Graphene Nanocoating: High Quality and Stability upon Several Stressors.

Titanium implants present 2 major drawbacks-namely, the long time needed for osseointegration and the lack of inherent antimicrobial properties. Surface modifications and coatings to improve biomaterials can lose their integrity and biological potential when exposed to stressful microenvironments. Graphene nanocoating (GN) can be deposited onto actual-size dental and orthopedic implants. It has antiadhesive properties and can enhance bone formation in vivo. However, its ability to maintain structural integrity and quality when challenged by biologically relevant stresses remains largely unknown. GN was produced by chemical vapor deposition and transferred to titanium via a polymer-assisted transfer technique. GN has high inertness and did not increase expression of inflammatory markers by macrophages, even in the presence of lipopolysaccharides. It kept high coverage at the top tercile of tapered dental implant collars after installation and removal from bone substitute and pig maxilla. It also resisted microbiologically influenced corrosion, and it maintained very high coverage area and quality after prolonged exposure to biofilms and their removal by different techniques. Our findings show that GN is unresponsive to harsh and inflammatory environments and that it maintains a promising level of structural integrity on the top tercile of dental implant collars, which is the area highly affected by biofilms during the onset of implant diseases. Our findings open the avenues for the clinical studies required for the use of GN in the development of implants that have higher osteogenic potential and are less prone to implant diseases.

Probiotic therapy for periodontal and peri-implant health - silver bullet or sham?

Probiotics are thought to be beneficial microbes that influence health-related outcomes through host immunomodulation and modulation of the bacteriome. Its reported success in the treatment of gastrointestinal disorders has led to further research on its potential applicability within the dental field due to similarities such as a polymicrobial aetiology and disease associated microbial-shifts. Although the literature is replete with studies demonstrating its efficacy, the use of probiotics in dentistry continues to polarise opinion. Here, we explore the evidence for probiotics and its effect on periodontal and peri-implant health. MEDLINE, EMBASE, and CENTRAL were systemically searched from June 2010 to June 2020 based on a formulated search strategy. Of 1,956 potentially relevant articles, we selected 27 double-blinded randomised clinical trials in the areas of gingivitis, periodontitis, residual pockets during supportive periodontal therapy, and peri-implant diseases, and reviewed their efficacy in these clinical situations. We observed substantial variation in treatment results and protocols between studies. Overall, the evidence for probiotic therapy for periodontal and peri-implant health appears unconvincing. The scarcity of trials with adequate power and follow-up precludes any meaningful clinical recommendations. Thus, the routine use of probiotics for these purposes are currently unsubstantiated. Further multi-centre trials encompassing a standardised investigation on the most promising strains and administration methods, with longer observation times are required to confirm the benefits of probiotic therapy for these applications.

Subgingival Microbiota during Healthy Pregnancy and Pregnancy Gingivitis.

INTRODUCTION: Previous studies have largely explored the microbial composition and pathogenesis of pregnancy gingivitis. However, the patterns of microbial colonization during pregnancy in the absence of pregnancy gingivitis have rarely been studied. Characterization of the oral microbiome in pregnant women with healthy gingiva is an important initial step in understanding the role of the microbiome in progression to pregnancy gingivitis. OBJECTIVES: In this study, we compared the oral microbiome of pregnant women without gingivitis (healthy pregnancy) with pregnant women having gingivitis and nonpregnant healthy women to understand how pregnancy modifies the oral microbiome and induces progression to pregnancy gingivitis. METHODS: Subgingival plaque samples were collected from Chinese pregnant women with gingivitis (n = 10), healthy pregnant women (n = 10), and nonpregnant healthy women (n = 10). The Illumina MiSeq platform was used to perform 16S rRNA gene sequencing targeting the V4 region. RESULTS: The alpha and beta diversity was significantly different between pregnant and nonpregnant women, but minimal differences were observed between pregnant women with and without gingivitis. Interestingly, the oral bacterial community showed higher abundance of pathogenic taxa during healthy pregnancy as compared with nonpregnant women despite similar gingival and plaque index scores. However, when compared with overt pregnancy gingivitis, pathogenic taxa were less abundant during healthy pregnancy. PICRUSt analysis (phylogenetic investigation of communities by reconstruction of unobserved states) also suggested no difference in the functional capabilities of the microbiome during pregnancy, irrespective of gingival disease status. However, metabolic pathways related to amino acid metabolism were significantly increased in healthy pregnant women as compared with nonpregnant women. CONCLUSION: The presence of pathogenic taxa in healthy pregnancy and pregnancy gingivitis suggests that bacteria may be necessary for initiating disease development but progression to gingivitis may be influenced by the host environmental factors. More efforts are required to plan interventions aimed at sustaining health before the appearance of overt gingivitis. KNOWLEDGE TRANSFER STATEMENT: The results of this study draw attention to the importance of oral health maintenance during pregnancy, as women without any prenatal oral conditions are predisposed to the risk of developing pregnancy gingivitis. Hence, it is important to incorporate comprehensive assessment of oral health in the prenatal health care schedules. Pregnant woman should be screened for oral risks, counseled on proper oral hygiene and expected oral changes, and referred for dental treatment, when necessary.

Multi-omics Analyses Reveal Synergistic Carbohydrate Metabolism in Streptococcus mutans-Candida albicans Mixed-Species Biofilms.

Candida albicans, a major opportunistic fungal pathogen, is frequently found together with Streptococcus mutans in dental biofilms associated with severe childhood caries (tooth decay), a prevalent pediatric oral disease. However, the impact of this cross-kingdom relationship on C. albicans remains largely uncharacterized. Here, we employed a novel quantitative proteomics approach in conjunction with transcriptomic profiling to unravel molecular pathways of C. albicans when cocultured with S. mutans in mixed biofilms. RNA sequencing and iTRAQ (isobaric tags for relative and absolute quantitation)-based quantitative proteomics revealed that C. albicans genes and proteins associated with carbohydrate metabolism were significantly enhanced, including sugar transport, aerobic respiration, pyruvate breakdown, and the glyoxylate cycle. Other C. albicans genes and proteins directly and indirectly related to cell morphogenesis and cell wall components such as mannan and glucan were also upregulated, indicating enhanced fungal activity in mixed-species biofilm. Further analyses revealed that S. mutans-derived exoenzyme glucosyltransferase B (GtfB), which binds to the fungal cell surface to promote coadhesion, can break down sucrose into glucose and fructose that can be readily metabolized by C. albicans, enhancing growth and acid production. Altogether, we identified key pathways used by C. albicans in the mixed biofilm, indicating an active fungal role in the sugar metabolism and environmental acidification (key virulence traits associated with caries onset) when interacting with S. mutans, and a new cross-feeding mechanism mediated by GtfB that enhances C. albicans carbohydrate utilization. In addition, we demonstrate that comprehensive transcriptomics and quantitative proteomics can be powerful tools to study microbial contributions which remain underexplored in cross-kingdom biofilms.

Is the oral fungal pathogen Candida albicans a cariogen?

Pathobiology of dental caries is complex. Data from recent molecular microbiologic studies have further redefined the role of the oral microbiome in the etiology of dental caries. This new information challenges the conventional view on the hegemony of classic cariogenic prokaryotes such as Streptococcus mutans in caries etiology, and raises the intriguing possibility of the participation of the eukaryotic oral fungal pathogen Candida in the caries process. The virulence attributes of Candida species such as their acidogenicity and aciduric nature, the ability to develop profuse biofilms, ferment and assimilate dietary sugars, and produce collagenolytic proteinases are all indicative of their latent cariogenic potential. Based on the above, oral candidal counts have been used by some as a caries risk indicator. On the contrary, other studies suggest that Candida is merely a passenger extant in an acidic cariogenic milieu, and not a true pathogen. In this review, we critically examine the varying roles of Candida, and traditionally accepted cariogens such as the mutans group of streptococci in the pathobiology of dental caries. The weight of available data tends to imply that Candida may play a pivotal role as a secondary agent perpetuating the carious process, especially in dentinal caries.

Bacterial GtfB Augments Candida albicans Accumulation in Cross-Kingdom Biofilms.

Streptococcus mutans is a biofilm-forming oral pathogen commonly associated with dental caries. Clinical studies have shown that S. mutans is often detected with Candida albicans in early childhood caries. Although the C. albicans presence has been shown to enhance bacterial accumulation in biofilms, the influence of S. mutans on fungal biology in this mixed-species relationship remains largely uncharacterized. Therefore, we aimed to investigate how the presence of S. mutans influences C. albicans biofilm development and coexistence. Using a newly established haploid biofilm model of C. albicans, we found that S. mutans augmented haploid C. albicans accumulation in mixed-species biofilms. Similarly, diploid C. albicans also showed enhanced biofilm formation in the presence of S. mutans. Surprisingly, the presence of S. mutans restored the biofilm-forming ability of C. albicans bcr1Δ mutant and bcr1Δ/Δ mutant, which is known to be severely defective in biofilm formation when grown as single species. Moreover, C. albicans hyphal growth factor HWP1 as well as ALS1 and ALS3, which are also involved in fungal biofilm formation, were upregulated in the presence of S. mutans. Subsequently, we found that S. mutans-derived glucosyltransferase B (GtfB) itself can promote C. albicans biofilm development. Interestingly, GtfB was able to increase the expression of HWP1, ALS1, and ALS3 genes in the C. albicans diploid wild-type SC5314 and bcr1Δ/Δ, leading to enhanced fungal biofilms. Hence, the present study demonstrates that a bacterial exoenzyme (GtfB) augments the C. albicans counterpart in mixed-species biofilms through a BCR1-independent mechanism. This novel finding may explain the mutualistic role of S. mutans and C. albicans in cariogenic biofilms.

Effect of culture media and nutrients on biofilm growth kinetics of laboratory and clinical strains of Enterococcus faecalis.

OBJECTIVE: Enterococcus faecalis is a bacterial pathogen that is often associated with endodontic infections. Biofilm formation is a key virulence attribute in the pathogenicity of E. faecalis. In the present study, we comprehensively examined the effect of various culture media and nutrients on the development of E. faecalis biofilms. DESIGN: A reference strain and a clinical isolate of E. faecalis were used in all experiments for comparison. Commonly used liquid culture media with different nutrient compositions were used to support the development of E. faecalis biofilms in a time-dependent assay. E. faecalis biofilms were quantified by colony forming unit (CFU) and crystal violet (CV) assays. Biofilm architecture and cellular viability were evaluated by scanning electron microscopy and confocal laser scanning microscopy. RESULTS: Growth kinetics evaluated by CFU and CV assays and by microscopy showed that E. faecalis biofilms reached maturity at 72h. "Pg broth" (Tryptic Soy Broth with yeast extract, hemen and vitamin K) promoted E. faecalis biofilm formation more than Brain Heart Infusion broth or Tryptic Soy Broth. Addition of 2% glucose enhanced biofilm formation. Thus, it seems that nutrients such as hemen, vitamin K and glucose are important for E. faecalis for the formation of biofilms. CONCLUSION: The present study demonstrated that nutrient-rich media containing glucose enhances the formation of E. faecalis biofilms, which exhibit maturation at 72h.

Endodontic bacteria from primary and persistent endodontic lesions in Chinese patients as identified by cloning and 16S ribosomal DNA gene sequencing.

BACKGROUND: Few literatures pertain to the 16S ribosomal DNA (16S rDNA) analysis of bacteria contributing to primary and persistent endodontic lesions, with no information available for the Chinese population. As such, we investigated endodontic bacteria associated with primary and persistent endodontic lesions in adult Chinese patients living in Beijing, China using 16S rDNA gene sequencing techniques. METHODS: Endodontic microbial samples were obtained from fourteen adult Chinese patients and subjected to DNA extraction. Pllymerase chain reaction (PCR) products were cloned and 100 clones from each generated library were randomly selected. Purified plasmid DNA with 16S rDNA gene inserts was sequenced, and the sequences were searched against GenBank databases using the BLASTN algorithm. Only significant identification with the highest-scored BLAST result and 99% minimum similarity was considered for phylotyping. RESULTS: More than 150 taxa were obtained. Primary endodontic infection was mainly associated with Burkholderia cepacia, Actinomyces, Aranicola spp. and Streptococcus sanguinis, whilst Burkholderia cepacia was predominant in the persistent endodontic infections. CONCLUSION: There is a difference in the species profile associated with endodontic infections of Chinese patients living in Beijing in comparison to other geographical or ethnic reports.

Biocide resistance of Candida and Escherichia coli biofilms is associated with higher antioxidative capacities.

BACKGROUND: Most clinical guidelines for the use of biocides have been developed for planktonic micro-organisms, but in nature, most micro-organisms live as surface-adherent communities or biofilms. AIM: To evaluate the effectiveness of commonly used biocides against Escherichia coli and Candida spp. in three distinct growth phases: planktonic, adhesion and biofilm. METHODS: Ultrastructural, architectural and cellular viability changes following a 5 min exposure to biocide were monitored by scanning electron microscopy and confocal laser scanning microscopy using fluorescent dyes. Comparative transcript expression of the antioxidants SOD1 and CAT1 in the planktonic and biofilm phases was evaluated using quantitative real-time polymerase chain reaction. FINDINGS: E. coli and Candida spp. in the planktonic phase were susceptible to all the tested biocides at the recommended concentrations. However, early adhesion and late biofilm phases of both were less susceptible to the biocides, and exceeded the recommended concentrations on several occasions. A short period of biocide exposure failed to fully eradicate the adherent microbial cells, and they recovered from the biocide challenge, forming biofilm on the biocide-treated surfaces. The biofilm phase showed higher expression of SOD1 and CAT1. CONCLUSION: The recommended concentrations of biocides for clinical disinfection in the hospital setting may not fully eradicate the adhesion or biofilm phases of E. coli and Candida spp. Higher antioxidative capacities in microbial biofilms may be responsible for the resistance of biofilms against clinical biocides.

Antifungal susceptibility and virulence attributes of bloodstream isolates of Candida from Hong Kong and Finland.

Candida bloodstream infection has dramatically increased in the last decade due to the growing number of immunocompromised populations worldwide. In this study, we evaluated the antifungal susceptibility profiles and virulence attributes of Candida bloodstream isolates (CBIs) derived from Hong Kong and Finland, information which are vital for devising empirical clinical strategies. Susceptibility testing of a wide range of antifungals including fluconazole, itraconazole, voriconazole, ketoconazole, 5-fluorocytosine, amphotericin B and caspofungin was performed. Haemolytic activity and secretion of proteinase of CBIs were also examined. All CBIs derived from Hong Kong were susceptible to all the antifungals tested whilst some CBIs from Finland were resistant to azoles and caspofungin. C. albicans, C. glabrata and C. tropicalis showed higher haemolytic activity whereas C. parapsilosis and C. guilliermondii were non-haemolytic in general. Proteinase activity of the Finland C. albicans isolates was significantly higher than the Hong Kong isolates. Our data provide a glimpse of the possible evolutionary changes in pathogenic potential of Candida that may be occurring in different regions of the world. Therefore, continuous surveillance and availability of local data should be taken into consideration when treating candidemia patients.

Transcriptional regulation of drug-resistance genes in Candida albicans biofilms in response to antifungals.

Biofilm formation is a major virulence attribute of Candida albicans and is directly associated with therapeutic failure. One method by which Candida acquires antifungal resistance is the expression of drug-resistance genes. This study aimed to evaluate the transcriptional regulation of several genes associated with antifungal resistance of C. albicans under planktonic, recently adhered and biofilm growth modes and in C. albicans biofilms in response to antifungal agents. Initially, the antifungal susceptibility of C. albicans cultures in different growth modes was evaluated by standard antifungal susceptibility testing. Next, to assess CDR1, CDR2, MDR1, ERG11, FKS1 and PIL1 expression, RNA was harvested from cells in each growth mode, and from biofilms after drug treatment, and subjected to quantitative real-time RT-PCR (qRT-PCR). Biofilm C. albicans was more resistant to antifungals than recently adhered cells and stationary-phase planktonic cultures. Transcriptional expression of CDR1, CDR2, MDR1, ERG11 and FKS1 was lower in recently adhered C. albicans than in the stationary-phase planktonic cultures. In contrast, PIL1 levels were significantly increased in recently adhered and biofilm modes of growth. The expression of MDR1 in biofilms greatly increased on challenge with amphotericin B but not with the other drugs tested (P<0.01). ERG11 was significantly upregulated by ketoconazole (P<0.01). Caspofungin and amphotericin B significantly upregulated FKS1 expression, whereas they significantly downregulated PIL1 expression (P<0.01). These results indicate that the expression of drug-resistance genes is associated with higher drug resistance of Candida biofilms, and lay a foundation for future large-scale genome-wide expression analysis.

The antimicrobial efficacy of Fructus mume extract on orthodontic bracket: a monospecies-biofilm model study in vitro.

OBJECTIVE: the aim of this study was to evaluate the antimicrobial efficacy of Traditional Chinese Medicine Fructus mume on a monospecies-biofilm model established on orthodontic brackets in vitro. MATERIALS AND METHODS: the antimicrobial effect of Fructus mume aqueous extract on the planktonic Streptococcus mutans (S. mutans) was tested by microdilution method (MIC). The cell viability of S. mutans biofilm on Damon3 MX bracket (Ormco, USA) after exposed to Fructus mume extract was quantified by XTT reduction assay. Visualization of the samples was performed by fluorescence microscope and confocal laser scanning microscopy (CLSM). RESULTS: HPLC analysis revealed that the main compounds of Fructus mume are organic acids. The MIC of Fructus mume extract on the planktonic S. mutans was 50mg/mL. The optical density (OD) values, measured by XTT reduction assay from S. mutans biofilms after 1-min exposure to different test agents, demonstrated that the cell viability of S. mutans biofilms exposed to 250mg/mL Fructus mume extract

Ultrastructure and morphology of biofilms on thermoplastic orthodontic appliances in 'fast' and 'slow' plaque formers.

The aim of this study was to investigate the morphological features and distribution of biofilms on Invisalign orthodontic appliances, in a sample of 'slow' and 'fast' plaque formers using scanning electron microscopy (SEM). Fifty-six Chinese male/female volunteers (aged 19-39 years) were screened for their plaque-forming rate using the plaque percentage index (PPI) coupled with digital photography and computer-based image analysis, after a period of 48 hours of abstinence from oral hygiene procedures. Eleven volunteers (seven males/four females) representing the lowest and highest ends of the plaque formation spectrum were chosen as slow and fast plaque formers, respectively. The subjects wore a full-coverage splint appliance, in which four tiles of Invisalign material were embedded. These tiles were collected at intervals of 1, 3, 6, 12, 24, and 48 hours, as well as 3, 7, and 14 days, immediately fixed in 10 per cent paraformaldehyde in 0.2 M cacodylate buffer solution and prepared for SEM. The surface configuration of the Invisalign appliance was visualized, as well as the chronological pattern of biofilm formation. Significance between fast and slow plaque formers was determined using a Student's t-test. Colonization appeared to centre initially on the raised edges or textured surfaces of the appliance, and initial adhesion was quicker and more abundant in the fast plaque-forming group. In the later stages of biofilm development, both groups showed no discernible differences in biofilm accrual on the surfaces, but the fast group displayed a more complex biofilm structure. More recessed and sheltered areas of the appliance, such as the cusp tips and attachment dimples, harboured more biofilm than the flat surfaces. Hence, it seems that the novel Invisialign orthodontic appliance is a useful tool to investigate the features of biofilm formation in time-course studies.

Antimicrobial activity of Chinese medicine herbs against common bacteria in oral biofilm. A pilot study.

Twenty traditional Chinese medicines (TCM) were evaluated for their antimicrobial activity against four common oral bacteria. TCMs were tested for sensitivity against Streptococcus mitis, Streptococcus sanguis, Streptococcus mutans and Porphyromonas gingivalis. Aliquots of suspension of each bacterial species were inoculated onto a horse blood agar plate with TCMs soaked separately on 6mm paper disks. The plates were incubated for 48h anaerobically and the mean diameters of growth inhibition of three different areas obtained. 0.2% (w/v) chlorhexidine was used as a positive control. Broth microdilution assay was used to determine minimum inhibitory concentration and minimum bactericidal concentration. Fructus armeniaca mume was effective against all four bacteria. Thirteen TCMs demonstrated antimicrobial activity against Porphyromonas gingivalis, including Cortex magnoliae officinalis, Cortex phellodendri, Flos caryophylli, Flos lonicerae japonicae, Fructus armeniaca mume, Fructus forsythiae suspensae, Herba cum radice violae yedoensitis, Herba menthae haplocalycis, Pericarpium granati, Radix et rhizoma rhei, Radix gentianae, Ramulus cinnamomi cassia and Rhizoma cimicifugae. Cortex phellodendri showed antimicrobial activity against Streptococcus mutans, while Radix et rhizoma rhei was effective against Streptococcus mitis and Streptococcus sanguis. Fructus armeniaca mume had inhibitory effects against Streptococcus mitis, Streptococcus sanguis, Streptococcus mutans and Porphyromonas gingivalis in vitro.

Photodynamic inactivation of Candida albicans by BAM-SiPc.

Photodynamic therapy is a treatment that combines the use of three non-toxic components, viz. photosensitiser, light and oxygen to cause localised oxidative photodamage. In the present study, the antifungal effect of the photosensitiser, BAM-SiPc, an unsymmetrical bisamino phthalocyanine, was investigated. BAM-SiPc was effective in photo-inactivating Candida albicans in a dose-dependent manner. The cell viability as determined by the clonogenic assay was reduced to c. 10% at 0.02 micromol l(-1) BAM-SiPc with a total fluence of 12 J cm(-2) at a cell density of 10(7) cells ml(-1). A short incubation time of 5-15 min was sufficient to allow the photosensitiser to exert its optimal antifungal activity. Microscopical analysis showed that BAM-SiPc was effectively internalised by the fungal cells. Photodynamic treatment led to an increase in the intracellular reactive oxygen species level and disturbed the membrane integrity of the fungal cells.

Architectural analysis, viability assessment and growth kinetics of Candida albicans and Candida glabrata biofilms.

The human fungal pathogen Candida is able to form biofilms in almost all the medical devices in current use. Indeed, biofilm formation is a major virulence attribute of microorganisms and account for a majority of human infections. Therefore, understanding processes appertaining to biofilm development is an important prerequisite for devising new strategies to prevent or eradicate biofilm-related infections. In the present study we used an array of both conventional and novel analytical tools to obtain a comprehensive view of Candida biofilm development. Enumeration of colony forming units, colorimetric (XTT) assay, Scanning Electron Microscopy (SEM) and novel Confocal Laser Scanning Microscopy (CLSM) coupled with COMSTAT software analyses were utilised to evaluate growth kinetics; architecture and viability of biofilms of a reference (ATCC) and a clinical strain each of two Candida species, C. albicans and C. glabrata. Biofilm growth kinetics on a polystyrene substrate was evaluated from the initial adhesion step (1.5 h) up to 72 h. These analyses revealed substantial inter- and intra-species differences in temporal organisation of Candida biofilm architecture, spatiality and cellular viability, while reaching maturity within a period of 48 h, on a polystyrene substrate. There were substantial differences in the growth kinetics upon methodology, although general trend seemed to be the same. Detailed architectural analysis provided by COMSTAT software corroborated the SEM and CSLM views. These analyses may provide a strong foundation for down stream molecular work of fungal biofilms.

Community lifestyle of Candida in mixed biofilms: a mini review.

Candida is the most common human fungal pathogen that causes a variety of afflictions from superficial mucosal infections to deep mycoses. Biofilm formation is a major virulence factor of Candida, and more than 300 articles have been published on Candida biofilms over the past two decades. However, most of these data are on monospecies biofilms of Candida, and information on mixed-species Candida biofilms or bacteria-Candida combinations is still scarce. Yet, in nature, the yeast exist in a mixed milieu either in the oral cavity or in other habitats with a multitude of bacteria colonising mucosal surfaces within a shared community. This mini review describes the current knowledge on candidal-candidal or bacterial-candidal interactions in mixed-species biofilms. The underlying mechanisms of these interactions appear to depend on several factors relating to biofilm development, such as species and strains of organisms, nutritional factors, aerobiosis and related environmental factors. Although the fundamental nature of these interactions appears to be commensalism and antagonism, the emerging evidence based on novel molecular, proteomic and imaging tools indicates these biological mechanisms to be far more complex than hitherto recognised. Demystifying the mechanisms underlying the growth and development of mixed-species communities involving Candida will undoubtedly yield useful data for the effective management of microbial infections in general.