Anti-bacterial and anti-biofilm activities of arachidonic acid against the cariogenic bacterium Streptococcus mutans.

Streptococcus mutans is a Gram-positive, facultative anaerobic bacterium, which causes dental caries after forming biofilms on the tooth surface while producing organic acids that demineralize enamel and dentin. We observed that the polyunsaturated arachidonic acid (AA) (ω-6; 20:4) had an anti-bacterial activity against S. mutans, which prompted us to investigate its mechanism of action. The minimum inhibitory concentration (MIC) of AA on S. mutans was 25 µg/ml in the presence of 5% CO2, while it was reduced to 6.25-12.5 µg/ml in the absence of CO2 supplementation. The anti-bacterial action was due to a combination of bactericidal and bacteriostatic effects. The minimum biofilm inhibitory concentration (MBIC) was the same as the MIC, suggesting that part of the anti-biofilm effect was due to the anti-bacterial activity. Gene expression studies showed decreased expression of biofilm-related genes, suggesting that AA also has a specific anti-biofilm effect. Flow cytometric analyses using potentiometric DiOC2(3) dye, fluorescent efflux pump substrates, and live/dead SYTO 9/propidium iodide staining showed that AA leads to immediate membrane hyperpolarization, altered membrane transport and efflux pump activities, and increased membrane permeability with subsequent membrane perforation. High-resolution scanning electron microscopy (HR-SEM) showed remnants of burst bacteria. Furthermore, flow cytometric analysis using the redox probe 2',7'-dichlorofluorescein diacetate (DCFHDA) showed that AA acts as an antioxidant in a dose-dependent manner. α-Tocopherol, an antioxidant that terminates the radical chain, counteracted the anti-bacterial activity of AA, suggesting that oxidation of AA in bacteria leads to the production of cytotoxic radicals that contribute to bacterial growth arrest and death. Importantly, AA was not toxic to normal Vero epithelial cells even at 100 µg/ml, and it did not cause hemolysis of erythrocytes. In conclusion, our study shows that AA is a potentially safe drug that can be used to reduce the bacterial burden of cariogenic S. mutans.

Polyglactin 910 Meshes Coated with Sustained-Release Cannabigerol Varnish Inhibit Staphylococcus aureus Biofilm Formation and Macrophage Cytokine Secretion: An In Vitro Study.

Synthetic surgical meshes are commonly used in abdominal wall reconstruction surgeries to strengthen a weak abdominal wall. Common mesh-related complications include local infection and inflammatory processes. Because cannabigerol (CBG) has both antibacterial and anti-inflammatory properties, we proposed that coating VICRYL (polyglactin 910) mesh with a sustained-release varnish (SRV) containing CBG would prevent these complications. We used an in vitro infection model with Staphylococcus aureus and an in vitro inflammation model of lipopolysaccharide (LPS)-stimulated macrophages. Meshes coated with either SRV-placebo or SRV-CBG were exposed daily to S. aureus in tryptic soy medium (TSB) or macrophage Dulbecco's modified eagle medium (DMEM). Bacterial growth and biofilm formation in the environment and on the meshes were assessed by changes in optical density, bacterial ATP content, metabolic activity, crystal violet staining, spinning disk confocal microscopy (SDCM), and high-resolution scanning electron microscopy (HR-SEM). The anti-inflammatory effect of the culture medium that was exposed daily to the coated meshes was analyzed by measuring the release of the cytokines IL-6 and IL-10 from LPS-stimulated RAW 264.7 macrophages with appropriate ELISA kits. Additionally, a cytotoxicity assay was performed on Vero epithelial cell lines. We observed that compared with SRV-placebo, the segments coated with SRV-CBG inhibited the bacterial growth of S. aureus in the mesh environment for 9 days by 86 ± 4% and prevented biofilm formation and metabolic activity in the surroundings for 9 days, with respective 70 ± 2% and 95 ± 0.2% reductions. The culture medium that was incubated with the SRV-CBG-coated mesh inhibited LPS-induced secretion of IL-6 and IL-10 from the RAW 264.7 macrophages for up to 6 days without affecting macrophage viability. A partial anti-inflammatory effect was also observed with SRV-placebo. The conditioned culture medium was not toxic to Vero epithelial cells, which had an IC50 of 25 µg/mL for CBG. In conclusion, our data indicate a potential role of coating VICRYL mesh with SRV-CBG in preventing infection and inflammation in the initial period after surgery.

Anti-Bacterial and Anti-Biofilm Activities of Anandamide against the Cariogenic Streptococcus mutans.

Streptococcus mutans is a cariogenic bacterium in the oral cavity involved in plaque formation and dental caries. The endocannabinoid anandamide (AEA), a naturally occurring bioactive lipid, has been shown to have anti-bacterial and anti-biofilm activities against Staphylococcus aureus. We aimed here to study its effects on S. mutans viability, biofilm formation and extracellular polysaccharide substance (EPS) production. S. mutans were cultivated in the absence or presence of various concentrations of AEA, and the planktonic growth was followed by changes in optical density (OD) and colony-forming units (CFU). The resulting biofilms were examined by MTT metabolic assay, Crystal Violet (CV) staining, spinning disk confocal microscopy (SDCM) and high-resolution scanning electron microscopy (HR-SEM). The EPS production was determined by Congo Red and fluorescent dextran staining. Membrane potential and membrane permeability were determined by diethyloxacarbocyanine iodide (DiOC2(3)) and SYTO 9/propidium iodide (PI) staining, respectively, using flow cytometry. We observed that AEA was bactericidal to S. mutans at 12.5 µg/mL and prevented biofilm formation at the same concentration. AEA reduced the biofilm thickness and biomass with concomitant reduction in total EPS production, although there was a net increase in EPS per bacterium. Preformed biofilms were significantly affected at 50 µg/mL AEA. We further show that AEA increased the membrane permeability and induced membrane hyperpolarization of these bacteria. AEA caused S. mutans to become elongated at the minimum inhibitory concentration (MIC). Gene expression studies showed a significant increase in the cell division gene ftsZ. The concentrations of AEA needed for the anti-bacterial effects were below the cytotoxic concentration for normal Vero epithelial cells. Altogether, our data show that AEA has anti-bacterial and anti-biofilm activities against S. mutans and may have a potential role in preventing biofilms as a therapeutic measure.

Sinonasal Stent Coated with Sustained-Release Varnish of Mometasone Furoate Inhibits Pro-Inflammatory Cytokine Release from Macrophages: An In Vitro Study.

The aim of the study was to develop a sustained-release varnish (SRV) containing mometasone furoate (MMF) for sinonasal stents (SNS) to reduce mucosa inflammation in the sinonasal cavity. The SNS' segments coated with SRV-MMF or an SRV-placebo were incubated daily in a fresh DMEM at 37 °C for 20 days. The immunosuppressive activity of the collected DMEM supernatants was tested on the ability of mouse RAW 264.7 macrophages to secrete the cytokines' tumor necrosis factor α (TNFα) and interleukin (IL)-10 and IL-6 in response to lipopolysaccharide (LPS). The cytokine levels were determined by respective Enzyme-Linked Immunosorbent Assays (ELISAs). We found that the daily amount of MMF released from the coated SNS was sufficient to significantly inhibit LPS-induced IL-6 and IL-10 secretion from the macrophages up to days 14 and 17, respectively. SRV-MMF had, however, only a mild inhibitory effect on LPS-induced TNFα secretion as compared to the SRV-placebo-coated SNS. In conclusion, the coating of SNS with SRV-MMF provides a sustained delivery of MMF for at least 2 weeks, maintaining a level sufficient for inhibiting pro-inflammatory cytokine release. This technological platform is, therefore, expected to provide anti-inflammatory benefits during the postoperative healing period and may play a significant role in the future treatment of chronic rhinosinusitis.

The Antibacterial Effect of Cannabigerol toward Streptococcus mutans Is Influenced by the Autoinducers 21-CSP and AI-2.

Bacteria can communicate through an intercellular signaling system referred to as quorum sensing (QS). The QS system involves the production of autoinducers that interact with their respective receptors, leading to the induction of specific signal transduction pathways. The QS systems of the oral cariogenic Streptococcus mutans regulate the maturation of biofilms and affect its virulent properties. We have previously shown that the non-psychoactive compound cannabigerol (CBG) of the Cannabis sativa L. plant has anti-bacterial and anti-biofilm activities towards S. mutans. Here we were interested in investigating the effect of the two QS systems ComCDE and LuxS on the susceptibility of S. mutans to CBG and the anti-QS activities of CBG. This was assessed by using various comCDE and luxS mutant strains and complementation with the respective autoinducers, competence stimulating peptide (CSP) and (S)-4,5-dihydroxy-2,3-pentandione (DPD, pre-AI-2). We found that S. mutans comCDE knockout strains were more sensitive to the anti-bacterial actions of CBG compared to the WT strain. Exogenously added 21-CSP prevented the anti-bacterial actions caused by CBG on the ΔcomC, ΔcomE and ΔluxS mutants, while having no effect on the susceptibility of the WT and ΔcomCDE strains to CBG. Exogenously added DPD increased the susceptibility of WT and ΔluxS to CBG. Vice versa, CBG significantly reduced the 21-CSP-induced expression of comCDE genes and ComE-regulated genes and suppressed the expression of luxS with concomitant reduction in AI-2 production. DPD induced the expression of comCDE genes and ComE-regulated genes, and this induction was repressed by CBG. 21-CSP alone had no significant effect on luxS gene expression, while ΔcomCDE strains showed reduced AI-2 production. In conclusion, our study shows that the susceptibility of S. mutans to CBG is affected by the ComCDE and LuxS QS pathways, and CBG is a potential anti-QS compound for S. mutans. Additionally, we provide evidence for crosstalk between the ComCDE and LuxS QS systems.

Improved Anti-Biofilm Effect against the Oral Cariogenic Streptococcus mutans by Combined Triclosan/CBD Treatment.

Streptococcus mutans is a Gram-positive bacterium highly associated with dental caries, and it has a strong biofilm-forming ability, especially in a sugar-rich environment. Many strategies have been undertaken to prevent dental caries by targeting these bacteria. Recently, we observed that a sustained-release varnish containing triclosan and cannabidiol (CBD) was more efficient than each compound alone in preventing biofilm formation by the fungus Candida albicans, which is frequently involved in oral infections together with S. mutans. It was therefore inquiring to study the effect of this drug combination on S. mutans. We observed that the combined treatment of triclosan and CBD had stronger anti-bacterial and anti-biofilm activity than each compound alone, thus enabling the use of lower concentrations of each drug to achieve the desired effect. The combined drug treatment led to an increase in the SYTO 9low, propidium iodide (PI)high bacterial population as analyzed by flow cytometry, indicative for bacteria with disrupted membrane. Both triclosan and CBD induced membrane hyperpolarization, although there was no additive effect on this parameter. HR-SEM images of CBD-treated bacteria show the appearance of elongated and swollen bacteria with several irregular septa structures, and upon combined treatment with triclosan, the bacteria took on a swollen ellipse and sometimes oval morphology. Increased biofilm formation was observed at sub-MIC concentrations of each compound alone, while combining the drugs at these sub-MIC concentrations, the biofilm formation was prevented. The inhibition of biofilm formation was confirmed by CV biomass staining, MTT metabolic activity, HR-SEM and live/dead together with exopolysaccharide (EPS) staining visualized by spinning disk confocal microscopy. Importantly, the concentrations required for the anti-bacterial and anti-biofilm activities toward S. mutans were non-toxic to the normal Vero epithelial cells. In conclusion, the data obtained in this study propose a beneficial role of combined triclosan/CBD treatment for potential protection against dental caries.

Anti-Bacterial Effect of Cannabidiol against the Cariogenic Streptococcus mutans Bacterium: An In Vitro Study.

Dental caries is caused by biofilm-forming acidogenic bacteria, especially Streptococcus mutans, and is still one of the most prevalent human bacterial diseases. The potential use of cannabidiol (CBD) in anti-bacterial therapies has recently emerged. Here we have studied the anti-bacterial and anti-biofilm activity of CBD against S. mutans. We measured minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC). The bacterial growth and changes in pH values were measured in a kinetic study. The biofilm biomass was assessed by Crystal Violet staining and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) metabolic assay. Spinning Disk Confocal Microscopy (SDCM) was used to assess biofilm structure, bacterial viability and extracellular polysaccharide (EPS) production. CBD inhibited S. mutans planktonic growth and biofilm formation in a dose-dependent manner, with similar MIC and MBIC values (5 µg/mL). CBD prevented the bacteria-mediated reduction in pH values that correlated with bacterial growth inhibition. SDCM showed a decrease of 50-fold in live bacteria and EPS production. CBD significantly reduced the viability of preformed biofilms at 7.5 µg/mL with an 80 ± 3.1% reduction of metabolic activity. At concentrations above 20 µg/mL, there was almost no bacterial recovery in the CBD-treated preformed biofilms even 48 h after drug withdrawal. Notably, precoating of the culture plate surfaces with CBD prior to incubation with bacteria inhibited biofilm development. Additionally, CBD was found to induce membrane hyperpolarization in S. mutans. Thus, CBD affects multiple processes in S. mutans including its cariogenic properties. In conclusion, we show that CBD has a strong inhibitory effect against cariogenic bacteria, suggesting that it is a potential drug adjuvant for reducing oral pathogenic bacterial load as well as protecting against dental caries.

Corrigendum to "Sustained-Release Fillers for Dentin Disinfection: An Ex Vivo Study".

[This corrects the article DOI: 10.1155/2019/2348146.].

Prolonged Inhibition of Streptococcus mutans Growth and Biofilm Formation by Sustained Release of Chlorhexidine from Varnish Coated Dental Abutments: An in Vitro Study.

Background: It has been confirmed that bacterial biofilm covering dental implants is the main microbial source causing preimplant infectious and inflammatory diseases. The purpose of this study was to evaluate the antibacterial/antibiofilm effect of chlorhexidine, incorporated into a sustained-release varnish of chlorhexidine (SRV-CHX) coating, on dental abutments. Materials and Methods: Three kinds of dental abutments were used: a high-performance semi-crystalline engineering thermoplastic polyetheretherketone (PEAK) healing abutment, a titanium healing abutment, and a titanium permanent abutment. These abutments were coated with SRV-CHX or SRV-placebo and exposed daily to fresh cultures of Streptococcus mutans. The effect of SRV-CHX on S. mutans growth on agar plates was studied by measuring the zone of inhibition (ZOI) around each tested abutment every day for a period of 36 days. Biofilm formation on the SRV-CHX/placebo-coated abutments was detected using confocal laser scanning microscopy (CLSM) and high-resolution scanning electron microscopy (HR-SEM), energy dispersive X-ray analysis (EDX), and monitored by crystal violet (CV) staining. Results: SRV-CHX-coated abutments 2 and 3 were able to inhibit S. mutans growth for 34 days, while abutment 1 inhibited growth for 32 days. Abutment-associated biofilm formation was notably inhibited by SRV-CHX coating after 13 days of incubation with S. mutans. Finally, the biofilm formed around SRV-CHX-coated abutments was completely inhibited up to 12 days of abutment exposure to S. mutans. Conclusion: Coating of dental abutments with SRV-CHX demonstrated long-term effective inhibition of S. mutans growth and biofilm formation on the abutment surface.

An open-source computational tool for measuring bacterial biofilm morphology and growth kinetics upon one-sided exposure to an antimicrobial source.

Bacillus subtilis biofilms are well known for their complex and highly adaptive morphology. Indeed, their phenotypical diversity and intra-biofilm heterogeneity make this gram-positive bacterium the subject of many scientific papers on the structure of biofilms. The "robustness" of biofilms is a term often used to describe their level of susceptibility to antimicrobial agents and various mechanical and molecular inhibition/eradication methods. In this paper, we use computational analytics to quantify Bacillus subtilis morphological response to proximity to an antimicrobial source, in the form of the antiseptic chlorhexidine. Chlorhexidine droplets, placed in proximity to Bacillus subtilis macrocolonies at different distances result in morphological changes, quantified using Python-based code, which we have made publicly available. Our results quantify peripheral and inner core deformation as well as differences in cellular viability of the two regions. The results reveal that the inner core, which is often characterized by the presence of wrinkled formations in the macrocolony, is more preserved than the periphery. Furthermore, the paper describes a crescent-shaped colony morphology which occurs when the distance from the chlorhexidine source is 0.5 cm, as well as changes observed in the growth substrate of macrocolonies exposed to chlorhexidine.

Potential Combinatory Effect of Cannabidiol and Triclosan Incorporated into Sustained Release Delivery System against Oral Candidiasis.

Candida albicans is a common fungal pathogen. Biofilm formation on various surfaces is an important determinant of C. albicans pathogenicity. Our previous results demonstrated the high potential of cannabidiol (CBD) to affect C. albicans biofilms. Based on these data, we investigated the possibility of incorporating CBD and/or triclosan (an antimicrobial agent that is widely utilized in dentistry) in a sustained-release varnish (SRV) (SRV-CBD, SRV-triclosan) to increase their pharmaceutical potential against C. albicans biofilm, as well as that of the mixture of the agents into SRV (SRV-CBD/triclosan). The study was conducted in a plastic model, on agar, and in an ex vivo tooth model. Our results demonstrated strong antibiofilm activity of SRV-CBD and SRV-triclosan against C. albicans in all tested models. Both formulations were able to inhibit biofilm formation and to remove mature fungal biofilm. In addition, SRV-CBD and SRV-triclosan altered C. albicans morphology. Finally, we observed a dramatic enhancement of antibiofilm activity when combined SRV-CBD/triclosan was applied. In conclusion, we propose that incorporation of CBD or triclosan into SRV is an effective strategy to fight fungal biofilms. Importantly, the data demonstrate that our CBD/triclosan varnish is safe, and is not cytotoxic for normal mammalian cells. Furthermore, we propose that CBD and triclosan being in mixture in SRV exhibit complementary antibiofilm activity, and thus can be explored for further development as a potential treatment against fungal infections.

Targeting the Achilles' Heel of Multidrug-Resistant Staphylococcus aureus by the Endocannabinoid Anandamide.

Antibiotic-resistant Staphylococcus aureus is a major health issue that requires new therapeutic approaches. Accumulating data suggest that it is possible to sensitize these bacteria to antibiotics by combining them with inhibitors targeting efflux pumps, the low-affinity penicillin-binding protein PBP2a, cell wall teichoic acid, or the cell division protein FtsZ. We have previously shown that the endocannabinoid Anandamide (N-arachidonoylethanolamine; AEA) could sensitize drug-resistant S. aureus to a variety of antibiotics, among others, through growth arrest and inhibition of drug efflux. Here, we looked at biochemical alterations caused by AEA. We observed that AEA increased the intracellular drug concentration of a fluorescent penicillin and augmented its binding to membrane proteins with concomitant altered membrane distribution of these proteins. AEA also prevented the secretion of exopolysaccharides (EPS) and reduced the cell wall teichoic acid content, both processes known to require transporter proteins. Notably, AEA was found to inhibit membrane ATPase activity that is necessary for transmembrane transport. AEA did not affect the membrane GTPase activity, and the GTPase cell division protein FtsZ formed the Z-ring of the divisome normally in the presence of AEA. Rather, AEA caused a reduction in murein hydrolase activities involved in daughter cell separation. Altogether, this study shows that AEA affects several biochemical processes that culminate in the sensitization of the drug-resistant bacteria to antibiotics.

Targeting the Holy Triangle of Quorum Sensing, Biofilm Formation, and Antibiotic Resistance in Pathogenic Bacteria.

Chronic and recurrent bacterial infections are frequently associated with the formation of biofilms on biotic or abiotic materials that are composed of mono- or multi-species cultures of bacteria/fungi embedded in an extracellular matrix produced by the microorganisms. Biofilm formation is, among others, regulated by quorum sensing (QS) which is an interbacterial communication system usually composed of two-component systems (TCSs) of secreted autoinducer compounds that activate signal transduction pathways through interaction with their respective receptors. Embedded in the biofilms, the bacteria are protected from environmental stress stimuli, and they often show reduced responses to antibiotics, making it difficult to eradicate the bacterial infection. Besides reduced penetration of antibiotics through the intricate structure of the biofilms, the sessile biofilm-embedded bacteria show reduced metabolic activity making them intrinsically less sensitive to antibiotics. Moreover, they frequently express elevated levels of efflux pumps that extrude antibiotics, thereby reducing their intracellular levels. Some efflux pumps are involved in the secretion of QS compounds and biofilm-related materials, besides being important for removing toxic substances from the bacteria. Some efflux pump inhibitors (EPIs) have been shown to both prevent biofilm formation and sensitize the bacteria to antibiotics, suggesting a relationship between these processes. Additionally, QS inhibitors or quenchers may affect antibiotic susceptibility. Thus, targeting elements that regulate QS and biofilm formation might be a promising approach to combat antibiotic-resistant biofilm-related bacterial infections.

Cannabigerol Effect on Streptococcus mutans Biofilms-A Computational Approach to Confocal Image Analysis.

Biofilms are complex bacterial structures in which bacterial cells thrive as a community. Many bacterial species, including pathogens, form biofilms of high complexity and adaptability to a wide range of environmental conditions. One example of these is Streptococcus mutans, a gram-positive bacterium that has been associated with caries. Cannabigerol, a non-psychoactive cannabinoid, has been shown to affect S. mutans biofilms. In order to better characterize the effect of cannabigerol on biofilms of S. mutans, this paper provides a series of computational assays for biofilm analysis, applied on confocal images of S. mutans biofilms treated with cannabigerol. Confocal images are ubiquitous in biofilm analysis-they are often used to visualize the complex structure and molecular composition of biofilm macrocolonies. In this article, we demonstrate how confocal imaging data can be used to reveal more comprehensive insights into biofilm structure and measure specific anti-biofilm effects. This is accomplished by a series of computational assays, each focusing on a different aspect of biofilm structure.

Anti-Microbial Activity of Phytocannabinoids and Endocannabinoids in the Light of Their Physiological and Pathophysiological Roles.

Antibiotic resistance has become an increasing challenge in the treatment of various infectious diseases, especially those associated with biofilm formation on biotic and abiotic materials. There is an urgent need for new treatment protocols that can also target biofilm-embedded bacteria. Many secondary metabolites of plants possess anti-bacterial activities, and especially the phytocannabinoids of the Cannabis sativa L. varieties have reached a renaissance and attracted much attention for their anti-microbial and anti-biofilm activities at concentrations below the cytotoxic threshold on normal mammalian cells. Accordingly, many synthetic cannabinoids have been designed with the intention to increase the specificity and selectivity of the compounds. The structurally unrelated endocannabinoids have also been found to have anti-microbial and anti-biofilm activities. Recent data suggest for a mutual communication between the endocannabinoid system and the gut microbiota. The present review focuses on the anti-microbial activities of phytocannabinoids and endocannabinoids integrated with some selected issues of their many physiological and pharmacological activities.

Sustained release varnish containing chlorhexidine for prevention of Streptococcus mutans biofilm formation on voice prosthesis surface: an in vitro study / Barniz de liberación sostenida que contiene clorhexidina para la prevención de la formación de biopelículas de Streptococcus mutans en la superficie de la prótesis de voz: un estudio in vitro

Objectives: In this study, we aimed to develop a novel, sustained release varnish (SRV) for voice prostheses (VP) releasing chlorhexidine (CHX), for the prevention of biofilm formation caused by the common oral bacteria Streptococcus mutans on VP surfaces. Methods: This study was performed in an in vitro model as a step towards future in vivo trials. VPs were coated with a SRV containing CHX (SRV-CHX) or SRV alone (placebo-SRV) that were daily exposed to S. mutans. The polymeric materials of SRV were composed of ethylcellulose and PEG-400. Biofilm formation was assessed by DNA quantification (qPCR), crystal violet staining, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and kinetics experiments. Results: The amount of DNA in the biofilms formed by S. mutans on VP surfaces coated once with SRV-CHX (1.024 ± 0.218 ng DNA/piece) was 58.5 ± 8.8% lower than that of placebo-SRV-coated VPs (2.465 ± 0.198 ng DNA/piece) after a 48-h exposure to S. mutans (p = 0.038). Reduced biofilm mass on SRV-CHX-coated VPs was visually confirmed by CLSM and SEM. CV staining of SRV-CHX single-coated VPs that have been exposed to S. mutans nine times showed a 98.1 ± 0.2% reduction in biofilm mass compared to placebo-SRV-coated VPs (p = 0.003). Kinetic experiments revealed that SRV-CHX triple-coated VPs could delay bacterial growth for 23 days. Conclusions: Coating VPs with SRV-CHX has an inhibitory effect on biofilm formation and prevents bacterial growth in their vicinities. This study is a proof-of-principle that paves the way for developing new clinical means for reducing both VPs’ bacterial biofilm formation and device failure.(AU)

Sustained release varnish containing chlorhexidine for prevention of Streptococcus mutans biofilm formation on voice prosthesis surface: an in vitro study.

OBJECTIVES: In this study, we aimed to develop a novel, sustained release varnish (SRV) for voice prostheses (VP) releasing chlorhexidine (CHX), for the prevention of biofilm formation caused by the common oral bacteria Streptococcus mutans on VP surfaces. METHODS: This study was performed in an in vitro model as a step towards future in vivo trials. VPs were coated with a SRV containing CHX (SRV-CHX) or SRV alone (placebo-SRV) that were daily exposed to S. mutans. The polymeric materials of SRV were composed of ethylcellulose and PEG-400. Biofilm formation was assessed by DNA quantification (qPCR), crystal violet staining, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and kinetics experiments. RESULTS: The amount of DNA in the biofilms formed by S. mutans on VP surfaces coated once with SRV-CHX (1.024 ± 0.218 ng DNA/piece) was 58.5 ± 8.8% lower than that of placebo-SRV-coated VPs (2.465 ± 0.198 ng DNA/piece) after a 48-h exposure to S. mutans (p = 0.038). Reduced biofilm mass on SRV-CHX-coated VPs was visually confirmed by CLSM and SEM. CV staining of SRV-CHX single-coated VPs that have been exposed to S. mutans nine times showed a 98.1 ± 0.2% reduction in biofilm mass compared to placebo-SRV-coated VPs (p = 0.003). Kinetic experiments revealed that SRV-CHX triple-coated VPs could delay bacterial growth for 23 days. CONCLUSIONS: Coating VPs with SRV-CHX has an inhibitory effect on biofilm formation and prevents bacterial growth in their vicinities. This study is a proof-of-principle that paves the way for developing new clinical means for reducing both VPs' bacterial biofilm formation and device failure.

Characterization of the Oral Microbiome Among Children With Type 1 Diabetes Compared With Healthy Children.

Aim: Current microbiome profiling of type 1 diabetes mellitus (T1D) patients is mostly limited to gut microbiome. We characterized the oral microbiome associated with T1D in children after the onset of the disease and explored its relationship with oral physiological factors and dental status. Methods: This cohort study comprised 37 children aged 5-15 years with T1D and 29 healthy children matched in age and gender. Unstimulated whole saliva was collected from diabetic and non-diabetic children, in the morning after brushing their teeth and a fasting period of at least 1 h before sampling. 16S rRNA gene-based analysis was performed by Powersoil Pro kit by Qiagen and Phusion High-Fidelity PCR Master Mix. Oral physiological and dental parameters studied included decayed, missing, and filled teeth index, salivary flow rate, and salivary pH, glucose, calcium, phosphate, and urea levels. Results: Of the identified 105 different genera and 211 different species, the most abundant genera were Streptococcus, Prevotella, Veillonella, Haemophilus, and Neisseria. Streptococcus was more abundant in T1D children. The diabetes group had 22 taxa at the genus level and 33 taxa at the species level that were not present in the control group and the control group exhibited 6 taxa at the genus level and 9 taxa at the species level that did not exist in the diabetes group. In addition, Catonella, Fusobacterium, and Mogibacterium differed between healthy and T1D subjects. Eight species and eight subspecies were significantly more abundant among healthy children than in T1D children. Porphyromonas and Mogibacterium genera were significantly correlated with salivary parameters. We found similarities between taxa revealed in the present study and those found in gut microbiome in type 1 diabetes mellitus according to gutMDisorder database. Conclusions: Salivary microbiome analysis revealed unique microbial taxa that differed between T1D children and healthy subjects. Several genera found in the saliva of T1D children were associated with gut microbiome in T1D individuals.

Sinonasal Stent Coated with Slow-Release Varnish of Chlorhexidine Has Sustained Protection against Bacterial Biofilm Growth in the Sinonasal Cavity: An In Vitro Study.

The aim of the study was to develop a sustained-release varnish (SRV) containing chlorhexidine (CHX) for sinonasal stents (SNS) to reduce bacterial growth and biofilm formation in the sinonasal cavity. Segments of SNS were coated with SRV-CHX or SRV-placebo and exposed daily to bacterial cultures of Staphylococcus aureus subsp. aureus ATCC 25923 or Pseudomonas aeruginosa ATCC HER-1018 (PAO1). Anti-bacterial effects were assessed by disc diffusion assay and planktonic-based activity assay. Biofilm formation on the coated stents was visualized by confocal laser scanning microscopy (CLSM) and high-resolution scanning electron microscopy (HR-SEM). The metabolic activity of the biofilms was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method. Disc diffusion assay showed that SRV-CHX-coated SNS segments inhibited bacterial growth of S. aureussubsp. aureus ATCC 25923 for 26 days and P. aeruginosa ATCC HER-1018 for 19 days. CHX was released from coated SNS segments in a pH 6 medium up to 30 days, resulting in growth inhibition of S. aureussubsp. aureus ATCC 25923 for 22 days and P. aeruginosa ATCC HER-1018 for 24 days. The MTT assay showed a reduction of biofilm growth on the coated SNS by 69% for S. aureussubsp. aureus ATCC 25923 and 40% for P. aeruginosa ATCC HER-1018 compared to the placebo stent after repeated exposure to planktonic growing bacteria. CLSM and HR-SEM showed a significant reduction of biofilm formation on the SRV-CHX-coated SNS segments. Coating of SNS with SRV-CHX maintains a sustained delivery of CHX, providing an inhibitory effect on the bacterial growth of S. aureussubsp. aureus ATCC 25923 and P. aeruginosa ATCC HER-1018 for approximately 3 weeks.

Anti-Biofilm Activity of Cannabigerol against Streptococcus mutans.

Streptococcus mutans is a common cariogenic bacterium in the oral cavity involved in plaque formation. Previous studies showed that Cannabigerol (CBG) has bacteriostatic and bacteriocidic activity against S. mutans. The aim of the present study was to study its effect on S. mutans biofilm formation and dispersion. S. mutans was cultivated in the presence of CBG, and the resulting biofilms were examined by CV staining, MTT assay, qPCR, biofilm tracer, optical profilometry, and SEM. Gene expression was determined by real-time qPCR, extracellular polysaccharide (EPS) production was determined by Congo Red, and reactive oxygen species (ROS) were determined using DCFH-DA. CBG prevented the biofilm formation of S. mutans shown by reduced biofilm biomass, decreased biofilm thickness, less EPS production, reduced DNA content, diminished metabolic activity, and increased ROS levels. CBG altered the biofilm roughness profile, resulting in a smoother biofilm surface. When treating preformed biofilms, CBG reduced the metabolic activity of S. mutans with a transient effect on the biomass. CBG reduced the expression of various genes involved in essential metabolic pathways related to the cariogenic properties of S. mutans biofilms. Our data show that CBG has anti-biofilm activities against S. mutans and might be a potential drug for preventive treatment of dental caries.