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Candida albicans is an opportunistic yeast capable of causing a wide range of mucosal, cutaneous, and systemic infections. However, therapeutic strategies are limited to a few antifungal agents. Inorganic nanoparticles have been investigated as carrier systems for antifungals as potential new treatments. In this study, we focused on the antifungal activity of gold nanorods, a specific rod-shaped gold nanoparticle, produced by green synthesis using resveratrol as a metal-reducing agent. The synthesis method resulted in stable control nanoparticles (AuNp) and resveratrol-coated gold nanoparticles (AuNpRSV) with medium sizes of 32.4 × 15.9 nm for AuNp, and 33.5 × 15.3 nm for AuNpRSV. Both AuNp and AuNpRSV inhibited the C. albicans grown at 2.46 µg/mL, exhibited fungicidal effects at 4.92 µg/mL, and significantly decreased filamentation, biofilm viability, reactive oxygen species production and ergosterol levels of C. albicans . In addition, exposure to AuNpRSV reduced the ability of C. albicans to grow in the presence of cell membrane stressors. Transmission electron microscopy revealed enlargement of the cell wall and retraction of the cell membrane after treatment with AuNp and AuNpRSV. Promisingly, in vivo toxicity analysis demonstrated that both nanoparticles maintained the full viability of Galleria mellonella larvae at 49.20 µg/mL. In conclusion, both gold nanoparticles exhibited antifungal activity; however, these effects were enhanced by AuNpRSV. Altogether, AuNps and AuNpRSVs are potential antifungal agents for the treatment of C. albicans infections.

Cryptococcosis is an invasive mycosis caused mainly by Cryptococcus gattii and C. neoformans and is treated with amphotericin B (AMB), fluconazole and 5-fluorocytosine. However, antifungal resistance, limited and toxic antifungal arsenal stimulate the search for therapeutic strategies such as drug repurposing. Among the repurposed drugs studied, the selective serotonin reuptake inhibitors (SSRIs) have shown activity against Cryptococcus spp. However, little is known about the antifungal effect of duloxetine hydrochloride (DH), a selective serotonin and norepinephrine reuptake inhibitor (SSNRI), against C. neoformans and C. gattii. In this study, DH inhibited the growth of several C. neoformans and C. gattii strains at concentrations ranging from 15.62 to 62.50 µg/mL. In addition, DH exhibited fungicidal activity ranging from 15.62 to 250 µg/mL. In biofilm, DH treatment reduced Cryptococcus spp. biomass at a level comparable to AMB, with a significant reduction (85%) for C. neoformans biofilms. The metabolic activity of C. neoformans and C. gattii biofilms decreased significantly (99%) after treatment with DH. Scanning electron micrographs confirmed the anti-biofilm activity of DH, as isolated cells could be observed after treatment. In conclusion, DH showed promising antifungal activity against planktonic cells and biofilms of C. neoformans and C. gattii, opening perspectives for further studies with DH in vivo., (© 2024. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

This study investigated the antimicrobial activity of surface pre-reacted glass ionomer eluate (S-PRG) against oral microcosm biofilms collected from the oral cavity of patients. Dental biofilm samples were collected from three volunteers to form microcosm biofilms in vitro . Initially, screening tests were carried out to determine the biofilm treatment conditions with S-PRG eluate. The effects of a daily treatment for 5 min using three microcosm biofilms from different patients was then evaluated. For this, biofilms were formed on tooth enamel specimens for 120 h. Biofilms treated with 100% S-PRG for 5 min per day for 5 days showed a reduction in the number of total microorganisms, streptococci and mutans streptococci. SEM images confirmed a reduction in the biofilm after treatment. Furthermore, S-PRG also reduced lactic acid production. It was concluded that S-PRG eluate reduced the microbial load and lactic acid production in oral microcosm biofilms, reinforcing its promising use as a mouthwash agent.

Fungal infections can cause significant patient morbidity and mortality. Nanoparticle therapeutics have the potential to improve treatment of these infections. Here we report the development of liposomal nanoparticles incorporating anidulafungin, a potent antifungal, with the goal of increasing its solubility and aiding in localization to fungi. Liposomes were fabricated with three concentrations of anidulafungin yielding monodisperse ~100 nm unilamellar vesicles. All three formulations inhibited planktonic Candida albicans growth at a minimum inhibitory concentration equivalent to free drug. All three formulations also disrupted preformed C. albicans biofilms, reducing fungal burden by as much as 99%, exhibiting superior biofilm disruption compared with free drug. Liposome formulations tested in vivo in C. albicans infected Galleria mellonella wax moth larvae demonstrated increased survival compared to free drug equivalents, leading to a survival of 33 to 67% of larvae over 7 days depending on the liposome utilized compared with only 25% survival of larvae administered free drug. Liposomal formulations along with free anidulafungin did not cause red blood cell lysis. Ultimately, the liposome formulations reported here increased anidulafungin solubility, displayed promising efficacy against planktonic and biofilm C. albicans, and improved the survival of C. albicans-infected G. mellonella compared to free anidulafungin., (© 2020 Wiley Periodicals, Inc.)

Fungi of the genus Candida are important etiological agents of superficial and life-threatening infections in individuals with a compromised immune system. One of the main characteristics of Candida is its ability to form highly drug tolerance biofilms in the human host. Biofilms are a dynamic community of multiple cell types whose formation over time is orchestrated by a network of transcription regulators. In this brief review, we provide an update of the processes involved in biofilm formation by Candida spp. (formation, treatment, and control), as well as the transcriptional circuitry that regulates its development and interactions with other microorganisms. Candida albicans is known to build mixed species biofilms with other Candida species and with various other bacterial species in different host niches. Taken together, these properties play a key role in Candida pathogenesis. In addition, this review gathers recent studies with new insights and perspectives for the treatment and control of Candida biofilms.

Kyotorphin (KTP, l-tyrosyl-l-arginine) is an endogenous analgesic neuropeptide first isolated from bovine brain in 1979. Previous studies have shown that kyotorphins possess anti-inflammatory and antimicrobial activity. Six kyotorphins-KTP-NH 2 , KTP-NH 2 -DL, ibuprofen-conjugated KTP (IbKTP), IbKTP-NH 2 , N-methyl-D-Tyr-L-Arg, and N-methyl-L-Tyr-D-Arg-were designed and synthesized to improve lipophilicity and resistance to enzymatic degradation. This study assessed the antimicrobial and antibiofilm activity of these peptides. The antifungal activity of kyotorphins was determined in representative strains of Candida species, including Candida albicans ATCC 10231, Candida krusei ATCC 6258, and six clinical isolates-Candida dubliniensis 19-S, Candida glabrata 217-S, Candida lusitaniae 14-S, Candida novergensis 51-S, Candida parapsilosis 63, and Candida tropicalis 140-S-obtained from the oral cavity of HIV-positive patients. The peptides were synthesized by standard solution or solid-phase synthesis, purified by RP-HPLC (purity >95 %), and characterized by nuclear magnetic resonance. The results of the broth microdilution assay and scanning electron microscopy showed that IbKTP-NH 2 presented significant antifungal activity against Candida strains and antibiofilm activity against the clinical isolates. The absence of toxic activity and survival after infection was assessed after injecting the peptide in larvae of Galleria mellonella as experimental infection model. Furthermore, IbKTP-NH 2 had strong antimicrobial activity against multidrug-resistant bacteria and fungi and was not toxic to G. mellonella larvae up to a concentration of 500 mM. These results suggest that IbKTP-NH 2 , in addition to its known effect on cell membranes, can elicit a cellular immune response and, therefore, is promising for biomedical application., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Surface pre-reacted glass-ionomer (S-PRG) is a bioactive filler produced by PRG technology, which is applied to various dental materials. The inhibitory effects of S-PRG eluate against Candida , the most common fungal oral pathogen, were investigated. Minimum inhibitory concentrations (MIC) and anti-biofilm activities were tested against Candida albicans , Candida glabrata , Candida krusei , and Candida tropicalis . For the in vivo study, Galleria mellonella was used as a model to evaluate the effects of S-PRG on toxicity, hemocyte counts and candidiasis. The MIC of S-PRG ranged from 5 to 40% (v/v). S-PRG eluate exhibited anti-biofilm activity for all the Candida species tested. Furthermore, injection of S-PRG eluate into G. mellonella was not toxic to the larvae and protected G. mellonella against experimental candidiasis. In addition, S-PRG eluate inhibited biofilm formation by C. albicans , C. glabrata , C. krusei , and C. tropicalis and exerted protective effects on G. mellonella against experimental candidiasis in vivo .

Introduction: This study evaluated the efficacy of photodynamic inactivation (PDI) with hematoporphyrin IX (H) and modified hematoporphyrin IX (MH) at 10 μmol/L, using a blue light-emitting diode (LED), fluence of 75 J/cm, 2 over planktonic cultures and biofilm of Streptococcus mutans (UA 159)., Methods: Suspensions containing 107 cells/mL were tested under different experimental conditions: a) H and LED (H+L+), b) MH and LED (MH+L+), c) only LED (P-L+), d) only H (H+L-), e) only MH (MH+L-), and f) control group, no LED or photosensitizer treatment (P-L-). The study also evaluated the effect of PDI on S mutans biofilm on metallic or ceramic brackets bonded on specimens of human teeth. The strains were seeded onto Mitis salivarius-bacitracin-sacarose agar to determine the number of colony-forming units., Results: H and MH under LED irradiation were effective on planktonic cultures (P <0.0001). H and MH (H+L+ and MH+L+) caused a reduction of 3.80 and 6.78 log 10  CFU/mL. PDI with the use of H or MH and LED exerted a strong antimicrobial effect over S mutans showing 54% and 100% reduction, respectively. PDI on S mutans biofilm on metallic and ceramic brackets with the use of H was not effective (P = 0.0162, P = 0.1669), however, MH caused a significant reduction of 44% and 53% of the cell count on metallic and ceramic brackets, respectively (P = 0.0020, P = 0.004)., Conclusions: In vitro planktonic cultures with the use of H or MH and LED exerted significant antimicrobial activity. No effect was observed on S mutans biofilm on either bracket type with the use of H, MH showed better results, suggesting a promising use against dental caries and white spot lesions., (Copyright © 2018 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.)

Cryptococcosis is an opportunistic or primary fungal infection considered to be the most prevalent fatal fungal disease worldwide. Owing to the limited number of available drugs, it is necessary to search for novel antifungal compounds. In the present work, we assessed the antifungal efficacy of three thiazole derivatives (1, 2, and 3). We conducted in vitro and in vivo assays to investigate their effects on important virulence factors, such as capsule and biofilm formation. In addition, the phagocytosis index of murine macrophages exposed to compounds 1, 2, and 3 and the in vivo efficacy of 1, 2, and 3 in Galleria mellonella infected with Cryptococcus spp. were evaluated. All compounds exhibited antifungal activity against biofilms and demonstrated a reduction in biofilm metabolic activity by 43-50% for C. gattii and 26-42% for C. neoformans. Thiazole compounds promoted significant changes in the capsule thickness of C. gattii compared to that of C. neoformans. Further examination of these compounds suggests that they can improve the phagocytosis process of peritoneal murine macrophages in vitro, causing an increase in the phagocytosis rate. Survival percentage was examined in the invertebrate model Galleria mellonella larvae, and only compound 3 could increase the survival at doses of 5 mg/kg after infection with C. gattii (P = .0001) and C. neoformans (P = .0007), similar to fluconazole at 10 mg/kg. The results demonstrated that thiazole compounds, mainly compound 3, have potential to be used for future studies in the search for new therapeutics for cryptococcosis.

Photodynamic therapy (PDT) can be used for the control of oral pathogens and different photosensitizers (PS) have been investigated. This study evaluated the efficacy of PDT against Streptococcus mutans biofilms using two second-generation PS derived from chlorin: Photoditazine® (PDZ) and Fotoenticine® (FTC). These PS were compared to methylene blue (MB), a dye with proven antimicrobial activity against S. mutans. Suspensions of S. mutans were cultured in contact with bovine tooth disks for biofilm formation. After 48 h, the biofilms were treated with PDZ (0.6 mg/mL), FTC (0.6 mg/mL) or MB (1 mg/mL) and submitted to laser irradiation (660 nm, 50 mW/cm 2 ). The biofilms were quantified by the determination of CFU/mL count and analyzed by scanning electron microscopy (SEM). All PS used for PDT reduced the number of S. mutans, with a statistically significant difference compared to the untreated groups. PDT achieved microbial reductions of 4 log with MB and 6 log with PDZ, while the use of FTC resulted in the complete elimination of S. mutans biofilms. SEM analysis confirmed the CFU/mL results, showing that all PS, particularly FTC, were able to detach the biofilms and to eliminate the bacteria. In conclusion, PDT mediated by chlorin-type PS exhibited greater antimicrobial activity against S. mutans than MB-mediated PDT, indicating that these PS can be useful for the control of dental caries., (Copyright © 2018. Published by Elsevier B.V.)

Probiotics can release bioactive substances that can inhibit the growth and biofilm formation of pathogenic microorganisms such as Streptococcus mutans. In this context, we evaluated whether the supernatants of Lactobacillus strains isolated from caries-free subjects can inhibit S. mutans, one of the most important bacteria for dental caries. First, the supernatants of 22 Lactobacillus strains were screened for antibacterial activity against S. mutans in planktonic cultures. All 22 Lactobacillus strains studied (100%) showed antibacterial activity. Thereafter, the Lactobacillus strains with the greatest reductions in the planktonic S. mutans cultures were tested on biofilms. The L. fermentum 20.4, L. paracasei 11.6, L. paracasei 20.3 and L. paracasei 25.4 strains could significantly reduce the number of S. mutans cells in biofilms formed in hydroxyapatite (p < 0.05). This reduction was also confirmed by scanning electron microscopy analysis and was not caused by the decreased pH value in the medium (p > 0.05). In addition, the supernatants of these probiotic strains could also reduce the total biomass of S. mutans biofilms (p < 0.05). In conclusion, most of the Lactobacillus strains tested have some antibacterial activity against S. mutans. L. fermentum 20.4, L. paracasei 11.6, L. paracasei 20.3 and L. paracasei 25.4 produce bioactive substances that caused a significant reduction in S. mutans biofilms., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The objective of this study was to evaluate the effects of photodynamic inactivation (PDI) on Candida albicans biofilms, evaluating its effects on gene expression of ALS3, HWP1, BCR1, TEC1, CPH1, and EFG1 by yeast. Three samples of C. albicans were used in this study: a clinical sample from a patient with HIV (39S), a clinical sample from a patient with denture stomatitis lesion (Ca30), and a standard strain ATCC 18804. The quantification of gene expression was related to the production of those genes in the samples referred above using quantitative polymerase chain reaction (qPCR) assay in real time. The photosensitizer methylene blue at 300 uM and erythrosine at 400 uM, sensitized with low-power laser (visible red, 660 nm) and green LED (532 nm), respectively, were used for PDI. Four groups of each sample and PDI protocol were evaluated: (a) P+L+: sensitization with the photosensitizer and irradiation with light, (b) P+L-: only treatment with the photosensitizer, (c) P-L+: only irradiation with light, and (d) P-L-: without sensitization with the dye and absence of light. The results were analyzed by t test, with a significance level of 5%. The photodynamic inactivation was able to reduce the expression of all genes for both treatments, laser and LED. The fold-decrease for the genes ALS3, HWP1, BCR1, TEC1, CPH1, and EFG1 were 0.73, 0.39, 0.77, 0.71, 0.67, and 0.60 for laser, respectively, and 0.66, 0.61, .050, 0.43, 0.54, and 0.66 for LED, respectively. It could be concluded that PDI showed a reduction in the expression of C. albicans genes, suggesting its virulence decrease.

This study isolated Lactobacillus strains from caries-free subjects and evaluated the inhibitory effects directly on three strains of C. albicans, two clinical strains and one reference strain. Thirty Lactobacillus strains were isolated and evaluated for antimicrobial activity against in vitro C. albicans biofilms. L. paracasei 28.4, L. rhamnosus 5.2 and L. fermentum 20.4 isolates exhibited the most significant inhibitory activity against C. albicans. Co-incubation between these microorganisms resulted in deterrence of biofilm development and retardation of hyphal formation. The hindrance of biofilm development was characterized by the downregulated expression of C. albicans biofilm-specific genes (ALS3, HWP1, EFG1 and CPH1). L. paracasei 28.4, L. rhamnosus 5.2 and L. fermentum 20.4 demonstrated the ability to exert antifungal activity through the inhibition of C. albicans biofilms.

The characterization of Candida albicans strains with different degrees of virulence became very useful to understand the mechanisms of fungal virulence. Then, the objective of this study was to assess and compare the temporal profiles of biofilms formation, gene expression of ALS1, ALS3, HWP1, BCR1, EFG1, TEC1, SAP5, PLB2 and LIP9 and virulence in Galleria mellonella of C. albicans ATCC18804 and a clinical sample isolated from an HIV-positive patient (CA60). Although the CFU/mL counting was higher in biofilms formed in vitro by ATCC strain, the temporal profile of the analysis of the transcripts of the C. albicans strains was elevated to Ca60 compared to strain ATCC, especially in the genes HWP1, ALS3, SAP5, PLB2 and LIP9 (up regulation). Ca60 was more pathogenic for G. mellonella in the survival assay (p = 0.0394) and hemocytes density (p = 0.0349), agreeing with upregulated genes that encode the expression of hyphae and hydrolase genes of Ca60. In conclusion, the C. albicans strains used in this study differ in the amount of biofilm formation, virulence in vivo and transcriptional profiles of genes analyzed that can change factors associated with colonization, proliferation and survival of C. albicans at different niches. SAP5 and HWP1 were the genes more expressed in the formation of biofilm in vitro.

Previous studies have been suggested that photodynamic therapy (PDT) can be used as an adjuvant treatment for denture stomatitis. In this study, we evaluated the effects of multiple sessions of PDT on Candida glabrata biofilms in specimens of polymerized acrylic resin formed after 5 days. Subsequently, four applications of PDT were performed on biofilms in 24-h intervals (days 6-9). Also, we evaluated two types of PDT, including application of laser and methylene blue or light-emitting diode (LED) and erythrosine. The control groups were treated with physiological solution. The effects of PDT on biofilm were evaluated after the first and fourth application of PDT. The biofilm analysis was performed by counting the colony-forming units. The results showed that between the days 6 and 9, the biofilms not treated by PDT had an increase of 5.53 to 6.05 log (p = 0.0271). Regarding the treatments, after one application of PDT, the biofilms decreased from 5.53 to 0.89 log. When it was done four applications, the microbial reduction ranged from 6.05 log to 0.11 log. We observed that one application of PDT with laser or LED caused a reduction of 3.36 and 4.64 compared to the control groups, respectively (p = 0.1708). When it was done four applications of PDT, the reductions achieved were 1.57 for laser and 5.94 for LED (p = 0.0001). It was concluded that repeated applications of PDT on C. glabrata biofilms showed higher antimicrobial activity compared to single application. PDT mediated by LED and erythrosine was more efficient than the PDT mediated by laser and methylene blue.

The increase in survival and resistance of microorganisms organized in biofilms demonstrates the need for new studies to develop therapies able to break this barrier, such as photodynamic therapy, which is characterized as an alternative, effective, and non-invasive treatment. The objective was to evaluate in vitro the effect of antimicrobial photodynamic therapy on heterotypic biofilms of Candida albicans and Bacillus atrophaeus using rose bengal (12.5 μM) and light-emitting diode (LED) (532 nm and 16.2 J). We used standard strains of B. atrophaeus (ATCC 9372) and C. albicans (ATCC 18804). The biofilm was formed in the bottom of the plate for 48 h. For the photodynamic therapy (PDT) experimental groups, we added 100 μL of rose bengal with LED (P+L+), 100 μL of rose bengal without LED (P+L-), 100 μL of NaCl 0.9 % solution with LED (P-L+), and a control group without photosensitizer or LED (P-L-). The plates remained in agitation for 5 min (pre-irradiation) and were irradiated with LED for 3 min, and the biofilm was detached using an ultrasonic homogenizer for 30 s. Serial dilutions were plated in BHI agar and HiChrom agar and incubated at 37 °C/48 h. There was a reduction of 33.92 and 29.31 % of colony-forming units per milliliter (CFU/mL) for C. albicans and B. atrophaeus, respectively, from the control group to the group subjected to PDT. However, statistically significant differences were not observed among the P+L+, P+L-, P-L+, and P-L- groups. These results suggest that antimicrobial photodynamic therapy using rose bengal (12.5 μM) with a pre-irradiation period of 5 min and LED for 3 min was not enough to cause a significant reduction in the heterotypic biofilms of C. albicans and B. atrophaeus.

Objective: The present study aimed to evaluate the interactions between the species Candida albicans, Candida krusei and Candida glabrata in monotypic and mixed biofilm models formed in vitro as well as the relative expression of the ALS1, ALS3, HWP1, BCR1, EFG1, TEC1, SAP5, PLB2 and LIP9 genes., Material and Methods: Mixed (C. albicans/C. krusei and C. albicans/C. glabrata) and monotypic biofilms were cultured for 0, 12 and 24h. Gene expression was analyzed in the same biofilm model in which the number of CFU/mL was counted., Results: The C. albicans CFU/mL values were lower at the 12 and 24h time points in the mixed biofilms compared with the monotypic biofilms, and decreases of 56.23% and 64.4% in C. albicans were observed when this species was associated with C. glabrata and C. krusei, respectively. In the presence of C. krusei, the expression of the ALS3, HWP1, BCR1, EFG1 and TEC1 genes of C. albicans was completely inhibited, indicating both transcriptome and the phenotypic antagonism between these two species, but genes related to the secretion of enzymes were stimulated. In the presence of C. glabrata, C. albicans showed a similar gene expression profile to that obtained in association with C. krusei, though it was altered to a lesser degree., Conclusion: We conclude that C. krusei and C. glabrata may alter or inhibit the mechanisms involved in the in vitro adherence and formation of C. albicans biofilms, influencing the pathogenicity of this species and suggesting a competitive interaction with C. krusei and C. glabrata in biofilm formation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Streptococcus mutans and Candida albicans are found together in the oral biofilms on dental surfaces, but little is known about the ecological interactions between these species. Here, we studied the effects of S. mutans UA159 on the growth and pathogencity of C. albicans. Initially, the effects of S. mutans on the biofilm formation and morphogenesis of C. albicans were tested in vitro. Next, we investigate the influence of S. mutans on pathogenicity of C. albicans using in vivo host models, in which the experimental candidiasis was induced in G. mellonella larvae and analyzed by survival curves, C. albicans count in hemolymph, and quantification of hyphae in the host tissues. In all the tests, we evaluated the direct effects of S. mutans cells, as well as the indirect effects of the subproducts secreted by this microorganism using a bacterial culture filtrate. The in vitro analysis showed that S. mutans cells favored biofilm formation by C. albicans. However, a reduction in biofilm viable cells and inhibition of hyphal growth was observed when C. albicans was in contact with the S. mutans culture filtrate. In the in vivo study, injection of S. mutans cells or S. mutans culture filtrate into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, a reduction in hyphal formation was observed in larval tissues when C. albicans was associated with S. mutans culture filtrate. These findings suggest that S. mutans can secrete subproducts capable to inhibit the biofilm formation, morphogenesis and pathogenicity of C. albicans, attenuating the experimental candidiasis in G. mellonella model.

Most Candida infections are related to microbial biofilms often formed by the association of different species. The objective of this study was to evaluate the interactions between Candida albicans and non-albicans species in biofilms formed in vitro. The non-albicans species studied were:Candida tropicalis, Candida glabrata and Candida krusei. Single and mixed biofilms (formed by clinical isolates of C. albicans and non-albicans species) were developed from standardized suspensions of each strain (10(7) cells/mL), on flat-bottom 96-well microtiter plates for 48 hour. These biofilms were analyzed by counting colony-forming units (CFU/mL) in Candida HiChrome agar and by determining cell viability, using the XTT 2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide colorimetric assay. The results for both the CFU/mL count and the XTT colorimetric assay showed that all the species studied were capable of forming high levels of in vitro biofilm. The number of CFU/mL and the metabolic activity of C. albicans were reduced in mixed biofilms with non-albicans species, as compared with a single C. albicans biofilm. Among the species tested, C. krusei exerted the highest inhibitory action against C. albicans. In conclusion, C. albicans established antagonistic interactions with non-albicans Candida species in mixed biofilms.

This study evaluated the action of Pfaffia paniculata K., Juglans regia L., and Rosmarius officinalis L. extracts against planktonic form and biofilm of Klebsiella pneumoniae (ATCC 4352). Minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) values were determined for each extract by microdilution broth method, according to Clinical and Laboratory Standards Institute. Next, antimicrobial activity of the extracts on biofilm was analyzed. For this, standardized suspension at 10 7  UFC/mL of K. pneumoniae was distributed into 96-well microplates ( n = 10) and after 48 h at 37°C and biofilm was subjected to treatment for 5 min with the extracts at a concentration of 200 mg/mL. ANOVA and Tukey tests (5%) were used to verify statistical significant reduction ( p < 0.05) of planktonic form and biofilm . P paniculata K., R. officinalis L., and J. regia L. showed reductions in biomass of 55.6, 58.1, and 18.65% and cell viability reduction of 72.4, 65.1, and 31.5%, respectively. The reduction obtained with P. paniculata and R. officinalis extracts was similar to the reduction obtained with chlorhexidine digluconate 2%. In conclusion, all extracts have microbicidal action on the planktonic form but only P. paniculata K. and R. officinalis L. were effective against biofilm., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

Candida species are major microorganisms isolated in denture stomatitis (DS), an inflammatory process of the mucosa underlying removable dental prostheses, and express a variety of virulence factors that can increase their pathogenicity. The potential of Photodynamic inactivation (PDI) in planktonic culture, biofilms and virulence factors of Candida strains was evaluated. A total of 48 clinical Candida isolates from individuals wearing removable maxillary prostheses with DS were included in the study. The effects of erythrosine (ER, 200 μM) and a green LED (λ 532 ± 10 nm, 237 mW/cm(2) and 42.63 J/cm(2)) in a planktonic culture were evaluated. The effect of the addition of ER at a concentration of 400 μM together with a green LED was evaluated in biofilms. The virulence factors of all of the Candida strains were evaluated before and after the PDI process in cells derived from biofilm and planktonic assays. All of the Candida species were susceptible to ER and green LED. However, the biofilm structures were more resistant to PDI than the planktonic cultures. PDI also promoted slight reductions in most of the virulence factors of C. albicans and some of the Candida tropicalis strains. These results suggest that the addition of PDI is effective for reducing yeasts and may also reduce the virulence of certain Candida species and decrease their pathogenicity., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this study, we evaluated the interactions between Candida albicans, Candida krusei and Candida glabrata in mixed infections. Initially, these interactions were studied in biofilms formed in vitro. CFU/mL values of C. albicans were lower in mixed biofilms when compared to the single biofilms, verifying 77% and 89% of C. albicans reduction when this species was associated with C. glabrata and C. krusei, respectively. After that, we expanded this study for in vivo host models of experimental candidiasis. G. mellonella larvae were inoculated with monotypic and heterotypic Candida suspensions for analysis of survival rate and quantification of fungal cells in the haemolymph. In the groups with single infections, 100% of the larvae died within 18 h after infection with C. albicans. However, interaction groups achieved 100% mortality after 72 h of infection by C. albicans-C. glabrata and 96 h of infection by C. albicans-C. krusei. C. albicans CFU/mL values from larvae hemolymph were lower in the interacting groups compared with the monoespecies group after 12 h of infection. In addition, immunosuppressed mice were also inoculated with monotypic and heterotypic microbial suspensions to induce oral candidiasis. C. albicans CFU/mL values recovered from oral cavity of mice were higher in the group with single infection by C. albicans than the groups with mixed infections by C. albicans-C. glabrata and C. albicans-C. krusei. Moreover, the group with single infection by C. albicans had a higher degree of hyphae and epithelial changes in the tongue dorsum than the groups with mixed infections. We concluded that single infections by C. albicans were more harmful for animal models than mixed infections with non-albicans species, suggesting that C. albicans establish competitive interactions with C. krusei and C. glabrata during biofilm formation and development of experimental candidiasis.

With the increasing number of strains of Candida ssp. resistant to antifungal agents, the accomplishment of researches that evaluate the effects of new therapeutic methods, like photodynamic inactivation (PDI), becomes important and necessary. Thus, the objective of this study was to verify the effects of the PDI on Candida albicans biofilms, evaluating their effects on the expression of the gene hydrolytic enzymes aspartyl proteinase (SAP5), lipase (LIP9), and phospholipase (PLB2). Clinical strains of C. albicans (n = 9) isolated from patient bearers of the virus HIV and a pattern strain ATCC 18804 were used. The quantification of gene expression was related to the production of hydrolytic enzymes using the quantitative polymerase chain reaction (qPCR) assay. For PDI, we used laser-aluminum-gallium arsenide low power (red visible, 660 nm) as a light source and the methylene blue at 300 μM as a photosensitizer. We assessed two experimental groups for each strain: (a) PDI: sensitization with methylene blue and laser irradiation and (b) control: without sensitization with methylene blue and light absence. The PDI decreased gene expression in 60 % of samples for gene SAP5 and 50 % of the samples decreased expression of LIP9 and PLB2. When we compared the expression profile for of each gene between the treated and control group, a decrease in all gene expression was observed, however no statistically significant difference (Tukey's test/p = 0.12). It could be concluded that PDI (photosensitization with methylene blue followed by low-level laser irradiation) showed a slight reduction on the expression of hydrolytic enzymes of C. albicans, without statistical significance.

Background: The search for alternative therapies for oral candidiasis is a necessity and the use of medicinal plants seems to be one of the promising solutions. The objective of this study was to evaluate the in vitro and in vivo effects of the essential oil of Melaleuca alternifolia on Candida albicans., Methods: The minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) of M. alternifolia were determined by the broth microdilution assay. For the in vivo study, twelve immunosuppressed mice with buccal candidiasis received topical applications of M. alternifolia with MBEC. After treatment, yeasts were recovered from the mice and quantified (CFU/mL). Mice were killed for morphologic analysis of the tongue dorsum by optical and scanning electron microscopy. Data were analyzed using Student's t test or Mann-Whitney test., Results: The MIC of M. alternifolia was 0.195% and the MBEC was 12.5%. Treatment with M. alternifolia achieved a 5.33 log reduction in C. albicans and reduced the microscopic lesions of candidiasis., Conclusions: M. alternifolia oil at a 12.5% was effective to eradicate a C. albicans biofilm formed in vitro and to reduce yeasts of C. albicans in an immunosuppressed mouse model.

Candida albicans is classified into different serotypes according to cell wall mannan composition and cell surface hydrophobicity. Since the effectiveness of photodynamic therapy (PDT) depends on the cell wall structure of microorganisms, the objective of this study was to compare the sensitivity of in vitro biofilms of C. albicans serotypes A and B to antimicrobial PDT. Reference strains of C. albicans serotype A (ATCC 36801) and serotype B (ATCC 36802) were used for the assays. A gallium-aluminum-arsenide laser (660 nm) was used as the light source and methylene blue (300 μM) as the photosensitizer. After biofilm formation on the bottom of a 96-well microplate for 48 h, each Candida strain was submitted to assays: PDT consisting of laser and photosensitizer application (L + P+), laser application alone (L + P-), photosensitizer application alone (L-P+), and application of saline as control (L-P-). After treatment, biofilm cells were scraped off and transferred to tubes containing PBS. The content of the tubes was homogenized, diluted, and seeded onto Sabouraud agar plates to determine the number of colony-forming units (CFU/mL). The results were compared by analysis of variance and Tukey test (p < 0.05). The two strains studied were sensitive to PDT (L + P+), with a log reduction of 0.49 for serotype A and of 2.34 for serotype B. Laser application alone only reduced serotype B cells (0.53 log), and the use of the photosensitizer alone had no effect on the strains tested. It can be concluded that in vitro biofilms of C. albicans serotype B were more sensitive to PDT.

Context: Teeth with periapical lesion usually present external root resorption around the apical foramen. These areas facilitate adhesion and co-aggregation of microorganisms developing biofilms. Up to the present moment, there is no methodology in the literature that enables the in vitro evaluation of endodontic irrigants and intracanal dressings on biofilms located in apical external root resorptions of human teeth., Aims: This study aimed to describe a new in vitro methodology for Enterococcus faecalis biofilm development in external apical reportion areas of human extracted teeth in different periods of time., Settings and Design: In vitro qualitative laboratory study., Subjects and Methods: Thirty roots from human extracted teeth presenting external apical resorption had their root canal diameters standardized by means of instrumentation. Next, the roots were randomly divided into three groups (n = 30) according to E. faecalis strains (ATCC 29212) exposure time as follows: Group T5, with 5-day exposure; Group T10, with 10-day exposure, and Group T15, with 15-day exposure. The roots were attached to 24-well culture plates so that only their apices could be in contact with bacteria for induction of biofilm formation. At the end of these exposure times, the roots were qualitatively evaluated with scanning electron microscope to observe the presence of biofilm in external resorptions around the apical foramen., Results: It was found that microorganisms were present in all exposure times, although structures suggesting the presence of biofilm with great conglomerate of bacteria showing structures similar to polysaccharide extensions were observed at the 10 th day of exposure., Conclusions: By means of this new methodology, it was possible to observe biofilm formation in the areas of external apical resorption after 10 days of exposure.

Candida albicans is an opportunistic yeast that can cause oral candidosis through the formation of a biofilm, an important virulence factor that compromises the action of antifungal agents. The objective of this study was to compare the effect of rose bengal (RB)- and eosin Y (EY)-mediated photodynamic inactivation (PDI) using a green light-emitting diode (LED; 532 ± 10 nm) on planktonic cells and biofilms of C. albicans (ATCC 18804). Planktonic cultures were treated with photosensitizers at concentrations ranging from 0.78 to 400 μM, and biofilms were treated with 200 μM of photosensitizers. The number of colony-forming unit per milliliter (CFU/mL) was compared by analysis of variance and Tukey's test (P ≤ 0.05). After treatment, one biofilm specimen of the control and PDI groups were examined by scanning electron microscopy. The photosensitizers (6.25, 25, 50, 200, and 400 μM of EY, and 6.25 μM of RB or higher) significantly reduced the number of CFU/mL in the PDI groups when compared to the control group. With respect to biofilm formation, RB- and EY-mediated PDI promoted reductions of 0.22 log10 and 0.45 log10, respectively. Scanning electron microscopy showed that the two photosensitizers reduced fungal structures. In conclusion, EY- and RB-mediated PDI using LED irradiation significantly reduced C. albicans planktonic cells and biofilms.

The purpose of this study was to evaluate specific effects of photodynamic inactivation (PDI) using erythrosine (ER) and Rose Bengal (RB) photosensitizers and a blue light-emitting diode (LED) on the viability of Streptococcus mutans and Streptococcus sanguinis biofilms. Biofilms were grown in acrylic disks immersed in broth to production of biofilms, inoculated with microbial suspension (10(6) cells/mL) and incubated for 48 h. After the formation of biofilms, the effects of the photosensitizers ER and RB at a concentration of 5 μM for 5 min and blue LED (455 ± 20 nm) for 180 s, photosensitizers alone and conjugated were evaluated. Next, the disks were placed in tubes with sterile physiological solution (0.9 % sodium chloride) and sonicated for to disperse the biofilms. Tenfold serial dilutions were carried and aliquots seeded in brain heart infusion agar which were then incubated for 48 h. Then the numbers colony-forming units per milliliter (CFU/mL; log10) were counted and analyzed statistically (ANOVA, Tukey test, P ≤ 0.05). Significant decreases in the viability of all microorganisms were observed for biofilms exposed to PDI mediated by both photosensitizers. The reductions with RB and ER were, 0.62 and 0.52 log10 CFU mL(-1) for S. mutans biofilms (p=0.001), and 0.95 and 0.88 log10 CFU mL(-1) for S. sanguinis biofilms (p=0.001), respectively. The results showed that biofilms formed in vitro by S. mutans and S. sanguinis, were sensitive to PDI using a blue LED associated with photosensitizers ER or RB, indicating its use in the control of caries and periodontal diseases.

Objectives: The organization of biofilms in the oral cavity gives them added resistance to antimicrobial agents. The action of phenothiazinic photosensitizers on oral biofilms has already been reported. However, the action of the malachite green photosensitizer upon biofilm-organized microorganisms has not been described. The objective of the present work was to compare the action of malachite green with the phenothiazinic photosensitizers (methylene blue and toluidine blue) on Staphylococcus aureus and Escherichia coli biofilms., Methods: The biofilms were grown on sample pieces of acrylic resin and subjected to photodynamic therapy using a 660-nm diode laser and photosensitizer concentrations ranging from 37.5 to 3000 μM. After photodynamic therapy, cells from the biofilms were dispersed in a homogenizer and cultured in Brain Heart Infusion broth for quantification of colony-forming units per experimental protocol. For each tested microorganism, two control groups were maintained: one exposed to the laser radiation without the photosensitizer (L+PS-) and other treated with the photosensitizer without exposure to the red laser light (L-PS+). The results were subjected to descriptive statistical analysis., Results: The best results for S. aureus and E. coli biofilms were obtained with photosensitizer concentrations of approximately 300 μM methylene blue, with microbial reductions of 0.8-1.0 log(10); 150 μM toluidine blue, with microbial reductions of 0.9-1.0 log(10); and 3000 μM malachite green, with microbial reductions of 1.6-4.0 log(10)., Conclusion: Greater microbial reduction was achieved with the malachite green photosensitizer when used at higher concentrations than those employed for the phenothiazinic dyes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The aim of this study was to evaluate the effects of photodynamic therapy (PDT) using rose bengal or erythrosine with light emitting diode (LED) on Candida albicans planktonic cultures and biofilms. Seven C. albicans clinical strains and one standard strain (ATCC 18804) were used. Planktonic cultures and biofilms of each C. albicans strain were submitted to the following experimental conditions: (a) treatment with rose bengal and LED (RB+L+); (b) treatment with erythrosine and LED (E+L+); and (c) control group, without LED irradiation or photosensitiser treatment (P-L-). After irradiation of the planktonic cultures and biofilms, the cultures were seeded onto Sabouraud dextrose agar (37 °C at 48 h) for counting of colony-forming units (CFU ml(-1) ) followed by posterior anova and Tukey's test analyses (P < 0.05). The biofilms were analysed using scanning electron microscopy (SEM). The results revealed a significant reduction of planktonic cultures (3.45 log(10) and 1.97 log(10) ) and of biofilms (<1 log(10) ) for cultures that were subjected to PDT mediated using either erythrosine or rose bengal, respectively. The SEM data revealed that the PDT was effective in reducing and destroying of C. albicans blastoconidia and hyphae. The results show that erythrosine- and rose bengal-mediated PDT with LED irradiation is effective in treating C. albicans., (© 2011 Blackwell Verlag GmbH.)

Due to the increase in life expectancy, new treatments have emerged which, although palliative, provide individuals with a better quality of life. Artificial saliva is a solution that contains substances that moisten a dry mouth, thus mimicking the role of saliva in lubricating the oral cavity and controlling the existing normal oral microbiota. This study aimed to assess the influence of commercially available artificial saliva on biofilm formation by Candida albicans. Artificial saliva I consists of carboxymethylcellulose, while artificial saliva II is composed of glucose oxidase, lactoferrin, lysozyme and lactoperoxidase. A control group used sterile distilled water. Microorganisms from the oral cavity were transferred to Sabouraud Dextrose Agar and incubated at 37 °C for 24 hours. Colonies of Candida albicans were suspended in a sterile solution of NaCl 0.9%, and standardisation of the suspension to 106 cells/mL was achieved. The acrylic discs, immersed in artificial saliva and sterile distilled water, were placed in a 24-well plate containing 2 mL of Sabouraud Dextrose Broth plus 5% sucrose and 0.1 mL aliquot of the Candida albicans suspension. The plates were incubated at 37 °C for 5 days, the discs were washed in 2 mL of 0.9% NaCl and placed into a tube containing 10 mL of 0.9% NaCl. After decimal dilutions, aliquots of 0.1 mL were seeded on Sabouraud Dextrose Agar and incubated at 37 °C for 48 hours. Counts were reported as CFU/mL (Log10). A statistically significant reduction of 29.89% (1.45 CFU/mL) of Candida albicans was observed in saliva I when compared to saliva II (p = 0.002, considering p≤0.05).

The effect of erythrosine- and LED-mediated photodynamic therapy (PDT) on planktonic cultures and biofilms of Candida albicans and Candida dubliniensis was evaluated. Planktonic cultures of standardized suspensions (10(6)cells/mL) of C. albicans and C. dubliniensis were treated with erythrosine concentrations of 0.39-200 μM and LEDs in a 96-well microtiter plate. Biofilms formed by C. albicans and C. dubliniensis in the bottom of a 96-well microtiter plate were treated with 400 μM erythrosine and LEDs. After PDT, the biofilms were analysed by scanning electron microscopy (SEM). The antimicrobial effect of PDT against planktonic cultures and biofilms was verified by counting colony-forming units (CFU/mL), and the data were submitted to analysis of variance and the Tukey test (P<0.05). C. albicans and C. dubliniensis were not detectable after PDT of planktonic cultures with erythrosine concentrations of 3.12 μM or higher. The CFU/mL values obtained from biofilms were reduced 0.74 log(10) for C. albicans and 0.21 log(10) for C. dubliniensis. SEM revealed a decrease in the quantity of yeasts and hyphae in the biofilm after PDT. In conclusion, C. albicans and C. dubliniensis were susceptible to erythrosine- and LED-mediated PDT, but the biofilms of both Candida species were more resistant than their planktonic counterparts., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The purpose of this study was to evaluate specific effects of photodynamic inactivation (PDI) using methylene blue as photosensitizer and low-power laser irradiation on the viability of single-, dual-, and three-species biofilms formed by C. albicans, S. aureus, and S. mutans. Biofilms were grown in acrylic discs immersed in sterile brain heart infusion broth (BHI) containing 5% sucrose, inoculated with microbial suspension (10(6) cells/ml) and incubated for 5 days. On the fifth day, the effects of the methylene blue (MB) photosensitizer at a concentration of 0.1 mg/ml for 5 min and InGaAlP laser (660 nm) for 98 s, alone and conjugated were evaluated. Next, the discs were placed in tubes with sterile physiological solution [0.9% sodium chloride (NaCl)] and sonicated for to disperse the biofilms. Ten-fold serial dilutions were carried and aliquots seeded in selective agar, which were then incubated for 48 h. Then the numbers CFU/ml (log(10)) were counted and analyzed statistically (ANOVA, Tukey test, p < 0.05). Scanning electron microscopy (SEM) on discs treated with PDI and control biofilms groups was performed. Significant decreases in the viability of all microorganisms were observed for biofilms exposed to PDI mediated by MB dye. Reductions (log(10)) of single-species biofilms were greater (2.32-3.29) than the association of biofilms (1.00-2.44). Scanning electron microscopy micrographs suggested that lethal photosensitization occurred predominantly in the outermost layers of the biofilms. The results showed that PDI mediated by MB dye, might be a useful approach for the control of oral biofilms.