Growth Suppression of Oral Squamous Cell Carcinoma Cells by Lactobacillus Acidophilus.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) is a highly aggressive form of oral cancer. Probiotic lactobacilli have demonstrated anticancer effects, whilst their interaction with Streptococcus mutans in this context remains unexplored. The objective of this study was to investigate the antiproliferative effect of Lactobacillus acidophilus on OSCC and to understand the effect of S mutans on OSCCs and whether it affects the antiproliferative potential of L acidophilus when co-exposed to OSCC. METHODS: The human head and neck squamous cell carcinoma cells of the oral cavity (HNO97 cell line) were exposed to cultures of L acidophilus and S mutans separately and in combination. Further, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess the viability of HNO97 cells. Bacterial adhesion to HNO97 cells was examined by confocal microscopy and apoptosis by Nexin staining. To understand the underlying mechanism of apoptosis, expression of the tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) gene and protein were determined by real-time polymerase chain reaction and quantitative enzyme-linked immunosorbent assay, respectively. RESULTS: A significant decrease (53%-56%) in the viability of HNO97 cells on exposure to L acidophilus, S mutans, and the 2 species together demonstrated the antiproliferative activity of L acidophilus and S mutans. Both bacteria showed adhesion to HNO97 cells. The expression of the TRAIL gene increased 5-fold in HNO97 cells on treatment with L acidophilus and S mutans, which further increased to ∼17-fold with both species present. Expression levels of the TRAIL protein were significantly (P < .05) increased in bacteria-treated cell lysates. Further, bacteria-treated HNO97 cells exhibited lower live and intact cell percentages with higher proportions of cells in early and late apoptotic stages. CONCLUSIONS: L acidophilus exhibits the antiproliferative activity against OSCC cells possibly partially via a TRAIL-induced mechanism of apoptosis, which is not affected by the presence of S mutans. These findings may encourage further investigation into the possible therapeutic application of probiotic L acidophilus in OSCC.

Hygiene practices and antibiotic resistance among dental and medical students: a comparative study.

PURPOSE: Healthcare students' hand and smartphone hygiene is critical due to potential pathogenic and antibiotic-resistant bacteria transmission. This study evaluates hygiene practices in medical and dental students at Kuwait University, exploring antibiotic resistance gene prevalence. METHODS: Swab samples were collected from the hands and smartphones of 32 medical and 30 dental students. These samples were cultured on Columbia Blood Agar and McConkey Agar plates to quantify bacterial colony-forming units (CFUs). The extracted DNA from these colonies underwent RT-PCR to identify antibiotic resistance genes, including tem-1, shv, blaZ, and mecA. Additionally, a questionnaire addressing hygiene practices was distributed post-sample collection. RESULTS: Medical students exhibited more frequent hand hygiene compared to dental students (P ≤ 0.0001). Although significantly fewer bacterial CFUs were found on medical students' smartphones (mean = 35 ± 53) than dental students' (mean = 89 ± 129) (P ≤ 0.05), no significant differences were observed in CFU counts on their hands (medical: mean = 17 ± 37; dental: mean = 96 ± 229). Detection of at least one of the targeted antibiotic resistance genes on medical (89% hands, 52% smartphones) and dental students' (79% hands, 63% smartphones) was not statistically significant. However, the prevalence of two genes, tem-1 and shv, was significantly higher on medical students' hands (78% and 65%, respectively) than on dental students' hands (32% and 28%, respectively). CONCLUSION: Clinically significant prevalence of antibiotic resistance genes were found on medical and dental students' hands and smartphones, emphasizing the importance of ongoing education regarding hand hygiene and smartphone disinfection. This continuous reinforcement in the curriculum is crucial to minimizing the risk of cross-contamination.

Secretome analysis and virulence assessment in Abiotrophia defectiva.

Background: Abiotrophia defectiva, although infrequently occurring, is a notable cause of culture-negative infective endocarditis with limited research on its virulence. Associated with oral infections such as dental caries, exploring its secretome may provide insights into virulence mechanisms. Our study aimed to analyze and characterize the secretome of A. defectiva strain CCUG 27639. Methods: Secretome of A. defectiva was prepared from broth cultures and subjected to mass spectrometry and proteomics for protein identification. Inflammatory potential of the secretome was assessed by ELISA. Results: Eighty-four proteins were identified, with diverse subcellular localizations predicted by PSORTb. Notably, 20 were cytoplasmic, 12 cytoplasmic membrane, 5 extracellular, and 9 cell wall-anchored proteins. Bioinformatics tools revealed 54 proteins secreted via the 'Sec' pathway and 8 via a non-classical pathway. Moonlighting functions were found in 23 proteins, with over 20 exhibiting potential virulence properties, including peroxiredoxin and oligopeptide ABC transporter substrate-binding protein. Gene Ontology and KEGG analyses categorized protein sequences in various pathways. STRING analysis revealed functional protein association networks. Cytokine profiling demonstrated significant proinflammatory cytokine release (IL-8, IL-1ß, and CCL5) from human PBMCs. Conclusions: Our study provides a comprehensive understanding of A. defectiva's secretome, laying the foundation for insights into its pathogenicity.

The potential of nano graphene oxide and chlorhexidine composite membranes for use as a surface layer in functionally graded membranes for periodontal lesions.

Membranes have been used for treating periodontal defects and play a crucial role in guided bone regeneration applications. Nano graphene oxide have been exploited in tissue engineering due to its biomechanical properties. Its composite formulations with hydroxyapatite and chitosan with controlled degradation could aid in becoming part of a surface layer in a functionally graded membrane. The aim of the study was to synthesize chitosan and composite formulations of nano graphene oxide, hydroxyapatite and chlorhexidine digluconate using solvent casting technique and to characterize the physiochemical, mechanical, water vapor transmission rate (barrier), degradation and antimicrobial potential of the membranes. Altogether four different membranes were prepared (CH, CCG, 3511 and 3322). Results revealed the chemical interactions of hydroxyapatite, chitosan and nanographene oxide due to inter and intra molecular hydrogen bonding. The tensile strength of 3322 (33.72 ± 6.3 MPa) and 3511 (32.06 ± 5.4 MPa) was higher than CH (27.46 ± 9.6 MPa). CCG showed the lowest water vapor transmission rate (0.23 ± 0.01 g/h.m2) but the highest weight loss at day 14 (76.6 %). 3511 showed a higher drug release after 72 h (55.6 %) Significant biofilm growth inhibition was observed for all membranes. 3511 showed complete inhibition against A. actinomycetemcomitans. Detailed characterization of the synthesized membranes revealed that 3511 composite membrane proved to be a promising candidate for use as a surface layer of membranes for guided bone regeneration of periodontal lesions.

Sugar Substitute Stevia Inhibits Biofilm Formation, Exopolysaccharide Production, and Downregulates the Expression of Streptococcal Genes Involved in Exopolysaccharide Synthesis.

BACKGROUND: Acid production by sucrose fermentation disturbs the balance in dental plaque by lowering the oral pH. As a consequence of the profound effect of sucrose on caries initiation and progression, many studies have been directed towards finding non-cariogenic artificial sweeteners that can be used as a substitute to sucrose. Existing literature shows that dietary sucrose upregulates the expression of biofilm associated genes involved in exopolysaccharide (EPS) production. OBJECTIVE: In this study, we aimed to investigate the effect of the sugar substitute stevia on biofilm formation, EPS secretion, and streptococcal genes encoding glucan-binding proteins (Gbps) and glucosyltransferases (Gtfs), which are essential for the synthesis of EPS. MATERIALS AND METHODS: Streptococcus mutans and Streptococcus gordonii were grown as biofilm cultures with or without stevia and sucrose. Biomass was quantified for biofilm and EPS production by crystal violet staining and the phenol-sulfuric acid method, respectively. Expression of gtfB and gbpB genes was studied by RT-PCR. RESULTS: The quantities of biofilm were significantly lower when grown in the presence of stevia compared to sucrose in both species (p < 0.05). The proportion of EPS in the biofilm pellet decreased with increasing concentrations of stevia in both species but remained nearly unchanged with sucrose with respect to the control. In both streptococcal species, exposure of stevia decreased the expression of gtfB and gbpB genes compared to sucrose (p < 0.05). In comparison to the untreated control, the expression was decreased in the presence of stevia in both species, while it increased 2.5- to 4-fold in S. mutans and 1.5- to 2.5-fold in S. gordonii in the presence of sucrose. CONCLUSION: The ability of stevia to inhibit biofilm formation, reduce EPS production, and downregulate the expression of gtfB and gbpB genes in S. mutans and S. gordonii may have potential therapeutic applications in controlling dental plaques and caries.

In vitro investigation of the impact of contemporary restorative materials on cariogenic bacteria counts and gene expression.

OBJECTIVES: The aim of this study was to compare the antibacterial effect of different fluoride-containing and bioactive restorative materials, and their effect on the expression of specific biofilm-associated genes and therefore the caries process. MATERIALS AND METHODS: The restorative materials utilized in this study included: 1. Filtek Z250, 2. Fuji II LC, 3. Beautifil II, 4. ACTIVA, and 5. Biodentine. For each material, disc-shaped specimens were prepared. The inhibitory effects against Streptococcus mutans, Lactobacillus acidophilus, and Leptotrichia shahii were tested. After incubation for 24 h and 1 week, colony-forming units (CFUs) were enumerated. From the plates dedicated for biomass quantification and RNA purification, the target glucosyltransferase B (gtfB) and glucan-binding protein B (gbpB) genes were chosen for S. mutans. For L. acidophilus, a gene involved in exopolysaccharide synthesis (epsB) was chosen. RESULTS: Except for Filtek Z250, all four materials showed statistically significant inhibitory effects on the biofilms of all three species. When biofilms were grown in the presence of the same four materials, the expression of S. mutans gtfB and gbpB genes, was significantly reduced. For L. acidophilus, the decrease in the expression of gtfB gene in the presence of ACTIVA was the highest change seen. The epsB gene expression also decreased. Compared to fluoride-releasing materials, bioactive materials had more inhibitory effect against L. acidophilus, both at 24 h and 1 week. CONCLUSIONS: Both fluoride releasing and bioactive materials exhibited a significant inhibitory effect on the biofilm growth. The expression of the targeted biofilm-associated genes was downregulated by both material groups. CLINICAL SIGNIFICANCE: The findings from this study give insight into the antibacterial effect of fluoride-containing and bioactive materials which would help to reduce the chances for secondary caries and therefore increase the lifetime of dental restorations placed for patients.

Anticandidal Activity of Capsaicin and Its Effect on Ergosterol Biosynthesis and Membrane Integrity of Candida albicans.

Oral candidiasis is an infection of the oral cavity commonly caused by Candida albicans. Endodontic treatment failure has also been found to be persistent from C. albicans in the root canal system. Despite the availability of antifungal drugs, the management of Candida oral infection is difficult as it exhibits resistance to a different class of antifungal drugs. Therefore, it is necessary to discover new antifungal compounds to cure fungal infections. This study aimed to examine the antifungal susceptibility of Capsaicin, an active compound of chili pepper. The susceptibility of Capsaicin and Fluconazole was tested against the Candida species by the CLSI (M27-A3) method. The effect of Capsaicin on the fungal cell wall was examined by the ergosterol inhibitory assay and observed by the scanning electron micrograph. The MIC range of Capsaicin against Candida isolates from oral (n = 30), endodontic (n = 8), and ATCC strains (n = 2) was 12.5−50 µg/mL. The MIC range of Fluconazole (128- 4 µg/mL) significantly decreased (2- to 4-fold) after the combination with Capsaicin (MIC/4) (p < 0.05). Capsaicin (at MIC) significantly reduced the mature biofilm of C. albicans by 70 to 89% (p < 0.01). The ergosterol content of the cell wall decreased significantly with the increase in the Capsaicin dose (p < 0.01). Capsaicin showed high sensitivity against the hyphae formation and demonstrated a more than 71% reduction in mature biofilm. A fluorescence microscopy revealed the membrane disruption of Capsaicin-treated C. albicans cells, whereas a micrograph of electron microscopy showed the distorted cells' shape, ruptured cell walls, and shrinkage of cells after the release of intracellular content. The results conclude that Capsaicin had a potential antifungal activity that inhibits the ergosterol biosynthesis in the cell wall, and therefore, the cells' structure and integrity were disrupted. More importantly, Capsaicin synergistically enhanced the Fluconazole antifungal activity, and the synergistic effect might be helpful in the prevention of Fluconazole resistance development and reduced drug-dosing.

Proteomic analysis of the periodontal pathogen Prevotella intermedia secretomes in biofilm and planktonic lifestyles.

Prevotella intermedia is an important species associated with periodontitis. Despite the remarkable clinical significance, little is known about the molecular basis for its virulence. The aim of this study was to characterize the secretome of P. intermedia in biofilm and planktonic life mode. The biofilm secretome showed 109 proteins while the planktonic secretome showed 136 proteins. The biofilm and the planktonic secretomes contained 17 and 33 signal-peptide bearing proteins, 13 and 18 lipoproteins, respectively. Superoxide reductase, sensor histidine kinase, C40 family peptidase, elongation factor Tu, threonine synthase etc. were unique to biofilm. Of the ~ 30 proteins with predicted virulence potential from biofilm and planktonic secretomes, only 6 were common between the two groups, implying large differences between biofilm and planktonic modes of P. intermedia. From Gene Ontology biofilm secretome displayed a markedly higher percent proteins compared to planktonic secretome in terms of cellular amino acid metabolic process, nitrogen compound metabolic process etc. Inflammatory cytokine profile analysis revealed that only the biofilm secretome, not the planktonic one, induced important cytokines such as MIP-1α/MIP-1ß, IL-1ß, and IL-8. In conclusion, the revealed differences in the protein profiles of P. intermedia biofilm and planktonic secretomes may trigger further questions about molecular mechanisms how this species exerts its virulence potential in the oral cavity.

The Effect of Benzyl Isothiocyanate on the Expression of Genes Encoding NADH Oxidase and Fibronectin-Binding Protein in Oral Streptococcal Biofilms.

Recent studies have shown that antimicrobial treatment results in up- or down regulation of several virulence-associated genes in bacterial biofilms. The genes encoding NADH oxidase (nox) and fibronectin-binding protein (fbp) are known to play important roles in biofilm growth of some oral bacterial species. The objective was to study the effect of benzyl isothiocyanate (BITC), an antimicrobial agent from Miswak plant, on the expression of nox and fbp genes in some oral streptococci. The biofilms were treated with BITC and mRNA expression of nox and fbp genes was measured by comparative ΔΔCt method. The highest amount of biofilm mass was produced by A. defectiva, followed by S. gordonii, S. mutans, G. elegans and G. adiacens. Upon treatment with BITC, S. gordonii biofilms showed highest folds change in mRNA expression for both fbp and nox genes followed by S. mutans, A. defectiva, and G. adiacens. G. elegans mRNA levels for nox were extremely low. In conclusion, BITC treatment of the biofilms caused an upregulation of biofilm-associated genes fbp and nox genes in most of the tested species suggesting the significance of these genes in biofilm lifestyle of these oral bacteria and needs further investigation to understand if it contributes to antimicrobial resistance.

Different scaling and root planing strategies in Turkish patients with aggressive periodontitis: A randomized controlled clinical trial.

OBJECTIVES: The aim of this study was to compare clinical, cytokine and microbiological responses after quadrant-based scaling and root planing (Q-SRP), full-mouth SRP (FM-SRP) and full-mouth disinfection (FMD) in patients with generalized aggressive periodontitis (GAgP), which is currently termed as generalized stage-III and grade-C periodontitis. METHODS: Forty-two patients with GAgP were randomly assigned into groups as Q-SRP, FM-SRP or FMD with chlorhexidine. Clinical parameters were recorded, and gingival crevicular fluid (GCF) and subgingival plaque samples were collected at baseline, 3 and 6 months after treatment. GCF levels of interleukin (IL)-1ß and IL-17 were analysed using ELISA. Quantities of six bacterial species were determined using qPCR. RESULTS: Clinical parameters improved significantly in all groups at 3 and 6 months (p < 0.05). Percentage of sites with probing depth >6 mm was lower in the FMD than Q-SRP group at 3 and 6 months (p < 0.05). FMD showed significantly higher percentage of pocket closure compared with Q-SRP and FM-SRP at both 3 and 6 months after treatment (p < 0.05). The IL-1ß levels decreased only in the FMD group (p < 0.05), whereas no changes were found in IL-17 levels in any group. The levels of five out of six bacterial species decreased at 3 and/or 6 months only in the FMD group (p < 0.05). CONCLUSIONS: The FMD treatment appears to offer superior outcome than Q-SRP and could be the first choice for patients with GAgP.

Biofilm growth and microbial contamination of dental unit waterlines at Kuwait University dental center.

Biofilm formation in dental unit waterlines and the resulting microbial contamination of the water in the system has become a significant problem. Contaminated water in the dental units is a major concern in dental clinics due to potential risk of causing infections particularly in elderly and immunocompromised patients. The aim of this study was at first to determine microbial contamination of the dental unit waterlines and then to study the efficacy of a comprehensive disinfection protocol on decreasing the microbial load. Water samples were collected before and after disinfection procedure from handpieces and water storage bottles from the dental units, a small 1-cm tubing was cut from each unit and subjected to microbiological culture on different growth media. Identification of the predominant species was achieved by 16S rRNA gene sequencing. Microbial growth was observed in samples collected from all dental units. Upon disinfection procedure, microbial contamination in the water samples and in the tubing surfaces was significantly reduced (P > 0.05). 16S rRNA gene sequencing revealed the presence of several species belonging to the genera Staphylococcus, Corynebacterium and Roseomonas, some of which are implicated in human infections. Aggravation of the biofilm growth on the tubing surfaces and the microbial contamination in the water can be effectively controlled by implementing appropriate and routine disinfection protocols. This may help protect the dental unit staff and the patients being exposed to the risk of infections.

Locally isolated broad host-range bacteriophage kills methicillin-resistant Staphylococcus aureus in an in vivo skin excisional wound model in mice.

From the perspective of an ever-increasing multidrug resistance among bacterial pathogens, bacteriophages are receiving renewed interest as potential alternative to antibiotics. We investigated the potential of a locally isolated species-specific phage against Staphylococcus aureus infection in a skin excisional wound model in mice. A significant time-dependent increase (P < 0.05) in wound healing was observed in the phage-treated mice groups. The animals treated with the phage ΦDMSA-2 exhibited a faster re-epithelialization and faster tissue re-modelling. Bacterial load in the infected tissue in all phage-treated groups diminished. The mean ± SD CFU per ml decreased from 3.3 × 108 ± 3.5 × 106 at day 1-1.43 × 103 ± 8.48 × 102 at day 16 (P < 0.05). The highest reduction in the bacterial load was observed in G5 (povidine-iodine treated) and G6 (povidine iodine + phage 107 PFU) groups as no bacterial counts were detected by day 12 in both groups. Interestingly, group G3, which was treated with a lower phage concentration (5 × 106 PFU), resulted in total clearing of the inoculated bacteria by day 16; while bacterial counts were still detected by that time in group G4, which was treated with a higher phage concentration of 107 PFU. Animals from phage-treated group G3 survived 100%, while those from the infected wound control group survived at a rate of 34% at day 9 and reached 0% by the end of day 22 (P < 0.001). The data from this study convincingly showed that phage treatment of the S. aureus-infected wounds resulted in a faster wound healing and a 100% survival of the animals. The results emphasize the utility of locally isolated species-specific phages in treatment against multidrug-resistant MRSA infections.

In vitro Characterization of Biofilm Formation in Prevotella Species.

Background: Periodontitis, a chronic inflammatory oral infection is the outcome of disturbances in the homeostasis of the oral biofilm microbiota. A number of studies have found the occurrence of Prevotella species in elevated levels in periodontitis compared to healthy subjects. Even though different aspects of Prevotella as part of oral biofilm have been studied, in vitro biofilms formed by these species have not been characterized systematically. The objective of this study was to characterize biofilms formed by several Prevotella species and further to assess biofilm inhibition and detachment of preformed biofilms. Methods: Biofilms were grown in 24-well plates containing brucella broth in anaerobic conditions for 3 days, and were quantified using crystal violet staining. Images of SYTO 9 Green fluorescent stained biofilms were captured using confocal microscopy. Biofilm inhibition and detachment by proteinase and DNase I was tested. The biochemical characterization included quantification of proteins and DNA in the biofilms and biofilm-supernatants. Results: Prevotella loescheii, Prevotella oralis and Prevotella nigrescens showed highest biofilm formation. P. nigrescens formed significantly higher amounts of biofilms than P. loescheii (P = 0.005) and P. oralis (P = 0.0013). Inhibition of biofilm formation was significant only in the case of P. oralis when treated with proteinase (P = 0.037), whereas with DNase I treatment, the inhibition was not significant (P = 0.531). Overall, proteinase was more effective in biofilm detachment than DNase I. Protein and DNA content were higher in biofilm than the supernatant with the highest amounts found in P. nigrescens biofilm and supernatants. P. oralis biofilms appeared to secrete large amounts of proteins extracellularly into the biofilm-supernatants. Conclusion: Significant differences among Prevotella species to form biofilms may imply their variable abilities to get integrated into oral biofilm communities. Of the species that were able to grow as biofilms, DNase I and proteinase inhibited the biofilm growth or were able to cause biofilm detachment.

A clinical investigation into the efficacy of miswak chewing sticks as an oral hygiene aid: A crossover randomized trial.

OBJECTIVES: This study evaluates the effect of using miswak chewing sticks on dental plaque, selected oral microbiota and gingival inflammation among patients with gingivitis. METHODS: The study was a single-centre, single-examiner blind, randomized, crossover study. Twenty healthy participants were randomly assigned into two equal groups (n = 10). Group 1 were instructed to use both toothbrush and miswak (TB+M) for the first 2 weeks from baseline (T1) and only toothbrush for the next 2 weeks (T2); and Group 2, only TB during T1 and TB+M during T2. Gingival index (GI), plaque index (PI) and bleeding on probing (BOP) were evaluated at baseline (T0), T1 and T2 visits. Supra-gingival plaque samples were taken at T0, T1 and T2. Quantification of Streptococcus mutans and Aggregatibacter actinomycetemcomitans from the supra-gingival plaque samples were performed by real-time polymerase chain reaction (qPCR). RESULTS: The scores of GI, PI and BOP had significantly improved for both groups between T0 and T1. A significantly greater reduction in the percentage of sites with BOP was observed for TB+M group compared with TB group (TB+M group: from 32.2 to 14.93; TB group: from 34.00 to 26.0; p = .014). At T2, TB+M group had significant improvements (p < .05) in the PI, GI and BOP scores compared with TB group. There was no significant difference in the microbial counts of S. mutans and A. actinomycetemcomitans between the two groups at the end of the study period. CONCLUSIONS: Oral hygiene and gingival health may be improved by complementing miswak chewing sticks with toothbrushing.

Dental plaque microbiota profiles of children with caries-free and caries-active dentition.

OBJECTIVE: Microbiota comparisons between healthy and diseased dental tissues have accentuated the importance of cultivating and identifying bacterial species that play a role in the initiation and progression of dental caries. The objective of this study was to evaluate the bacterial community composition in caries-active and caries-free children. METHODS: Supragingival plaque samples were collected from 64 caries-active and 64 caries-free Middle Eastern children. The hypervariable V3-V4 of the bacterial 16S rRNA gene was sequenced with Human Oral Microbe Identification using Next Generation Sequencing. Microbial community structure and composition analyses were performed by processing operational taxonomic units. Bioinformatic analyses, including analysis of similarity, alpha and beta diversities, and principal coordinate analysis, were carried out. RESULTS: Diversity indices did not find differences between the caries-active and caries-free groups (p > 0.05). Similarity analysis demonstrated that the microbiota composition did not differ between the two groups. Comparative analysis at the species level revealed a significantly higher relative abundance of Leptotrichia shahii, Prevotella melaninogenica, Veillonella dispar, Leptotrichia HOT 498, and Streptococcus mutans in caries-active children (p < 0.05). Corynebacterium matruchotii, Lautropia mirabilis, Neisseria elongata, and Corynebacterium durum were relatively more abundant in the caries-free group (p < 0.05). Species belonging to the Leptotrichia, Prevotella, and Veillonella genera were significantly predominant in the caries-active subjects. CONCLUSION: In view of the lack of a clear association between Corynebacterium spp. and dental caries status in the literature, the predominance of these species in caries-free children warrants further research to understand their possible role in a health-associated microbial community. CLINICAL SIGNIFICANCE: Understanding the relationship between specific bacteria present in dental biofilms and health and disease is essential for preventing and combating dental caries. Using advanced next generation sequencing techniques, the present study demonstrated the complexity of the caries microbiome and identified species/genera whose virulence or protective properties should be further explored.

Proteomics of extracellular vesicles produced by Granulicatella adiacens, which causes infective endocarditis.

When oral bacteria accidentally enter the bloodstream due to transient tissue damage during dental procedures, they have the potential to attach to the endocardium or an equivalent surface of an indwelling prosthesis and cause infection. Many bacterial species produce extracellular vesicles (EVs) as part of normal physiology, but also use it as a virulence strategy. In this study, it was hypothesized that Granulicatella adiacens produce EVs that possibly help it in virulence. Therefore, the objectives were to isolate and characterize EVs produced by G. adiacens and to investigate its immune-stimulatory effects. The reference strain G. adiacens CCUG 27809 was cultured on chocolate blood agar for 2 days. From subsequent broth culture, the EVs were isolated using differential centrifugation and filtration protocol and then observed using scanning electron microscopy. Proteins in the vesicle preparation were identified by nano LC-ESI-MS/MS. The EVs proteome was analyzed and characterized using different bioinformatics tools. The immune-stimulatory effect of the EVs was studied via ELISA quantification of IL-8, IL-1ß and CCL5, major proinflammatory cytokines, produced from stimulated human PBMCs. It was revealed that G. adiacens produced EVs, ranging in diameter from 30 to 250 nm. Overall, G. adiacens EVs contained 112 proteins. The proteome consists of several ribosomal proteins, DNA associated proteins, binding proteins, and metabolic enzymes. It was also shown that these EVs carry putative virulence factors including moonlighting proteins. These EVs were able to induce the production of IL-8, IL-1ß and CCL5 from human PBMCs. Further functional characterization of the G. adiacens EVs may provide new insights into virulence mechanisms of this important but less studied oral bacterial species.

Biofilm growth and IL-8 & TNF-α-inducing properties of Candida albicans in the presence of oral gram-positive and gram-negative bacteria.

BACKGROUND: Interaction of C. albicans with oral bacteria is crucial for its persistence, but also plays a potential role in the infection process. In the oral cavity, it grows as part of dental plaque biofilms. Even though growth and interaction of C. albicans with certain bacterial species has been studied, little is known about its biofilm growth in vitro in the simultaneous presence of Gram-negative and Gram-positive bacteria. The aim was to evaluate the growth of C. albicans in polymicrobial biofilms comprising oral Gram-negative and Gram-positive bacteria. Further, we also aimed to assess the potential of C. albicans in the Candida-bacteria polymicrobial biofilm to elicit cytokine gene expression and cytokine production from human blood cells. RESULTS: C. albicans cell counts increased significantly up to 48 h in polymicrobial biofilms (p < 0.05), while the bacterial counts in the same biofilms increased only marginally as revealed by qPCR absolute quantification. However, the presence of bacteria in the biofilm did not seem to affect the growth of C. albicans. Expression of IL-8 gene was significantly (p < 0.05) higher upon stimulation from biofilm-supernatants than from biofilms in polymicrobial setting. On the contrary, TNF-α expression was significantly higher in biofilms than in supernatants but was very low (1-4 folds) in the monospecies biofilm of C. albicans. ELISA cytokine quantification data was in agreement with mRNA expression results. CONCLUSION: Persistence and enhanced growth of C. albicans in polymicrobial biofilms may imply that previously reported antagonistic effect of A. actinomycetemcomitans was negated. Increased cytokine gene expression and cytokine production induced by Candida-bacteria polymicrobial biofilms and biofilm supernatants suggest that together they possibly exert an enhanced stimulatory effect on IL-8 and TNF-α production from the host.

Real-time polymerase chain reaction quantification of the salivary levels of cariogenic bacteria in patients with orthodontic fixed appliances.

AIM: The aim was to investigate the salivary detection frequencies and quantities of caries-associated bacteria from patients with orthodontic brackets. METHODS: Patients wearing orthodontic brackets (n = 40, mean age = 26 years) and healthy controls without brackets (n = 40, mean age = 17 years) were enrolled in the study. Saliva samples from each patient was collected. After DNA purification, target species comprising streptococci and a Lactobacillus species were detected and quantified from the samples using polymerase chain reaction (PCR) and real-time quantitative PCR. RESULTS: Detection frequencies did not differ between the orthodontic patients and the control subjects for any target species except for Streptococcus sobrinus, which showed significantly lower detection rates in orthodontic patients (p < .05). Lactobacillus casei and Streptococcus gordonii were found at the highest detection frequencies with both species being detected in 38 (95%) of the saliva samples of orthodontic patients. Similarly, L. casei and Streptococcus salivarius were the species with highest detection frequencies (35, 87.5%) in the control subjects. Real-time PCR revealed that Streptococcus mutans and S. salivarius quantities were significantly higher in orthodontic patients than in the control subjects (p < .05). CONCLUSIONS: Application of orthodontic brackets for 12 months leads to increased salivary levels of cariogenic bacteria and may serve as a potential risk factor for caries initiation.

Proteomic Analysis and Virulence Assessment of Granulicatella adiacens Secretome.

Despite reports on the occurrence of Granulicatella adiacens in infective endocarditis, few mechanistic studies on its virulence characteristics or pathogenicity are available. Proteins secreted by this species may act as determinants of host-microbe interaction and play a role in virulence. Our aim in this study was to investigate and functionally characterize the secretome of G. adiacens. Proteins in the secretome preparation were digested by trypsin and applied to nanoLC-ESI-MS/MS. By using a combined mass spectrometry and bioinformatics approach, we identified 101 proteins. Bioinformatics tools predicting subcellular localization revealed that 18 of the secreted proteins possessed signal sequence. More than 20% of the secretome proteins were putative virulence proteins including serine protease, superoxide dismutase, aminopeptidase, molecular chaperone DnaK, and thioredoxin. Ribosomal proteins, molecular chaperones, and glycolytic enzymes, together known as "moonlighting proteins," comprised fifth of the secretome proteins. By Gene Ontology analysis, more than 60 proteins of the secretome were grouped in biological processes or molecular functions. KEGG pathway analysis disclosed that the secretome consisted of enzymes involved in biosynthesis of antibiotics. Cytokine profiling revealed that secreted proteins stimulated key cytokines, such as IL-1ß, MCP-1, TNF-α, and RANTES from human PBMCs. In summary, the results from the current investigation of the G. adiacens secretome provide a basis for understanding possible pathogenic mechanisms of G. adiacens.

In vivo antimicrobial activity of silver diammine fluoride on carious lesions in dentin.

Minimally invasive treatment protocols may leave a residual layer of carious dentin, which requires treatment for the inhibition of bacterial growth beneath restorations. We aimed to examine the in vivo effect of silver diammine fluoride (SDF) and SDF + potassium iodide (KI) application on bacteria present in deep carious lesions. We studied the in vivo efficacy in five patients, each of which had five carious lesions. Dentin samples taken before and after treatment were subjected to microbial analyses. Following treatment with SDF, the median colony-forming unit (CFU) counts per mg of dentin reduced from 9 × 105 to 1.6 × 102 (P < 0.05), and following that with SDF + KI, the counts decreased from 2.9 × 105 to 9.2 × 10 (P < 0.05). The use of chlorhexidine gluconate (CHX) reduced CFU counts from 1.1 × 105 to 4.8 × 102 (P < 0.05). In four of the five patients, no CFUs were found on mitis salivarius-bacitracin agar with respect to SDF or SDF + KI application. For CHX, the median CFU count before treatment was 1.6 × 103 and that after treatment was 1.1 × 102. SDF completely inhibited mutans streptococci growth in four of the five patients, while the growth of anaerobes was not completely inhibited.