Difference in formation of a dental multi-species biofilm according to substratum direction.

OBJECTIVES: The aim of this study was to investigate the difference in dental biofilm formation according to substratum direction, using an artificial biofilm model. METHODS: A three-species biofilm, consisting of Streptococcus mutans, Streptococcus oralis, and Actinomyces naeslundii, was formed on saliva-coated hydroxyapatite (sHA) discs oriented in three directions: downward (the discs placed in the direction of gravity), vertical (the discs placed parallel to the direction of gravity), and upward (the discs placed in opposite direction of gravity). The biofilms at 22 h and 46 h of age were analyzed using microbiological and biochemical methods, fluorescence-based assays, and scanning electron microscopy to investigate difference in bacterial adhesion, early and mature biofilm formation. RESULTS: The biofilms formed in the upward direction displayed the most complex structure, with the highest number and biovolume of bacteria, as well as the lowest pH conditions at both time points. The vertical and downward directions, however, had only scattered and small bacterial colonies. In the 22-h-old biofilms, the proportion of S. oralis was similar to, or slightly higher than, that of S. mutans in all directions of substratum surfaces. However, in the 46-h-old biofilms, S. mutans became the dominant bacteria in all directions, especially in the vertical and upward directions. CONCLUSIONS: The direction of the substratum surface could impact the proportion of bacteria and cariogenic properties of the multi-species biofilm. Biofilms in an upward direction may exhibit a higher cariogenic potential, followed by those in the vertical and downward directions, which could be related to gravity.

Weighted gene co-expression network analysis (WGCNA) to explore genes responsive to Streptococcus oralis biofilm and immune infiltration analysis in human gingival fibroblasts cells.

The correlation between oral bacteria and dental implants failure has been reported. However, the effect and mechanism of bacteria during dental implants is unclear. In this study, we explored key genes and candidate gene clusters in human gingival fibroblasts (HGF) cells in response to Streptococcus oralis biofilm through weighted gene co-expression network analysis (WGCNA) and differential genes analysis using gene expression matrix, GSE134481, downloaded from the Gene Expression Omnibus (GEO) database. We obtained 325 genes in the module significantly associated with S. oralis infection and 113 differentially expressed genes (DEGs) in the S. oralis biofilm; 62 DEGs indicated significant correlation with S. oralis injury. Multiple immune pathways, such as the tumor necrosis factor (TNF) signaling pathway, were considerably enriched. We obtained a candidate genes cluster containing 12 genes - IL6, JUN, FOS, CSF2, HBEGF, EDN1, CCL2, MYC, NGF, SOCS3, CXCL1, and CXCL2; we observed 5 candidate hub genes associated with S. oralis infection - JUN, IL6, FOS, MYC, and CCL2. The fraction of macrophage M0 cells was significantly increased in biofilm treatment compared with control; expression of FOS and MYC was significantly positively correlated with macrophage M0 cells. Our findings present a fierce inflammation changes in the transcript level of HGF in response to S. oralis.

Local Bacteriotherapy - a promising preventive tool in recurrent respiratory infections.

INTRODUCTION: Children with recurrent respiratory infections (RRI) represent a social issue for the economic burden and the negative family impact. Local Bacteriotherapy is an attractive therapeutic strategy that could be potentially effective in preventing infections. The current article remarks on the existing evidence of preventing RRI by Local Bacteriotherapy. AREAS COVERED: The literature search methodology was based on the articles cited by PubMed from 1980 to 2020. Respiratory infections include rhino-pharyngitis, otitis media, rhinosinusitis, pharyngo-tracheitis, bronchitis, and pneumonia. Several studies were performed to investigate the effects of Local Bacteriotherapy in children with RRI. Both intranasal and oral Local Bacteriotherapy were evaluated. The findings showed that Local Bacteriotherapy significantly reduced the number of RI episodes, their severity, the use of antibiotics, and school absences. EXPERT OPINION: Local Bacteriotherapy is a promising approach to RRI prevention and could be a profitable strategy to contrast infections in the future.

Streptococcus mutans adhesion force sensing in multi-species oral biofilms.

Bacteria utilize chemical and mechanical mechanisms to sense their environment, to survive hostile conditions. In mechanical sensing, intra-bilayer pressure profiles change due to deformation induced by the adhesion forces bacteria experience on a surface. Emergent properties in mono-species Streptococcus mutans biofilms, such as extracellular matrix production, depend on the adhesion forces that streptococci sense. Here we determined whether and how salivary-conditioning film (SCF) adsorption and the multi-species nature of oral biofilm influence adhesion force sensing and associated gene expression by S. mutans. Hereto, Streptococcus oralis, Actinomyces naeslundii, and S. mutans were grown together on different surfaces in the absence and presence of an adsorbed SCF. Atomic force microscopy and RT-qPCR were used to measure S. mutans adhesion forces and gene expressions. Upon SCF adsorption, stationary adhesion forces decreased on a hydrophobic and increased on a hydrophilic surface to around 8 nN. Optical coherence tomography showed that triple-species biofilms on SCF-coated surfaces with dead S. oralis adhered weakly and often detached as a contiguous sheet. Concurrently, S. mutans displayed no differential adhesion force sensing on SCF-coated surfaces in the triple-species biofilms with dead S. oralis, but once live S. oralis were present S. mutans adhesion force sensing and gene expression ranked similar as on surfaces in the absence of an adsorbed SCF. Concluding, live S. oralis may enzymatically degrade SCF components to facilitate direct contact of biofilm inhabitants with surfaces and allow S. mutans adhesion force sensing of underlying surfaces to define its appropriate adaptive response. This represents a new function of initial colonizers in multi-species oral biofilms.

In Vitro Effects of Streptococcus oralis Biofilm on Peri-Implant Soft Tissue Cells.

Human gingival epithelial cells (HGEps) and fibroblasts (HGFs) are the main cell types in peri-implant soft tissue. HGEps are constantly exposed to bacteria, but HGFs are protected by connective tissue as long as the mucosa-implant seal is intact. Streptococcus oralis is one of the commensal bacteria, is highly abundant at healthy implant sites, and might modulate soft tissue cells-as has been described for other streptococci. We have therefore investigated the effects of the S. oralis biofilm on HGEps and HGFs. HGEps or HGFs were grown separately on titanium disks and responded to challenge with S. oralis biofilm. HGFs were severely damaged after 4 h, exhibiting transcriptional inflammatory and stress responses. In contrast, challenge with S. oralis only induced a mild transcriptional inflammatory response in HGEps, without cellular damage. HGFs were more susceptible to the S. oralis biofilm than HGEps. The pro-inflammatory interleukin 6 (IL-6) was attenuated in HGFs, as was interleukin 8 (CXCL8) in HGEps. This indicates that S. oralis can actively protect tissue. In conclusion, commensal biofilms can promote homeostatic tissue protection, but only if the implant-mucosa interface is intact and HGFs are not directly exposed.

Role of glucosyltransferase R in biofilm interactions between Streptococcus oralis and Candida albicans.

Streptococcal glucosyltransferases (Gtf) synthesize α-glucan exopolymers which contribute to biofilm matrix. Streptococcus oralis interacts with the opportunistic pathogen Candida albicans to form hypervirulent biofilms. S. oralis 34 has a single gtf gene (gtfR). However, the role of gtfR in single and mixed species biofilms with C. albicans has never been examined. A gtfR deletion mutant, purified GtfR, and recombinant GtfR glucan-binding domain were tested in single and mixed biofilms on different substrata in vitro. A mouse oral infection model was also used. We found that in single species biofilms growing with sucrose on abiotic surfaces S. oralis gtfR increased biofilm matrix, but not bacterial biomass. In biofilms with C. albicans, S. oralis encoding gtfR showed increased bacterial biomass on all surfaces. C. albicans had a positive effect on α-glucan synthesis, and α-glucans increased C. albicans accretion on abiotic surfaces. In single and mixed infection of mice receiving sucrose S. oralis gtfR enhanced mucosal burdens. However, sucrose had a negative impact on C. albicans burdens and reduced S. oralis burdens in co-infected mice. Our data provide new insights on the GtfR-mediated interactions between the two organisms and the influence of biofilm substratum and the mucosal environment on these interactions.

Commensal and pathogenic biofilms differently modulate peri-implant oral mucosa in an organotypic model.

The impact of oral commensal and pathogenic bacteria on peri-implant mucosa is not well understood, despite the high prevalence of peri-implant infections. Hence, we investigated responses of the peri-implant mucosa to Streptococcus oralis or Aggregatibacter actinomycetemcomitans biofilms using a novel in vitro peri-implant mucosa-biofilm model. Our 3D model combined three components, organotypic oral mucosa, implant material, and oral biofilm, with structural assembly close to native situation. S. oralis induced a protective stress response in the peri-implant mucosa through upregulation of heat shock protein (HSP70) genes. Attenuated inflammatory response was indicated by reduced cytokine levels of interleukin-6 (IL-6), interleukin-8 (CXCL8), and monocyte chemoattractant protein-1 (CCL2). The inflammatory balance was preserved through increased levels of tumor necrosis factor-alpha (TNF-α). A. actinomycetemcomitans induced downregulation of genes important for cell survival and host inflammatory response. The reduced cytokine levels of chemokine ligand 1 (CXCL1), CXCL8, and CCL2 also indicated a diminished inflammatory response. The induced immune balance by S. oralis may support oral health, whereas the reduced inflammatory response to A. actinomycetemcomitans may provide colonisation advantage and facilitate later tissue invasion. The comprehensive characterisation of peri-implant mucosa-biofilm interactions using our 3D model can provide new knowledge to improve strategies for prevention and therapy of peri-implant disease.

Putative Microbial Population Shifts Attributable to Nasal Administration of Streptococcus salivarius 24SMBc and Streptococcus oralis 89a.

Changes in bacterial composition of nasal microbiota may alter the host's susceptibility to several infectious and allergic diseases such as chronic rhinosinusitis and allergic rhinitis. The aim of this study was to evaluate the effects of 1-week administration of a probiotic product, composed by a combination of Streptococcus salivarius 24SMBc and Streptococcus oralis 89a, on the nostril microbiota. Differences in the nasal microbiota composition were investigated by using a next-generation sequencing approach. A strong and significant decrease in Staphylococcus aureus abundance was detected immediately after the bacterial administration. Moreover, comparing the microbial networks of nostril microbiota before and 1 month after the end of treatment, we detected an increase in the total number of both bacterial nodes and microbial correlations, with particular regard to the beneficial ones. Furthermore, a less abundance of microbial genera commonly associated to potential harmful bacteria has been observed. These results suggest a potential ability of S. salivarius 24SMBc and S. oralis 89a to regulate and reorganize the nasal microbiota composition, possibly favoring those microorganisms that may be able to limit the overgrowth of potential pathogens.

Probiotics Streptococcus salivarius 24SMB and Streptococcus oralis 89a interfere with biofilm formation of pathogens of the upper respiratory tract.

BACKGROUND: Infections of the ears, paranasal sinuses, nose and throat are very common and represent a serious issue for the healthcare system. Bacterial biofilms have been linked to upper respiratory tract infections and antibiotic resistance, raising serious concerns regarding the therapeutic management of such infections. In this context, novel strategies able to fight biofilms may be therapeutically beneficial and offer a valid alternative to conventional antimicrobials. Biofilms consist of mixed microbial communities, which interact with other species in the surroundings and communicate through signaling molecules. These interactions may result in antagonistic effects, which can be exploited in the fight against infections in a sort of "bacteria therapy". Streptococcus salivarius and Streptococcus oralis are α-hemolytic streptococci isolated from the human pharynx of healthy individuals. Several studies on otitis-prone children demonstrated that their intranasal administration is safe and well tolerated and is able to reduce the risk of acute otitis media. The aim of this research is to assess S. salivarius 24SMB and S. oralis 89a for the ability to interfere with biofilm of typical upper respiratory tract pathogens. METHODS: To investigate if soluble substances secreted by the two streptococci could inhibit biofilm development of the selected pathogenic strains, co-cultures were performed with the use of transwell inserts. Mixed-species biofilms were also produced, in order to evaluate if the inhibition of biofilm formation might require direct contact. Biofilm production was investigated by means of a spectrophotometric assay and by confocal laser scanning microscopy. RESULTS: We observed that S. salivarius 24SMB and S. oralis 89a are able to inhibit the biofilm formation capacity of selected pathogens and even to disperse their pre-formed biofilms. Diffusible molecules secreted by the two streptococci and lowered pH of the medium revealed to be implied in the mechanisms of anti-biofilm activity. CONCLUSIONS: S. salivarius 24SMB and S. oralis 89a possess desirable characteristics as probiotic for the treatment and prevention of infections of the upper airways. However, the nature of the inhibition appear to be multifactorial and additional studies are required to get further insights.

Surface properties of dental zirconia ceramics affected by ultrasonic scaling and low-temperature degradation.

Zirconia (3Y-TZP) dental prostheses are widely used in clinical dentistry. However, the effect of ultrasonic scaling performed as a part of professional tooth cleaning on 3Y-TZP dental prostheses, especially in conjunction with low-temperature degradation (LTD), has not been fully investigated. The present study aimed to evaluate the influence of ultrasonic scaling and LTD on the surface properties of 3Y-TZP in relation to bacterial adhesion on the treated surface. 3Y-TZP specimens (4 × 4 × 2 mm) were polished and then subjected to autoclaving at 134°C for 100 h to induce LTD, followed by 10 rounds of ultrasonic scaling using a steel scaler tip for 1 min each. Surface roughness, crystalline structure, wettability, and hardness were analyzed by optical interferometry, X-ray diffraction analysis, contact angle measurement, and nano-indentation technique, respectively. Subsequently, bacterial adhesion onto the treated 3Y-TZP surface was evaluated using Streptococcus mitis and S. oralis. The results demonstrated that the combination of ultrasonic scaling and LTD significantly increased the Sa value (surface roughness parameter) of the polished 3Y-TZP surface from 1.6 nm to 117 nm. LTD affected the crystalline structure, causing phase transformation from the tetragonal to the monoclinic phase, and decreased both the contact angle and surface hardness. However, bacterial adhesion was not influenced by these changes in surface properties. The present study suggests that ultrasonic scaling may be acceptable for debridement of 3Y-TZP dental prostheses because it did not facilitate bacterial adhesion even in the combination with LTD, although it did cause slight roughening of the surface.

Role of Neuraminidase-Producing Bacteria in Exposing Cryptic Carbohydrate Receptors for Streptococcus gordonii Adherence.

Streptococcus gordonii is an early colonizer of the oral cavity. Although a variety of S. gordonii adherence mechanisms have been described, current dogma is that the major receptor for S. gordonii is sialic acid. However, as many bacterial species in the oral cavity produce neuraminidase that can cleave terminal sialic acid, it is unclear whether S. gordonii relies on sialic acid for adherence to oral surfaces or if this species has developed alternative binding strategies. Previous studies have examined adherence to immobilized glycoconjugates and identified binding to additional glycans, but no prior studies have defined the contribution of these different glycan structures in adherence to oral epithelial cells. We determined that the majority of S. gordonii strains tested did not rely on sialic acid for efficient adherence. In fact, adherence of some strains was significantly increased following neuraminidase treatment. Further investigation of representative strains that do not rely on sialic acid for adherence revealed binding not only to sialic acid via the serine-rich repeat protein GspB but also to ß-1,4-linked galactose. Adherence to this carbohydrate occurs via an unknown adhesin distinct from those utilized by Streptococcus oralis and Streptococcus pneumoniae Demonstrating the potential biological relevance of binding to this cryptic receptor, we established that S. oralis increases S. gordonii adherence in a neuraminidase-dependent manner. These data suggest that S. gordonii has evolved to simultaneously utilize both terminal and cryptic receptors in response to the production of neuraminidase by other species in the oral environment.

Streptococcus oralis maintains homeostasis in oral biofilms by antagonizing the cariogenic pathogen Streptococcus mutans.

Bacteria residing in oral biofilms live in a state of dynamic equilibrium with one another. The intricate synergistic or antagonistic interactions between them are crucial for determining this balance. Using the six-species Zürich "supragingival" biofilm model, this study aimed to investigate interactions regarding growth and localization of the constituent species. As control, an inoculum containing all six strains was used, whereas in each of the further five inocula one of the bacterial species was alternately absent, and in the last, both streptococci were absent. Biofilms were grown anaerobically on hydroxyapatite disks, and after 64 h they were harvested and quantified by culture analyses. For visualization, fluorescence in situ hybridization and confocal laser scanning microscopy were used. Compared with the control, no statistically significant difference of total colony-forming units was observed in the absence of any of the biofilm species, except for Fusobacterium nucleatum, whose absence caused a significant decrease in total bacterial numbers. Absence of Streptococcus oralis resulted in a significant decrease in Actinomyces oris, and increase in Streptococcus mutans (P < .001). Absence of A. oris, Veillonella dispar or S. mutans did not cause any changes. The structure of the biofilm with regards to the localization of the species did not result in observable changes. In summary, the most striking observation of the present study was that absence of S. oralis resulted in limited growth of commensal A. oris and overgrowth of S. mutans. These data establish highlight S. oralis as commensal keeper of homeostasis in the biofilm by antagonizing S. mutans, so preventing a caries-favoring dysbiotic state.

Differential oxidative stress tolerance of Streptococcus mutans isolates affects competition in an ecological mixed-species biofilm model.

Streptococcus mutans strongly influences the development of pathogenic biofilms associated with dental caries. Our understanding of S. mutans behaviour in biofilms is based on a few well-characterized laboratory strains; however, individual isolates vary widely in genome content and virulence-associated phenotypes, such as biofilm formation and environmental stress sensitivity. Using an ecological biofilm model, we assessed the impact of co-cultivation of several S. mutans isolates with Streptococcus oralis and Actinomyces naeslundii on biofilm composition following exposure to sucrose. The laboratory reference strain S. mutans UA159 and clinical isolates Smu44 (most aciduric), Smu56 (altered biofilm formation) and Smu81 (more sensitive to oxidative stress) were used. Our data revealed S. mutans isolates varied in their ability to compete and become dominant in the biofilm after the addition of sucrose, and this difference correlated with sensitivity to H2 O2 produced by S. oralis. Smu81 was particularly sensitive to H2 O2 and could not compete with S. oralis in mixed-species biofilm, despite forming robust biofilms on its own. Thus, diminished oxidative stress tolerance in S. mutans isolates can impair their ability to compete in complex biofilms, even in the presence of sucrose, which could influence the progression of a healthy biofilm community to one capable of causing disease.

Synergistic antibiotic activity against planktonic and biofilm-embedded Streptococcus agalactiae, Streptococcus pyogenes and Streptococcus oralis.

OBJECTIVES: To determine the antimicrobial activity against streptococcal biofilm in species mostly isolated from implant-associated infections and examine the effect of enzyme treatment of biofilm on the antimicrobial activity of different antibiotics. METHODS: The activities of fosfomycin, rifampicin, benzylpenicillin, daptomycin, gentamicin, levofloxacin, proteinase K and their combinations on planktonic and/or biofilm-embedded standard laboratory strains of Streptococcus agalactiae, Streptococcus pyogenes and Streptococcus oralis were investigated in vitro by standard methods and isothermal microcalorimetry. RESULTS: MIC values obtained for the tested antimicrobials against planktonic bacteria ranged from 0.016 to 128 mg/L for the three species tested. Higher antibiotic concentrations were usually required to reduce biofilm in comparison with planktonic bacteria, with the exception of gentamicin, for which similar concentrations (4-16 mg/L) exerted an effect on both planktonic and biofilm cells. A synergistic effect against the streptococcal biofilm of the three species was observed when gentamicin was combined with benzylpenicillin or with rifampicin. Moreover, antibiotic concentrations comparable to the MIC observed against planktonic cells induced a strong reduction of viable bacteria in proteinase K pre-treated biofilm. CONCLUSIONS: This study shows that the combination of gentamicin with either benzylpenicillin or rifampicin exerts a synergistic effect against biofilms produced by the tested streptococci strains in vitro. Our results also suggest that coupling a dispersal agent with conventional antibiotics may facilitate their access to the bacteria within the biofilm. In vivo and clinical studies are needed in order to confirm whether such a strategy may be effective in the treatment of implant-associated infections caused by streptococci.

Electron Transport Chain Is Biochemically Linked to Pilus Assembly Required for Polymicrobial Interactions and Biofilm Formation in the Gram-Positive Actinobacterium Actinomyces oris.

The Gram-positive actinobacteria Actinomyces spp. are key colonizers in the development of oral biofilms due to the inherent ability of Actinomyces to adhere to receptor polysaccharides on the surface of oral streptococci and host cells. This receptor-dependent bacterial interaction, or coaggregation, requires a unique sortase-catalyzed pilus consisting of the pilus shaft FimA and the coaggregation factor CafA forming the pilus tip. While the essential role of the sortase machine SrtC2 in pilus assembly, biofilm formation, and coaggregation has been established, little is known about trans-acting factors contributing to these processes. We report here a large-scale Tn5 transposon screen for mutants defective in Actinomyces oris coaggregation with Streptococcus oralis We obtained 33 independent clones, 13 of which completely failed to aggregate with S. oralis, and the remainder of which exhibited a range of phenotypes from severely to weakly defective coaggregation. The former had Tn5 insertions in fimA, cafA, or srtC2, as expected; the latter were mapped to genes coding for uncharacterized proteins and various nuo genes encoding the NADH dehydrogenase subunits. Electron microscopy and biochemical analyses of mutants with nonpolar deletions of nuo genes and ubiE, a menaquinone C-methyltransferase-encoding gene downstream of the nuo locus, confirmed the pilus and coaggregation defects. Both nuoA and ubiE mutants were defective in oxidation of MdbA, the major oxidoreductase required for oxidative folding of pilus proteins. Furthermore, supplementation of the ubiE mutant with exogenous menaquinone-4 rescued the cell growth and pilus defects. Altogether, we propose that the A. oris electron transport chain is biochemically linked to pilus assembly via oxidative protein folding.IMPORTANCE The Gram-positive actinobacterium A. oris expresses adhesive pili, or fimbriae, that are essential to biofilm formation and Actinomyces interactions with other bacteria, termed coaggregation. While the critical role of the conserved sortase machine in pilus assembly and the disulfide bond-forming catalyst MdbA in oxidative folding of pilins has been established, little is known about other trans-acting factors involved in these processes. Using a Tn5 transposon screen for mutants defective in coaggregation with Streptococcus oralis, we found that genetic disruption of the NADH dehydrogenase and menaquinone biosynthesis detrimentally alters pilus assembly. Further biochemical characterizations determined that menaquinone is important for reactivation of MdbA. This study supports the notion that the electron transport chain is biochemically linked to pilus assembly in A. oris via oxidative folding of pilin precursors.

S. oralis activates the Efg1 filamentation pathway in C. albicans to promote cross-kingdom interactions and mucosal biofilms.

Candida albicans and Streptococcus oralis are ubiquitous oral commensal organisms. Under host-permissive conditions these organisms can form hypervirulent mucosal biofilms. C. albicans biofilm formation is controlled by 6 master transcriptional regulators: Bcr1, Brg1, Efg1, Tec1, Ndt80, and Rob1. The objective of this work was to test whether any of these regulators play a role in cross-kingdom interactions between C. albicans and S. oralis in oral mucosal biofilms, and identify downstream target gene(s) that promote these interactions. Organotypic mucosal constructs and a mouse model of oropharyngeal infection were used to analyze mucosal biofilm growth and fungal gene expression. By screening 6 C. albicans transcription regulator reporter strains we discovered that EFG1 was strongly activated by interaction with S. oralis in late biofilm growth stages. EFG1 gene expression was increased in polymicrobial biofilms on abiotic surfaces, mucosal constructs and tongue tissues of mice infected with both organisms. EFG1 was required for robust Candida-streptococcal biofilm growth in organotypic constructs and mouse oral tissues. S. oralis stimulated C. albicans ALS1 gene expression in an EFG1-dependent manner, and Als1 was identified as a downstream effector of the Efg1 pathway which promoted C. albicans-S. oralis coaggregation interactions in mixed biofilms. We conclude that S. oralis induces an increase in EFG1 expression in C. albicans in late biofilm stages. This in turn increases expression of ALS1, which promotes coaggregation interactions and mucosal biofilm growth. Our work provides novel insights on C. albicans genes which play a role in cross-kingdom interactions with S. oralis in mucosal biofilms.

Bactericidal effect of photocatalytically-active nanostructured TiO2 surfaces on biofilms of the early oral colonizer, Streptococcus oralis.

This study evaluated the photocatalytic bactericidal effect of nanostructured anatase-rich titanium dioxide (TiO2 ) on microbial biofilms. Commercially pure titanium discs were spin-coated with photocatalytic TiO2 nanoparticles (P25). Uncoated discs were used as control (CTRL). Half of the CTRL and half of the P25-coated surfaces were coated with purified saliva (SAL) to give four different groups (CTRL, CTRL + SAL, P25 and P25 + SAL). Streptococcus oralis were allowed to form biofilms on the discs for 18 h and non-adherent cells were rinsed off. Bacterial viability was assessed at time 0 with Live/Dead BacLight staining and epifluorescence microscopy. The remaining discs were divided into a non-UV group and UVA-irradiated (+UV) group (irradiation time, 6 or 24 h). Thereafter, viability was assessed as above. Viability at time 0 was high and no dead cells were seen on any of the surfaces, even after 24 h, in the absence of UVA. However, after 24 h of exposure, the proportion of viable cells was reduced by 40% on the P25 discs compared to 0 and 6 h, and this effect was enhanced with a salivary pellicle. Members of mixed species biofilms differ in their susceptibility to the bactericidal effect of the surfaces tested and further investigations are needed to optimize the conditions. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2321-2328, 2017.

Microarray analysis of macrophage response to infection with Streptococcus oralis reveals the immunosuppressive effect of hydrogen peroxide.

Oral streptococci including mitis group streptococci are commensal residents and are also the first to colonize the oral cavity. However, various species of these oral streptococci have the potential to invade the host and occasionally lead to severe infectious disease such as cardiovascular diseases. Oral streptococci have close interactions with the host immune system including macrophages at the oral mucosal surface. One notable common trait of oral streptococcus including Streptococcus oralis (S. oralis) is the production of hydrogen peroxide (H2O2). Using a comprehensive microarray approach, we sought to understand the innate immune response profiling affected by H2O2 production from oral streptococci. We compared the gene expression patterns of macrophages infected with S. oralis wild type (WT) and streptococcal pyruvate oxidase knockout (SpxB-KO), a strain that does not produce H2O2. We found that H2O2 from S. oralis suppressed proinflammatory gene expression such as TNF-α, that is induced in response to infection, and activated the cellular stress genes such as Egr-1 in response to oxidative stress. A comparative gene ontology analysis of S. oralis WT and SpxB-KO strains revealed that during infection, down regulated genes were closely related to the processes involved in the host defense reaction and up regulated genes were related with the cellular stress responses. Using qPCR analysis, we also confirmed the same pattern of expression changes such as TNF-α, IL-6 and Egr-1. Furthermore, supernatant from SpxB-KO could not suppress the expression of TNF-α in macrophages stimulated with LPS. These findings suggested that H2O2 production from S. oralis leads to the suppression of inflammatory responses and NF-κB signaling pathways in macrophages as well as the induction of the oxidative stress response. We concluded that streptococcal H2O2 production has the beneficial effects of modulating the innate immune response, thereby stabilizing streptococcal colonization at the mucosal surface and even in the bloodstream leading to cardiovascular disease after invasion, in addition to the commensal role to compete other bacterial species as initial colonizer at oral cavity.

Interactions between Streptococcus oralis, Actinomyces oris, and Candida albicans in the development of multispecies oral microbial biofilms on salivary pellicle.

The fungus Candida albicans is carried orally and causes a range of superficial infections that may become systemic. Oral bacteria Actinomyces oris and Streptococcus oralis are abundant in early dental plaque and on oral mucosa. The aims of this study were to determine the mechanisms by which S. oralis and A. oris interact with each other and with C. albicans in biofilm development. Spatial distribution of microorganisms was visualized by confocal laser scanning microscopy of biofilms labeled by differential fluorescence or by fluorescence in situ hybridization (FISH). Actinomyces oris and S. oralis formed robust dual-species biofilms, or three-species biofilms with C. albicans. The bacterial components tended to dominate the lower levels of the biofilms while C. albicans occupied the upper levels. Non-fimbriated A. oris was compromised in biofilm formation in the absence or presence of streptococci, but was incorporated into upper biofilm layers through binding to C. albicans. Biofilm growth and hyphal filament production by C. albicans was enhanced by S. oralis. It is suggested that the interkingdom biofilms are metabolically coordinated to house all three components, and this study demonstrates that adhesive interactions between them determine spatial distribution and biofilm architecture. The physical and chemical communication processes occurring in these communities potentially augment C. albicans persistence at multiple oral cavity sites.

Streptococcus oralis Neuraminidase Modulates Adherence to Multiple Carbohydrates on Platelets.

Adherence to host surfaces is often mediated by bacterial binding to surface carbohydrates. Although it is widely appreciated that some bacterial species express glycosidases, previous studies have not considered whether bacteria bind to multiple carbohydrates within host glycans as they are modified by bacterial glycosidases. Streptococcus oralis is a leading cause of subacute infective endocarditis. Binding to platelets is a critical step in disease; however, the mechanisms utilized by S. oralis remain largely undefined. Studies revealed that S. oralis, like Streptococcus gordonii and Streptococcus sanguinis, binds platelets via terminal sialic acid. However, unlike those organisms, S. oralis produces a neuraminidase, NanA, which cleaves terminal sialic acid. Further studies revealed that following NanA-dependent removal of terminal sialic acid, S. oralis bound exposed ß-1,4-linked galactose. Adherence to both these carbohydrates required Fap1, the S. oralis member of the serine-rich repeat protein (SRRP) family of adhesins. Mutation of a conserved residue required for sialic acid binding by other SRRPs significantly reduced platelet binding, supporting the hypothesis that Fap1 binds this carbohydrate. The mechanism by which Fap1 contributes to ß-1,4-linked galactose binding remains to be defined; however, binding may occur via additional domains of unknown function within the nonrepeat region, one of which shares some similarity with a carbohydrate binding module. This study is the first demonstration that an SRRP is required to bind ß-1,4-linked galactose and the first time that one of these adhesins has been shown to be required for binding of multiple glycan receptors.