Enhanced activity of antifungal drugs using natural phenolics against yeast strains of Candida and Cryptococcus

Aims: Determine whether certain, natural phenolic compounds enhance activity of commercial antifungal drugs against yeast strains of Candida and Cryptococcus neoformans. Methods and Results: Twelve natural phenolics were examined for fungicidal activity against nine reference strains of Candida and one of C. neoformans. Six compounds were selected for synergistic enhancement of antifungal drugs, amphotericin B (AMB), fluconazole (FLU) and itraconazole (ITR). Matrix assays of phenolic and drug combinations conducted against one reference strain, each, of Candida albicans and C. neoformans showed cinnamic and benzoic acids, thymol, and 2,3‐ and 2,5‐dihydroxybenzaldehydes (‐DBA) had synergistic interactions depending upon drug and yeast strain. 2,5‐DBA was synergistic with almost all drug and strain combinations. Thymol was synergistic with all drugs against Ca. albicans and with AMB in C. neoformans. Combinations of benzoic acid or thymol with ITR showed highest synergistic activity. Of 36 combinations of natural product and drug tested, none were antagonistic. Conclusions: Relatively nontoxic natural products can synergistically enhance antifungal drug activity, in vitro. Significance and Impact of the Study: This is a proof‐of‐concept, having clinical implications. Natural chemosensitizing agents could lower dosages needed for effective chemotherapy of invasive mycoses. Further studies against clinical yeast strains and use of animal models are warranted.     

The isolation,characterization and antifungal susceptibilities of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> from bird excreta in Klang Valley,Malaysia

The occurrence of Cryptococcus neoformans in bird excreta in Klang valley, Malaysia was determined in this study. Of 544 samples of bird excreta collected from a local zoo, pet shops and public areas, 20 strains of C. neoformans were isolated. All C. neoformans strains were serotype A and thus identified as C. neoformans variety grubii. All did not produce color changes on canavanine–glycine–bromothymol blue agar. All were of α-mating types, as determined by a pheromone-specific PCR assay. The antifungal susceptibility testing using agar diffusion method Neo-sensitabs showed that all were susceptible to amphotericin B, fluconazole and itraconazole.     

Cryptococcus neoformans: Comparisons of in vitro antifungal susceptibilities of serotypes AD and BC

Thirty-nine isolates of Cryptococcus neoformans, nineteen serotype AD and twenty serotype BC, were assayed for susceptibility to eight antifungal agents using an in vitro agar dilution assay. Media employed were Kimmig agar and yeast nitrogen base supplemented with 10% glucose. The antifungal agents used were ketoconazole, amphotericin B, 5-fluorocytosine, nystatin, miconazole, BAY N 7133, ICI 153,066, and itraconazole. No clinically significant differences in in vitro minimum inhibitory concentrations were detected between serotypes AD and BC against any of the compounds tested. An adverse medium effect was observed in two of the assays, but the outcome of the AD/BC comparison was not affected. This is the first report in which the in vitro antifungal susceptibilities of Cryptococcus neoformans serotypes are analyzed.     

小檗碱抗新生隐球菌活性和作用机制

【目的】研究小檗碱对新生隐球菌的抗菌活性及其作用机制。【方法】采用微量肉汤稀释法测定小檗碱对新生隐球菌标准菌株和临床分离菌株的最小抑菌浓度,通过棋盘法测定小檗碱与氟康唑、两性霉素B的协同作用,测定小檗碱对隐球菌重要毒力因子的表达,以及对巨噬细胞和隐球菌互作的影响,采用隐球菌感染大蜡螟模型测定小檗碱的体内杀菌活性。【结果】小檗碱是一种杀真菌化合物,在测试的菌株中,最小抑菌浓度(minimum inhibitory concentration, MIC)范围为8-16μg/mL。亚致死剂量小檗碱能够抑制隐球菌荚膜大小、产黑色素能力和有性生殖能力,并能增强巨噬细胞的杀菌能力。锌指转录因子Nrg1介导了上述重要的过程。在隐球菌感染动物模型中,小檗碱能够延长感染大蜡螟的存活时间。【结论】小檗碱在体内外具有优异的抗隐球菌活性,有望作为抗隐球菌药物开发的起始化合物。     

Antifungal Activity,Antibiofilm and Association Studies with O-Alkylamidoximes against Cryptococcus spp.

This is the first study that describes the antifungal and anti-biofilm potential of O-alkylamidoximes against strains of Cryptococcus neoformans and Cryptococcus gattii. In vitro tests have shown that O-alkylamidoximes are capable of inhibiting fungal growth and biofilm formation of the C. neoformans and C. gattii strains, suggesting, from molecular docking, the potential for interaction with the Hsp90. The associations between O-alkylamidoximes and amphotericin B were beneficial. Therefore, O-alkylamidoximes can be a useful alternative to contribute to the limited arsenal of drugs, since they showed a powerful action against the primary agents of Cryptococcosis.     

Effects of Pimenta pseudocaryophyllus (Gomes) L. R. Landrum, on Melanized and Non-melanized Cryptococcus neoformans

In the present study, the in vitro susceptibility and capsular width from both melanized and non-melanized Cryptococcus neoformans cells in the presence of Pimenta pseudocaryophyllus crude extract were determined. The results were compared with those obtained for voriconazole and amphotericin B. Melanization was obtained in minimal medium broth with the addition of L-dopa, and the antifungal susceptibility tests were performed using the broth microdilution method. Capsular width of 30 cells of each one of the isolates in medium with crude extracts of P. pseudocaryophyllus or voriconazole or amphotericin B at a concentration corresponding to 0.5?times the minimal inhibitory concentration (MIC) was measured, and the mean was calculated. The MICs and minimal fungicidal concentrations (MFCs) for plant extract and voriconazole were identical for both melanized and non-melanized C. neoformans isolates, but for amphotericin, the MFCs for melanized cells were up to 8?times higher than for non-melanized cells. The capsular width of C. neoformans cells was smaller (p?<?0.001) in the presence crude extract of P. pseudocaryophyllus and of voriconazole regardless melanization. The findings of capsule alterations of C. neoformans verified in this study provide fertile ways for future research into the effects of antifungal agents on the pathogenesis of cryptococcosis.     

Comparison of Different In Vitro Tests to Detect <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> Not Susceptible to Amphotericin B

Infections due to Cryptococcus neoformans cause severe disease, mostly in AIDS patients. The antifungal drug recommended for the initial treatment of these infections is amphotericin B with or without flucytosine, but treatment failure occurs, associated with high mortality. Thus, antifungal susceptibility testing is needed. However, the in vitro susceptibility tests available for C. neoformans are not useful to detect isolates that are not susceptible to antifungal agents such as amphotericin B. The aims of the present study were: (1) to determine and compare the in vitro activity of amphotericin B against C. neoformans clinical isolates by using different dilution and diffusion methods; (2) to evaluate the concordance among the methods used and the reference method; (3) to evaluate which method could be the best to correlate with the clinical outcome. The reference method EDef 7.2 from the European Committee on Antimicrobial Susceptibility Testing and commercial Etest strips were used to determine the minimal inhibitory concentration against amphotericin B. curves, minimal fungicidal concentration, and a disk diffusion method were also developed to evaluate the cidal activity of amphotericin B. The time–kill curve assay showed correlation (p < 0.05) with clinical outcome, whereas EDef 7.2, minimal fungicidal concentration, Etest, and disk diffusion showed no correlation (p > 0.05). Thus, the time–kill curve assay could be a potential tool to guide a more efficient treatment when amphotericin B is used.     

Itraconazole vs amphotericin B: in vitro comparative evaluation of the minimal inhibitory concentration (MIC) against clinically isolated yeasts

Itraconazole is a triazole compound which, following several clinical trials, has begun to be used for therapy of mycotic infections. This new drug, with a broad-spectrum antifungal activity, can be orally administered. The Authors studied the in vitro susceptibility to amphotericin B and itraconazole of the following clinical isolates of pathogenic yeasts: 100 Candida albicans, 20 C. tropicalis, 20 C. parapsilosis, 8 C. guilliermondii, 6 C. pseudotropicalis, 24 Torulopsis glabrata and 16 Cryptococcus neoformans.Serial two-fold dilutions, from 100 g/ml to 0.04 g/ml, of each drug were prepared in Yeast Nitrogen Base + Glucose 5%, after dissolving the itraconazole in dimethylsulfoxide (DMSO) and amphotericin B in 5% glucose solution. Amphotericin B (MIC₉₀: 3.12 g/ml) was found to have an average in vitro MIC six-fold lower than itraconazole (MIC₉₀: 25 g/ml).Thus, even though itraconazole is active, amphotericin B remains one of the most effective of the antifungal drugs.     

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.     

DNA fingerprinting pattern and susceptibility to antifungal drugs in Cryptococcus neoformans variety grubii isolates from Barcelona city and rural environmental samples

Cryptococcus neoformans var. grubii (serotype A) was isolated from 12 soil samples mixed with pigeon droppings (16.9%) from 71 soil samples in Barcelona and rural areas of Catalonia. C. neoformans was not isolated from indoor dust and Eucalyptus debris. PCR fingerprinting was performed in 22 representative isolates and all of them corresponded to the VNI pattern. Susceptibility testing for the 22 isolates of C. neoformans var. grubii showed that all of them were susceptible to amphotericin B. Three isolates presented MICs (Minimal Inhibitory Concentrations) ≥ 1 μg/ml to Itraconazole, five MICs ≥ 1 μg/ml to ketoconazole and four were fluconazole resistant, (MICs ≥ 64 μg/ml), while three of them were shown to have MICs ≥ 1 μg/ml to voriconazole. In spite that all isolates presented the same DNA fingerprinting pattern, the susceptibility to antifungals is very variable. The possibility of acquiring cryptococcosis infection with primarily resistant environment strains is feasible.     

Cryptococcal therapies and drug targets: the old,the new and the promising

Half a century after the introduction of Amphotericin B the management of cryptococcosis remains unsatisfactory. The disease, caused primarily by the two fungal species Cryptococcus neoformans and Cryptococcus gattii, remains responsible for considerable morbidity and mortality despite standard medical care. Current therapeutic options are limited to Amphotericin B, azoles and 5‐flucytosine. However, this organism has numerous well‐characterized virulence mechanisms that are amenable to pharmacological interference and are thus potential therapeutic targets. Here, we discuss existing approved antifungal drugs, resistance mechanisms to these drugs and non‐standard antifungal drugs that have potential in treatment of cryptococcosis, including immunomodulatory strategies that synergize with antifungal drugs, such as cytokine administration or monoclonal antibodies. Finally, we summarize attempts to target well‐described virulence factors of Cryptococcus, the capsule or fungal melanin. This review emphasizes the pressing need for new therapeutic alternatives for cryptococcosis.     

Serotypes and mating types of clinical strains ofCryptococcus neoformans isolated in Taiwan

Twenty-one strains ofCryptococcus neoformans isolated from patients in Taiwan were characterized for serotypes and mating types. Slide agglutination test was performed with 8 factor-specific sera (Iatron Company, Japan) to determine the serotypes. Wheat bran agar (WBA) and malt extract agar (MEA, Wickerham) media were used for the mating tests. Twenty of the isolates were of serotype A, and one was serotype B. Except for 2 strains of serotype A, all of the serotype A strains mated withFilobasidiella neoformans var.neoformans, mating type a. The only serotype B strain mated withF. neoformans var.bacillispora mating type a in MEA medium. These data revealed the low prevalence (1/21; 4.8%) ofC. neoformans var.gattii in Taiwan, a subtropically located island.     

Serotypes of Cryptococcus neoformans isolated from patients prior to and during the AIDS era in Thailand

One hundred and eighty-seven strains of Cryptococcus neoformans isolated from patients in Thailand were charcterized by biochemical varieties relating to serogroups. Canavanine-glycine-bromothymol blue (CGB) agar was used for differentiating the varieties of C. neoformans. Slide agglutination tests were performed with Crypto Check (Iatron, Inc., Tokyo) to determine their serotypes. Fifty-five percent (10 out of 18) of the pre-AIDS isolates were serotype B, 28% were serotype A, 5% were serotype D, and an unexpected 11% (2 out of 18) were serotype C. These are the first to be recorded in Asia. In contrast, among the 169 clinical isolates obtained between January 1993 and March 1995 (AIDS epidemic), serotype A was outstandingly predominant-93% (157 out of 169), serotype B was relatively low (3.6%) and both serotypes D and AD were 1.8%. The pattern of serotypes of the 59 isolates from known HIV-positive patients was closely similar to the total isolates during the AIDS epidemic. In determining the varieties of C. neoformans by CGB, only 1 of the 187 isolates gave a false reaction. On the basis of our findings, we believe that in the pre-AIDS era either C. neoformans var. gattii serorype B or serotype C were the common causative agents of cryptococcosis in Thailand. The advent of AIDS changed the pattern of serotypes with serotype A becoming predominant as has been reported world wide.     

Amphotericin B synergy testing by the FCST

The flow cytofluorometric susceptibility test (FCST) was incorporated into two in vitro synergy assays of amphotericin B-drug combinations. The FCST checkerboard assay and the FCST concentration-effect assay were developed to detect subtle modulation of amphotericin B fungicidal effects onCandida albicans andCryptococcus neoformans. Amphotericin B × cyclosporine A was a synergistic fungicidal combination against bothC. albicans andC. neoformans. Amphotericin B×gentamicin and amphotericin B×ketoconazole were synergistic combinations againstC. neoformans. In all cases tested, the synergy was effective when 0.50–0.62 g amphotericin B/ml was used with>-0.50 g of the other drug/ml. The fungicidal effect of 1.00 g amphotericin B/ml overwhelmed the synergistic effects. A number of other drug combinations were additive, autonomous, or antagonistic.     

Fluconazole Non-susceptible <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>, Relapsing/Refractory Cryptococcosis and Long-term Use of Liposomal Amphotericin B in an AIDS Patient

The treatment of cryptococcosis is hampered by inefficacy or intolerance to the recommended antifungal agents. A patient diagnosed with AIDS had multiple relapses of cryptococcal infection, which became refractory to antifungal agents during the course of therapy. During the follow-up, the patient developed renal toxicity due to amphotericin B use and non-susceptibility of isolated Cryptococcus neoformans to fluconazole was detected. Thereafter, antifungal treatment was performed exclusively with liposomal amphotericin B, reaching a cumulative dose of 19,180 mg over 46 months. The final relapse of cryptococcosis occurred during the maintenance phase with liposomal formulation in a once-weekly dose. Measurement of the minimum serum concentrations of amphotericin B, determined sequentially before and after this relapse, suggested the importance of monitoring drug levels when the liposomal formulation is used for a long period.     

Cryptococcus neoformans varieties as agents of cryptococcosis in Brazil

The study of the clinical isolates of Cryptococcus neoformans from 83 Brazilian patients with disseminated cryptococcosis showed that 75 were C. neoformans var. neoformans and 8 were var. gattii. Twenty-seven isolates were serotyped; all 19 var. neoformans were serotype A and all 8 var. gattii were serotype B. The correlation of the varieties of C. neoformans with the presence or not of hosts predisposing conditions to the mycosis showed that: (1) cryptococcosis caused by gattii variety occurred in 7 (58.3%) of the 12 nonimmunosuppressed patients, and (2) cryptococcosis caused by neoformans variety occurred in 65 (98.5%) of the 66 AIDS patients and in all 5 patients with other immunosuppressive conditions. The comparison of the distribution of the gattii and neoformans varieties between the nonimmunosup-pressed and immunosuppressed patients showed a significant statistical difference (p < 0.01).     

Toward a clinical antifungal peptoid: Investigations into the therapeutic potential of AEC5

Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningitis in immunocompromised individuals. Existing antifungal treatment plans have high mammalian toxicity and increasing drug resistance, demonstrating the dire need for new, nontoxic therapeutics. Antimicrobial peptoids are one alternative to combat this issue. Our lab has recently identified a tripeptoid, AEC5, with promising efficacy and selectivity against C. neoformans. Here, we report studies into the broad-spectrum efficacy, killing kinetics, mechanism of action, in vivo half-life, and subchronic toxicity of this compound. Most notably, these studies have demonstrated that AEC5 rapidly reduces fungal burden, killing all viable fungi within 3 hours. Additionally, AEC5 has an in vivo half-life of 20+ hours and no observable in vivo toxicity following 28 days of daily injections. This research represents an important step in the characterization of AEC5 as a practical treatment option against C. neoformans infections.     

Anticandidial and anticryptococcal activity of <Emphasis Type="Italic">Euphorbia fusiformis</Emphasis>, a rare medicinal plant

The present work demonstrates the screening of extracts of the rare medicinal herb Euphorbia fusiformis for antifungal activity. The main aim was to investigate its antifungal properties against Candida albicans and Cryptococcus neoformans, the causative agents of human candidiasis and cryptococcosis, respectively. Aqueous and organic solvent extracts from the leaves and rootstock of the plant were tested against the fungi by the well-in-agar method. Almost all the organic solvent extracts exhibited an inhibitory effect against C. albicans and to some extent on C. neoformans, except for the aqueous extracts, which had no effect. The combined formulations of the extracts also had better activity against C. albicans than C. neoformans. This study thus concludes by demonstrating the antifungal properties of E. fusiformis and also the potential research in identifying the active principles, which may have future therapeutic value.     

Hydroxyaldimines as potent in vitro anticryptococcal agents

Cryptococcosis, a fungal infection that affects both immunocompromised and immunocompetent individuals, contributes to increasing indices of mortality and morbidity. The development of resistance by Cryptococcus spp., the limited number of commercial antifungal drugs and the various side effects of these drugs cause the treatment of cryptococcosis to be a challenge. The in vitro anticryptococcal activity of nine hydroxyaldimines was evaluated against 24 strains of Cryptococcus spp. Antifungal susceptibility was evaluated using a broth microdilution assay following the Clinical and Laboratory Standards Institute guidelines, using fluconazole as a positive control. Parameters such as the minimum inhibitory concentration and the minimum fungicidal concentration (MIC and MFC, respectively) were also determined. Antiproliferative activity on the normal cell line VERO was assessed 48 h post‐compound exposure to determine the selectivity index (SI) of the hydroxyaldimines and fluconazole. All hydroxyaldimines were active against Cryptococcus spp. strains. Compounds 3A9 and 3B7 were the most potent against the Cryptococcus gattii and Cryptococcus neoformans strains. Selectivity indices also revealed that 3B10, 3C3, 3D3 and 3D9 are good candidates for in vivo studies. The in vitro anticryptococcal activity of hydroxyaldimines against various strains of C. gattii and C. neoformans indicates the potential of this class of molecules as lead compound for the development of selective and efficient anticryptococcal agents.

Significance and Impact of the Study

The effectiveness of hydroxyaldimines for inhibition of Cryptococcus spp. growth and their low toxicity against healthy monkey kidney epithelial cells makes them promising lead compounds for the design of new anticryptococcal agents.