Oral streptococci S. anginosus and S. mitis induce distinct morphological, inflammatory, and metabolic signatures in macrophages.

Oral streptococci, key players in oral biofilm formation, are implicated in oral dysbiosis and various clinical conditions, including dental caries, gingivitis, periodontal disease, and oral cancer. Specifically, Streptococcus anginosus is associated with esophageal, gastric, and pharyngeal cancers, while Streptococcus mitis is linked to oral cancer. However, no study has investigated the mechanistic links between these Streptococcus species and cancer-related inflammatory responses. As an initial step, we probed the innate immune response triggered by S. anginosus and S. mitis in RAW264.7 macrophages. These bacteria exerted time- and dose-dependent effects on macrophage morphology without affecting cell viability. Compared with untreated macrophages, macrophages infected with S. anginosus exhibited a robust proinflammatory response characterized by significantly increased levels of inflammatory cytokines and mediators, including TNF, IL-6, IL-1ß, NOS2, and COX2, accompanied by enhanced NF-κB activation. In contrast, S. mitis-infected macrophages failed to elicit a robust inflammatory response. Seahorse Xfe96 analysis revealed an increased extracellular acidification rate in macrophages infected with S. anginosus compared with S. mitis. At the 24-h time point, the presence of S. anginosus led to reduced extracellular itaconate, while S. mitis triggered increased itaconate levels, highlighting distinct metabolic profiles in macrophages during infection in contrast to aconitate decarboxylase expression observed at the 6-h time point. This initial investigation highlights how S. anginosus and S. mitis, two Gram-positive bacteria from the same genus, can prompt distinct immune responses and metabolic shifts in macrophages during infection.IMPORTANCEThe surge in head and neck cancer cases among individuals devoid of typical risk factors such as Human Papilloma Virus (HPV) infection and tobacco and alcohol use sparks an argumentative discussion around the emerging role of oral microbiota as a novel risk factor in oral squamous cell carcinoma (OSCC). While substantial research has dissected the gut microbiome's influence on physiology, the oral microbiome, notably oral streptococci, has been underappreciated during mucosal immunopathogenesis. Streptococcus anginosus, a viridans streptococci group, has been linked to abscess formation and an elevated presence in esophageal cancer and OSCC. The current study aims to probe the innate immune response to S. anginosus compared with the early colonizer Streptococcus mitis as an important first step toward understanding the impact of distinct oral Streptococcus species on the host immune response, which is an understudied determinant of OSCC development and progression.

Streptococcus Species Abundance in the Gut Is Linked to Subclinical Coronary Atherosclerosis in 8973 Participants From the SCAPIS Cohort.

BACKGROUND: Gut microbiota have been implicated in atherosclerotic disease, but their relation with subclinical coronary atherosclerosis is unclear. This study aimed to identify associations between the gut microbiome and computed tomography-based measures of coronary atherosclerosis and to explore relevant clinical correlates. METHODS: We conducted a cross-sectional study of 8973 participants (50 to 65 years of age) without overt atherosclerotic disease from the population-based SCAPIS (Swedish Cardiopulmonary Bioimage Study). Coronary atherosclerosis was measured using coronary artery calcium score and coronary computed tomography angiography. Gut microbiota species abundance and functional potential were assessed with shotgun metagenomics sequencing of fecal samples, and associations with coronary atherosclerosis were evaluated with multivariable regression models adjusted for cardiovascular risk factors. Associated species were evaluated for association with inflammatory markers, metabolites, and corresponding species in saliva. RESULTS: The mean age of the study sample was 57.4 years, and 53.7% were female. Coronary artery calcification was detected in 40.3%, and 5.4% had at least 1 stenosis with >50% occlusion. Sixty-four species were associated with coronary artery calcium score independent of cardiovascular risk factors, with the strongest associations observed for Streptococcus anginosus and Streptococcus oralis subsp oralis (P<1×10-5). Associations were largely similar across coronary computed tomography angiography-based measurements. Out of the 64 species, 19 species, including streptococci and other species commonly found in the oral cavity, were associated with high-sensitivity C-reactive protein plasma concentrations, and 16 with neutrophil counts. Gut microbial species that are commonly found in the oral cavity were negatively associated with plasma indole propionate and positively associated with plasma secondary bile acids and imidazole propionate. Five species, including 3 streptococci, correlated with the same species in saliva and were associated with worse dental health in the Malmö Offspring Dental Study. Microbial functional potential of dissimilatory nitrate reduction, anaerobic fatty acid ß-oxidation, and amino acid degradation were associated with coronary artery calcium score. CONCLUSIONS: This study provides evidence of an association of a gut microbiota composition characterized by increased abundance of Streptococcus spp and other species commonly found in the oral cavity with coronary atherosclerosis and systemic inflammation markers. Further longitudinal and experimental studies are warranted to explore the potential implications of a bacterial component in atherogenesis.

Aspects of oral streptococcal metabolic diversity: Imagining the landscape beneath the fog.

It is widely acknowledged that the human-associated microbial community influences host physiology, systemic health, disease progression, and even behavior. There is currently an increased interest in the oral microbiome, which occupies the entryway to much of what the human initially encounters from the environment. In addition to the dental pathology that results from a dysbiotic microbiome, microbial activity within the oral cavity exerts significant systemic effects. The composition and activity of the oral microbiome is influenced by (1) host-microbial interactions, (2) the emergence of niche-specific microbial "ecotypes," and (3) numerous microbe-microbe interactions, shaping the underlying microbial metabolic landscape. The oral streptococci are central players in the microbial activity ongoing in the oral cavity, due to their abundance and prevalence in the oral environment and the many interspecies interactions in which they participate. Streptococci are major determinants of a healthy homeostatic oral environment. The metabolic activities of oral Streptococci, particularly the metabolism involved in energy generation and regeneration of oxidative resources vary among the species and are important factors in niche-specific adaptations and intra-microbiome interactions. Here we summarize key differences among streptococcal central metabolic networks and species-specific differences in how the key glycolytic intermediates are utilized.

Combined pH ratiometry and fluorescence lectin-binding analysis (pH-FLBA) for microscopy-based analyses of biofilm pH and matrix carbohydrates.

Bacterial biofilms have a complex and heterogeneous three-dimensional architecture that is characterized by chemically and structurally distinct microenvironments. Confocal microscopy-based pH ratiometry and fluorescence lectin-binding analysis (FLBA) are well-established methods to characterize pH developments and the carbohydrate matrix architecture of biofilms at the microscale. Here, we developed a combined analysis, pH-FLBA, to concomitantly map biofilm pH and the distribution of matrix carbohydrates in bacterial biofilms while preserving the biofilm microarchitecture. As a proof of principle, the relationship between pH and the presence of galactose- and fucose-containing matrix components was investigated in dental biofilms grown with and without sucrose. The pH response to a sucrose challenge was monitored in different areas at the biofilm base using the ratiometric pH-sensitive dye C-SNARF-4. Thereafter, the fucose- and galactose-specific fluorescently labeled lectins Aleuria aurantia lectin (AAL) and Morus nigra agglutinin G (MNA-G) were used to visualize carbohydrate matrix components in the same biofilm areas and their immediate surroundings. Sucrose during growth significantly decreased biofilm pH (P < 0.05) and increased the amounts of both MNA-G- and AAL-targeted matrix carbohydrates (P < 0.05). Moreover, it modulated the biofilm composition towards a less diverse community dominated by streptococci, as determined by 16S rRNA gene sequencing. Altogether, these results suggest that the production of galactose- and fucose-containing matrix carbohydrates is related to streptococcal metabolism and, thereby, pH profiles in dental biofilms. In conclusion, pH-FLBA using lectins with different carbohydrate specificities is a useful method to investigate the association between biofilm pH and the complex carbohydrate architecture of bacterial biofilms.IMPORTANCEBiofilm pH is a key regulating factor in several biological and biochemical processes in environmental, industrial, and medical biofilms. At the microscale, microbial biofilms are characterized by steep pH gradients and an extracellular matrix rich in carbohydrate components with diffusion-modifying properties that contribute to bacterial acid-base metabolism. Here, we propose a combined analysis of pH ratiometry and fluorescence lectin-binding analysis, pH-FLBA, to concomitantly investigate the matrix architecture and pH developments in microbial biofilms, using complex saliva-derived biofilms as an example. Spatiotemporal changes in biofilm pH are monitored non-invasively over time by pH ratiometry, while FLBA with lectins of different carbohydrate specificities allows mapping the distribution of multiple relevant matrix components in the same biofilm areas. As the biofilm structure is preserved, pH-FLBA can be used to investigate the in situ relationship between the biofilm matrix architecture and biofilm pH in complex multispecies biofilms.

Isolation of phages against Streptococcus species in the oral cavity for potential control of dental diseases and associated systemic complications.

Dental infections and systemic complications caused by Streptococcus species in the oral cavity are increasingly exhibiting resistance to commonly used antibiotics, posing a potential threat to global public health. Phage therapy may offer a superior alternative, given that bacteriophages can be easily isolated and rapidly replicate in large numbers. In this study, six Streptococcus species from the oral cavity were characterized. Bacteriophages isolated from wastewater using five of these species as hosts produced plaques ranging from 0.2 to 2.4 mm in size. The phages demonstrated stability within a temperature range of 4 â„ƒ to 37 â„ƒ. However, at temperatures exceeding 45 â„ƒ, a noticeable reduction in bacteriophage titer was observed. Similarly, the phages showed greater stability within a pH range of 5 to 10. The isolated phages exhibited latency periods ranging from 15 to 20 min and had burst sizes varying from 10 to 200 viral particles. This study supports the potential use of bacteriophages in controlling infections caused by Streptococcus species.

Odontogenic infections in the antibiotic era: approach to diagnosis, management, and prevention.

PURPOSE: The prevalence of odontogenic infections remains one of the highest in the world. If untreated, odontogenic infections can break through the limitation, disseminate to other organs or spaces, and cause high mortality rates. However, it is still difficult to rapidly target limited or disseminated infections in clinical practice. The type of disseminated odontogenic infections and the responsible bacteria have not been described in detail. METHODS: Search databases (e.g., PubMed, MEDLINE, Web of Science, Embase) for reports published from 2018.1 to 2022.9. Use search strategies: ("odontogenic infections" OR "pulpitis" OR "periapical lesions" OR "periodontal diseases") AND ("disseminated infections" OR "complication"). RESULTS: Fourteen different types of disseminated odontogenic infections, most of which are polymicrobial infections, can spread through the body either direct or through hematogenous diffusion. Multiple microbial infections can be more invasive in the transmission of infection. Secondary infections are commonly associated with bacteria like Fusobacterium spp., Streptococcus spp., Peptostreptococcus spp., Prevotella spp., and Staphylococcus spp. Antibiotics with broad-spectrum activity are fundamental as first-line antimicrobial agents based on the microorganisms isolated from disseminated infections. CONCLUSION: This review elaborates on the epidemiology, microorganisms, risk factors, and dissemination routes, and provides evidence-based opinions on the diagnosis, multidisciplinary management, and prevention of odontogenic infections for dentists and clinicians.

Production improvement of an antioxidant in cariogenic Streptococcus mutans UA140.

AIMS: This study aimed to improve the production of mutantioxidin, an antioxidant encoded by a biosynthetic gene cluster (mao) in Streptococcus mutans UA140, through a series of optimization methods. METHOD AND RESULTS: Through the construction of mao knockout strain S. mutans UA140∆mao, we identified mutantioxidin as the antioxidant encoded by mao and verified its antioxidant activity through a reactive oxygen species (ROS) tolerance assay. By optimizing the culture medium and fermentation time, 72 h of fermentation in chemically defined medium (CDM) medium was determined as the optimal fermentation conditions. Based on two promoters commonly used in Streptococcus (ldhp and xylS1p), eight promoter refactoring strains were constructed, nevertheless all showed impaired antioxidant production. In-frame deletion and complementation experiments demonstrated the positive regulatory role of mao1 and mao2, on mao. Afterward, the mao1 and mao2, overexpression strain S. mutans UA140/pDL278:: mao1mao2, were constructed, in which the production of mutantioxidin was improved significantly. CONCLUSIONS: In this study, through a combination of varied strategies such as optimization of fermentation conditions and overexpression of regulatory genes, production of mutantioxidin was increased by 10.5 times ultimately.

High Adherence of Oral Streptococcus to Polylactic Acid Might Explain Implant Infections Associated with PLA Mesh Implantation.

The aim of this study was to evaluate and compare the biofilm formation properties of common pathogens associated with implant-related infections on two different implant material types. Bacterial strains tested in this study were Staphylococcus aureus, Streptococcus mutans, Enterococcus faecalis, and Escherichia coli. Implant materials tested and compared were PLA Resorb × polymer of Poly DL-lactide (PDLLA) comprising 50% poly-L-lactic acid and 50% poly-D-lactic acid) and Ti grade 2 (tooled with a Planmeca CAD-CAM milling device). Biofilm assays were done with and without saliva treatment to evaluate the effect of saliva on bacterial adhesion and to mimic the intraoral and extraoral surgical routes of implant placement, respectively. Five specimens of each implant type were tested for each bacterial strain. Autoclaved material specimens were first treated with 1:1 saliva-PBS solution for 30 min, followed by washing of specimens and the addition of bacterial suspension. Specimens with bacterial suspension were incubated for 24 h at 37 °C for biofilm formation. After 24 h, non-adhered bacteria were removed, and specimens were washed, followed by removal and calculation of adhered bacterial biofilm. S. aureus and E. faecalis showed more attachment to Ti grade 2, whereas S. mutans showed higher adherence to PLA in a statistically significant manner. The salivary coating of specimens enhanced the bacterial attachment by all the bacterial strains tested. In conclusion, both implant materials showed significant levels of bacterial adhesion, but saliva treatment played a vital role in bacterial attachment, therefore, saliva contamination of the implant materials should be minimized and considered when placing implant materials inside the body.

Increased IL-12p70 and IL-8 Produced by Monocytes in Response to Streptococcus spp. and Actinomyces spp. Causals of Endodontic Primary Infections.

We sought to evaluate the effect of endodontic-causative microorganisms of primary infections on mononuclear cells such as CD14+, CD4+, CD8+, CD19+ and Tregs Foxp3+. Facultative anaerobic microorganisms were isolated from radicular conducts and peripheral blood samples, which were taken from patients with primary infections. Cellular cultures were performed with peripheral blood mononuclear cells (PBMC) with and without Actinomyces spp. and Streptococcus spp. during 48, 72, and 96 h of contact in culture (concentration 5 × 105 cells/well) in a round plate bound with 48 wells. Later, PBMC was collected for analysis by flow cytometry, with the monoclonal antibodies αCD14, αCD4, αCD8, αCD19 and αFoxp3, and acquired using an FACSCanto II cytometer. The supernatant of cellular cultures was analyzed for the quantification of inflammatory cytokines. Data analysis was performed in FlowJo v10.8.2 and FCAPArray software, and statistical analysis was performed using GraphPad v5.0. software. We observed an increase in the percentage of CD14+ cells in patients at different hours of cellular culture in the presence of both Actinomyces spp. and Streptococcus spp. microorganisms, compared to healthy controls. This study demonstrates the role played by the innate immune system in the pathogeny of endodontic primary infections, explaining the effects that generate the more common microorganisms in this oral pathology.

Oral microbial communities in 5-year-old children with versus without dental caries.

BACKGROUND: Caries in young children has received more and more attention. The study of the oral microbiota may help to understand the polymicrobial etiology of dental caries. OBJECTIVES: To investigate the diversity and structure of microbial communities in saliva samples from 5-year-old children with versus without dental caries. METHODS: A total of 36 saliva samples were collected from 18 children with high caries (HB group) and from 18 children without caries (NB group). Then, 16S rDNA was amplified from bacterial samples using polymerase chain reaction, and high-throughput sequencing was performed using Illumina Novaseq platforms. RESULTS: Sequences were clustered into operational taxonomic units (OTUs), which were distributed among 16 phyla, 26 classes, 56 orders, 93 families, 173 genera, and 218 species. Firmicutes, Bacteroides, Proteobacteria, Actinobacteria, Fusobacteria, Patescibacteria, Epsilonbacteraeota, Cyanobacteria, Acidobacteria and Spirochaetes were basically the same in different groups, but their relative abundances were different. The core microbiome was defined as the species from 218 shared microbial taxa. The alpha diversity test showed that there were no significant differences in microbial abundance and diversity between the high caries and no caries groups. The results from principal coordinate analysis (PCoA) and hierarchical clustering showed that the two groups had similar microorganisms. The biomarkers of different groups were defined by LEfSe analysis to identify potential caries-related and health-related bacteria. Co-occurrence network analysis of dominant genera showed that oral microbial communities in the no caries group were more complex and aggregated than those in the high caries group. Finally, the PICRUSt algorithm was used to predict the function of the microbial communities from saliva samples. The obtained results showed that mineral absorption was greater in the no caries group than in the high caries group. BugBase was used to determine phenotypes present in microbial community samples. The obtained results showed that Streptococcus was greater in the high caries group than in the no caries group. CONCLUSION: Findings of this study provide a comprehensive understanding of the microbiological etiology of dental caries in 5-year-old children and are expected to provide new methods for its prevention and treatment.

Associated Bacterial Coinfections in COVID-19-Positive Patients.

Background and Objectives: The aim of this study was to identify specific rhino- and oropharyngeal microbiological pathogens as well as associated comorbidities that favor SARS-CoV-2 infection and corelate them. Materials and Methods: This prospective clinical study enrolled 61 patients (28 COVID-19-positive and 33 controls) who were tested for other comorbidities and co-existence of associated oral pathogenic microbiota. Results: A total of 247 bacterial isolates were identified in the bacterial cultures in both groups. Viral hepatitis type A was more prevalent in the COVID-19-positive group (p = 0.026), as was the presence of oral candidiasis (p = 0.006). In the control group, a moderate direct relationship was observed between the Beta hemolytic streptococcus group G and dermatitis, and strong direct relationships were observed between the Beta hemolytic streptococcus group G and external otitis, Streptococcus pyogenes and dental alveolitis, and Streptococcus pyogenes and chronic lymphocytic leukemia. In the test group, strong direct relationships were observed between Hemophilus influenzae and pulmonary thromboembolism; Staphylococcus aureus and autoimmune thyroiditis; post-viral immunosuppression, chronic coronary syndrome, and hypernatremia; Beta hemolytic streptococcus group C and rheumatoid polyneuropathy; Beta hemolytic streptococcus group G and hyperkalemia, hypothyroidism, secondary anemia, and splenomegaly; and active oral candidiasis and SARS-CoV-2 viral pneumonia. The following relationships were strong, but inverse: Beta hemolytic streptococcus group G and acute respiratory failure, and active oral candidiasis and SARS-CoV-2 viral bronchopneumonia. Conclusions: Briefly, COVID-19-positive patients have the predisposition to build up associated comorbidities and coinfections, which can be the expression of the immune burden that this virus generates to the host.

Oral microbiome characteristics in children with and without early childhood caries.

OBJECTIVE: Early childhood caries (ECC) negatively affects children's growth due to its close relation to an imbalance of the oral microbiota. This study aimed to evaluate the distribution of the oral microbiota in children with ECC and healthy individuals. METHODS: The oral microbiota of 20 children with dental caries from both carious teeth (CC cohort) and healthy teeth (CH cohort), and the oral microbiota of 20 healthy control children (HH cohort) were subjected to 16S rDNA sequencing. RESULTS: The results revealed significant differences between the microbial structure of the CC and CH cohorts of every child with ECC. The most common microbes were Streptococcus, Neisseria, Leptotrichia, Lautropia and Haemophilus. Specifically, the CC cohort contained Lactobacillus, Veillonella, and Prevotella 7, the CH cohort contained Actinomyces, Bifidobacterium and Abiotrophia, and the HH cohort mainly contained Neisseria, Leptotrichia, Porphyromonas and Gemella. Lastly, we established a random forest model consisting of 10 genera (Prevotella 7, Actinobacillus, etc.) which demonstrated promising clinical diagnostic ability (area under the curve (AUC) = 89.8%). These findings indicate that oral microbiota can potentially be used as therapeutic targets or diagnostic markers for the early prediction and prevention of caries in children.

Oral Commensal Streptococci: Gatekeepers of the Oral Cavity.

Oral commensal streptococci are primary colonizers of the oral cavity. These streptococci produce many adhesins, metabolites, and antimicrobials that modulate microbial succession and diversity within the oral cavity. Often, oral commensal streptococci antagonize cariogenic and periodontal pathogens such as Streptococcus mutans and Porphyromonas gingivalis, respectively. Mechanisms of antagonism are varied and range from the generation of hydrogen peroxide, competitive metabolite scavenging, the generation of reactive nitrogen intermediates, and bacteriocin production. Furthermore, several oral commensal streptococci have been shown to alter the host immune response at steady state and in response to oral pathogens. Collectively, these features highlight the remarkable ability of oral commensal streptococci to regulate the structure and function of the oral microbiome. In this review, we discuss mechanisms used by oral commensal streptococci to interact with diverse oral pathogens, both physically and through the production of antimicrobials. Finally, we conclude by exploring the critical roles of oral commensal streptococci in modulating the host immune response and maintaining health and homeostasis.

Optimization and Evaluation of the 30S-S11 rRNA Gene for Taxonomic Profiling of Oral Streptococci.

Dental caries is a multifactorial disease driven by interactions between the highly complex microbial biofilm community and host factors like diet, oral hygiene habits, and age. The oral streptococci are one of the most dominant members of the plaque biofilm and are implicated in disease but also in maintaining oral health. Current methods used for studying the supragingival plaque community commonly sequence portions of the16S rRNA gene, which often cannot taxonomically resolve members of the streptococcal community past the genus level due to their sequence similarity. The goal of this study was to design and evaluate a more reliable and cost-effective method to identify oral streptococci at the species level by applying a new locus, the 30S-S11 rRNA gene, for high-throughput amplicon sequencing. The study results demonstrate that the newly developed single-copy 30S-S11 gene locus resolved multiple amplicon sequence variants (ASVs) within numerous species, providing much improved taxonomic resolution over 16S rRNA V4. Moreover, the results reveal that different ASVs within a species were found to change in abundance at different stages of caries progression. These findings suggest that strains of a single species may perform distinct roles along a biochemical spectrum associated with health and disease. The improved identification of oral streptococcal species will provide a better understanding of the different ecological roles of oral streptococci and inform the design of novel oral probiotic formulations for prevention and treatment of dental caries. IMPORTANCE The microbiota associated with the initiation and progression of dental caries has yet to be fully characterized. Although much insight has been gained from 16S rRNA hypervariable region DNA sequencing, this approach has several limitations, including poor taxonomic resolution at the species level. This is particularly relevant for oral streptococci, which are abundant members of oral biofilm communities and major players in health and caries disease. Here, we develop a new method for taxonomic profiling of oral streptococci based on the 30S-S11 rRNA gene, which provides much improved resolution over 16S rRNA V4 (resolving 10 as opposed to 2 species). Importantly, 30S-S11 can resolve multiple amplicon sequence variants (ASVs) within species, providing an unprecedented insight into the ecological progression of caries. For example, our findings reveal multiple incidences of different ASVs within a species with contrasting associations with health or disease, a finding that has high relevance toward the informed design of prebiotic and probiotic therapy.

Reclassification of Streptococcus ilei as a later heterotypic synonym of Streptococcus koreensis based on whole-genome sequence analysis.

The genus Streptococcus, a member of family Streptococcaceae, is known for its wide range of industrial, clinical and human relevance. Among the species of genus Streptococcus two members, namely Streptococcus koreensis and Streptococcus ilei, were isolated from subgingival dental plaque and human small intestinal fluid, respectively. The 16S rRNA gene sequence similarity of the type strains of these members shows a similarity of 99.87%. In this study, we performed a systematic study to clarify the taxonomic assignment of these two species. Genome similarity assessment based on whole-genome sequence information such as average nucleotide identity using orthoANI and fastANI, digital DNA-DNA hybridization value between S. koreensis and S. ilei were 96.31, 96.60, 86.4 and 97.63, respectively. All these genome similarity values clearly exceeded the species delineation cutoffs. Phylogenetic assessment using 16S rRNA gene and whole-genome information using PhyloPhlAn, which uses around 400 conserved genes across bacterial phyla, provides additional evidence for these members forming a monophyletic clade in the phylogenetic tree. Pan genome analysis suggests a very large core genome (n = 1374) and the presence of no unique gene between the genomes of S. koreensis and S. ilei. Additionally, we found highly syntenic genomes of type strains of these two species. Based on these evidences, we propose S. ilei should be reclassified as a later heterotypic synonym of S. koreensis.

Testing of candidate probiotics to prevent dental caries induced by Streptococcus mutans in a mouse model.

AIMS: We evaluated two species of human oral commensal streptococci in protection against dental caries induced by Streptococcus mutans. METHODS AND RESULTS: Candidate probiotics, Streptococcus sp. A12, Streptococcus sanguinis BCC23 and an arginine deiminase mutant of BCC23 (∆arcADS) were tested for their ability to reduce S. mutans-induced caries in an established mouse model. Mice were colonized with a probiotic, challenged with S. mutans, then intermittently reinoculated with a probiotic strain. Oral colonization of each strain and autochthonous bacteria was assessed by quantitative polymerase chain reaction. Both BCC23 strains, but not A12, were associated with markedly reduced sulcal caries, persistently colonized mucosal and dental biofilms, and significantly lowered S. mutans counts. All three strains enhanced mucosal colonization of autochthonous bacteria. In a follow-up experiment, when S. mutans was established first, dental and mucosal colonization of S. mutans was unaltered by a subsequent challenge with either BCC23 strain. Results between BCC23 and BCC23 ∆arcADS were equivalent. CONCLUSIONS: BCC23 is a potential probiotic to treat patients at high caries risk. Its effectiveness is independent of ADS activity, but initial dental cleaning to enhance establishment in dental biofilms may be required. SIGNIFICANCE AND IMPACT OF THE STUDY: In vivo testing of candidate probiotics is highly informative, as effectiveness is not always reflected by genotype or in vitro behaviours.

Tea extracts inhibit the attachment of streptococci to oral/dental substrata by reducing hydrogen bonding energies.

Previous work in the authors' lab demonstrated that tea extracts significantly suppressed streptococcal colonization of abiotic substrata by coating the bacterial cell surfaces with tea components. In this study, the physico-chemical mechanisms by which the tea coating inhibits cellular attachment are demonstrated. The changes in the cell surface physico-chemical properties of streptococci, induced by tea extracts, were measured. Using these results, surface interaction energies were calculated between streptococcal cells and hard surfaces (glass, stainless steel, hydroxyapatite and titanium) within the cellular attachment system exploiting the extended Derjaguin-Landau-Verwey-Overbeek theory. The net energy outcomes were compared with experiment results of attachment assays to validate the predictability of the model. The results showed that the tea extracts inhibited the attachment of the bacteria by 11.1%-91.5%, and reduced the interaction energy by 15.4%-94.9%. It was also demonstrated that the abilities of the bacteria to attach to hard surfaces correlated well with their net interaction energies. The predominant interaction in the systems was found to be hydrogen bonding. In conclusion, tea extracts suppress streptococcal attachment to hard substrata by limiting the formation of hydrogen bonds.

Comparative evaluation of the antibacterial effect of Allium sativum, calcium hydroxide and their combination as intracanal medicaments in infected mature anterior teeth: A randomized clinical trial.

AIM: The purpose of this study was to compare the antibacterial effects of Allium sativum (garlic extract), calcium hydroxide (Ca [OH]2 ) and their combination as intracanal medicaments in infected mature anterior teeth using real-time PCR. METHODOLOGY: This prospective double-blind, controlled, parallel, superiority, randomized clinical trial was carried out on 66 permanent, necrotic incisors associated with asymptomatic apical periodontitis in 66 male patients. Patients were randomly divided into three groups (n = 22) according to the intracanal medications used. After access preparation, four microbiological samples (S) were taken using sterile absorbent paper points as follows: S1: before canal instrumentation and S2: after cleaning and shaping. The third sample (S3) and fourth sample (S4) were taken after the placement of the tested intracanal medications into their corresponding canals for 7 and 14 days, respectively. Total DNA was extracted from microbiological samples and relative quantitative real-time PCRs were done to quantify the relative gene expression fold change (FC) for Enterococcus faecalis and Streptococcus species. At significance level p ≤ .05, the data were statistically analysed in SPSS software using Kruskal-Wallis and Freidman's tests, followed by Dunn-Bonferroni post hoc test for pairwise comparisons. RESULTS: Both bacterial mean FC decreased significantly after mechanical instrumentation (S1 to S2) in all groups. However, no statistically significant differences were found after intracanal medicament placement (from S2 to S3 and from S3 to S4) except in the garlic group. Garlic significantly reduced Enterococcus faecalis FC in S3 and S4 when compared to Ca (OH)2 and Ca (OH)2 + garlic combination. However, garlic and Ca (OH)2 reduced Streptococcus bacteria in S3 similarly. Whilst in S4, garlic showed significantly more reduction than Ca (OH)2 . The combination of Ca (OH)2 with garlic extract showed the least significant bacterial reduction. CONCLUSION: Within the study limitations, garlic intracanal medicament has a comparable anti-Streptococcus efficiency to Ca (OH)2 , whilst it is more effective against Enterococcus faecalis species. When Ca (OH)2 and garlic are combined, their antibacterial effectiveness is reduced. Increasing the time of application for tested intracanal medicaments by more than one week has no additional antibacterial effectiveness.

Streptococcus species enriched in the oral cavity of patients with RA are a source of peptidoglycan-polysaccharide polymers that can induce arthritis in mice.

OBJECTIVES: Analysis of oral dysbiosis in individuals sharing genetic and environmental risk factors with rheumatoid arthritis (RA) patients may illuminate how microbiota contribute to disease susceptibility. We studied the oral microbiota in a prospective cohort of patients with RA, first-degree relatives (FDR) and healthy controls (HC), then genomically and functionally characterised streptococcal species from each group to understand their potential contribution to RA development. METHODS: After DNA extraction from tongue swabs, targeted 16S rRNA gene sequencing and statistical analysis, we defined a microbial dysbiosis score based on an operational taxonomic unit signature of disease. After selective culture from swabs, we identified streptococci by sequencing. We examined the ability of streptococcal cell walls (SCW) from isolates to induce cytokines from splenocytes and arthritis in ZAP-70-mutant SKG mice. RESULTS: RA and FDR were more likely to have periodontitis symptoms. An oral microbial dysbiosis score discriminated RA and HC subjects and predicted similarity of FDR to RA. Streptococcaceae were major contributors to the score. We identified 10 out of 15 streptococcal isolates as S. parasalivarius sp. nov., a distinct sister species to S. salivarius. Tumour necrosis factor and interleukin 6 production in vitro differed in response to individual S. parasalivarius isolates, suggesting strain specific effects on innate immunity. Cytokine secretion was associated with the presence of proteins potentially involved in S. parasalivarius SCW synthesis. Systemic administration of SCW from RA and HC-associated S. parasalivarius strains induced similar chronic arthritis. CONCLUSIONS: Dysbiosis-associated periodontal inflammation and barrier dysfunction may permit arthritogenic insoluble pro-inflammatory pathogen-associated molecules, like SCW, to reach synovial tissue.

Acid tolerance in early colonizers of oral biofilms.

BACKGROUND: In caries, low pH drives selection and enrichment of acidogenic and aciduric bacteria in oral biofilms, and development of acid tolerance in early colonizers is thought to play a key role in this shift. Since previous studies have focussed on planktonic cells, the effect of biofilm growth as well as the role of a salivary pellicle on this process is largely unknown. We explored acid tolerance and acid tolerance response (ATR) induction in biofilm cells of both clinical and laboratory strains of three oral streptococcal species (Streptococcus gordonii, Streptococcus oralis and Streptococcus mutans) as well as two oral species of Actinomyces (A. naeslundii and A. odontolyticus) and examined the role of salivary proteins in acid tolerance development. METHODS: Biofilms were formed on surfaces in Ibidi® mini flow cells with or without a coating of salivary proteins and acid tolerance assessed by exposing them to a challenge known to kill non-acid tolerant cells (pH 3.5 for 30 min) followed by staining with LIVE/DEAD BacLight and confocal scanning laser microscopy. The ability to induce an ATR was assessed by exposing the biofilms to an adaptation pH (pH 5.5) for 2 hours prior to the low pH challenge. RESULTS: Biofilm formation significantly increased acid tolerance in all the clinical streptococcal strains (P < 0.05) whereas the laboratory strains varied in their response. In biofilms, S. oralis was much more acid tolerant than S. gordonii or S. mutans. A. naeslundii showed a significant increase in acid tolerance in biofilms compared to planktonic cells (P < 0.001) which was not seen for A. odontolyticus. All strains except S. oralis induced an ATR after pre-exposure to pH 5.5 (P < 0.05). The presence of a salivary pellicle enhanced both acid tolerance development and ATR induction in S. gordonii biofilms (P < 0.05) but did not affect the other bacteria to the same extent. CONCLUSIONS: These findings suggest that factors such as surface contact, the presence of a salivary pellicle and sensing of environmental pH can contribute to the development of high levels of acid tolerance amongst early colonizers in oral biofilms which may be important in the initiation of caries.