The natural compound magnolol affects growth, biofilm formation, and ultrastructure of oral Candida isolates.

The incidence of oral candidosis has increased in recent years due to the escalation in HIV-infection, cancer treatments, organ transplantation, and diabetes. In addition, corticosteroid use, dentures, and broad-spectrum antibiotic use have also contributed to the problem. Treatment of oral candidosis has continued to be problematic because of the potential toxicity of antifungals in clinical use, and, above all, development of drug resistance among patients. In this study, the antifungal effect of magnolol was investigated against 64 strains of Candida spp. (four standard and 60 oral isolates) through minimum inhibitory concentration (MIC) and growth curve assays. Insight into the mechanisms of the antifungal action has been gained through ultrastructural studies using confocal scanning laser microscopy (CSLM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Molecular docking was done for predicting the interactions of magnolol with ergosterol at supramolecular level. The toxicity of magnolol on human erythrocytes was measured by in vitro hemolytic assay. MIC values of magnolol ranged from 16-64 µg/ml, respectively. All tested isolates showed a marked sensitivity towards magnolol in growth curve assays. Biofilm results suggested that magnolol showed strong anti-biofilm activity. The results obtained for four different Candida spp. demonstrated that MBIC values of magnolol showed the average biofilm inhibition by 69.5%, respectively. CLSM experiments showed that cells exposed to magnolol (MIC) exhibited cell membrane disruption. SEM analysis of magnolol treated cells resulted in deformed cells. TEM micrographs showed rupturing of the cell wall and plasma membrane, releasing the intracellular content, and swelling of the cell wall. Hemolytic activity of magnolol is 11.9% at its highest MIC compared to an activity level of 25.4% shown by amphotericin B (Amp B) at 1 µg/ml. Lipinski's parameters calculated for magnolol suggested its good oral bioavailability. Docking studies indicated that magnolol might be interacting with ergosterol in the fungal cell membranes. Together, the present study provides enough evidence for further work on magnolol so that better strategies could be employed to treat oral candidosis.

Caspofungin-induced in-vitro post-antifungal effect and its impact on adhesion related traits of oral Candida dubliniensis and Candida albicans isolates.

Adhesion to buccal epithelial cells (BEC) and denture acrylic surfaces (DAS), germ tube (GT) formation and cell surface hydrophobicity (CSH) are all virulence traits involved in the pathogenicity of Candida. Post-antifungal effect (PAFE) also have a bearing on pathogenicity and virulence of Candida. Candida dubliniensis is associated with oral and systemic candidosis, which can be managed with caspofungin. There is no published information on caspofungin-induced PAFE and its impact on adhesion traits of C. dubliniensis isolates. Thus, the purpose of this investigation was to determine the in vitro duration of PAFE on 20 C. dubliniensis isolates following transient exposure to caspofungin. Furthermore the impacts of caspofungin-induced PAFE on adhesion to BEC and DAS, GT formation and CSH of these isolates were also determined. After establishing the minimum inhibitory concentration (MIC) of caspofungin, C. dubliniensis isolates were exposed to sub-lethal concentrations (×3 MIC) of caspofungin for 1 hr. Thereafter the duration of PAFE, adhesion to BEC and DAS, GT formation and CSH were determined by previously described in-vitro assays. MIC (µg/mL) of C. dubliniensis isolates to caspofungin ranged from 0.004 to 0.19. Caspofungin-induced mean PAFE on C. dubliniensis isolates was 2.17 hr. Exposure to caspofungin suppressed the ability of C. dubliniensis isolates to adhere to BEC and DAS, form GT and CSH by 69.97%, 71.95%, 90.06% and 32.29% (P < 0.001 for all), respectively. Thus, transient exposure of C. dubliniensis isolates to caspofungin produces an antifungal effect not only by suppressing its growth but also by altering its adhesion traits.

Organic acids from lignocellulose: Candida lignohabitans as a new microbial cell factory.

Biorefinery applications require microbial cell factories for the conversion of various sugars derived from lignocellulosic material into value-added chemicals. Here, the capabilities of the yeast Candida lignohabitans to utilize a range of such sugars is characterized. Substrates efficiently converted by this yeast include the pentoses xylose and arabinose. Genetic engineering of C. lignohabitans with the isolated endogenous GAP promoter and GAP terminator was successful. GFP expression was used as a proof of functionality for the isolated transcription elements. Expression of lactate dehydrogenase and cis-aconitate decarboxylase resulted in stable and reproducible production of lactic acid and itaconic acid, respectively. The desired organic acids were accumulated converting pure sugars as well as lignocellulosic hydrolysates. C. lignohabitans proved therefore to be a promising reliable microbial host for production of organic acids from lignocellulosic material.

Controlling the Interaction between Fluorescent Gold Nanoclusters and Biointerfaces for Rapid Discrimination of Fungal Pathogens.

Nondestructive detection and discrimination of fungal pathogens is essential for rapid and precise treatment, which further effectively prevents antifungal resistance from overused drugs. In this work, fluorescent gold nanoclusters served as the basis for discriminating Candida species. Varied on surface ligands, these gold nanoclusters demonstrated different optical properties as a result of the perturbation effects of ligands. The biointerface interaction between the surface ligands of gold nanoclusters and the cell walls of Candida species can be constructed, and their restriction on ligands perturbation effect produced enhanced fluorescence signals. Owing to the variation of the cell wall composition, cells of different Candida species demonstrated different degrees of association with the gold nanoclusters, leading to discriminable amounts of fluorescence enhancements. The reverse signal response from these gold nanoclusters gives rise to a synergistic and effective assay that allows identification of Candida species.

Microflora of the oral cavity in patients with xerostomia.

OBJECTIVE: The aim of this study was to evaluate the dependence of the condition of the microflora of the oral cavity on the etiology of xerostomia, patients' sex, age, degree of hyposalivation, and duration of the sense of dryness. MATERIAL AND METHODS: A total of 64 patients with complaints of oral dryness referred to the Clinic of Oral and Dental Diseases, Hospital of Lithuanian University of Health Sciences, for consultation during the period from 2003 to 2005 were selected for the study. The etiological factors of xerostomia were as follows: radiotherapy (PRT) to the maxillofacial area, Sjögren's syndrome (SS), and xerogenic medications, tricyclic antidepressants (TCAs). RESULTS: There were 50 women and 14 men. Their mean age was 60.5 ± 1.6 years. All the patients in the PRT group had high counts of Candida spp. as compared with percentages of patients in the TCA and SS groups (100% vs. 66.7% and 56.2%, P<0.05). Patients' age and sex in different etiology groups had no significant impact on the condition of their oral microflora. There were equal percentages of patients with deficient and normal salivation in the TCA group (44% in both the groups; P<0.01). All the patients in the PRT group had pronounced hyposalivation (P<0.002). A significantly greater percentage of patients with severely reduced salivation had high counts of Lactobacillus spp. (P<0.01). Significantly greater percentages of patients with the clinical duration of xerostomia of up to 6 months had high counts of Lactobacillus spp. and Candida spp. colonies. CONCLUSIONS: In patients with xerostomia, the condition of the microflora of the oral cavity and impairment of major salivary gland function varied according to the etiology of the disease. The level of hyposalivation and the duration of xerostomia were found to have a significant impact on the microflora of the oral cavity.

Erythematous oral candidiasis in patients with controlled type II diabetes mellitus and complete dentures.

Diabetes mellitus (DM) is a systemic condition characterized by a deficient sugar metabolism, which affects the immune system and favors the development of yeasts. The aim of the present study was to perform biochemical, morphological, exoenzyme analyses of Candida species and the molecular identification (DNA) of C. albicans in patients with type II diabetes mellitus. The exoenzyme quantification was compared to non-diabetic patients as controls. Two hundred and seventy-four patients who make use of complete dentures were evaluated, 28 of whom had diabetes and erythematous oral candidiasis. Other thirty patients presented the same clinical feature but without diabetes. Samples were isolated for biochemical identification (auxonogram), morphological identification (production of germ tubes) and PCR molecular identification (DNA). The capability of the Candida samples in producing phospholipases and proteinases was also determined. The diabetic patients had a greater diversity of Candida species (Fischer's exact test, P = 0.04). The production of proteinases by C. albicans in patients with diabetes was greater than in the control group (unpaired "t" test P < 0.003). However, there was no difference between groups for phospholipase production (unpaired "t" test P > 0.05). It was concluded that patients with controlled DM exhibited systemic conditions predisposing C. albicans proteinase increased production.

Colony morphologies, species, and biotypes of yeasts from thrush and denture stomatitis.

OBJECTIVE: To study the species and phenotypic characteristics of yeasts, i.e. colony morphology, biotypes, and biotype relatedness, and the oral distribution of yeasts, in thrush and denture stomatitis. MATERIAL AND METHODS: Yeast colony morphology was observed under a stereo-microscope and photographed with a digital camera. Genus, species, and biotypes of the yeast isolates were identified by using a commercial kit, ID 32C. Yeast biotype dendrograms were generated by Spotfire software and SPSS 15.0 for Windows. RESULTS: Multiple colony morphologies were observed among the yeasts from both thrush and denture stomatitis. One genus, 6 species, and 21 biotypes were identified among the yeasts from thrush, while 2 genera, 7 species, and 20 biotypes were identified among the yeasts from denture stomatitis. Considerable similarities in predominant species, biotypes, and biotype clustering profiles were shown among the yeasts from thrush and denture stomatitis. However, Candida dubliniensis was identified exclusively in subgingival areas and biotype 7347340215 of C. albicans was identified more frequently in palate and sulci in thrush. CONCLUSIONS: A diversity of species and phenotypes was found among the yeasts in thrush and denture stomatitis. Candidal commensals were predominant in thrush and denture stomatitis, but the observation of divergent Candida species and biotypes, constituting 23% of all the yeast isolates, should not be ignored.

Effectiveness of different cleaning agents against the colonization of Candida spp and the in vitro detection of the adherence of these yeast cells to denture acrylic surfaces.

PURPOSE: The aim of this study is to examine the effect Klorhex and Fittydent, which are used as cleaning agents on the adhesion of Candida on the surfaces of acrylic denture and palatal mucosa. In addition, ability of yeasts to adhere to acrylic strips was evaluated after applying these agents in vitro. MATERIALS AND METHODS: Each group of 15 patients cleaned their dentures with either Klorhex or with Fittydent. The control group cleaned their dentures with water. RESULTS: It was found that 62.2% of the patients had colonies of Candida species on their palatal mucosa which was reduced to 51.1% after using these cleaning agents. The colonization rate with Candida spp on their dentures was reduces from 82.2% to 68.8% using these cleaning agents. The mean adhesion value of the Candida strains isolated from the acrylic strips were found to be 75 cell/strip prior to applying the Klorhex and Fittydent and 37.5 cell/strip and 15 cell/strip after applying these agents, respectively. CONCLUSION: These results showed that Klorhex and Fittydent have a certain preventive effect on the colonization rate of Candida spp on the surface of these dentures, the palatal mucosa, as well as on the acrylic strips in vitro.

Improvement on D-xylose to Xylitol Biotransformation by Candida guilliermondii Using Cells Permeabilized with Triton X-100 and Selected Process Conditions.

Cells of Candida guilliermondii permeabilized with Triton X-100 were able to efficiently produce xylitol from a medium composed only by D-xylose and MgCl2·6H2O in potassium phosphate buffer, at 35 °C and pH 6.5. Under these conditions, the results were similar to those obtained when cofactor and co-substrate or nutrients were added to the medium (about 95 % D-xylose was assimilated producing 42 g/L of xylitol, corresponding to 0.80 g/g yield and 2.65 g/L h volumetric productivity). Furthermore, the permeabilized cells kept the D-xylose assimilation in about 90 % and the xylitol production in approx. 40 g/L during three bioconversion cycles of 16 h each. These values are highly relevant when compared to others reported in the literature using enzyme technology and fermentative process, thereby demonstrating the effectiveness of the proposed method. The present study reveals that the use of permeabilized cells is an interesting alternative to obtain high xylitol productivity using low cost medium formulation. This approach may allow the future development of xylitol production from xylose present in lignocellulosic biomass, with additional potential for implementation in biorefinery strategies.

Antibiofilm activity of carboxymethyl chitosan on the biofilms of non-Candida albicans Candida species.

Although most cases of candidiasis have been attributed to Candida albicans, non-C. albicans Candida species have been isolated in increasing numbers in patients. In this study, we determined the inhibition of carboxymethyl chitosan (CM-chitosan) on single and mixed species biofilm of non-albicans Candida species, including Candida tropicalis, Candida parapsilosis, Candida krusei and Candida glabrata. Biofilm by all tested species in microtiter plates were inhibited nearly 70%. CM-chitosan inhibited mixed species biofilm in microtiter plates and also on medical materials surfaces. To investigate the mechanism, the effect of CM-chitosan on cell viability and biofilm growth was employed. CM-chitosan inhibited Candida planktonic growth as well as adhesion. Further biofilm formation was inhibited with CM-chitosan added at 90min, 12h or 24h after biofilm initiation. CM-chitosan was not only able to inhibit the metabolic activity of Candida cells, but was also active upon the establishment and the development of biofilms.

Reduced adherence of Candida to silane-treated silicone rubber.

Silicone rubber is widely used in the construction of medical devices that can provide an essential role in the treatment of human illness. However, subsequent microbial colonization of silicone rubber can result in clinical infection or device failure. The objective of this study was to determine the effectiveness of a novel silane-treated silicone rubber in inhibiting microbial adherence and material penetration. Test material was prepared by a combination of argon plasma discharge treatment and fluorinated silane coupling. Chemicophysical changes were then confirmed by X-ray photoelectron spectroscopy, contact-angle measurement, and atomic force microscopy. Two separate adherence assays and a material penetration assay assessed the performance of the new material against four strains of Candida species. Results showed a significant reduction (p < 0.01) of Candida albicans GDH 2346 adherence to silane-treated silicone compared with untreated controls. This reduction was still evident after the incorporation of saliva into the assay. Adherence inhibition also occurred with Candida tropicalis MMU and Candida krusei NCYC, although this was assay dependent. Reduced penetration of silane-treated silicone by Candida was evident when compared to untreated controls, plaster-processed silicone, and acrylic-processed silicone. To summarize, a novel silicone rubber material is described that inhibits both candidal adherence and material penetration. The clinical benefit and performance of this material remains to be determined.

Comparison of the plaque microflora in immunodeficient and immunocompetent dental patients.

The nature and extent of the immune dysfunctions in 20 immunodeficient patients, as well as the immunocompetence of 22 control subjects, were verified by cell-mediated responsiveness and immunoglobulin quantitations. Comparisons of the microbial composition of supragingival plaque between the two populations showed that a greater number of immunodeficient than control subjects harbored Candida sp. and Staphylococcus sp. Conversely, a lower number of immunodeficient than control subjects harbored Streptococcus mutans. Also, patients with immune dysfunctions had a lower dental caries experience than their immunocompetent counterparts.

Xylitol production from sugarcane bagasse hydrolyzate in fluidized bed reactor. Effect of air flowrate.

Cells of Candida guilliermondii immobilized onto porous glass spheres were cultured batchwise in a fluidized bed bioreactor for xylitol production from sugarcane bagasse hemicellulose hydrolyzate. An aeration rate of only 25 mL/min ensured minimum yields of xylose consumption (0.60) and biomass production (0.14 g(DM)/g(Xyl)), as well as maximum xylitol yield (0.54 g(Xyt)/g(Xyl)) and ratio of immobilized to total cells (0.83). These results suggest that cell metabolism, although slow because of oxygen limitation, was mainly addressed to xylitol production. A progressive increase in the aeration rate up to 140 mL/min accelerated both xylose consumption (from 0.36 to 0.78 g(Xyl)/L.h) and xylitol formation (from 0.19 to 0.28 g(Xyt)/L.h) but caused the fraction of immobilized to total cells and the xylitol yield to decrease up to 0.22 and 0.36 g(Xyt)/g(Xyl), respectively. The highest xylitol concentration (17.0 g(Xyt)/L) was obtained at 70 mL/min, but the specific xylitol productivity and the xylitol yield were 43% and 22% lower than the corresponding values obtained at the lowest air flowrate, respectively. The concentrations of consumed substrates and formed products were used in material balances to evaluate the xylose fractions consumed by C. guilliermondii for xylitol production, complete oxidation through the hexose monophosphate shunt, and cell growth. The experimental data collected at variable oxygen level allowed estimating a P/O ratio of 1.35 mol(ATP)/mol(O) and overall ATP requirements for biomass growth and maintenance of 3.4 mol(ATP)/C-mol(DM).

Immobilization of Candida bombicola cells on free-standing organic-gold nanoparticle membranes and their use as enzyme sources in biotransformations.

Preparation of chemically functionalized biocompatible surfaces is of current interest, with application in the immobilization of various bioactive species such as DNA, enzymes, whole cells, etc. We report herein the one-step synthesis of a self-supporting gold nanoparticle membrane, its surface modification, and application in the immobilization of Candida bombicola (yeast) cells. The gold nanoparticle membrane is prepared by the spontaneous reduction of aqueous chloroaurate ions by a diamine at a liquid-liquid interface. The gold nanoparticles in the polymeric membrane may be capped with octadecylamine (ODA) molecules, thereby rendering the nanoparticle membrane hydrophobic. Exposure of the hydrophobized organic-gold nanoparticle membrane to C. bombicola yeast cells results in their binding to the membrane, possibly through nonspecific interactions such as hydrophobic interactions between the yeast cell walls and the ODA molecules. The enzyme cytochrome P450 present in the yeast cells immobilized on the organic-gold nanoparticle membrane was then used in the transformation of the arachidonic acid (AA) to sophorolipids followed by acid hydrolysis to form 20-hydroxyeicosatetraneoic acid (20-HETE). The organic-gold nanoparticle membrane-C. bombicola bioconjugate could be easily separated from the reaction medium and reused a number of times.

Adhesion of Candida biofilm cells to human epithelial cells and polystyrene after treatment with silver nanoparticles.

This study investigated the adhesion to human epithelial cells and polystyrene surface of viable yeasts recovered from Candida biofilms treated with silver nanoparticles (SN). Biofilm resuspended Candida cells were added to HeLa cells or to empty wells of microtiter plates and the adhesion was verified using crystal violet staining. The adhesion of Candida cells was significantly reduced, mainly when biofilms were pretreated with 54 µg/mL SN. These new findings allow to conclude that SN may induce changes in viable yeasts, which can decrease the dissemination of Candida infections, mainly in susceptible patients.

Self-aggregated nanoparticles based on amphiphilic poly(lactic acid)-grafted-chitosan copolymer for ocular delivery of amphotericin B.

BACKGROUND: The purpose of this study was to develop a self-aggregated nanoparticulate vehicle using an amphiphilic poly(lactic acid)-grafted-chitosan (PLA-g-CS) copolymer and to evaluate its potential for ocular delivery of amphotericin B. METHODS: A PLA-g-CS copolymer was synthesized via a "protection-graft-deprotection" procedure and its structure was confirmed by Fourier transform infrared spectroscopy, (1)H nuclear magnetic resonance, and X-ray diffraction spectra. Amphotericin B-loaded nanoparticles based on PLA-g-CS (AmB/PLA-g-CS) were prepared by the dialysis method and characterized for particle size, zeta potential, and encapsulation efficiency. Studies of these AmB/PLA-g-CS nanoparticles, including their mucoadhesive strength, drug release properties, antifungal activity, ocular irritation, ocular pharmacokinetics, and corneal penetration were performed in vitro and in vivo. RESULTS: Fourier transform infrared spectroscopy, (1)H nuclear magnetic resonance, and X-ray diffraction spectra showed that the PLA chains were successfully grafted onto chitosan molecules and that crystallization of chitosan was suppressed. The self-aggregated PLA-g-CS nanoparticles had a core-shell structure with an average particle size of approximately 200 nm and zeta potentials higher than 30 mV. Amphotericin B was incorporated into the hydrophobic core of the nanoparticles with high encapsulation efficiency. Sustained drug release from the nanoparticles was observed in vitro. The ocular irritation study showed no sign of irritation after instillation of the PLA-g-CS nanoparticles into rabbit eyes. The minimal inhibitory concentration of the AmB/PLA-g-CS nanoparticles showed antifungal activity similar to that of free amphotericin B against Candida albicans. The in vivo ocular pharmacokinetic study suggested that the PLA-g-CS nanoparticles have the advantage of prolonging residence time at the ocular surface. The corneal penetration study showed that the PLA-g-CS nanoparticles could penetrate into the cornea. CONCLUSION: Our results suggest that this nanoparticulate vehicle based on a PLA-g-CS copolymer might be a promising system for effective ocular delivery of amphotericin B.

Synthesis of duloxetine intermediate (S)-3-Chloro-1-(2-thienyl)-1-propanol with liquid-core immobilized Candida pseudotropicalis 104.

(S) -3-Chloro-1-(2-thienyl)-1-propanol was synthesized by the asymmetric reduction of 3-chloro-1-(2-thienyl)propanone with liquid-core immobilized Candida pseudotropicalis 104. The optimum time was 28 h for the re-cultivation of immobilized cells. The optimum film solvent for the liquid-core capsule was 0.3 % chitosan (M (w) 1.0 × 10(5)). Conversion decreased with the increase of the liquid-core capsule diameter and with the addition of more substrates at the same reduction time. The immobilized cells show good reduction ability in a potassium phosphate buffer (pH 6.6~7.2). The material outside the spread speed of immobilized cells was not restricted when the shaking speed was higher than 160 r/min. Liquid-core immobilized cells can be reused 11 times. Compared with the batch reduction, the continuous reduction of 3-chloro-1-(2-thienyl)propanone in the membrane reactor with liquid-core immobilized cells as catalyst can relieve the inhibition from a high-concentration substrate. Conversion and enantiometric excess of (S)-3-chloro-1-(2-thienyl)-1-propanol reached 100 % and >99 % in a continuous reduction of 12 g/L 3-chloro-1-(2-thienyl)propanone for 10 days.

Oral epithelial atypical changes in apparently healthy oral mucosa exposed to smoking, alcohol, peppers and hot meals, using the AgNOR and Papanicolaou staining techniques.

To evaluate cytological atypical changes in apparently healthy oral mucosa exposed to smoking, alcohol, hot meals, and peppers using the AgNOR and Papanicolaou methods. A total of 180 individuals were evaluated, of which 60 were smokers, 34 were alcohol users, 52 were habitual peppers and hot meal (exposed) consumers, 24 were non-exposed, and 10 were patients with Oral Squamous Cell Carcinoma (OSCC), as an internal control. Cytological materials were obtained by brushing of buccal mucosa, on the border of the tongue and on the floor of the mouth, and participants underwent the Papanicolaou test for cytological changes and AgNOR staining for evaluation of the mean number of AgNOR dots per nucleus. SPSS program was used to perform the Pearson chi-square test. The 95% confidence level, Odds Ratio (OR), and the 95% Confidence Intervals (CI) were used. The features of cytological atypia were verified among 10 individuals, including 5 smokers, 2 alcohol users, 2 hot meals and peppers consumers, and one non-exposed. For atypia among tobacco smokers, the adjusted Odds Ratio (OR) and the 95% CI were found to be 2 (0.246-16.24). Increased keratinization was detected among 27 (45%) of the smokers (P < 0.0001), 17 (32.7%) of the pepper and hot meals consumers (P < 0.005), 4 (11.8%) of the alcohol consumers, and among 2 (3.7%) of the non-exposed group. Statistical analyses revealed a greater mean number of AgNORs per nucleus in smokers (3.68) followed by (2.82) alcohol consumers, compared to the habitual peppers and hot meal consumers (2.28) and the non-exposed group (2.00). What's more, 80% of the smears with cytological atypia were identified with 6 +/- 2 AgNOR mean count. The increase of the variables suggests that the evaluation of epithelial atypical changes in individuals exposed to smoking and alcohol carcinogens may be a useful screening tool. While hot meals and peppers did not seem to be a risk for oral mucosal proliferation, they increased the potency of keratinization and infection.