Regulatory Effect of Irresistin-16 on Competitive Dual-Species Biofilms Composed of Streptococcus mutans and Streptococcus sanguinis

Based on the ecological plaque hypothesis, suppressing opportunistic pathogens within biofilms, rather than killing microbes indiscriminately, could be a biofilm control strategy for managing dental caries. The present study aimed to evaluate the effects of irresistin-16 (IRS-16) on competitive dual-species biofilms, which consisted of the conditional cariogenic agent Streptococcus mutans (S. mutans) and oral commensal bacteria Streptococcus sanguinis (S. sanguinis). Bacterial growth and biofilm formation were monitored using growth curve and crystal violet staining, respectively. The microbial proportion was determined using fluorescence in situ hybridization. A 2, 5-diphenyltetrazolium bromide assay was used to measure the metabolic activity of biofilms. Bacterial/extracellular polysaccharide (EPS) dyeing, together with water-insoluble EPS measurements, were used to estimate EPS synthesis. A lactic acid assay was performed to detect lactic acid generation in biofilms. The cytotoxicity of IRS-16 was evaluated in mouse fibroblast L929 cells using a live/dead cell viability assay and cell counting kit-8 assay. Our results showed that IRS-16 exhibited selective anti-biofilm activity, leading to a remarkable survival disadvantage of S. mutans within competitive dual-species biofilms. In addition, the metabolic activity, EPS synthesis, and acid generation of dual-species biofilms were significantly reduced by IRS-16. Moreover, IRS-16 showed minimal cytotoxicity against mouse fibroblast L929 cells. In conclusion, IRS-16 exhibited remarkable regulatory effects on dual-species biofilms composed of S. mutans and S. sanguinis with low cytotoxicity, suggesting that it may have potential for use in caries management through ecological biofilm control.

Phytochemical constituents and antibacterial activities of indigenous chewing stick (Anogeissus leiocarpus) stem

The stems from Anogeissus leiocarpus are commonly used as chewing sticks in Northern Nigeria. If properly used, the chewing sticks have proven to be effective in removing dental plaque due to mechanical cleaning and enhanced salivation. Chewing sticks from other plants have been shown to display antimicrobial activities against a broad spectrum of microorganisms. However, there is limited information available in Northern Nigeria on the chemical composition, antimicrobial properties and the ability of the plants under study to prevent bacterial adhesion to tooth surface. Therefore, the purpose of this study was to ascertain the phytochemical and antibacterial properties of Anogeissus leiocarpus and correlate the results obtained to their ethnomedicinal uses as chewing sticks. Powdered stem was exhaustively extracted using methanol at room temperature for 72 hours. Antibacterial activities of the methanol extract was assessed using the agar well diffusion methods against the oral pathogens, Staphylococcus aureus, Streptococcus salivarius, Streptococcus pyogenes, Streptococcus mutans and Streptococcus sanguinis. Acute toxicity study was achieved using Lorke method. Phytochemicals which include flavonoid, steroid, triterpenes, alkaloids, tannins, carbohydrate, glycoside, phenols were detected in the extracts while anthraquinones was absent. The antibacterial results revealed that, the methanol extract had promising antibacterial activity. S. aureuswas found to be the most susceptible bacteria at 500mg/ml with inhibition of 22 mm, Streptococcus salivarius, Streptococcus pyogenes and Streptococcus sanguinis were inhibited at 16 mm while Streptococcus mutans showed inhibition of 14 mm. The extract have MIC and MBC of 31.25 mg/ml and 62.5 mg/ml respectively against all the tested clinical isolates. The LD50 of Anogeissus leiocarpus was found to be greater than 5000 mg/kg and could be considered safe for consumption.

Antibacterial potential of strawberries and basil extracts combination against Streptococcus sanguinis (ATCC 10556)

Introduction: Streptococcus sanguinis is a commensal microorganism as well as a pioneer colony in forming dental plaque. Oral biofilm formation can be prevented by a mechanical cleaning procedure followed by the use of mouthwash. The current gold standard for mouthwash is chlorhexidine. Nevertheless, it has side effects that are not recommended for long-term use. Previous studies had proven that herbal-based mouthwashes such as basil leaves (Ocimum basilicum) and strawberry fruit (Fragaria x ananassa) have been shown to have antibacterial properties. The effectivity of antibacterial activity phenomenon in combined extracts has been reported in other studies. This research aims to observe the antibacterial potential of the F. x ananassa and O. basilicum extract combinations against S. sanguinis (ATCC 10556). Methods: The sample of this study was a combination of F. x ananassa and O. basilicum extract, which initially screened for their antibacterial activities. Antibacterial activities of F. x ananassa and O. basilicum extracts against S. sanguinis were observed using Kirby Bauer method, while Minimum Inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) by serial microdilution method. The 2% concentration from each extract was combined in 1:1, 1:2, and 2:1 volume ratio variations then tested for inhibitory zones, MIC, and MBC. Results: F. x ananassa extract had 0.125% and 0.25% for MIC and MBC respectively, while O. basilicum extract showed the value of MIC and MBC as 0.031% and 0.063% against S. sanguinis (ATCC 10556). The extract combinations in 1:1, 1:2, and 2:1 volume ratio variations showed 0.016% for MIC and 0.031% for MBC. Conclusions: It was concluded that combining extracts of 2 % F. x ananassa and 2% O. basilicum in various ratios were observably to have the antibacterial potential against S. sanguinis (ATCC 10556).

Type IV Pili of Streptococcus sanguinis Contribute to Pathogenesis in Experimental Infective Endocarditis

This work provides evidence that type IV pili produced by Streptococcus sanguinis SK36 are critical to the ability of these bacteria to attach to and colonize the aortic heart valve (endocarditis). We found that an S. sanguinis type IV pili mutant strain was defective in causing platelet-dependent aggregation in a 24-h infection assay but not in a 1-h platelet aggregation assay, suggesting that the type IV pili act at later stages of vegetation development.

Spontaneous Mutants of Streptococcus sanguinis with Defects in the Glucose-PTS Show Enhanced Post-Exponential Phase Fitness

Genetic truncations in a gene encoding a putative glucose-PTS protein ( manL , EIIAB Man ) were identified in subpopulations of two separate laboratory stocks of Streptococcus sanguinis SK36; the mutants had reduced PTS activities on glucose and other monosaccharides. To understand the emergence of these mutants, we engineered deletion mutants of manL and showed that the ManL-deficient strain had improved bacterial viability in stationary phase and was better able to inhibit the growth of the dental caries pathogen Streptococcus mutans . Transcriptional analysis and biochemical assays suggested that the manL mutant underwent reprograming of central carbon metabolism that directed pyruvate away from production of lactate, increasing production of hydrogen peroxide (H 2 O 2 ) and excretion of pyruvate. Addition of pyruvate to the medium enhanced the survival of SK36 in overnight cultures. Meanwhile, elevated pyruvate levels were detected in the cultures of a small, but significant percentage (∼10%), of clinical isolates of oral commensal bacteria. Furthermore, the manL mutant showed higher expression of the arginine deiminase system than the wild type, which enhanced the ability of the mutant to raise environmental pH when arginine was present. To our surprise, significant discrepancies in genome sequence were identified between strain SK36 obtained from ATCC and the sequence deposited in GenBank. As the conditions that are likely associated with the emergence of spontaneous manL mutations, i.e. excess carbohydrates and low pH, are those associated with caries development, we propose that the glucose-PTS strongly influences commensal-pathogen interactions by altering the production of ammonia, pyruvate, and H 2 O 2 . Importance A health-associated dental microbiome provides a potent defense against pathogens and diseases. Streptococcus sanguinis is an abundant member of a health-associated oral flora that antagonizes pathogens by producing hydrogen peroxide. There is a need for a better understanding of the mechanisms that allow bacteria to survive carbohydrate-rich and acidic environments associated with the development of dental caries. We report the isolation and characterization of spontaneous mutants of S. sanguinis with impairment in glucose transport. The resultant reprograming of central metabolism in these mutants reduced the production of lactic acid and increased pyruvate accumulation; the latter enables these bacteria to better cope with hydrogen peroxide and low pH. The implications of these discoveries in the development of dental caries are discussed.

Spontaneous Mutants of Streptococcus sanguinis with Defects in the Glucose-PTS Show Enhanced Post-Exponential Phase Fitness

Genetic truncations in a gene encoding a putative glucose-PTS protein (manL, EIIABMan) were identified in subpopulations of two separate laboratory stocks of Streptococcus sanguinis SK36; the mutants had reduced PTS activities on glucose and other monosaccharides. Using an engineered mutant of manL and its complemented derivative, we showed that the ManL-deficient strain had improved bacterial viability in stationary phase and was better able to inhibit the growth of the dental caries pathogen Streptococcus mutans. Transcriptional analysis and biochemical assays suggested that the manL mutant underwent reprograming of central carbon metabolism that directed pyruvate away from production of lactate, increasing production of hydrogen peroxide (H2O2) and excretion of pyruvate. Addition of pyruvate to the medium enhanced the survival of SK36 in overnight cultures. Meanwhile, elevated pyruvate levels were detected in the cultures of a small, but significant percentage (~10%), of clinical isolates of oral commensal bacteria. Furthermore, the manL mutant showed higher expression of the arginine deiminase system than the wild type, which enhanced the ability of the mutant to raise environmental pH when arginine was present. Significant discrepancies in genome sequence were identified between strain SK36 obtained from ATCC and the sequence deposited in GenBank. As the conditions that are likely associated with the emergence of spontaneous manL mutations, i.e. excess carbohydrates and low pH, are those associated with caries development, we propose that the glucose-PTS strongly influences commensal-pathogen interactions by altering the production of ammonia, pyruvate, and H2O2.

Differential Response of Oral Mucosal and Gingival Cells to Corynebacterium durum, Streptococcus sanguinis, and Porphyromonas gingivalis Multispecies Biofilms

Polymicrobial interactions with oral mucosal surfaces determine the health status of the host. While a homeostatic balance provides protection from oral disease, a dysbiotic polymicrobial community promotes tissue destruction and chronic oral diseases. How polymicrobial communities transition from a homeostatic to a dysbiotic state is an understudied process. Thus, we were interested to investigate this ecological transition by focusing on biofilm communities containing high abundance commensal species and low abundance pathobionts to characterize the host-microbiome interactions occurring during oral health. To this end, a multispecies biofilm model was examined using the commensal species Corynebacterium durum and Streptococcus sanguinis and the pathobiont Porphyromonas gingivalis. We compared how both single and multispecies biofilms interact with different oral mucosal and gingival cell types, including the well-studied oral keratinocyte cell lines OKF4/TERT-1and hTERT TIGKs as well as human primary periodontal ligament cells. While single species biofilms of C. durum, S. sanguinis, and P. gingivalis are all characterized by unique cytokine responses for each species, multispecies biofilms elicited a response resembling S. sanguinis single species biofilms. One notable exception is the influence of P. gingivalis upon TNF-α and Gro-α production in hTERT TIGKs cells, which was not affected by the presence of other species. This study is also the first to examine the host response to C. durum. Interestingly, C. durum yielded no notable inflammatory responses from any of the tested host cells, suggesting it functions as a true commensal species. Conversely, S. sanguinis was able to induce expression and secretion of the proinflammatory cytokines IL-6 and IL-8, demonstrating a much greater inflammatory potential, despite being health associated. Our study also demonstrates the variability of host cell responses between different cell lines, highlighting the importance of developing relevant in vitro models to study oral microbiome-host interactions.