Study of the Structure of Hyperbranched Polyglycerol Coatings and their Anti-Biofouling and Anti-Thrombotic Applications.

Whilst blood-contacting materials are widely deployed in medicine in vascular stents, catheters and cannulas, devices fail in-situ because of thrombosis and restenosis. Furthermore, microbial attachment and biofilm formation is not an uncommon problem for medical devices. Even incremental improvements in hemocompatible materials could provide significant benefits for patients in terms of safety and patency as well as substantial cost savings.Herein, we describe a novel but simple strategy for coating a range of medical materials, that can be applied to objects of complex geometry, involving plasma-grafting of an ultra-thin hyperbranched polyglycerol coating (HPG). Plasma activation creates highly reactive surface oxygen moieties that readily react with glycidol. Irrespective of the substrate, coatings are uniform and pinhole free, comprising O-C-O repeats, with HPG chains packing in a fashion that holds reversibly binding proteins at the coating surface.In vitro assays with planar test samples show that HPG prevents platelet adhesion and activation, as well as reducing (>3log) bacterial attachment and preventing biofilm formation. Ex vivo and preclinical studies show that HPG-coated nitinol stents do not elicit thrombosis or restenosis, nor complement or neutrophil activation. Subcutaneous implantation of HPG coated disks under the skin of mice showed no evidence of toxicity nor inflammation. This article is protected by copyright. All rights reserved.

The Effect of Chitosan Incorporation on Physico-Mechanical and Biological Characteristics of a Calcium Silicate Filling Material.

OBJECTIVES: A tricalcium silicate-based cement, Biodentine™, has displayed antibiofilm activity when mixed with chitosan powder. This study aimed to assess the effect of chitosan incorporation on the physico-mechanical and biological properties of Biodentine™. METHODS: In this study, medium molecular weight chitosan powder was incorporated into Biodentine™ in varying proportions (2.5 wt%, 5 wt%, 10 wt%, and 20 wt%). The setting time was determined using a Vicat apparatus, solubility was assessed by calculating weight variation after water immersion, radiopacity was evaluated and expressed in millimeters of aluminum, the compressive strength was evaluated using an Instron testing machine, and the microhardness was measured with a Vickers microhardness tester. In addition, surface topography of specimens was analyzed using scanning electron microscopy, and the effect of chitosan on the viability of human embryonic kidney (HEK 293) cells was measured by a colorimetric MTT assay. RESULTS: Incorporation of 2.5 wt% and 5 wt% chitosan powder delivered an advantage by speeding up the setting time of Biodentine material. However, the incorporation of chitosan compromised all other material properties and the crystalline structure in a dose-dependent manner. The chitosan-modified material also showed significant decreases in the proliferation of the HEK 293 cells, signifying decreased biocompatibility. SIGNIFICANCE: Chitosan incorporation into calcium silicate materials adversely affects the physical and biological properties of the material. Despite the increased antimicrobial activity of the modified material, the diminution in these properties is likely to reduce its clinical value.

Informed development of a multi-species biofilm in chronic obstructive pulmonary disease.

Recent evidence indicates that microbial biofilm aggregates inhabit the lungs of COPD patients and actively contribute towards chronic colonization and repeat infections. However, there are no contextually relevant complex biofilm models for COPD research. In this study, a meta-analysis of the lung microbiome in COPD was used to inform development of an optimized biofilm model composed of genera highly associated with COPD. Bioinformatic analysis showed that although diversity matrices of COPD microbiomes were similar to healthy controls, and internal compositions made it possible to accurately differentiate between these cohorts (AUC = 0.939). Genera that best defined these patients included Haemophilus, Moraxella and Streptococcus. Many studies fail to account for fungi; therefore, Candida albicans was included in the creation of an interkingdom biofilm model. These organisms formed a biofilm capable of tolerating high concentrations of antimicrobial therapies with no significant reductions in viability. However, combined therapies of antibiotics and an antifungal resulted in significant reductions in viable cells throughout the biofilm (p < 0.05). This biofilm model is representative of the COPD lung microbiome and results from in vitro antimicrobial challenge experiments indicate that targeting both bacteria and fungi in these interkingdom communities will be required for more positive clinical outcomes.

To update or not to update the ESCMID guidelines for the diagnosis and treatment of biofilm infections - That is the question! The opinion of the ESGB board.

Background: The work on the ESGB guidelines for diagnosis and treatment of biofilm infections began in 2012 and the result was published in 2014. The guidelines have been and still are frequently cited in the literature proving its usefulness for people working with biofilm infections. At the ESGB Biofilm conference in Mallorca 2022 (Eurobiofilms2022) the board of the ESGB decided to evaluate the 2014-guidelines and relevant publications since 2014 based on a lecture given at the Eurobiofilms2022. Guideline methods: The Delphi method for working on production of guidelines and the current ESCMID rules for guidelines are presented. The criteria for evaluation of relevant literature are very strict and especially for treatment, most clinicians and regulatory authorities require convincing results from Level I (randomized controlled trials) publications to justify changes of treatments. The relevant new biofilm literature and the relevant biofilm presentations from the Eurobiofilms meetings and ECCMID conferences was used for evaluating the contemporary relevance of the ESGB 2014 guidelines. Diagnosis of biofilm infections: Several infectious diseases have been recognized as biofilm infections since 2014, but the diagnostic methods and therapeutic strategies are still the same as recommended in the 2014 ESGB guidelines which are summarized in this opinion paper. Treatment of biofilm infections: Some promising new in vitro and in vivo (animal experiments) observations and reports for therapy of biofilm infections are mentioned, but they still await clinical trials. Conclusion: The interim opinion at the present time (2022) is therefore, that the guidelines do not need revision now, but there is a need for survey articles discussing new methods of diagnosis and treatment of biofilm infections in order - hopefully - to give inspiration to conduct clinical trials which may lead to progress in diagnosis and treatment of patients with biofilm infections.

Dual nanocarrier of chlorhexidine and fluconazole: Physicochemical characterization and effects on microcosm biofilms and oral keratinocytes.

OBJECTIVES: This study assembled and characterized a dual nanocarrier of chlorhexidine (CHX) and fluconazole (FLZ), and evaluated its antibiofilm and cytotoxic effects. METHODS: CHX and FLZ were added to iron oxide nanoparticles (IONPs) previously coated by chitosan (CS) and characterized by physical-chemical analyses. Biofilms from human saliva supplemented with Candida species were grown (72 h) on glass discs and treated (24 h) with IONPs-CS carrying CHX (at 39, 78, or 156 µg/mL) and FLZ (at 156, 312, or 624 µg/mL) in three growing associations. IONPs and CS alone, and 156 µg/mL CHX + 624 µg/mL FLZ (CHX156-FLZ624) were tested as controls. Next, microbiological analyses were performed. The viability of human oral keratinocytes (NOKsi lineage) was also determined (MTT reduction assay). Data were submitted to ANOVA or Kruskal-Wallis, followed by Fisher's LSD or Tukey's tests (α=0.05). RESULTS: Nanocarriers with spherical-like shape and diameter around 6 nm were assembled, without compromising the crystalline property and stability of IONPs. Nanocarrier at the highest concentrations was the most effective in reducing colony-forming units of Streptococcus mutans, Lactobacillus spp., Candida albicans, and Candida glabrata. The other carriers and CHX156-FLZ624 showed similar antibiofilm effects, and significantly reduced lactic acid production (p<0.001). Also, a dose-dependent cytotoxic effect against oral keratinocytes was observed for the dual nanocarrier. IONPs-CS-CHX-FLZ and CHX-FLZ significantly reduced keratinocyte viability at CHX and FLZ concentrations ≥7.8 and 31.25 µg/mL, respectively (p<0.05). CONCLUSION: The nanotherapy developed outperformed the effect of the combination CHX-FLZ on microcosm biofilms, without increasing the cytotoxic effect of the antimicrobials administered. CLINICAL SIGNIFICANCE: The dual nanocarrier is a promising topically-applied therapy for the management of oral candidiasis considering that its higher antibiofilm effects allow the use of lower concentrations of antimicrobials than those found in commercial products.

Evaluation of Metabolomics as Diagnostic Targets in Oral Squamous Cell Carcinoma: A Systematic Review.

In recent years, high-throughput technologies have facilitated the widespread use of metabolomics to identify biomarkers and targets for oral squamous cell carcinoma (OSCC). As a result, the primary goal of this systematic review is to identify and evaluate metabolite biomarkers and their pathways for OSCC that featured consistently across studies despite methodological variations. Six electronic databases (Medline, Cochrane, Web of Science, CINAHL, ProQuest, and Embase) were reviewed for the longitudinal studies involving OSCC patients and metabolic marker analysis (in accordance with PRISMA 2020). The studies included ranged from the inception of metabolomics in OSCC (i.e., 1 January 2007) to 30 April 2023. The included studies were then assessed for their quality using the modified version of NIH quality assessment tool and QUADOMICS. Thirteen studies were included after screening 2285 studies. The majority of the studies were from South Asian regions, and metabolites were most frequently derived from saliva. Amino acids accounted for more than quarter of the detected metabolites, with glutamate and methionine being the most prominent. The top dysregulated metabolites indicated dysregulation of six significantly enriched pathways including aminoacyl-tRNA biosynthesis, glutathione metabolism and arginine biosynthesis with the false discovery rate (FDR) <0.05. Finally, this review highlights the potential of metabolomics for early diagnosis and therapeutic targeting of OSCC. However, larger studies and standardized protocols are needed to validate these findings and make them a clinical reality.

Investigating the chemical pathway to the formation of a single biofilm using infrared spectroscopy.

Diagnosing biofilm infections has remained a constant challenge for the last 50 years. Existing diagnostic methods struggle to identify the biofilm phenotype. Moreover, most methods of biofilm analysis destroy the biofilm making the resultant data interpretation difficult. In this study we introduce Fourier Transform Infra-Red (FTIR) spectroscopy as a label-free, non-destructive approach to monitoring biofilm progression. We have utilised FTIR in a novel application to evaluate the chemical composition of bacterial biofilms without disrupting the biofilm architecture. S. epidermidis (RP62A) was grown onto calcium fluoride slides for periods of 30 min-96 h, before semi-drying samples for analysis. We report the discovery of a chemical marker to distinguish between planktonic and biofilm samples. The appearance of new proteins in biofilm samples of varying maturity is exemplified in the spectroscopic data, highlighting the potential of FTIR for identifying the presence and developmental stage of a single biofilm.

Antifungal therapy of Candida biofilms: Past, present and future.

Virtually all Candida species linked to clinical candidiasis are capable of forming highly resistant biofilms on different types of surfaces, which poses an additional significant threat and further complicates therapy of these infections. There is a scarcity of antifungal agents, and their effectiveness, particularly against biofilms, is limited. Here we provide a historical perspective on antifungal agents and therapy of Candida biofilms. As we reflect upon the past, consider the present, and look towards the future of antifungal therapy of Candida biofilms, we believe that there are reasons to remain optimistic, and that the major challenges of Candida biofilm therapy can be conquered within a reasonable timeframe.

Staphylococcus aureus strains exhibit heterogenous tolerance to direct cold atmospheric plasma therapy.

The global clinical and socioeconomic impact of chronic wounds is substantial. The main difficulty that clinicians face during the treatment of chronic wounds is the risk of infection at the wound site. Infected wounds arise from an accumulation of microbial aggregates in the wound bed, leading to the formation of polymicrobial biofilms that can be largely resistant to antibiotic therapy. Therefore, it is essential for studies to identify novel therapeutics to alleviate biofilm infections. One innovative technique is the use of cold atmospheric plasma (CAP) which has been shown to possess promising antimicrobial and immunomodulatory properties. Here, different clinically relevant biofilm models will be treated with cold atmospheric plasma to assess its efficacy and killing effects. Biofilm viability was assessed using live dead qPCR, and morphological changes associated with CAP evaluated using scanning electron microscopy (SEM). Results indicated that CAP was effective against Candida albicans and Pseudomonas aeruginosa, both as mono-species biofilms and when grown in a triadic model system. CAP also significantly reduced viability in the nosocomial pathogen, Candida auris. Staphylococcus aureus Newman exhibited a level of tolerance to CAP therapy, both when grown alone or in the triadic model when grown alongside C. albicans and P. aeruginosa. However, this degree of tolerance exhibited by S. aureus was strain dependent. At a microscopic level, biofilm treatment led to subtle changes in morphology in the susceptible biofilms, with evidence of cellular deflation and shrinkage. Taken together, these results indicate a promising application of direct CAP therapy in combatting wound and skin-related biofilm infections, although biofilm composition may affect the treatment efficacy.

Cold Atmospheric Plasma-Activated Composite Hydrogel for an Enhanced and On-Demand Delivery of Antimicrobials.

We present the concept of a versatile drug-loaded composite hydrogel that can be activated using an argon-based cold atmospheric plasma (CAP) jet to deliver both a drug and CAP-generated molecules, concomitantly, in a tissue target. To demonstrate this concept, we utilized the antibiotic gentamicin that is encapsulated in sodium polyacrylate (PAA) particles, which are dispersed within a poly(vinyl alcohol) (PVA) hydrogel matrix. The final product is a gentamicin-PAA-PVA composite hydrogel suitable for an on-demand triggered release using CAP. We show that by activating using CAP, we can effectively release gentamicin from the hydrogel and also eradicate the bacteria effectively, both in the planktonic state and within a biofilm. Besides gentamicin, we also successfully demonstrate the applicability of the CAP-activated composite hydrogel loaded with other antimicrobial agents such as cetrimide and silver. This concept of a composite hydrogel is potentially adaptable to a range of therapeutics (such as antimicrobials, anticancer agents, and nanoparticles) and activatable using any dielectric barrier discharge CAP device.

Our current clinical understanding of Candida biofilms: where are we two decades on?

Clinically we have been aware of the concept of Candida biofilms for many decades, though perhaps without the formal designation. Just over 20 years ago the subject emerged on the back of progress made from the bacterial biofilms, and academic progress pace has continued to mirror the bacterial biofilm community, albeit at a decreased volume. It is apparent that Candida species have a considerable capacity to colonize surfaces and interfaces and form tenacious biofilm structures, either alone or in mixed species communities. From the oral cavity, to the respiratory and genitourinary tracts, wounds, or in and around a plethora of biomedical devices, the scope of these infections is vast. These are highly tolerant to antifungal therapies that has a measurable impact on clinical management. This review aims to provide a comprehensive overight of our current clinical understanding of where these biofilms cause infections, and we discuss existing and emerging antifungal therapies and strategies.

Limitations of using 16S rRNA microbiome sequencing to predict oral squamous cell carcinoma.

A new era of next-generation sequencing has changed our perception of the oral microbiome in health and disease, and with this there is a growing understanding that the oral microbiome is a contributing factor to oral squamous cell carcinoma (OSCC), a malignancy of the oral cavity. This study aimed to analyse the trends and relevant literature based on the 16S rRNA oral microbiome in head and neck cancer using next-generation sequencing technologies, and to conduct a meta-analysis of the studies with OSCC cases and healthy controls. A literature search using the databases Web of Science and PubMed was conducted in a scoping-like review to collect information based on the study design, and plots were generated using RStudio. We selected case-control studies using 16S rRNA oral microbiome sequencing analysis in OSCC cases versus healthy controls for re-analysis. Statistical analyses were conducted using R. Out of 916 original articles, we filtered and selected 58 studies for review, and 11 studies for meta-analysis. Differences between sampling type, DNA extraction methods, next-generation sequencing technology and region of the 16S rRNA were identified. No significant differences in the α- and ß-diversity between health and oral squamous cell carcinoma were observed (p < 0.05). Random Forest classification marginally improved predictability of four studies (training set) when split 80/20. We found an increase in Selenomonas, Leptotrichia and Prevotella species to be indicative of disease. A number of technological advances have been accomplished to study oral microbial dysbiosis in oral squamous cell carcinoma. There is a clear need for standardization of study design and methodology to ensure 16S rRNA outputs are comparable across the discipline in the hope of identifying 'biomarker' organisms for designing screening or diagnostic tools.

Longitudinal changes in subgingival biofilm composition following periodontal treatment.

BACKGROUND: Current periodontal treatment involves instrumentation using hand and/or ultrasonic instruments, which are used either alone or in combination based on patient and clinician preference, with comparable clinical outcomes. This study sought to investigate early and later changes in the subgingival biofilm following periodontal treatment, to identify whether these changes were associated with treatment outcomes, and to investigate whether the biofilm responded differently to hand compared with ultrasonic instruments. METHODS: This was a secondary-outcome analysis of a randomized-controlled trial. Thirty-eight periodontitis patients received full-mouth subgingival instrumentation using hand (n = 20) or ultrasonic instrumentation (n = 18). Subgingival plaque was sampled at baseline and 1, 7, and 90 days following treatment. Bacterial DNA was analyzed using 16S rRNA sequencing. Periodontal clinical parameters were evaluated before and after treatment. RESULTS: Biofilm composition was comparable in both (hand and ultrasonics) treatment groups at all time points (all genera and species; p[adjusted] > 0.05). Large-scale changes were observed within groups across time points. At days 1 and 7, taxonomic diversity and dysbiosis were reduced, with an increase in health-associated genera including Streptococcus and Rothia equating to 30% to 40% of the relative abundance. When reassessed at day 90 a subset of samples reformed a microbiome more comparable with baseline, which was independent of instrumentation choice and residual disease. CONCLUSIONS: Hand and ultrasonic instruments induced comparable impacts on the subgingival plaque microbiome. There were marked early changes in the subgingival biofilm composition, although there was limited evidence that community shifts associated with treatment outcomes.

An integrated transcriptomic and metabolomic approach to investigate the heterogeneous Candida albicans biofilm phenotype.

Candida albicans is the most prevalent and notorious of the Candida species involved in bloodstream infections, which is characterised by its capacity to form robust biofilms. Biofilm formation is an important clinical entity shown to be highly variable among clinical isolates. There are various environmental and physiological factors, including nutrient availability which influence the phenotype of Candida species. However, mechanisms underpinning adaptive biofilm heterogeneity have not yet been fully explored. Within this study we have profiled previously characterised and phenotypically distinct C. albicans bloodstream isolates. We assessed the dynamic susceptibility of these differing populations to antifungal treatments using population analysis profiling in addition to assessing biofilm formation and morphological changes. High throughput methodologies of RNA-Seq and LC-MS were employed to map and integrate the transcriptional and metabolic reprogramming undertaken by heterogenous C. albicans isolates in response to biofilm and hyphal inducing serum. We found a significant relationship between biofilm heterogeneity and azole resistance (P < 0.05). In addition, we observed that in response to serum our low biofilm forming (LBF) C. albicans exhibited a significant increase in biofilm formation and hyphal elongation. The transcriptional reprogramming of LBF strains compared to high biofilm forming (HBF) was distinct, indicating a high level of plasticity and variation in stress responses by heterogenous strains. The metabolic responses, although variable between LBF and HBF, shared many of the same responses to serum. Notably, a high upregulation of the arachidonic acid cascade, part of the COX pathway, was observed and this pathway was found to induce biofilm formation in LBF 3-fold. C. albicans is a highly heterogenous bloodstream pathogen with clinical isolates varying in antifungal tolerance and biofilm formation. In addition to this, C. albicans is capable of highly complex and variable regulation of transcription and metabolic pathways and heterogeneity across isolates further increases the complexity of these pathways. Here we have shown with a dual and integrated approach, the importance of studying a diverse panel of C. albicans isolates, which has the potential to reveal distinct pathways that can harnessed for drug discovery.

In vitro bacterial vaginosis biofilm community manipulation using endolysin therapy.

Bacterial vaginosis (BV) affects approximately 26% of women of childbearing age globally, presenting with 3-5 times increased risk of miscarriage and two-fold risk of pre-term birth. Antibiotics (metronidazole and clindamycin) are typically employed to treat BV; however the success rate is low due to the formation of recalcitrant polymicrobial biofilms. As a novel therapeutic, promising results have been obtained in vitro using Gardnerella endolysins, although to date their efficacy has only been demonstrated against simple biofilm models. In this study, a four-species biofilm was developed consisting of Gardnerella vaginalis, Fannyhessea vaginae, Prevotella bivia and Mobiluncus curtisii. Biofilms were grown in NYC III broth and treated using antibiotics and an anti-Gardnerella endolysin (CCB7.1) for 24 h. Biofilm composition, viability and structure were assessed using colony counts, live/dead qPCR and scanning electron microscopy. All species colonised biofilms to varying degrees, with G. vaginalis being the most abundant. Biofilm composition remained largely unchanged when challenged with escalated concentrations of conventional antibiotics. A Gardnerella-targeted endolysin candidate (CCB7.1) showed efficacy against several Gardnerella species planktonically, and significantly reduced viable G. vaginalis within polymicrobial biofilms at 1 to 4X pMIC (p < 0.05 vs. vehicle control). Collectively, this study highlights the resilience of biofilm-embedded pathogens against the currently used antibiotics and provides a polymicrobial model that allows for more effective pre-clinical screening of BV therapies. The Gardnerella-specific endolysin CCB7.1 demonstrated significant activity against G. vaginalis within polymicrobial biofilms, altering the overall community dynamic and composition.

Generation of Multispecies Oral Bacteria Biofilm Models.

It is well-recognized that oral biofilms that occur in health and disease have a polymicrobial composition, though these are poorly reflected in the literature, with many studies focussing on simple mono-species biofilm model systems. The utility of polymicrobial biofilm model systems is that they more accurately reflect the oral cavity and allow researchers to ask relevant questions in basic science studies, pharmaceutical screening, and investigating inflammatory interactions. Here we describe the detailed methodology of how to sequentially construct and maintain polymicrobial biofilm models pertinent to caries, periodontal disease, and denture stomatitis.

Acetylcholine Receptors in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are well known for their regenerative potential. Even though the ability of MSCs to proliferate and differentiate has been studied extensively, there remains much to learn about the signaling mechanisms and pathways that control proliferation and influence the differentiation phenotype. In recent years, there has been growing evidence for the utility of non-neuronal cholinergic signaling systems and that acetylcholine (ACh) plays an important ubiquitous role in cell-to-cell communication. Indeed, cholinergic signaling is hypothesized to occur in stem cells and ACh synthesis, as well as in ACh receptor (AChR) expression, has been identified in several stem cell populations, including MSCs. Furthermore, AChRs have been found to influence MSC regenerative potential. In humans, there are two major classes of AChRs, muscarinic AChRs and nicotinic AChRs, with each class possessing several subtypes or subunits. In this review, the expression and function of AChRs in different types of MSC are summarized with the aim of highlighting how AChRs play a pivotal role in regulating MSC regenerative function.

Meta-analysis of caries microbiome studies can improve upon disease prediction outcomes.

As one of the most prevalent infective diseases worldwide, it is crucial that we not only know the constituents of the oral microbiome in dental caries but also understand its functionality. Herein, we present a reproducible meta-analysis to effectively report the key components and the associated functional signature of the oral microbiome in dental caries. Publicly available sequencing data were downloaded from online repositories and subjected to a standardized analysis pipeline before analysis. Meta-analyses identified significant differences in alpha and beta diversities of carious microbiomes when compared to healthy ones. Additionally, machine learning and receiver operator characteristic analysis showed an ability to discriminate between healthy and disease microbiomes. We identified from importance values, as derived from random forest analyses, a group of genera, notably containing Selenomonas, Aggregatibacter, Actinomyces and Treponema, which can be predictive of dental caries. Finally, we propose the most appropriate study design for investigating the microbiome of dental caries by synthesizing the studies, which had the most accurate differentiation based on random forest modelling. In conclusion, we have developed a non-biased, reproducible pipeline, which can be applied to microbiome meta-analyses of multiple diseases, but importantly we have derived from our meta-analysis a key group of organisms that can be used to identify individuals at risk of developing dental caries based on oral microbiome inhabitants.

Investigating Dual-Species Candida auris and Staphylococcal Biofilm Antiseptic Challenge.

Candida auris can persistently colonize human skin, alongside a diverse bacterial microbiome. In this study we aimed to investigate the efficacy of antiseptic activities on dual-species interkingdom biofilms containing staphylococci to determine if antiseptic tolerance was negatively impacted by dual-species biofilms. Chlorhexidine, povidone iodine, and hydrogen peroxide (H2O2), were able to significantly reduce biofilm viable cell counts following exposure at 2%, 10%, and 3%, respectively. Notably, H2O2-treated biofilms were able to significantly recover and considerably repopulate following treatment. Fortunately, inter-kingdom interactions in dual-species biofilms of C. auris and staphylococci did not increase the tolerance of C. auris against antiseptics in vitro. These data indicate mixed infections are manageable with chlorhexidine and povidone iodine, but caution should be exercised in the consideration of H2O2.