Cellular apoptosis and necrosis as therapeutic targets for novel Eugenol Tosylate Congeners against Candida albicans.

Despite the rise of new Candida species, Candida albicans tops the list with high morbidity and mortality rates. To tackle this problem there is a need to explore new antifungals that could replace or augment the current treatment options. We previously reported that tosylation of eugenol on hydroxyl group resulted in molecules with enhanced antifungal potency. In line with that work, we synthesized new eugenol tosylate congeners (ETC-1-ETC-7) with different substituents on pendent sulfonyl group and tested their susceptibility against different fluconazole susceptible and resistant C. albicans strains. We evaluated physiology and mode of cell death in response to the most active derivatives by analyzing major apoptotic markers in yeast such as phosphatidylserine externalization, DNA fragmentation, mitochondrial depolarization and decrease in cytochrome c oxidase activity. The results demonstrated that all C. albicans strains were variably susceptible to the test compounds with MIC ranging from 0.125-512 µg/ml, and the most active compounds (ETC-5, ETC-6 and ETC-7) actuate apoptosis and necrosis in Candida cells in a dose-dependent manner via metacaspase-dependent pathway. Furthermore haemolytic assay showed low cytotoxicity effect of these ETCs. Overall the results indicated that ETCs exhibit potential antifungal activity against C. albicans by activating apoptotic and necrotic pathways.

Cranberry-derived proanthocyanidins induce a differential transcriptomic response within Candida albicans urinary biofilms.

Candida albicans is one of the most common causes of hospital-acquired urinary tract infections (UTIs). However, azoles are poorly active against biofilms, echinocandins do not achieve clinically useful urinary concentrations, and amphotericin B exhibits severe toxicities. Thus, novel strategies are needed to prevent Candida UTIs, which are often associated with urinary catheter biofilms. We previously demonstrated that cranberry-derived proanthocyanidins (PACs) prevent C. albicans biofilm formation in an in vitro urinary model. To elucidate functional pathways unique to urinary biofilm development and PAC inhibition, we investigated the transcriptome of C. albicans in artificial urine (AU), with and without PACs. C. albicans biofilm and planktonic cells were cultivated with or without PACs. Genome-wide expression analysis was performed by RNA sequencing. Differentially expressed genes were determined using DESeq2 software; pathway analysis was performed using Cytoscape. Approximately 2,341 of 6,444 total genes were significantly expressed in biofilm relative to planktonic cells. Functional pathway analysis revealed that genes involved in filamentation, adhesion, drug response and transport were up-regulated in urinary biofilms. Genes involved in carbon and nitrogen metabolism and nutrient response were down-regulated. In PAC-treated urinary biofilms compared to untreated control biofilms, 557 of 6,444 genes had significant changes in gene expression. Genes downregulated in PAC-treated biofilms were implicated in iron starvation and adhesion pathways. Although urinary biofilms share key features with biofilms formed in other environments, many genes are uniquely expressed in urinary biofilms. Cranberry-derived PACs interfere with the expression of iron acquisition and adhesion genes within urinary biofilms.

Iranian HIV/AIDS patients with oropharyngeal candidiasis: identification, prevalence and antifungal susceptibility of Candida species.

Oropharyngeal candidiasis is the commonest mucocutaneous infection in HIV-positive individuals. Herein, samples were taken from oral cavities of 150 HIV-infected patients and cultured on Sabouraud-dextrose agar; 89 (59·3%) of 150 patients had positive culture for Candida and presented clinical sign of classical oral candidiasis. Totally, 102 morphologically distinct colonies were isolated from Candida positive cultures and subsequently identified by polymerase chain reaction and sequencing assay, presenting the following frequency: 54 C. albicans (52·9%), 16 C. dubliniensis (15·7%), 12 C. tropicalis (11·8%), 9 C. glabrata (8·8%), 7 C. kefyr (6·9%) and 4 C. africana (3·9%). Additionally, multiple Candida species were co-isolated from 13·5% (12/89) patients. Regarding the antifungal susceptibility test, which was performed by CLSI protocol (M27-A3/M27-S3), all Candida isolates were susceptible to amphotericin B and caspofungin, while some of them were resistant to fluconazole (17·6%; 16 C. albicans, 1 C. dubliniensis and 1 C. glabrata), itraconazole (16·7%; 15 C. albicans, 1 C. dubliniensis and 1 C. tropicalis) and voriconazole (5·9%; 5 C. albicans and 1 C. tropicalis). Collectively, our findings reinforce the urgent necessity to find new therapeutic agents to treat oral candidiasis in HIV-positive patients, especially due to the high incidence of azole-resistant Candida strains and the increased frequency of non-C. albicans species. SIGNIFICANCE AND IMPACT OF THE STUDY: The Candida species recovered from oral cavity of 150 Iranian HIV/AIDS patients and their antifungal susceptibility profiles were reported. Candida albicans was the commonest Candida species, followed by C. dubliniensis, C. tropicalis, C. glabrata, C. kefyr and C. africana. All Candida isolates were susceptible to amphotericin B and caspofungin, while resistance to azoles was detected. The growing drug-resistance profile reported in clinical isolates of C. albicans and non-C. albicans strains is a serious problem in hospitals worldwide. Consequently, the suitable antifungal choice to treat the HIV/AIDS population with oral candidiasis needs to be rethought and new therapeutic options must urgently arise.

Perfil fitoquímico e determinação da atividade antimicrobiana de Syzygium cumini (L. ) Skeels (Myrtaceae) frente a microrganismos bucais / Phytochemical profile and determination of antimicrobial activity of Syzygiumcumini (L. ) Skeels (Myrtaceae) against oral microorganisms

RESUMO Este estudo teve como objetivo determinar o perfil fitoquímico e avaliar a atividade antimicrobiana in vitro do extrato etanólico da casca do caule de Syzygium cumini(L.) Skeels frente a microrganismos bucais. O perfil fitoquímico do extrato foi traçado através da determinação espectrofotométrica quantitativa para verificar o teor de taninos, flavonóides, saponinas e polifenóis. A atividade antimicrobiana foi determinada através da Concentração Inibitória Mínima (CIM), por meio da técnica de microdiluição em caldo, utilizando-se as seguintes linhagens de microrganismos: Streptococcus mutans (25175), Streptococcus oralis (10557) e Candida albicans (10231). Uma quantidade apreciável de fitocontituintes foi observada, especialmente de taninos (100,58 ± 1,81). Os extratos apresentaram atividade antimicrobiana inibindo o crescimento das linhagens em estudo, destacando-se essa atividade sobre o crescimento de C. albicans (CIM=250 µg/mL). Já as CIMs para Streptococcus foram baixas. Diante dos resultados expostos, pode-se concluir que o perfil fitoquímico foi traçado e que, dentre os microrganismos testados, o extrato etanólico da casca de S. cumini apresentou forte potencial de inibição sobre o crescimento de C. albicans e fraca inibição frente aos Streptococcus testados. Este estudo sugere que mais pesquisas devem ser realizadas dando continuidade à bioprospecção, por meio de análises experimentais com essa espécie vegetal, objetivando, no futuro, que essa planta possa ser utilizada clinicamente para tratar candidose bucal.

ABSTRACT This study aimed to determine the phytochemical profile and to evaluate the in vitro antimicrobial activity of the ethanol extract of stem bark of Syzygium cumini (L.) Skeels against oral microorganisms. The phytochemicalprofile of the extract was traced through a quantitative spectrophotometric determination in order to check the tannin, flavonoids, saponins, and polyphenols content. The antimicrobial activity was determined through minimum inhibitory concentration (MIC) by the broth microdilution technique, using the following strains of microorganisms: Streptococcus mutans (25175), Streptococcus oralis (10557) and Candida albicans (10231). An appreciable amount of fitocontituintes was observed, particularly the tannin (100.58 ± 1.81). The extracts showed antimicrobial activity, inhibiting the growth of the strains under study, with this activity being more intense on the growth of C. albicans ( MIC = 250 mg / mL). On the other hand, the MICs of the Streptococcus were low. In face of the mentioned results, we can conclude that the phytochemical profile was traced and that, among the tested microorganisms, the ethanol extract of S. cumini bark showed strong potential to inhibit the growth of C. albicans and weak inhibition against the Streptococcus tested. This study suggests that more research should be done by proceeding with the bioprospecting, through experimental tests with this plant`s species, aiming that in the future this substance can be used clinically for the treatment of oral candidiasis.

Impact of antifungal prophylaxis on colonization and azole susceptibility of Candida species.

Two large studies compared posaconazole and fluconazole or itraconazole for prophylaxis in subjects undergoing allogeneic hematopoietic stem cell transplantation or subjects with acute myelogenous leukemia. To assess the impact of prophylaxis on colonization and the development of resistance in Saccharomyces yeasts, identification and susceptibility testing were performed with yeasts cultured at regular intervals from mouth, throat, and stool samples. Prior to therapy, 34 to 50% of the subjects were colonized with yeasts. For all three drugs, the number of positive Candida albicans cultures decreased during drug therapy. In contrast, the proportion of subjects with positive C. glabrata cultures increased by two- and fourfold in the posaconazole and itraconazole arms, respectively. Likewise, in the fluconazole arm the proportion of subjects with positive C. krusei cultures increased twofold. C. glabrata was the species that most frequently exhibited decreases in susceptibility, and this trend did not differ significantly between the prophylactic regimens. For the subset of subjects from whom colonizing C. glabrata isolates were recovered at the baseline and the end of treatment, approximately 40% of the isolates exhibited more than fourfold increases in MICs during therapy. Molecular typing of the C. albicans and C. glabrata isolates confirmed that the majority of the baseline and end-of-treatment isolates were closely related, suggesting that they were persistent colonizers and not newly acquired. Overall breakthrough infections by Candida species were very rare (approximately 1%), and C. glabrata was the colonizing species that was the most frequently associated with breakthrough infections.

Differentiation of Candida dubliniensis from Candida albicans on rosemary extract agar and oregano extract agar.

Candida dubliniensis is a recently described pathogenic species which shares many phenotypic features with Candida albicans and therefore, may be misidentified in microbiological laboratories. Because molecular methods can be onerous and unfeasible in routine mycological laboratories with restricted budgets such as those in developing countries, phenotypic techniques have been encouraged in the development of differential media for the presumptive identification of these species. We examined the colony morphology and chlamydospore production of 30 C. dubliniensis isolates and 100 C. albicans isolates on two new proposed media: rosemary (Rosmarinus officinalis) extract agar (REA) and oregano (Origanum vulgare) extract agar (OEA). These substrates are traditionally used as spices and medicinal herbs. In both of these media, all C. dubliniensis isolates (100%) showed rough colonies with peripheral hyphal fringes and abundant chlamydospores after 24 to 48 hr of incubation at 25 degrees C. In contrast, under the same conditions, all isolates of C. albicans (100%) showed smooth colonies without hyphal fringes or chlamydospores. In conclusion, REA and OEA offer a simple, rapid, and inexpensive screening media for the differentiation of C. albicans and C. dubliniensis.

Characterization of switch phenotypes in Candida albicans biofilms.

The aim of this study was to characterize switch phenotypes in Candida albicans biofilms. Cells of Candida albicans 192887g biofilms (24 h) were resuspended and these together with their planktonic counterparts were separately inoculated on Lee's medium agar supplemented with arginine and zinc, at 25 degrees C for 9 days, for colony formation. The different switch phenotypes, as reflected by varying colony morphologies, were then examined for their (i) stability under various growth conditions, (ii) carbohydrate assimilation profiles, (iii) susceptibility to the polyene antifungal, nystatin, (iv) adhering and biofilm-forming ability, (v) filamentation, and (vi) growth rate in yeast nitrogen base medium supplemented with 100 mM glucose. Our data showed that the frequency of phenotypic switching in C. albicans biofilms was approximately 1%. Compared with the planktonic yeasts, cells derived from candidal biofilms generated one of the phenotypes less frequently (Chi-square-tests: P = 0.017). The five phenotypes derived from the biofilm growth demonstrated differing profiles for carbohydrate assimilation, adhesion, biofilm formation, filamentation, and growth rate. These findings reported here, for the first time, imply that phenotypic switching in the candidal biofilms differs from that in the planktonic growth, and affects multiple biological attributes.

Azole susceptibility patterns and genetic relationship among oral Candida strains isolated in the era of highly active antiretroviral therapy.

We performed a cross-sectional study to analyze patterns of azole susceptibility of oral isolates in the highly active antiretroviral therapy (HAART) era and compared current data with those obtained for isolates from 1994. We further identified patients with relapsing oral pharyngeal candidiasis (OPC) who had been included in a similar study in 1994. For these subjects, we compared the susceptibility pattern for the OPC isolates, and if a modification of azole resistance was observed, we analyzed the genotypic pattern for the 1994 and 2000 isolates to determine whether the dominant strain was closely related. We included 69 consecutive HIV-infected subjects with 137 episodes of OPC who were admitted to our ward from January to June 2000. Ninety-two strains (67%) and 21 non- strains (15%) were isolated. We identified 24 episodes of OPC caused by two different species. Compared with the pre-HAART era, the fluconazole resistance of isolates significantly decreased from 45% to 10% (p <.001). Itraconazole resistance decreased from 37% to 7% (p <.001). Ketoconazole resistance (3% in 1994) was not detected more. Nine patients whose isolate was susceptible to all azole drugs had a previous isolate resistant to all azole drugs. We observed that these isolates exhibited different fingerprint profiles. Our findings demonstrate that most cases of HIV-associated OPC observed in the HAART era are caused by azole-susceptible strains. The reversion of an isolate from azole resistant to azole susceptible is related to strain replacement.

Replacement of Candida albicans with C. dubliniensis in human immunodeficiency virus-infected patients with oropharyngeal candidiasis treated with fluconazole.

Candida dubliniensis is an opportunistic yeast that has been increasingly implicated in oropharyngeal candidiasis (OPC) in human immunodeficiency virus (HIV)-infected patients but may be underreported due to its similarity with Candida albicans. Although most C. dubliniensis isolates are susceptible to fluconazole, the inducibility of azole resistance in vitro has been reported. Thus, the use of fluconazole prophylaxis in the treatment of these patients may have contributed to the increasing rates of isolation of C. dubliniensis. In this study, yeast strains were collected from the oral cavities of HIV-infected patients enrolled in a longitudinal study of OPC. Patients received fluconazole for the suppression or treatment of OPC, and isolates collected at both study entry and end of study were chosen for analysis. Samples were plated on CHROMagar Candida medium for initial isolation and further identified by Southern blot analysis with the species-specific probes Ca3 (for C. albicans) and Cd25 (for C. dubliniensis). Fluconazole MICs were determined by using NCCLS methods. At study entry, susceptible C. albicans isolates were recovered from oral samples in 42 patients who were followed longitudinally (1 to 36 months). C. albicans strains from 12 of these patients developed fluconazole resistance (fluconazole MIC, >/=64 micro g/ml). C. dubliniensis was not detected at end of study in any of these patients. Of the remaining 30 patients, eight (27%) demonstrated a replacement of C. albicans by C. dubliniensis when a comparison of isolates obtained at baseline and those from the last culture was done. For the 22 of these 30 patients in whom no switch in species was detected, the fluconazole MICs for initial and end-of-study C. albicans isolates ranged from 0.125 to 2.0 micro g/ml. For the eight patients in whom a switch to C. dubliniensis was detected, the fluconazole MICs for C. dubliniensis isolates at end of study ranged from 0.25 to 64 micro g/ml: the fluconazole MICs for isolates from six patients were 0.25 to 2.0 micro g/ml and those for the other two were 32 and 64 micro g/ml, respectively. In conclusion, a considerable number of patients initially infected with C. albicans strains that failed to develop fluconazole resistance demonstrated a switch to C. dubliniensis. C. dubliniensis in this setting may be underestimated due to lack of identification and may occur due to the impact of fluconazole on the ecology of oral yeast species.

Effect of commercial ethanol propolis extract on the in vitro growth of Candida albicans collected from HIV-seropositive and HIV-seronegative Brazilian patients with oral candidiasis.

The present study assessed the susceptibility of Candida albicans strains, collected from HIV-positive patients with oral candidiasis, to a commercial 20% ethanol propolis extract (EPE) and compare it to the inhibitory action of the standardized antifungal agents nystatin (NYS), clotrimazole (CL), econazole (EC), and fluconazole (FL). Twelve C. albicans strains collected from HIV-positive patients with oral candidiasis were tested. The inhibition zones were measured with a pachimeter and the results are reported as means and standard deviation (M +/- SD). Data were analyzed statistically by the non-parametric Kruskal-Wallis test. EPE inhibited all the C. albicans strained tested. No significant difference was observed between the results obtained with NYS and EPE, while significant differences were observed between EPE and other antifungals. The C. albicans strains tested showed resistance to the remaining antifungal agents. The propolis extract used in this study inhibited the in vitro growth of C. albicans collected from HIV-seropositive Brazilian patients, creating/forming inhibition zones like those ones formed by NYS. This fact suggests that commercial EPE could be an alternative medicine in the treatment of candidiasis from HIV-positive patients. However, in vivo studies of the effect of EPE are needed to determine its possible effects on the oral mucosa.

Oral fungal colonization and oral candidiasis in renal transplant patients: the relationship to Miswak use.

OBJECTIVES: The aim of this study was to determine and compare the prevalence of oral candidal colonization and oral candidiasis in a group of medically stable renal transplant patients (RTPs) and age and sex-matched healthy control (HC) subjects. STUDY DESIGN: The oral cavities of 58 RTPs and 52 HC subjects were clinically examined for the presence of oral candidiasis. Oral fungal colonization was determined by using the concentrated oral rinse technique. RESULTS: Prevalence of oral fungal colonization was not significantly higher in RTPs than in HC subjects (74.1% vs 59.6%, respectively; P =.1), but the density of growth was significantly higher in RTPs (P <.0017). Oral candidiasis was diagnosed in 15.5% of RTPs but in none of HC subjects (P =.002). RTPs who used a chewing stick (Miswak: Salvadora persica ) for oral hygiene had a significantly lower prevalence of oral candidiasis (P =.04) compared with other RTPs. CONCLUSIONS: RTPs are at high risk of developing oral candidiasis. More clinical investigations are needed to determine the antimycotic effect of Miswak. Regular oral screening is recommended for RTPs.

DNA fingerprinting of serial Candida albicans isolates obtained during itraconazole prophylaxis in patients with AIDS.

During a randomized double-blind placebo-controlled study testing the efficacy of itraconazole for prophylaxis of systemic and mucosal fungal infections in patients with acquired immune deficiency syndrome, 298 patients were enrolled with 295 evaluable. Of those, 46 patients were considered prophylaxis failures because of recurrent oral or esophageal candidiasis. Oropharyngeal fungal cultures were taken at the time of suspected thrush or Candida esophagitis, but not at baseline. All of the Candida spp. isolates were cultured on CHROMagar Candida medium then identified using API 20 AUX strips. Antifungal susceptibility testing was performed following the National Committee for Clinical Laboratory Standards M-27A guidelines. Sequential isolates were genotyped using randomly amplified polymorphic DNA. Polymerase chain reaction fingerprints were generated using two repetitive sequence primers, (GGA)7 and (GACA)4. The study group consisted of 23 patients, nine from the itraconazole arm and 14 from the placebo arm, who were prophylaxis failures and had more than two C. albicans isolates. Five of 23 had isolates showing a > or =4-fold reduction in susceptibility; four of these patients were in the itraconazole prophylaxis arm and one was in the placebo arm. Three of the five had yeast isolations showing changes in banding patterns over time. Such changes may indicate genetic changes in the same strain that could be linked to acquired resistance to itraconazole, or acquisition of a new strain, or emergence of a previously minor component of the original population.

Characterization and quantitation of the pharmacodynamics of fluconazole in a neutropenic murine disseminated candidiasis infection model.

We determined the pharmacodynamic parameter and the magnitude of that parameter that was predictive of the efficacy of fluconazole in the treatment of disseminated candidiasis. We used a neutropenic murine model of disseminated Candida albicans infection to characterize the time course of activity of fluconazole. Quantitation of colony counts in kidneys after 24 h of therapy with a wide range of doses and three dosing intervals was used to determine the dose required to achieve 50% of the maximal effect (ED(50)). The ED(50) was similar for each of the dosing intervals studied, supporting the area under the concentration-time curve (AUC) MIC ratio as the parameter that predicts the efficacy of fluconazole. Similar studies were performed with C. albicans strains for which fluconazole MICs are in the susceptible-dose-dependent range (MICs, 16 to 32 mg/liter). We found that the magnitude of the AUC/MIC ratio required to reach the ED(50) was similar for all three organisms studied, ranging from 12 to 25. When the pharmacokinetics of fluconazole in humans are considered, these AUC/MIC ratios would support in vitro susceptibility breakpoints of 8 mg/liter for dosages of 200 mg/day and susceptibility breakpoints of 16 to 32 mg/liter for dosages of 400 to 800 mg/day.

[Genotype, serotype and sensitivity to fluconazole of Candida albicans strains isolated from HIV-positive patients]. / Génotype, sérotype et sensibilité au fluconazole de souches de Candida albicans isolées chez des patients VIH positif.

Genotype, serotype and susceptibility in vitro to fluconazole of 104 C. albicans strains isolated from HIV+ patients were studied. The possible correlations between genotype analysed by multilocus enzyme electrophoresis (MLEE) and phenotype of medical relevance (serotype and susceptibility to fluconazole) of Candida albicans isolated from these patients treated with fluconazole were evaluated by factorial correspondence analysis. No correlation was observed between genotype and in vitro or clinical response to fluconazole. In counterpart, serotype B C. albicans was associated with some multilocus genotypic patterns.

Oropharyngeal candidiasis in AIDS patients from Abidjan (Ivory Coast): antifungal susceptibilities and multilocus enzyme electrophoresis analysis of Candida albicans isolates.

Multilocus enzyme electrophoresis (MEE) and in vitro antifungal susceptibility testing were used to investigate the Candida albicans strain diversity in twenty nine AIDS patients from Abidjan (Ivory Coast). All patients were monitored for a first episode of oropharyngeal candidiasis and were randomly clustered into three groups of therapy: ketoconazole, amphotericin B or nystatin. Oral swabs were collected before every treatment, 14 and 30 days after the initiation of the therapy; a total of 67 isolates were investigated. No resistant or less susceptible isolate to any antifungal agent was found despite the emergence of clinical relapses, mainly for patients treated with nystatin or amphotericin B. The MEE analysis revealed 27 different electrophoretic types (ETs). Genetic distances between ETs were statistically analyzed and represented on a dendrogram. The 27 ETs clustered into three groups; in each group, ETs represented variants of the same strain. A segregation of the C. albicans isolates seemed to be as a function of the serotype.

Evaluation of the susceptibility of pathogenic Candida species to fluconazole. Fluconazole Global Susceptibility Study Group.

A fluconazole 25 microg disk diffusion test was used to test 2230 consecutively isolated Candida strains from 42 different hospital laboratories in 23 countries. Ninety seven percent of 1634 Candida albicans isolates and 83.4% of 596 non-Candida albicans isolates were susceptible to fluconazole, applying the proposed breakpoints (> or = 26 mm for susceptible strains and 18-25 mm for dose-dependent susceptible strains). This is the first hospital laboratory study to evaluate a large number and wide range of sequential Candida isolates from patients with all types of hospital infections. The fluconazole disk diffusion test appears to be a low-cost, reproducible, and accurate means of assessing the in vitro susceptibility of Candida isolates.

Resistance of Candida albicans to fluconazole during treatment of oropharyngeal candidiasis in a patient with AIDS: documentation by in vitro susceptibility testing and DNA subtype analysis.

We describe a patient with recurrent episodes of oropharyngeal candidiasis who required progressively higher doses of fluconazole to control and infection. The patient was treated for 14 infections over a 2-year period with doses of fluconazole that ranged from 100 to 800 mg per day. Clinical response, two methods of in vitro susceptibility testing, and molecular epidemiologic techniques were evaluated for 12 of the 14 episodes. Ultimately, the patient became unresponsive clinically to a dose of 800 mg of fluconazole per day. In vitro susceptibility testing of isolates obtained during these successive episodes of infection revealed the development of resistance to fluconazole, and molecular epidemiologic techniques confirmed the persistence of the same Candida albicans strain throughout all 12 episodes.