Comparison between allicin and fluconazole in Candida albicans biofilm inhibition and in suppression of HWP1 gene expression

Candida albicans is an opportunistic human pathogen with the ability to differentiate and grow in filamentous forms and exist as biofilms. The biofilms are a barrier to treatment as they are often resistant to the antifungal drugs. In this study, we investigated the antifungal activity of allicin, an active compound of garlic on various isolates of C. albicans. The effect of allicin on biofilm production in C. albicans as compared to fluconazole, an antifungal drug, was investigated using the tetrazolium (XTT) reduction-dependent growth and crystal violet assays as well as scanning electron microscopy (SEM). Allicin-treated cells exhibited significant reduction in biofilm growth (p<0.05) compared to fluconazole-treated and also growth control cells. Moreover, observation by SEM of allicin and fluconazole-treated cells confirmed a dose-dependent membrane disruption and decreased production of organisms. Finally, the expression of selected genes involved in biofilm formation such as HWP1 was evaluated by semi-quantitative RT-PCR and relative real time RT-PCR. Allicin was shown to down-regulate the expression of HWP1.     

Antifungal susceptibility of epigallocatechin 3-O-gallate (EGCg) on clinical isolates of pathogenic yeasts

This is the first report to investigate the antifungal susceptibility of 21 clinical isolates of seven Candida species to epigallocatechin 3-O-gallate (EGCg) and to compare with six antifungal agents, amphotericin B (AMPH), fluconazole (FLCZ), flucytosin (5FC), itraconazole (ITCZ), micafungin (MCFG), and miconazole (MCZ), using a method following the National Committee for Clinical Laboratory Standards (NCCLS) M27-A guidelines. Among the tested species, Candida glabrata exhibited the highest susceptibility to EGCg (MIC50, 0.5-1 microg/ml and MIC90, 1-2 microg/ml) compared favorably with FLCZ, although they were slightly less susceptible than to AMPH, 5FC, MCFG, ITCZ, and MCZ. Candida guilliemondii and Candida parapsilosis (MIC50, 1-4 microg/ml and MIC90, 2-16 microg/ml) were also susceptible to EGCg, although they appear to be slightly less susceptible to EGCg than C. glabrata and the other antifungal agents tested. Moreover, the susceptibility of Candida krusei strains (MIC50, 2 microg/ml and MIC90, 4-8 microg/ml) to EGCg was approximately 2- to 8-fold higher than those of 5FC and FLCZ. Our data indicate that EGCg can inhibit clinically pathogenic Candida species, although the concentrations of EGCg for antifungal susceptibility were slightly higher than those of tested antifungal agents on the whole. Based on these results, we suggest that EGCg may be effectively used as a possible agent or adjuvant for antifungal therapy in Candidiasis.     

Repurposing the FDA-approved anticancer agent ponatinib as a fluconazole potentiator by suppression of multidrug efflux and Pma1 expression in a broad spectrum of yeast species

Fungal infections have emerged as a major global threat to human health because of the increasing incidence and mortality rates every year. The emergence of drug resistance and limited arsenal of antifungal agents further aggravates the current situation resulting in a growing challenge in medical mycology. Here, we identified that ponatinib, an FDA-approved antitumour drug, significantly enhanced the activity of the azole fluconazole, the most widely used antifungal drug. Further detailed investigation of ponatinib revealed that its combination with fluconazole displayed broad-spectrum synergistic interactions against a variety of human fungal pathogens such as Candida albicans, Saccharomyces cerevisiae and Cryptococcus neoformans. Mechanistic insights into the mode of action unravelled that ponatinib reduced the efflux of fluconazole via Pdr5 and suppressed the expression of the proton pump, Pma1. Taken together, our study identifies ponatinib as a novel antifungal that enhances drug activity of fluconazole against diverse fungal pathogens.     

地塞米松影响念珠菌对抗真菌药物敏感性的实验研究

目的 探讨地塞米松在体外试验中是否影响念珠菌对抗真菌药物的敏感性,以了解糖皮质激素与抗真菌药物直接作用于念珠菌时是否存在相互作用。方法 用微量液体培养基稀释法分别测定26株白念珠菌与地塞米松（0．2mg／ml）共同孵育前、孵育24～48h及7d时氟康唑、伊曲康唑、两性霉素B的最低抑菌浓度（MIC）值,并作对照。结果 白念珠菌与地塞米松孵育24～48h后、孵育后第7d氟康唑和伊曲康唑的MIC值升高,分别与孵育前的MIC值存在统计学差异,但孵育24～48h后的MIC与孵育后第7d的MIC无统计学差异;白念珠菌与地塞米松共同孵育24～48h后两性霉素B的MIC值也较孵育前升高,但第7d的MIC值与孵育前无差异。结论 地塞米松可增加三种抗真菌药物对于白念珠菌的MIC,但三种抗真菌药物间存在差异,表明地塞米松对于氟康唑和伊曲康唑体外抗白念珠菌的活性有拮抗作用,但没有时间依赖性,地塞米松对于两性霉素B的影响较氟康唑和伊曲康唑小,且影响时间较短。     

Molecular Analysis and Susceptibility Profiling of Candida albicans Isolates from Immunocompromised Patients in South India

The genetic diversity and in vitro antifungal susceptibility profiles of 55 Candida albicans from immunocompromised patients were studied. PCR based analysis of the transposable intron in the 25S rDNA revealed 39 genotype A, 4 genotype B and 12 genotype C isolates. Serotype analysis categorized 52 isolates as serotype A and 3 as serotype B. All strains were susceptible to micafungin, 5-flucytosine and miconazole, whereas resistance against amphotericin B (3.6%), fluconazole (3.6%), itraconazole (7.3%) and voriconazole (5.5%) was observed. No association was seen between antifungal resistance and genotype/serotype status.     

Design, synthesis, and evaluations of antifungal activity of novel phenyl(2H-tetrazol-5-yl)methanamine derivatives

Fungal infections pose a continuous and serious threat to human health and life. The intrinsic resistance has been observed in many genera of fungi. Many fungal infections are caused by opportunistic pathogens that may be endogenous (Candida infections) or acquired from the environment (Cryptococcus and Aspergillus infections). So, new therapeutic strategies are needed to combat various fungal infections. Fluconazole shows good antifungal activity with relatively low toxicity and is preferred as first line antifungal therapy, but it has suffered from severe drug resistance. So, there is a need to design novel analogues by modification of fluconazole-like structure. A novel series of phenyl(2H-tetrazol-5-yl)methanamine derivatives were synthesized by reaction of α-amino nitrile with sodium azide and ZnCl₂ in presence of isopropyl alcohol. They were evaluated for antifungal activity against Candida albicans and Aspergillus niger and subjected to docking study against 1EA1.     

Correlation between broth microdilution and disk diffusion methods for antifungal susceptibility testing of caspofungin, voriconazole, amphotericin B, itraconazole and fluconazole against Candida glabrata

Candida glabrata is one of the most frequent organisms isolated from superficial and invasive fungal infections, after Candida albicans. This organism also exhibits intrinsically low susceptibility to azole antifungals and treatment often fails. The microdilution method is not very practical for use in routine susceptibility testing in the clinical laboratory, thus necessitating the use of other methods. In this study, we compared the in vitro activity of five antifungal agents in three different groups (echinocandin, polyene and azole) against 50 C. glabrata isolates by broth microdilution and disk diffusion methods recommended by Clinical Laboratory Standards Institute CLSI M27-A3 and CLSI M44-A, respectively. All the isolates were susceptible to amphotericin B (100%) and 98% of the isolates were susceptible to caspofungin by the broth microdilution method. Within the azole group drugs, voriconazole was the most active followed by fluconazole and itraconazole in vitro. The highest rate of resistance was obtained against itraconazole with a high number of isolates defined as susceptible-dose dependent or resistant. Although the disk diffusion method is easy to use in clinical laboratories, it shows very poor agreement with the reference method for fluconazole and itraconazole against C. glabrata (8% and 14%, respectively).     

Evolution of drug resistance in experimental populations of Candida albicans

Adaptation to inhibitory concentrations of the antifungal agent fluconazole was monitored in replicated experimental populations founded from a single, drug-sensitive cell of the yeast Candida albicans and reared over 330 generations. The concentration of fluconazole was maintained at twice the MIC in six populations; no fluconazole was added to another six populations. All six replicate populations grown with fluconazole adapted to the presence of drug as indicated by an increase in MIC; none of the six populations grown without fluconazole showed any change in MIC. In all populations evolved with drug, increased fluconazole resistance was accompanied by increased resistance to ketoconazole and itraconazole; these populations contained ergosterol in their cell membranes and were amphotericin sensitive. The increase in fluconazole MIC in the six populations evolved with drug followed different trajectories, and these populations achieved different levels of resistance, with distinct overexpression patterns of four genes involved in azole resistance: the ATP-binding cassette transporter genes, CDR1 and CDR2; the gene encoding the target enzyme of the azoles in the ergosterol biosynthetic pathway, ERG11; and the major facilitator gene, MDR1. Selective sweeps in these populations were accompanied by additional genomic changes with no known relationship to drug resistance: loss of heterozygosity in two of the five marker genes assayed and alterations in DNA fingerprints and electrophoretic karyotypes. These results show that chance, in the form of mutations that confer an adaptive advantage, is a determinant in the evolution of azole drug resistance in experimental populations of C. albicans.     

Antifungal drug resistance pattern of Candida. spp isolated from vaginitis in Ilam-Iran during 2013-2014

Vaginal Candidiasis is the most common and important opportunistic fungal infection in women. By increasing use of antifungal drugs in recent years, it has caused drug resistance. This study aims to evaluate antifungal drugs susceptibility of Candida. spp isolated of women with vaginitis from Ilam-Iran during 2013-2014. samples were collected and cultured from 385 women with vaginitis, then Candida.spp was diagnosed by standard method. Antifungal drug susceptibility test for nystatin 100 unit/disk, fluconazole 10µg/disk, itraconazole 10µg/disk, ketoconazole 10µg/disk, amphotericinB 20µg/disk, clotrimazole 10µg/disk, posaconazole 5µg/disk, and voriconazole 1µg/disk were carried out by M44-A method(CLSI). From all culture positive samples, 150 isolates were Candida albicans and 89 isolates were non-albicans. The resistance to fluconazole, itraconazole, ketoconazole, clotrimazole, voriconazole, posaconazole, nystatin and amphotericin B was 76%, 62%, 72%, 55%, 6%, 7%, 1% and 0%. The highest resistance was seen for fluconazole , itraconazole, and the highest susceptible was seen for nystatin and amphotericin B. These results indicate nystatin and amphotericin B can be used as the first line for empirical therapy of vaginal candidiasis in the district.     

Fluconazole downregulates metallothionein expression and increases copper cytotoxicity in Microsporum canis

Azole antifungals are widely used to treat infections with dermatophyte fungi. Whereas it is well established that this class of drugs interferes with fungal ergosterol synthesis, little is known about its potential other biological effects. Here we report the isolation and structural organization of Microsporum canis metallothionein gene and demonstrate that fluconazole is able to downregulate the baseline as well as copper-induced expression of this gene. Since this effect occurred within 30 min after exposure of the fungus to fluconazole, it is unlikely that it is due to impaired ergosterol synthesis. Our additional demonstration that fluconazole enhances copper toxicity for M. canis suggests that inhibition of metallothionein expression by fluconazole is biologically relevant and may represent an important additional mode of the antifungal action of this drug. Therefore our data indicate that antifungal effects of azole derivatives might not only be due to interference with cell wall synthesis but may also affect other biological circuits within the fungal cells.     

The activity of echinocandins, amphotericin B and voriconazole against fluconazole-susceptible and fluconazole-resistant Brazilian Candida glabrata isolates

The extensive use of azole antifungal agents has promoted the resistance of Candida spp to these drugs. Candida glabrata is a problematic yeast because it presents a high degree of primary or secondary resistance to fluconazole. In Brazil, C. glabrata has been less studied than other species. In this paper, we compared the activity of three major classes of antifungal agents (azoles, echinocandins and polyenes) against fluconazole-susceptible (FS) and fluconazole-resistant (FR) C. glabrata strains. Cross-resistance between fluconazole and voriconazole was remarkable. Among the antifungal agents, the echinocandins were the most effective against FS and FR C. glabrata and micafungin showed the lowest minimal inhibitory concentrations.     

Multicenter survey of in vitro antifungal resistance in yeasts of medical importance isolated from Spanish patients

Twelve Spanish laboratories collected 325 yeast clinical isolates during a 30 day's period, among them 224 Candida albicans, 30 Candida glabrata, and 27 Candida parapsilosis. In vitro antifungal susceptibility to amphotericin B, ketoconazole, fluconazole and itraconazole was determined by an agar diffusion test (Neo-Sensitabs, Rosco, Denmark). All the isolates tested were susceptible in vitroto amphotericin B and nearly all (97.2%) to itraconazole. In vitrosusceptibility to fluconazole and ketoconazole was high (90.2% and 91.4% of isolates, respectively) but showed variations depending on the species tested. Resistance to fluconazole and ketoconazole was low in C. albicans (4% and 3%, respectively), but 30% of Candida guilliermondii and 36% of C. glabrata isolates were resistant to fluconazole. Ketoconazole resistance was observed in 40% of C. glabrata, and 17% of Candida tropicalis. Resistance to antifungal drugs is very low in Spain and it is related to non-C. albicans isolates.     

Silver nanoparticles,a promising treatment against clinically important fluconazole-resistant Candida glabrata

Resistance to azole antifungal agents is a challenging limitation in Candida glabrata treatment. It is associated with decreased intracellular concentrations of antifungal agents as a result of overexpression of efflux pumps on the cellular plasma membranes. This work evaluates the potential of silver nanoparticles (AgNPs) to reverse the resistance of fungal cells to fluconazole. Silver nanoparticles were prepared using wet chemical method and characterised by UV-Vis spectrophotometry, dynamic light scattering, and zeta potential. Broth microdilution and pour plates methods were used to study the anticandidal activity using two C. glabrata fluconazole-resistant strains (DSY565 and CBS138) known to overexpress active efflux pumps, and a standard fluconazole sensitive strain ATCC 22553. Silver nanoparticles–fluconazole combinations decreased concentrations of fluconazole substantially without compromising the activity. These findings suggest that AgNPs enhance the efficacy of fluconazole and offer a promising application in therapy of C. glabrata infections.     

In vitro resistance to fluconazole and itraconazole in clinical isolates of Candida spp and Cryptococcus neoformans

An in vitro susceptibility testing of 181 strains of six species of Candida and 21 strains of Cryptococcus neoformans was carried out in order to investigate the resistance to new antifungal drugs. We have studied clinical isolates from 200 different patients of Hospital del Mar (Barcelona) and Hospital La Inmaculada (Almería). An agar diffusion method (NeoSensitabs, Rosco, Taastrup, Denmark), was employed with fluconazole, itraconazole, and reference drugs amphotericin B, flucytosine, tioconazole and ketoconazole. A high level of susceptibility was found for amphotericin B in C. neoformans strains while 19% of them were resistant to flucytosine. All the strains of C. neoformans and Candida guilliermondii were susceptible to the new azoles derivatives and also Candida parapsilosis and Candida albicans had a great susceptibility to this antifungals. A greater level of resistance was found for Candida krusei, Candida tropicalis and Candida glabrata to fluconazole, itraconazole and ketoconazole, but resistance to fluconazole and itraconazole is not always linked because ten resistant strains for fluconazole were susceptible to itraconazole, and two other resistant to itraconazole were susceptible to fluconazole.     

Effect of farnesol on Candida dubliniensis biofilm formation and fluconazole resistance

Candida dubliniensis and Candida albicans are dimorphic fungal species with a number of pathogenic capabilities, including biofilm formation, systemic infection and development of fluconazole resistance. In this study, the ability of farnesol to disrupt these virulence capabilities was investigated. Biofilm assessment and susceptibility studies indicated antifungal and antibiofilm properties for farnesol on both species with a disruptive effect on the cell membrane. Synergy testing of farnesol and fluconazole in resistant strains resulted in reversal of fluconazole resistance, indicating a potential application for farnesol as an adjuvant therapeutic agent.     

Medical importance of biofilms in Candida infections

Many Candida infections involve biofilm formation on implanted devices such as an indwelling catheter, a prosthetic heart valve or a denture. Candida biofilms can be formed in vitro using several model systems. In the simplest of these, organisms are grown on the surfaces of small discs of catheter material or denture acrylic. Biofilms of C. albicans prepared in this way consist of matrix-enclosed microcolonies containing yeasts, hyphae and pseudohyphae, arranged in a bilayer structure. Candida biofilms are resistant to a range of antifungal agents in current clinical use, including amphotericin B and fluconazole. Current research suggests that multiple mechanisms are involved in biofilm drug resistance.     

重症监护病房真菌谱及耐药性6年变化趋势

目的 调查我院重症监护室（ICU）近6 a临床分离真菌株的菌种结构及耐药性变化.方法 2007 ～2012年从我院1435例ICU患者中分离出的1 435株真菌均为首次分离的菌株.采用Cox-Stuart趋势检验回顾性分析了分离真菌株的菌种结构和主要真菌对常用抗真菌药物耐药率的变化趋势.结果 2007 ～2012年间,我院tCU真菌检出阳性标本总数从159株/a逐年增加至396株/a.其中,白念珠菌、近平滑念珠菌、曲霉的构成比波动范围分别为34.6％ ～ 50.3％、6.3％～9.6％、1.3％～3.2％,2010 ～2012年较2007 ～2009年构成比总体均有上升趋势,P=0.02.热带念珠菌、光滑念珠菌、克柔念珠菌构成比波动范围分别为13.6％ ～ 17.3％、9.4％ ～ 22.6％、1.4％ ～5.2％,P=0.18,构成比均无明显上升或下降趋势.2010 ～2012年较2007 ～2009年,白念珠菌对氟康唑和伊曲康唑耐药率的变化均呈降低趋势,P=0.02.热带念珠菌对氟康唑和伊曲康唑耐药率的变化均呈降低趋势,P =0.02;光滑念珠菌对伊曲康唑耐药率的变化总体呈上升趋势,P =0.02.结论 我院ICU近6 a来白念珠菌仍然为临床最常见的真菌分离株,白念珠菌、近平滑念珠菌及曲霉占总真菌分离株的百分比有上升趋势.白念珠菌及热带念珠菌对氟康唑和伊曲康唑耐药率均呈降低趋势,光滑念珠菌对伊曲康唑耐药率呈上升趋势.     

Evolutionary dynamics of Candida albicans during in vitro evolution

While mechanisms of resistance to major antifungal agents have been characterized in Candida albicans, little is known about the evolutionary trajectories during the emergence of drug resistance. Here, we examined the evolutionary dynamics of C. albicans that evolved in vitro in the presence or absence of fluconazole using the visualizing evolution in real-time (VERT) method, a novel experimental approach that facilitates the systematic isolation of adaptive mutants that arise in the population. We found an increase in the frequency of adaptive events in the presence of fluconazole compared to the no-drug controls. Analysis of the evolutionary dynamics revealed that mutations that led to increased drug resistance appeared frequently and that mutants with increased levels of resistance arose in independent lineages. Interestingly, most adaptive mutants with increased fitness in the presence of the drug did not exhibit a significant fitness decrease in the absence of the drug, supporting the idea that rapid resistance can arise from mutations in strains maintained in the population prior to exposure to the drug.     

Antifungal activity of immunosuppressants used alone or in combination with fluconazole

Fungal infections remain a challenge to clinicians due to the limited available antifungals. With the increasing use of antifungals in clinical practice, drug resistance has been emerging continuously, especially to fluconazole (FLC). Thus, a search for new antifungals and approaches to overcome antifungal resistance is needed. However, the development of new antifungals is usually costly and time consuming; discovering the antifungal activity of non-antifungal agents is one way to address these problems. Interestingly, some researchers have demonstrated that several classes of immunosuppressants (calcineurin inhibitors, glucocorticoids, etc) also displayed potent antifungal activity when used alone or in combination with antifungals, especially with FLC. Some of them could increase FLC's susceptibility against resistant Candida albicans significantly reversing fungal resistance to FLC. This article reviews the antifungal activities of immunosuppressants used alone or in combination with antifungals and their potential antifungal mechanisms that have been discovered so far. Although immunosuppressive agents have been identified as risk factors for fungal infection, we believe these findings are very important for overcoming drug resistance and developing new antifungals.