Biomaterials with antifungal strategies to fight oral infections.

Oral fungal infections pose a threat to human health and increase the economic burden of oral diseases by prolonging and complicating treatment. A cost-effective strategy is to try to prevent these infections from happening in the first place. With this purpose, biomaterials with antifungal properties are a crucial element to overcome fungal infections in the oral cavity. In this review, we go through different kinds of biomaterials and coatings that can be used to functionalize them. We also review their potential as a therapeutic approach in addition to prophylaxis, by going through traditional and alternative antifungal compounds, e.g., essential oils, that could be incorporated in them, to enhance their efficacy against fungal pathogens. We aim to highlight the potential of these technologies and propose questions that need to be addressed in prospective research. Finally, we intend to concatenate the key aspects and technologies on the use of biomaterials in oral health, to create an easy to find summary of the current state-of-the-art for researchers in the field.

Characterization of Oral Candida spp. Biofilms in Children and Adults Carriers from Eastern Europe and South America.

BACKGROUND: Candida albicans and non-Candida albicans Candida species (NCACs) are known to colonize and invade various tissues, including the oral mucosa. In this work, we aimed to characterize mature biofilms of several Candida spp. clinical isolates (n = 33) obtained from the oral mucosa of children, adults, and elders of Eastern Europe and South America. METHODS: Each strain was evaluated for its capacity to form biofilms in terms of total biomass using the crystal violet assay and for matrix components production (proteins and carbohydrates) using the BCA and phenol-sulfuric tests, respectively. The effect of different antifungals on biofilm formation was studied. RESULTS: in the children's group, a predominance of C. krusei (81%) was observed, while, among adults, the main species was C. albicans (59%). Most strains showed a reduced response to antimicrobial drugs when in biofilm form (p < 0.01). Moreover, it was observed that strains isolated from children produced more matrix, with higher levels of protein and polysaccharides. CONCLUSIONS: children were more likely to be infected by NCACs than adults. More importantly, these NCACs were able to form biofilms richer in matrix components. This finding is of clinical importance, particularly in pediatric care, since stronger biofilms are highly associated with antimicrobial resistance, recurrent infections, and higher therapeutic failure.

Combination of Farnesol with Common Antifungal Drugs: Inhibitory Effect against Candida Species Isolated from Women with RVVC.

Background and Objectives: Vulvovaginal candidiasis (VVC) is a mucous membrane infection, with an increased rate of antifungal resistance of Candida species. In this study, the in vitro efficacy of farnesol alone or in combination with traditional antifungals was assessed against resistant Candida strains recovered from women with VVC. Materials and Methods: Eighty Candida isolates were identified by multiplex polymerase chain reaction (PCR), and the antifungal susceptibility to amphotericin B (AMB), fluconazole (FLU), itraconazole (ITZ), voriconazole (VOR), clotrimazole (CTZ), and farnesol was tested by the standard microdilution method. The combinations of farnesol with each antifungal were calculated based on the fractional inhibitory concentration index (FICI). Result: Candida glabrata was the predominant species (48.75%) isolated from vaginal discharges, followed by C. albicans (43.75%), C. parapsilosis (3.75%), a mixed infection of C. albicans and C. glabrata (2.5%) and C. albicans and C. parapsilosis (1%). C. albicans and C. glabrata isolates had lower susceptibility to FLU (31.4% and 23.0%, respectively) and CTZ (37.1% and 33.3%, respectively). Importantly, there was "synergism" between farnesol-FLU and farnesol-ITZ against C. albicans and C. parapsilosis (FICI = 0.5 and 0.35, respectively), reverting the original azole-resistant profile. Conclusion: These findings indicate that farnesol can revert the resistance profile of azole by enhancing the activity of FLU and ITZ in resistant Candida isolates, which is a clinically promising result.

Photodynamic Inactivation Effectively Eradicates Candida auris Biofilm despite Its Interference with the Upregulation of CDR1 and MDR1 Efflux Genes.

Candida auris, in recent years, has emerged as a dangerous nosocomial pathogen. It represents a challenge for effective treatment because of its multiresistance. Photodynamic inactivation (PDI) is a promising way to solve problems with a wide range of resistant microorganisms. This study aimed to use PDI for the eradication of C. auris biofilms. Moreover, the regulation of the CDR1, CDR2, and MDR1 resistance genes was studied. Experiments were performed on 24 h biofilms formed by three clinical isolates of C. auris in vitro. PDI was performed in the presence of the photosensitizer methylene blue (0.25 mM) and samples were irradiated with a red laser (λ = 660 nm, 190 mW/cm2) for 79, 120, and 300 s. To confirm the PDI effect, confocal laser scanning microscopy was performed after treatment. Effective PDI was achieved in all strains. The highest inhibition was observed after 300 s irradiation, with over 90% inhibition compared with the non-irradiated control sample. PDI was observed to upregulate the expression of the CDR1 gene, but mainly the MDR1 gene. Despite this observation, PDI significantly decreased the survival of C. auris biofilm cells and proved to have great potential for the eradication of problematic resistant yeasts.

Overview on the Infections Related to Rare Candida Species.

Atypical Candida spp. infections are rising, mostly due to the increasing numbers of immunocompromised patients. The most common Candida spp. is still Candida albicans; however, in the last decades, there has been an increase in non-Candida albicans Candida species infections (e.g., Candida glabrata, Candida parapsilosis, and Candida tropicalis). Furthermore, in the last 10 years, the reports on uncommon yeasts, such as Candida lusitaniae, Candida intermedia, or Candida norvegensis, have also worryingly increased. This review summarizes the information, mostly related to the last decade, regarding the infections, diagnosis, treatment, and resistance of these uncommon Candida species. In general, there has been an increase in the number of articles associated with the incidence of these species. Additionally, in several cases, there was a suggestive antifungal resistance, particularly with azoles, which is troublesome for therapeutic success.

Farnesol Boosts the Antifungal Effect of Fluconazole and Modulates Resistance in Candida auris through Regulation of the CDR1 and ERG11 Genes.

Candida auris is considered a serious fungal pathogen frequently exhibiting a high resistance to a wide range of antifungals. In this study, a combination of the quorum-sensing molecule farnesol (FAR) and fluconazole (FLU) was tested on FLU-resistant C. auris isolates (C. auris S and C. auris R) compared to the susceptible C. auris H261. The aim was to assess the possible synergy between FAR and FLU, by reducing the FLU minimal inhibitory concentration, and to determine the mechanism underlying the conjunct effect. The results confirmed a synergic effect between FAR and FLU with a calculated FIC index of 0.75 and 0.4 for C. auris S and C. auris R, respectively. FAR modulates genes involved in azole resistance. When FAR was added to the cells in combination with FLU, a significant decrease in the expression of the CDR1 gene was observed in the resistant C. auris isolates. FAR seems to block the Cdr1 efflux pump triggering a restoration of the intracellular content of FLU. These results were supported by observed increasing accumulation of rhodamine 6G by C. auris cells. Moreover, C. auris treated with FAR showed an ERG11 gene down-regulation. Overall, these results suggest that FAR is an effective modulator of the Cdr1 efflux pump in C. auris and, in combination with FLU, enhances the activity of this azole, which might be a promising strategy to control infections caused by azole-resistant C. auris.

Prevalence and Antifungal Susceptibility Profile of Oral Candida spp. Isolates from a Hospital in Slovakia.

Oral fungal infections are a worldwide healthcare problem. Although Candida albicans is still the most common yeast involved in the infections of oral cavity, non-Candida albicans Candida species (NCACs) have been highly related to these infections, particularly in older, immunosuppressed or patients with long exposure to antimicrobial drugs. The goal of this work was to perform a quick epidemiological and mycological study on the oral samples collected from a laboratory of a hospital in Slovakia, for 60 days. The samples' identification was performed by Germ-tube formation test, CHROMID® Candida, Auxacolor 2, ID 32C automated method, and the antifungal susceptibility testing determined by E-test®. Results confirm that comparing with bacteria, yeasts still occur in the lower number, but there is a high rate of antifungal resistance (81.6%)­to, at least one drug­among the collected samples, particularly to azoles and 5'-FC, which is clinically noteworthy.

Application of probiotics in candidiasis management.

Candidiasis (e.g., oral, gastrointestinal, vaginal, urinary tract, systemic) is a worldwide growing problem, since antifungal resistance and immunosuppression states are rising. To address this problem, very few drugs are available for the treatment of Candida spp. infections. Therefore, novel therapeutic strategies are urgently required. Probiotics have been proposed for the prevention and treatment of bacterial infections due to their safety record and efficacy, however, little is still known about their potential role regarding fungal infections. The purpose of this review is to present an updated summary of the evidence of the antifungal effects of probiotics along with a discussion of their potential use as an alternative/complementary therapy against Candida spp. infections. Thus, we performed a literature search using appropriate keywords ("Probiotic + Candida", "Candidiasis treatment", and "Probiotic + candidiasis") to retrieve relevant studies (both preclinical and clinical) with special emphasis on the works published in the last 5 years. An increasing amount of evidence has shown the potential usefulness of probiotics in the management of oral and vulvovaginal candidiasis in recent years. Among other results, we found that, as for bacterial infections, Lactobacillus, Bifidobacterium, and Saccharomyces are the most studied and effective genus for this purpose. However, in other areas, particularly in skincandidiaisis, studies are low or lacking. Thus, further investigation is necessary including in vitro and in vivo studies to establish the usefulness of probiotics in the management of candidiasis.

Overview on the Prevalence of Fungal Infections, Immune Response, and Microbiome Role in COVID-19 Patients.

Patients with severe COVID-19, such as individuals in intensive care units (ICU), are exceptionally susceptible to bacterial and fungal infections. The most prevalent fungal infections are aspergillosis and candidemia. Nonetheless, other fungal species (for instance, Histoplasma spp., Rhizopus spp., Mucor spp., Cryptococcus spp.) have recently been increasingly linked to opportunistic fungal diseases in COVID-19 patients. These fungal co-infections are described with rising incidence, severe illness, and death that is associated with host immune response. Awareness of the high risks of the occurrence of fungal co-infections is crucial to downgrade any arrear in diagnosis and treatment to support the prevention of severe illness and death directly related to these infections. This review analyses the fungal infections, treatments, outcome, and immune response, considering the possible role of the microbiome in these patients. The search was performed in Medline (PubMed), using the words "fungal infections COVID-19", between 2020-2021.

Candida auris: A Quick Review on Identification, Current Treatments, and Challenges.

Candida auris is a novel and major fungal pathogen that has triggered several outbreaks in the last decade. The few drugs available to treat fungal diseases, the fact that this yeast has a high rate of multidrug resistance and the occurrence of misleading identifications, and the ability of forming biofilms (naturally more resistant to drugs) has made treatments of C. auris infections highly difficult. This review intends to quickly illustrate the main issues in C. auris identification, available treatments and the associated mechanisms of resistance, and the novel and alternative treatment and drugs (natural and synthetic) that have been recently reported.

Microbial interactions and immunity response in oral Candida species.

Oral candidiasis are among the most common noncommunicable diseases, related with serious local and systemic illnesses. Although these infections can occur in all kinds of patients, they are more recurrent in immunosuppressed ones such as patients with HIV, hepatitis, cancer or under long antimicrobial treatments. Candida albicans continues to be the most frequently identified Candida spp. in these disorders, but other non-C. albicans Candida are rising. Understanding the immune responses involved in oral Candida spp. infections is a key feature to a successful treatment and to the design of novel therapies. In this review, we performed a literature search in PubMed and WoS, in order to examine and analyze common oral Candida spp.-bacteria/Candida-Candida interactions and the host immunity response in oral candidiasis.

Farnesol and Tyrosol: Secondary Metabolites with a Crucial quorum-sensing Role in Candida Biofilm Development.

When living in biological and interactive communities, microorganisms use quorum-sensing mechanisms for their communication. According to cell density, bacteria and fungi can produce signaling molecules (e.g., secondary metabolites), which participate, for example, in the regulation of gene expression and coordination of collective behavior in their natural niche. The existence of these secondary metabolites plays a main role in competence, colonization of host tissues and surfaces, morphogenesis, and biofilm development. Therefore, for the design of new antibacterials or antifungals and understanding on how these mechanisms occur, to inhibit the secretion of quorum-sensing (e.g., farnesol and tyrosol) molecules leading the progress of microbial infections seems to be an interesting option. In yeasts, farnesol has a main role in the morphological transition, inhibiting hyphae production in a concentration-dependent manner, while tyrosol has a contrary function, stimulating transition from spherical cells to germ tube form. It is beyond doubt that secretion of both molecules by fungi has not been fully described, but specific meaning for their existence has been found. This brief review summarizes the important function of these two compounds as signaling chemicals participating mainly in Candida morphogenesis and regulatory mechanisms.

The Contribution of Photodynamic Inactivation vs. Corsodyl Mouthwash to the Control of Streptococcus mutans Biofilms.

This work compared the inhibition effect of the commercially available mouthwash Corsodyl, containing 0.1% chlorhexidine digluconate, and photodynamic inactivation (PDI) employing methylene blue (MB) with irradiation from a red laser on 24-h biofilms formed by Streptococcus mutans strains on hydroxyapatite surfaces. The cytotoxicity of Corsodyl and MB was evaluated by Galleria mellonella surviving assay. The viability of biofilm cells after exposure to mouthwash and PDI was determined by counting colony-forming units. The inhibitory effect of antimicrobial agents was confirmed by confocal scanning laser microscopy. MB did not exhibit a cytotoxic effect on larval survival. Non-diluted Corsodyl slightly decreased the survival of larvae. Using our PDI parameters achieved better inhibition than with non-PDI, proving a significant effect on the eradication of S. mutans biofilms and therefore could be an appropriate supplement for the eradication of dental caries.

Novel Therapies for Biofilm-Based Candida spp. Infections.

The presence of fungal infections continue to grow worldwide, mostly in immunosuppressed patients, and in individuals with continued antimicrobial treatments. Candida spp. are the most common yeasts involved in these disorders, being associated with a high rate of antifungal resistance and an increased ability to form biofilms, which make the treatment of these infections difficult. This review aims to present and discuss the main biofilm-related infections cause by several Candida spp. and novel therapies that are currently available in the clinical, scientific and academic environment. New drugs with promising antifungal activity, natural approaches (e.g. probiotics, essential oils, plant extracts, honey) and a final consideration on alternative methodologies, such as photodynamic therapy are presented and discussed.

Advances in Chemical and Biological Methods to Identify Microorganisms-From Past to Present.

Fast detection and identification of microorganisms is a challenging and significant feature from industry to medicine. Standard approaches are known to be very time-consuming and labor-intensive (e.g., culture media and biochemical tests). Conversely, screening techniques demand a quick and low-cost grouping of bacterial/fungal isolates and current analysis call for broad reports of microorganisms, involving the application of molecular techniques (e.g., 16S ribosomal RNA gene sequencing based on polymerase chain reaction). The goal of this review is to present the past and the present methods of detection and identification of microorganisms, and to discuss their advantages and their limitations.

Synergy Over Monotherapy.

Pathogenic fungi, as an increasing global threat to human health, represent a sizable risk. However, significant attention should also be paid to the yeast biofilms. One promising strategy for combating resistant microbes, as well as fungal biofilms, is to extend the lifespan and efficacy of our currently employed drugs by using combination therapy. Since the application of combined therapy of fungal infections is currently accepted, we have decided to verify the efficacy of derivative H in combination with fluconazole on C. albicans biofilm. The main advantage of synergy over monotherapy lies in reducing or even completely preventing the induction of resistance of fungal cells. We have decided to verify the derivative H (1,4-dihydropyridine-2,3,5-tricarboxylate), an intermediate of nilvadipine synthesis, in the resistance of C. albicans to fluconazole. Therefore, we have focused on the influence of derivative H on the gene expression of the main C. albicans adhesin (ALS3), which is important for the tissue colonization during the infection process. Our results show that the newly synthesized derivative H had an impact on biofilm eradication. The effect of biofilm diminution could, therefore, be explained as derivative H preventing the adherence of C. albicans cells. This study supports even more the attractiveness of this substance as a potential agent that could be used in synergy with commonly used azoles to treat various fungal infections.

Impact of Farnesol as a Modulator of Efflux Pumps in a Fluconazole-Resistant Strain of Candida albicans.

Aim: This work studied the impact of the quorum-sensing molecule, farnesol (FAR), on fluconazole (FLC)-resistant Candida albicans isolate CY 1123 compared with the susceptible standard strain C. albicans SC5314. The genes encoding efflux pumps belonging to the ATP-binding cassette (ABC) and major facilitator superfamilies, together with overexpression or point mutation of the ERG11 gene, are the main resistance mechanisms to azole antifungal drugs. Results: The upregulation of genes coding for CDR1, CDR2, and MDR1 were confirmed by qPCR with respect to the housekeeping gene ACT1 in the resistant strain. The contribution of the ERG11 gene was also observed. Markedly, increased pump activity (Cdr1 and/or Cdr2) in the CY 1123 strain was confirmed using diS-C3(3) assay. However, the addition of FAR to the yeasts diminished the difference in staining levels between the SC5314 and CY 1123 strains, demonstrating the concentration-dependent character that could be caused by an effective modulation of Cdr pumps. FAR (60 and 100 µM) was also able to decrease the minimal inhibitory concentrations (MIC50), denoting the inhibition of planktonic cells by 50%, from 8 to 4 µg/mL of FLC when the resistant strain CY 1123 was not cultivated with FLC. However, when it was exposed to 64 µg/mL of FLC, the MIC50 shifted from 64 to 8 µg/mL. Conclusion: Besides the many other effects of FAR on eukaryotic and prokaryotic cells, it also affects ABC efflux transporters, resulting in changes in resistance to azoles in C. albicans isolates. However, this effect is dependent on FAR concentrations.

The impact of farnesol in combination with fluconazole on Candida albicans biofilm: regulation of ERG20, ERG9, and ERG11 genes.

Farnesol (FAR) has already demonstrated an inhibitory effect on Candida albicans biofilm. The aim of this work was to determine the effectiveness of externally added FAR in combination with fluconazole (FLC) on Candida albicans biofilm and on regulation of the ergosterol genes ERG20, ERG9, and ERG11. The effectiveness of compounds was determined by MTT assay and evaluated by the minimal inhibitory concentrations reducing a sessile biofilm to 50% activity (0.5 µg/mL and 200 µmol/L for FLC and FAR, respectively). These concentrations as well as 30 and 100 µmol/L FAR were selected for a study of the effectiveness of the FAR/FLC combination. The reduction in biofilm robustness mainly caused by the presence of 200 µmol/L FAR-alone or in combination with FLC-was accompanied by a significant inhibition of the yeast-to-hyphae transition that was observed by light microscopy and CLSM. Results from qRT-PCR indicated that while 30 µmol/L FAR only slightly regulated the expression of all 3 genes in the 48-h biofilm, the presence of 200 µmol/L FAR downregulated all the tested genes. However, the addition of 0.5 µg/mL of FLC to the samples with 200 µmol/L FAR restored the downregulation of the ERG20 and ERG11 genes to the control level. Moreover, the gene ERG9 was slightly upregulated. In summary, FAR acted via multiple effects on the C. albicans biofilm, but only a higher concentration of FAR proved to be effective.

Employment of methylene blue irradiated with laser light source in photodynamic inactivation of biofilm formed by Candida albicans strain resistant to fluconazole.

A promising approach for the eradication of biofilm formed by the yeast Candida albicans seems to be photodynamic inactivation (PDI). This work presents a use of methylene blue (MB, 1 mM) irradiated with a red laser (output power 190 mW/cm2, wavelength 660 nm) for the eradication of a biofilm formed by the fluconazole-resistant (FLC-resistant) strain C. albicans CY 1123 compared to the standard strain C. albicans SC5314. The periods of irradiation corresponded to the fluence of 15, 23 and 57 J/cm2. Effectiveness of PDI was evident with following percentage of survived biofilm cells: 24.57, 23.46, and 22.29% for SC5314 and 40.28, 17.91, and 5.89% for CY 1123, respectively, compared to the samples without irradiation. Light and confocal laser scanning microscopy confirmed the effectiveness of PDI. However, the morphological form of C. albicans seems to play an important role as well, since prolonged duration of irradiation did not increase efficiency of PDI on C. albicans SC5314. An experiment with the yeast-to-hyphae transition revealed that the FLC-resistant strain expressed a markedly reduced capacity to form hyphae compared to SC5314. We summarized that PDI was effective on biofilm formed by the FLC-resistant strain, but resistance most likely did not play significant role in PDI. Additionally, we observed differences in susceptibility to PDI between biofilms composed of the mycelia and only of the yeasts, and finally, the employment of a laser in PDI enabled a decreasing period of irradiation while maintaining the high effectiveness of PDI.

Effectiveness of the Photoactive Dye Methylene Blue versus Caspofungin on the Candida parapsilosis Biofilm in vitro and ex vivo.

This research studied the effectiveness of the photoactive compound methylene blue (MB) activated with red LED light (576-672 nm) compared to that of caspofungin (CAS) on 1 Candida albicans and 3 Candida parapsilosis strains. Results were evaluated in terms of SMIC50 for CAS or in PDI (photodynamic inactivation)-SMIC50 for MB (minimal inhibitory concentration inhibiting sessile biofilm to 50% in comparison to the control without CAS or after irradiation in comparison to the control without MB). While all strains were susceptible to CAS in planktonic form, the SMIC50 was determined to be >16 µg mL(-1) when CAS was added to a 24 h biofilm. However, PDI-MIC50s (1.67 mW cm(-2) , fluence 15 J cm(-2) ) were 0.0075-0.03 mmol L(-1) . For biofilm, PDI-SMIC50s were in the range from 0.7 to 1.35 mmol L(-1) . MB concentration of 1 mmol L(-1) prevented a biofilm being formed ex vivo on mouse tongues after irradiation regardless of the application time, in contrast to CAS, which was only effective at a concentration of 16 µg mL(-1) when it was added at the beginning of biofilm formation. PDI seems to be a promising method for the prevention of microbial biofilms that do not respond significantly to conventional drugs.