Extracellular DNA Network Abundance and Architecture within Streptococcus gordonii Biofilms Reveals Modulatory Factors.

Extracellular DNA (eDNA) is an important component of biofilm matrix that serves to maintain biofilm structural integrity, promotes genetic exchange within the biofilm, and provides protection against antimicrobial compounds. Advances in microscopy techniques have provided evidence of the cobweb- or lattice-like structures of eDNA within biofilms from a range of environmental niches. However, methods to reliably assess the abundance and architecture of eDNA remain lacking. This study aimed to address this gap by development of a novel, high-throughput image acquisition and analysis platform for assessment of eDNA networks in situ within biofilms. Utilizing Streptococcus gordonii as the model, the capacity for this imaging system to reliably detect eDNA networks and monitor changes in abundance and architecture (e.g., strand length and branch number) was verified. Evidence was provided of a synergy between glucans and eDNA matrices, while it was revealed that surface-bound nuclease SsnA could modify these eDNA structures under conditions permissive for enzymatic activity. Moreover, cross talk between the competence and hexaheptapeptide permease systems was shown to regulate eDNA release by S. gordonii. This novel imaging system can be applied across the wider field of biofilm research, with potential to significantly advance interrogation of the mechanisms by which the eDNA network architecture develops, how it can influence biofilm properties, and how it may be targeted for therapeutic benefit. IMPORTANCE Extracellular DNA (eDNA) is critical for maintaining the structural integrity of many microbial biofilms, making it an attractive target for the management of biofilms. However, our knowledge and targeting of eDNA are currently hindered by a lack of tools for the quantitative assessment of eDNA networks within biofilms. Here, we demonstrate use of a novel image acquisition and analysis platform with the capacity to reliably monitor the abundance and architecture of eDNA networks. Application of this tool to Streptococcus gordonii biofilms has provided new insights into how eDNA networks are stabilized within the biofilm and the pathways that can regulate eDNA release. This highlights how exploitation of this novel imaging system across the wider field of biofilm research has potential to significantly advance interrogation of the mechanisms by which the eDNA network architecture develops, how it can influence biofilm properties, and how it may be targeted for therapeutic benefit.

E-Cigarette Aerosol Exposure Favors the Growth and Colonization of Oral Streptococcus mutans Compared to Commensal Streptococci.

E-cigarettes (e-cigs) have drastically increased in popularity during the last decade, especially among teenagers. While recent studies have started to explore the effect of e-cigs in the oral cavity, little is known about their effects on the oral microbiota and how they could affect oral health and potentially lead to disease, including periodontitis and head and neck cancers. To explore the impact of e-cigs on oral bacteria, we selected members of the genus Streptococcus, which are abundant in the oral cavity. We exposed the commensals Streptococcus sanguinis and Streptococcus gordonii and the opportunistic pathogen Streptococcus mutans, best known for causing dental caries, to e-liquids and e-cig aerosols with and without nicotine and with and without menthol flavoring and measured changes in growth patterns and biofilm formation. Our results demonstrate that e-cig aerosols hindered the growth of S. sanguinis and S. gordonii, while they did not affect the growth of S. mutans. We also show that e-cig aerosols significantly increased biofilm formation by S. mutans but did not affect the biofilm formation of the two commensals. We found that S. mutans exhibits higher hydrophobicity and coaggregation abilities along with higher attachment to OKF6 cells than S. sanguinis and S. gordonii. Therefore, our data suggest that e-cig aerosols have the potential to dysregulate oral bacterial homeostasis by suppressing the growth of commensals while enhancing the biofilm formation of the opportunistic pathogen S. mutans. This study highlights the importance of understanding the consequences of e-cig aerosol exposure on selected commensals and pathogenic species. Future studies modeling more complex communities will provide more insight into how e-cig aerosols and vaping affect the oral microbiota. IMPORTANCE Our study shows that e-cigarette aerosol exposure of selected bacteria known to be residents of the oral cavity hinders the growth of two streptococcal commensals while enhancing biofilm formation, hydrophobicity, and attachment for the pathogen S. mutans. These results indicate that e-cigarette vaping could open a niche for opportunistic bacteria such as S. mutans to colonize the oral cavity and affect oral health.

Streptococcus gordonii DL1 evades polymorphonuclear leukocyte-mediated killing via resistance to lysozyme.

Streptococcus gordonii is an etiological bacterial agent of infective endocarditis. Although the pathogenesis mechanisms are not well understood, the interaction between streptococci and phagocytes is considered important for the development of infective endocarditis. Previous studies show that some S. gordonii strains, including DL1, survive in polymorphonuclear leukocytes (PMNs), whereas other strains such as SK12 are sensitive to PMN-dependent killing. In this study, we assessed the differences between the sensitivity of S. gordonii DL1 and S. gordonii SK12 to PMN-dependent killing. S. gordonii DL1 showed a higher survival when treated with PMNs than SK12. Both S. gordonii DL1 and S. gordonii SK12 showed high resistance to low pH condition. Compared to S. gordonii SK12, S. gordonii DL1 was sensitive to hydrogen peroxide. However, the resistance of S. gordonii DL1 to the tested bactericidal agents, especially lysozyme, was higher than that of SK12. Furthermore, we performed a bactericidal assay by treating a mixture of S. gordonii DL1 and SK12 with PMNs. S. gordonii DL1 did not enhance the survival of S. gordonii SK12 exposed to PMNs. These results indicated that S. gordonii DL1 is resistant to bactericidal agents that degrade bacteria in phagolysosomes. In addition, there was no secretory factor involved in the resistance to bactericidal agents. The findings of this study may help develop treatments for infective endocarditis caused by S. gordonii.

Short-chain fatty acids inhibit the biofilm formation of Streptococcus gordonii through negative regulation of competence-stimulating peptide signaling pathway.

Streptococcus gordonii, a Gram-positive commensal bacterium, is an opportunistic pathogen closely related to initiation and progression of various oral diseases, such as periodontitis and dental caries. Its biofilm formation is linked with the development of such diseases by enhanced resistance against antimicrobial treatment or host immunity. In the present study, we investigated the effect of short-chain fatty acids (SCFAs) on the biofilm formation of S. gordonii. SCFAs, including sodium acetate (NaA), sodium propionate (NaP), and sodium butyrate (NaB), showed an effective inhibitory activity on the biofilm formation of S. gordonii without reduction in bacterial growth. SCFAs suppressed S. gordonii biofilm formation at early time points whereas SCFAs did not affect its preformed biofilm. A quorum-sensing system mediated by competence-stimulating peptide (CSP) is known to regulate biofilm formation of streptococci. Interestingly, SCFAs substantially decreased mRNA expression of comD and comE, which are CSP-sensor and its response regulator responsible for CSP pathway, respectively. Although S. gordonii biofilm formation was enhanced by exogenous synthetic CSP treatment, such effect was not observed in the presence of SCFAs. Collectively, these results suggest that SCFAs have an anti-biofilm activity on S. gordonii through inhibiting comD and comE expression which results in negative regulation of CSP quorum-sensing system. SCFAs could be an effective anti-biofilm agent against S. gordonii for the prevention of oral diseases.

Regulation of olfactomedin 4 by Porphyromonas gingivalis in a community context.

At mucosal barriers, the virulence of microbial communities reflects the outcome of both dysbiotic and eubiotic interactions with the host, with commensal species mitigating or potentiating the action of pathogens. We examined epithelial responses to the oral pathogen Porphyromonas gingivalis as a monoinfection and in association with a community partner, Streptococcus gordonii. RNA-Seq of oral epithelial cells showed that the Notch signaling pathway, including the downstream effector olfactomedin 4 (OLFM4), was differentially regulated by P. gingivalis alone; however, regulation was overridden by S. gordonii. OLFM4 was required for epithelial cell migratory, proliferative and inflammatory responses to P. gingivalis. Activation of Notch signaling was induced through increased expression of the Notch1 receptor and the Jagged1 (Jag1) agonist. In addition, Jag1 was released in response to P. gingivalis, leading to paracrine activation. Following Jag1-Notch1 engagement, the Notch1 extracellular domain was cleaved by P. gingivalis gingipain proteases. Antagonism by S. gordonii involved inhibition of gingipain activity by secreted hydrogen peroxide. The results establish a novel mechanism by which P. gingivalis modulates epithelial cell function which is dependent on community context. These interrelationships have relevance for innate inflammatory responses and epithelial cell fate decisions in oral health and disease.

Enhanced biofilm formation of Streptococcus gordonii with lipoprotein deficiency.

Streptococcus gordonii is a commensal Gram-positive bacterium that acts as an opportunistic pathogen that can cause apical periodontitis, endocarditis, and pneumonia. Biofilm formation of bacteria is important for the initiation and progression of such diseases. Although lipoproteins play key roles in physiological functions, the role of lipoproteins of S. gordonii in its biofilm formation has not been clearly understood. In this study, we investigated the role of lipoproteins of S. gordonii in the bacterial biofilm formation using its lipoprotein-deficient strain (Δlgt). The S. gordonii Δlgt exhibited increased biofilm formation on the human dentin slices or on the polystyrene surfaces compared to the wild-type strain, while its growth rate did not differ from that of the wild-type. In addition, the S. gordonii Δlgt strain exhibited the enhanced LuxS mRNA expression and AI-2 production, which is known to be a positive regulator of biofilm formation, compared to the wild-type. Concordantly, the augmented biofilm formation of S. gordonii Δlgt was attenuated by an AI-2 inhibitor, D-ribose. In addition, lipoproteins from purified S. gordonii inhibited the biofilm formation of S. gordonii wild-type and Δlgt. Taken together, these results suggest that lipoprotein-deficient S. gordonii form biofilms more effectively than the wild-type strain, which might be related to the AI-2 quorum-sensing system.

Amino Sugars Reshape Interactions between Streptococcus mutans and Streptococcus gordonii.

Amino sugars, particularly glucosamine (GlcN) and N-acetylglucosamine (GlcNAc), are abundant carbon and nitrogen sources supplied in host secretions and in the diet to the biofilms colonizing the human oral cavity. Evidence is emerging that these amino sugars provide ecological advantages to beneficial commensals over oral pathogens and pathobionts. Here, we performed transcriptome analysis on Streptococcus mutans and Streptococcus gordonii growing in single-species or dual-species cultures with glucose, GlcN, or GlcNAc as the primary carbohydrate source. Compared to glucose, GlcN caused drastic transcriptomic shifts in each species of bacteria when it was cultured alone. Likewise, cocultivation in the presence of GlcN yielded transcriptomic profiles that were dramatically different from the single-species results from GlcN-grown cells. In contrast, GlcNAc elicited only minor changes in the transcriptome of either organism in single- and dual-species cultures. Interestingly, genes involved in pyruvate metabolism were among the most significantly affected by GlcN in both species, and these changes were consistent with measurements of pyruvate in culture supernatants. Differing from what was found in a previous report, growth of S. mutans alone with GlcN inhibited the expression of multiple operons required for mutacin production. Cocultivation with S. gordonii consistently increased the expression of two manganese transporter operons (slo and mntH) and decreased expression of mutacin genes in S. mutans Conversely, S. gordonii appeared to be less affected by the presence of S. mutans but did show increases in genes for biosynthetic processes in the cocultures. In conclusion, amino sugars profoundly alter the interactions between pathogenic and commensal streptococci by reprogramming central metabolism.IMPORTANCE Carbohydrate metabolism is central to the development of dental caries. A variety of sugars available to dental microorganisms influence the development of caries by affecting the physiology, ecology, and pathogenic potential of tooth biofilms. Using two well-characterized oral bacteria, one pathogen (Streptococcus mutans) and one commensal (Streptococcus gordonii), in an RNA deep-sequencing analysis, we studied the impact of two abundant amino sugars on bacterial gene expression and interspecies interactions. The results indicated large-scale remodeling of gene expression induced by GlcN in particular, affecting bacterial energy generation, acid production, protein synthesis, and release of antimicrobial molecules. Our study provides novel insights into how amino sugars modify bacterial behavior, information that will be valuable in the design of new technologies to detect and prevent oral infectious diseases.

Uncovering Roles of Streptococcus gordonii SrtA-Processed Proteins in the Biofilm Lifestyle.

Streptococcus gordonii is a commensal oral organism. Harmless in the oral cavity, S. gordonii is an opportunistic pathogen. S. gordonii adheres to body surfaces using surface adhesive proteins (adhesins), which are critical to subsequent formation of biofilm communities. As in most Gram-positive bacteria, S. gordonii surface proteins containing the C-terminal LPXTG motif cleavage sequence are processed by sortase A (SrtA) to become covalently attached to the cell wall. To characterize the functional diversity and redundancy in the family of SrtA-processed proteins, an S. gordonii DL1 markerless deletion mutant library was constructed of each of the 26 putative SrtA-processed proteins. Each library member was evaluated for growth in rich medium, biofilm formation on plastic, saliva and salivary fractions, cell surface hydrophobicity (CSH), hemagglutination, and integration into an ex vivo plaque biofilm community. Library members were compared to the non-SrtA-processed adhesins AbpA and AbpB. While no major growth differences in rich medium were observed, many S. gordonii LPXTG/A proteins impacted biofilm formation on one or more of the substrates. Several mutants showed significant differences in hemagglutination, hydrophobicity, or fitness in the ex vivo plaque model. From the identification of redundant and unique functions in these in vitro and ex vivo systems, functional stratification among the LPXTG/A proteins is apparent.IMPORTANCES. gordonii interactions with its environment depend on the complement of cell wall proteins. A subset of these cell wall proteins requires processing by the enzyme sortase A (SrtA). The identification of SrtA-processed proteins and their functional characterization will help the community to better understand how S. gordonii engages with its surroundings, including other microbes, integrates into the plaque community, adheres to the tooth surface, and hematogenously disseminates to cause blood-borne infections. This study identified 26 putative SrtA-processed proteins through creation of a markerless deletion mutant library. The library was subject to functional screens that were chosen to better understand key aspects of S. gordonii physiology and pathogenesis.

Bacteria Modify Candida albicans Hypha Formation, Microcolony Properties, and Survival within Macrophages.

Phagocytic cells are crucial components of the innate immune system preventing Candida albicans mucosal infections. Streptococcus gordonii and Pseudomonas aeruginosa often colonize mucosal sites, along with C. albicans, and yet interkingdom interactions that might alter the survival and escape of fungi from macrophages are not understood. Murine macrophages were coinfected with S. gordonii or P. aeruginosa, along with C. albicans to evaluate changes in fungal survival. S. gordonii increased C. albicans survival and filamentation within macrophage phagosomes, while P. aeruginosa reduced fungal survival and filamentation. Coinfection with S. gordonii resulted in greater escape of C. albicans from macrophages and increased size of fungal microcolonies formed on macrophage monolayers, while coinfection with P. aeruginosa reduced macrophage escape and produced smaller microcolonies. Microcolonies formed in the presence of P. aeruginosa cells outside macrophages also had significantly reduced size that was not found with P. aeruginosa phenazine deletion mutants. S. gordonii cells, as well as S. gordonii heat-fixed culture supernatants, increased C. albicans microcolony biomass but also resulted in microcolony detachment. A heat-resistant, trypsin-sensitive pheromone processed by S. gordonii Eep was needed for these effects. The majority of fungal microcolonies formed on human epithelial monolayers with S. gordonii supernatants developed as large floating structures with no detectable invasion of epithelium, along with reduced gene expression of C. albicansHYR1, EAP1, and HWP2 adhesins. However, a subset of C. albicans microcolonies was smaller and had greater epithelial invasiveness compared to microcolonies grown without S. gordonii Thus, bacteria can alter the killing and escape of C. albicans from macrophages and contribute to changes in C. albicans pathogenicity.IMPORTANCECandida albicans is the predominant fungus colonizing the oral cavity that can have both synergistic and antagonistic interactions with other bacteria. Interkingdom polymicrobial associations modify fungal pathogenicity and are believed to increase microbial resistance to innate immunity. However, it is not known how these interactions alter fungal survival during phagocytic killing. We demonstrated that secreted molecules of S. gordonii and P. aeruginosa alter C. albicans survival within the phagosome of macrophages and alter fungal pathogenic phenotypes, including filamentation and microcolony formation. Moreover, we provide evidence for a dual interaction between S. gordonii and C. albicans such that S. gordonii signaling peptides can promote C. albicans commensalism by decreasing microcolony attachment while increasing invasion in epithelial cells. Our results identify bacterial diffusible factors as an attractive target to modify virulence of C. albicans in polymicrobial infections.

Porphyromonas gingivalis HmuY and Streptococcus gordonii GAPDH-Novel Heme Acquisition Strategy in the Oral Microbiome.

The oral cavity of healthy individuals is inhabited by commensals, with species of Streptococcus being the most abundant and prevalent in sites not affected by periodontal diseases. The development of chronic periodontitis is linked with the environmental shift in the oral microbiome, leading to the domination of periodontopathogens. Structure-function studies showed that Streptococcus gordonii employs a "moonlighting" protein glyceraldehyde-3-phosphate dehydrogenase (SgGAPDH) to bind heme, thus forming a heme reservoir for exchange with other proteins. Secreted or surface-associated SgGAPDH coordinates Fe(III)heme using His43. Hemophore-like heme-binding proteins of Porphyromonas gingivalis (HmuY), Prevotella intermedia (PinO) and Tannerella forsythia (Tfo) sequester heme complexed to SgGAPDH. Co-culturing of P. gingivalis with S. gordonii results in increased hmuY gene expression, indicating that HmuY might be required for efficient inter-bacterial interactions. In contrast to the DhmuY mutant strain, the wild type strain acquires heme and forms deeper biofilm structures on blood agar plates pre-grown with S. gordonii. Therefore, our novel paradigm of heme acquisition used by P. gingivalis appears to extend to co-infections with other oral bacteria and offers a mechanism for the ability of periodontopathogens to obtain sufficient heme in the host environment. Importantly, P. gingivalis is advantaged in terms of acquiring heme, which is vital for its growth survival and virulence.

Biofilm Interactions of Candida albicans and Mitis Group Streptococci in a Titanium-Mucosal Interface Model.

Streptococci from the mitis group (represented mainly by Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii) form robust biofilms with Candida albicans in different experimental models. These microorganisms have been found in polymicrobial biofilms forming on titanium biomaterial surfaces in humans with peri-implant disease. The purpose of this work was to study mutualistic interactions in biofilms forming on titanium and their effect on the adjacent mucosa, using a relevant infection model. Single and mixed biofilms of C. albicans and each Streptococcus species were grown on titanium disks. Bacterial and fungal biovolume and biomass were quantified in these biofilms. Organotypic mucosal constructs were exposed to preformed titanium surface biofilms to test their effect on secretion of proinflammatory cytokines and cell damage. C. albicans promoted bacterial biofilms of all mitis Streptococcus species on titanium surfaces. This relationship was mutualistic since all bacterial species upregulated the efg1 hypha-associated gene in C. albicans Mixed biofilms caused increased tissue damage but did not increase proinflammatory cytokine responses compared to biofilms comprising Candida alone. Interestingly, spent culture medium from tissues exposed to titanium biofilms suppressed Candida growth on titanium surfaces.IMPORTANCE Our findings provide new insights into the cross-kingdom interaction between C. albicans and Streptococcus species representative of the mitis group. These microorganisms colonize titanium-based dental implant materials, but little is known about their ability to cause inflammation and damage of the adjacent mucosal tissues. Using an in vitro biomaterial-mucosal interface infection model, we showed that mixed biofilms of each species with C. albicans enhance tissue damage. One possible mechanism for this effect is the increased fungal hypha-associated virulence gene expression we observed in mixed biofilms with these species. Interestingly, we also found that the interaction of multispecies biofilms with organotypic mucosal surfaces led to the release of growth-suppressing mediators of Candida, which may represent a homeostatic defense mechanism of the oral mucosa against fungal overgrowth. Thus, our findings provide novel insights into biofilms on biomaterials that may play an important role in the pathogenesis of mucosal infections around titanium implants.

Effects of chemical treatment as an adjunctive of air-abrasive debridement on restoring the surface chemical properties and cytocompatibility of experimentally contaminated titanium surfaces.

The aim of this study was to evaluate the effects of three different chemotherapeutic agents, following air-abrasive debridement, on surface chemical properties and cytocompatibility. Disks contaminated with Streptococcus gordonii biofilm were treated with air-abrasion and immersion in either 0.9% NaCl (Air + NaCl), 0.05% alkaline electrolyzed water (AEW) (Air + AEW), or 3% H2 O2 (Air + H2 O2 ). Noncontaminated and untreated titanium disks served as a control (As-polished). The efficacy of biofilm removal, magnitude of initial cytocompatibility toward human bone marrow mesenchymal stem cells, and surface chemical properties were determined. In all treatment groups, biofilms containing microorganisms were observed to be completely removed. The data showed discrepancies for cell affinities among treatment groups, whereby: (1) the number of cells attached to the Air + AEW treated surfaces was approximately two times greater than that to the Air + NaCl treated surfaces; and (2) cell spreading was significantly enhanced on the Air + AEW treated surfaces compared with the Air + NaCl or Air + H2 O2 treated surfaces. X-ray photoelectron spectroscopy data showed that the mean relative concentrations of nitrogen to titanium on the As-polished, Air + NaCl, Air + AEW, and Air + H2 O2 surfaces were 0.0079, 0.0237, 0.0071, and 0.0210, respectively, which would provide a clear understanding that these discrepancies could be attributed to sufficient removals of organic-nitrogen deposits at the same magnitude as the As-polished following the Air + AEW treatment. This study clarifies that chemical surface treatment with AEW, as an adjunctive to air-abrasive debridement may be beneficial in restoring surface properties for tissue integration. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:183-191, 2020.

Antimicrobial Efficacy Assessment of Human Derived Composite Amnion-Chorion Membrane.

Human derived composite amnion-chorion membrane (ACM) has been used to facilitate wound healing due to reported anti-inflammatory properties and promotion of cell proliferation. This study aimed to assess the antimicrobial properties of the ACM using novel methods to visualize the antimicrobial efficacy of membranes in situ at different time points. Porcine Pericardium Collagen Membranes (PPCM) served as membrane controls. Circular pieces of the membranes were used in three different assays: insert, agar contact and glass-bottom well assays. Streptococcus gordonii were spotted onto the membranes and the plates were subsequently centrifuged to ensure direct bacterial contact with the membranes in the insert and agar contact assays, thus better mimicking bacterial adherence in the oral cavity. After incubation at 37 °C for 8, 24, and 48 hours, the membranes were dyed with the Live/Dead BacLight Bacterial Viability fluorescence stain and analyzed via confocal microscopy. The results demonstrated that the ACM completely inhibited bacterial growth at all time points, whereas the PPCM did not demonstrate any antimicrobial properties. Within the limits of this study, the ACM showed extremely high antimicrobial efficacy against oral streptococci. In addition, our methods may be useful in assessing antimicrobial properties for biomaterials with minimum diffusion ability, when traditional assessment methods are not applicable.

Streptococcal peptides that signal Enterococcus faecalis cells carrying the pheromone-responsive conjugative plasmid pAM373.

Pheromone-mediated conjugative transfer of enterococcal plasmids can contribute to the dissemination of genes involved in antibiotic resistance, fitness, and virulence among co-residents of mixed microbial communities. We have previously shown that intergeneric signaling by the Streptococcus gordonii strain Challis heptapeptide s.g.cAM373 (SVFILAA) induces an aggregation substance-mediated mating response and facilitates plasmid transfer from Enterococcus faecalis cells carrying the pheromone-responsive plasmid pAM373 to both pheromone-producing and non-pheromone-producing oral streptococcal recipients. To further investigate the streptococcal pheromone-like peptides, s.g.cAM373-like sequences were identified in the signal sequences of streptococcal CamG lipoproteins and their abilities to induce a mating response in E. faecalis/pAM373 cells were examined. Synthetic heptamers with the consensus sequence (A/S)-(I/V)-F-I-L-(A/V/T)-(S/A) induced AS-mediated clumping. The conserved pheromone ABC transporter encoded by S. gordonii genome loci SGO_RS02660 and SGO_RS02665 was identified and confirmed to be required for s.g.cAM373 activity. Functional assays of culture supernatants from representative oral and blood isolates of S. gordonii showed that in addition to strains encoding s.g.cAM373, strain SK120, encoding the newly identified pheromone s.g.cAM373-V (SVFILVA), was able to induce enterococcal clumping, whereas strains SK6, SK8, SK9, and SK86 which encoded s.g.cAM373-T (SVFILTA) did not elicit a detectable mating response. Absence of pheromone activity in supernatants of heterologous hosts encoding its CamG precursor suggested that s.g.cAM373-T was not effectively processed and/or transported. Overall, these studies demonstrated the distribution of active pheromone peptides among strains of S. gordonii, and support a potential role for enterococcal-streptococcal communication in contributing to genetic plasticity in the oral metagenome.

The inhibitory effect of quaternary ammonium salt on bacteria in root canal.

Persisting apical periodontitis is a primary reason for multiple intervention in root canal. Persisting bacteria in root canal is related with the persisting infection. Despite the advancement in treatment strategies the persisting infection is a major challenge for endodontist. Here we tested two newly developed quaternary ammonium methacrylates (QAMs) against endodontic bacteria and their biofilms. Their antibacterial and antibiofilm efficiency were compared with chlorhexidine (CHX) and sodium hypochlorite (NaOCl). We measured the MIC, MBC and MBIC of DMADDM and DMAHDM respectively. We also detected the ratio of live/dead bacteria and bacterial composition in the biofilms treated by DMADDM and DMAHDM. We found that DMADDM and DMAHDM could inhibit the growth of bacteria and biofilms formation. The result showed that novel QAMs were remarkably efficient than CHX against biofilms. In addition, we found that Streptococcus gordonii (S. gordonii) and Enterococcus faecalis (E. faecalis) were frequent isolates after treatment with antimicrobial compounds.

Contributions of Candida albicans Dimorphism, Adhesive Interactions, and Extracellular Matrix to the Formation of Dual-Species Biofilms with Streptococcus gordonii.

Fungal and bacterial populations coexist in the oral cavity, frequently forming mixed-species biofilms that complicate treatment against polymicrobial infections. However, despite relevance to oral health, the bidirectional interactions between these microbial populations are poorly understood. In this study, we aimed to elucidate the mechanisms underlying the interactions between the fungal species Candida albicans and the bacterial species Streptococcus gordonii as they coexist in mixed-species biofilms. Specifically, the interactions of different C. albicans mutant strains deficient in filamentation (efg1Δ/Δ and brg1Δ/Δ), adhesive interactions (als3Δ/Δ and bcr1Δ/Δ), and production of matrix exopolymeric substances (EPS) (kre5Δ/Δ, mnn9Δ/Δ, rlm1Δ/Δ, and zap1Δ/Δ) were evaluated with S. gordonii under different conditions mimicking the environment in the oral cavity. Interestingly, our results revealed that growth of the biofilm-deficient C. albicansals3Δ/Δ and bcr1Δ/Δ mutant strains in synthetic saliva or with S. gordonii restored their biofilm-forming ability. Moreover, challenging previous observations indicating an important role of morphogenetic conversions in the interactions between these two species, our results indicated a highly synergistic interaction between S. gordonii and the C. albicans filamentation-deficient efg1Δ/Δ and brg1Δ/Δ deletion mutants, which was particularly noticeable when the mixed biofilms were grown in synthetic saliva. Importantly, dual-species biofilms were found to exhibit increase in antimicrobial resistance, indicating that components of the fungal exopolymeric material confer protection to streptococcal cells against antibacterial treatment. Collectively, these findings unravel a high degree of complexity in the interactions between C. albicans and S. gordonii in mixed-species biofilms, which may impact homeostasis in the oral cavity.IMPORTANCE Microbial communities have a great impact in health and disease. C. albicans interacts with multiple microorganisms in the oral cavity, frequently forming polymicrobial biofilms. We report on the synergistic interactions between C. albicans and the Gram-positive bacterium S. gordonii, for which we have examined the different contributions of adhesive interactions, filamentation, and the extracellular matrix to the formation of dual-species biofilms. Our results demonstrate that growth in the presence of the bacterium can restore the biofilm-forming ability of different C. albicans mutant strains with defects in adhesion and filamentation. The mixed-species biofilms also show high levels of resistance to antibacterial and antifungal antibiotics, and our results indicate that the fungal biofilm matrix protects bacterial cells within these mixed-species biofilms. Our observations add to a growing body of evidence indicating a high level of complexity in the reciprocal interactions and consortial behavior of fungal/bacterial biofilms.