Amphotericin B-metronidazole combination against Candida spp

Interaction between amphotericin B and metronidazole was studied against Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krusei, and Candida lusitaniae strains. Minimum inhibitory concentrations (MICs) of the drugs alone and in combination were determined by means of the checkerboard method on YNB supplemented agar. Minimum fungicidal concentrations (MFCs) were determined on Sabouraud dextrose agar. Based on the MIC and MFC values, fractionary indices were determined respectively for inhibitory and lethal activities of the amphotericin B-metronidazole combination. These indices showed occurrence of additive and synergistic interactions between the drugs, but the synergysm was predominant against the studied strains.     

The postantifungal and paradoxical effects of echinocandins against Candida spp

Echinocandins induce a postantifungal effect and a paradoxical effect. The postantifungal effect is a concentration-dependent process that allows for sustained kill of Candida spp. after relatively brief exposures to a compound. The paradoxical effect is growth that occurs at high echinocandin concentrations above the MIC. Paradoxical growth varies in terms of media, species, strain and type of echinocandin. The study by Shields et al. evaluated the impact of a brief exposure of caspofungin on paradoxical growth and postantifungal effects in Candida albicans isolates. In the postantifungal effect experiments, prolonged concentration-dependent killing occurred. Maximum postantifungal effects occurred with caspofungin exposures of 5 or 15 min. A brief exposure of caspofungin eliminated the paradoxical growth that was observed in the time-kill experiments. The report by Shields et al. illustrates that short exposures to an echinocandin may lead to prolonged postantifungal effects and furthers our understanding of the paradoxical effect in C. albicans.     

Adhesion in Candida spp

Microbial adherence is one of the most important determinants of pathogenesis, yet very few adhesins have been identified from fungal pathogens. Four structurally related adhesins, Hwp1, Ala1p/Als5p, Als1p, from Candida albicans and Epa1p from Candida glabrata, are members of a class of proteins termed glycosylphosphatidylinositol-dependent cell wall proteins (GPI-CWP). These proteins have N-terminal signal peptides and C-terminal features that mediate glycosylphosphatidylinositol (GPI) membrane anchor addition, as well as other determinants leading to attachment to cell wall glucan. While common signalP/GPI motifs facilitate cell surface expression, unique features mediate ligand binding specificities of adhesins. The first glimpse of structural features of putative adhesins has come from biophysical characterizations of the N-terminal domain of Als5p. One protein not in the GPI-CWP class that was initially described as an adhesin, Int1p, has recently been shown to be similar to Bud4p of Saccharomyces cerevisiae in primary amino acid sequence, in co-localizing with septins and in functioning in bud site selection. Progress in understanding the role of adhesins in oroesophageal candidiasis has been made for Hwp1 in a study using beige athymic and transgenic epsilon 26 mice that have combined defects in innate and acquired immune responses. Searches of the C. albicans genome for proteins in the GPI-CWP class has led to the identification of a subset of genes that will be the focus of future efforts to identify new Candida adhesins.     

Antifungal Activities of Diphenyl Diselenide alone and in Combination with Fluconazole or Amphotericin B against Candida glabrata

Here, we evaluated combinations of diphenyl diselenide [(PhSe)₂] with fluconazole and amphotericin B in a checkerboard assay against clinical Candida glabrata strains. Minimal inhibitory concentration (geometric mean) ranged from 0.25 to >64 (5.16 μg/mL) for (PhSe)₂, 1 to 32 (5.04 μg/mL) for fluconazole and 0.06 to 0.5 (0.18 μg/mL) for amphotericin B. Synergistic (76.66 %) and indifferent (23.34 %) interactions were observed for (PhSe)₂ + amphotericin B combination. (PhSe)₂ + fluconazole combination demonstrated indifferent (50 %) and antagonistic (40 %) interactions, whereas synergistic interactions were observed in 10 % of the isolates. New experimental in vivo protocols are necessary and will promote a better understanding of the antimicrobial activity of (PhSe)₂ against C. glabrata and its use as an adjuvant therapy with antifungal agents.     

In vitro activity of amphotericin B and anidulafungin against Candida spp. biofilms

Invasive infections caused by Candida spp. are increasing worldwide and are becoming an important cause of morbidity and mortality in immunocompromised patients. A large number of manifestations of candidiasis are associated with the formation of biofilms on inert or biological surfaces. Candida spp. biofilms are recalcitrant to treatment with conventional antifungal therapies. The aim of this study was dual 1) to determine the prevalence of biofilm producers among clinical isolates from catheter (16 C. albicans ) and blood culture (2 C. albicans and 30 C. tropicalis), and 2) to determine the activity of amphotericin B and anidulafungin against C. albicans and C. tropicalis biofilms of 24 and 48 hours of maturation. Biofilms were developed using a 96-well microtitre plate model and production and activity of antifungal agents against biofilms were determined by the tetrazolium (XTT) reduction assay. Of catheter and blood isolates, 62.5 and 56.25%, respectively, produced biofilms. By species, 68.42% of C. albicans and 53.33% of C. tropicalis were biofilm producers. C. albicans biofilms showed more resistance to amphotericin B and anidulafungin than their planktonic counterparts. Complete killing of biofilms was never achieved, even at the highest concentrations of the drugs tested. Anidulafungin displayed more activity than amphotericin B against C. albicans biofilms of 24 hours of maturation (GM MIC 0.354 vs. 0.686 microg/ml), but against C. tropicalis biofilms amphotericin B was more active (GM MIC 11.285 vs. 0.476 microg/ml). In contrast, against biofilms with 48 hours maturation, amphotericin B was more active against both species.     

In Vitro Effect of Amphotericin B on Candida albicans,Candida glabrata and Candida parapsilosis Biofilm Formation

Candida spp. biofilm is considered highly resistant to conventional antifungals. The aim of this study was to investigate the in vitro effect of amphotericin B on Candida spp. biofilms at different stages of maturation. We investigated the activity of amphotericin B against 78 clinical isolates of Candida spp., representing three species, growing as planktonic and sessile cells, by a widely accepted broth microdilution method. The in vitro effect on sessile cell viability was evaluated by MTT reduction assay. All examined strains were susceptible to amphotericin B when grown as free-living cells. At the early stages of biofilm maturation 96.7–100.0 % strains, depending on species, displayed amphotericin B sessile minimal inhibitory concentration (SMIC) ≤1 μg/mL. Mature Candida spp. biofilm of 32.1–90.0 % strains displayed amphotericin B SMIC ≤1 μg/mL. Based on these results, amphotericin B displays species- and strain-depending activity against Candida spp. biofilms.     

In vitro activity of the lipopeptide PAL-Lys-Lys-NH2, alone and in combination with antifungal agents, against clinical isolates of Candida spp

Candida albicans is known to be the organism most often associated with serious fungal infection, but other Candida spp. are emerging as clinical pathogens associated with opportunistic infections. Among antimycotic treatments, increasing attention is currently given to anti-infective drugs based upon naturally occurring peptides, such as the short lipopeptide palmitoyl PAL-Lys-Lys-NH2 (PAL). The aim of this study is to evaluate the activity of this peptide compared to the traditional antifungal agents Fluconazole (FLU), amphotericin B (AMB) and caspofungin (CAS) on Candida spp. 24 clinical isolates of Candida spp. were tested against PAL_, FLU, AMB and CAS using in vitro susceptibility tests, time killing and checkerboard assay. All of the drugs studied showed good activity against clinical isolates of candida; in particular CAS and AMB which have MICs value lower than PAL and FLU. Moreover we observed synergistic interactions for PAL/FLU (81.25%), PAL/AMB (75%) and particularly for PAL/CAS (87.5). We think that our results are interesting since synergy between PAL and CAS might be useful in clinic trails to treat invasive fungal infections.     

黄芩苷联合氟康唑对白念珠菌生物膜的抑制作用研究

目的：探讨中药有效成分黄芩苷（ baicalin,BA）联合氟康唑（ fluconazole,FLC）对白念珠菌（ Candida albicans,C． albicans）生物膜的抑制作用。方法通过棋盘法考察BA联合FLC对白念珠菌浮游菌与生物膜的部分抑菌浓度指数（ FI?CI）;通过时间?杀菌曲线检测两药联合对白念珠菌标准株（C．albicans SC5314）的杀菌作用;以XTT减低法和干重法检测两药联合对白念珠菌SC5314生物膜代谢及生物量的影响;采用扫描电镜（ Scanning electron microscopy,SEM）和激光共聚焦显微镜（ Confocal laser scanning microscopy,CLSM）观察两药联合对白念珠菌SC5314生物膜形态结构的影响;以水?烃法检测两药联合对白念珠菌SC5314生物膜细胞表面疏水性（ cell surface hydrophobicity,CSH）的影响;通过实时荧光定量PCR （ quan?titative real time PCR,qRT?PCR）检测两药联合对白念珠菌生物膜和CSH相关基因表达的影响。结果黄芩苷与氟康唑联用抗白念珠菌浮游菌的FICI介于0．28～0．75之间,对生物膜的FICI介于0．16～0．5之间,表现为协同作用;SEM和CLSM在生物膜结构上验证了两药的协同效果;两药联合可降低生物膜表面疏水性,以及使ALS1、ALS3、EAP1、SUN41和CSH1分别下调6％、51％、24％、13％和39％。结论黄芩苷具有协同氟康唑抗白念珠菌生物膜作用。     

米卡芬净对分离自中国的念珠菌和曲霉临床株体外抑菌活性的研究

目的了解新型抗真菌药物米卡芬净(micafungin,MFG)对分离自中国的念珠菌和曲霉临床株的体外抑菌活性。方法参照CLSI(Clinical and Laboratory Standards Institute,以前为NCCLS)制定的M27-A2和M38-A方案测定86株念珠菌和35株曲霉的最低抑菌浓度(MIC)或最低有效浓度(MEC)。结果MFG对大多数念珠菌属和曲霉属均有较好的抑菌作用。对念珠菌属的MIC90从高到低依次为:氟康唑(FLC)敏感的白念珠菌、热带念珠菌、光滑念珠菌为0.125μg/ml,FLC耐药和剂量依赖敏感株为0.25μg/ml,克柔念珠菌为0.5μg/ml,近平滑念珠菌8μg/ml,季也蒙念珠菌>16μg/ml。MFG对烟曲霉的MEC90为≤0.03μg/ml,对非烟曲霉的曲霉属MEC90为0.06μg/ml。MFG与唑类药物、两性霉素B(AMB)不存在交叉耐药,对FLC耐药的念珠菌、伊曲康唑耐药的曲霉、AMB不敏感的曲霉均有好的抑菌活性。结论MFG对多数念珠菌属和曲霉属(包括对唑类耐药和AMB不敏感的菌株)有较好的体外抑菌作用。     

Enhancement of Amphotericin B Activity Against Candida albicans by Superoxide Radical

This study aimed to examine the involvement of oxidative damage in amphotericin B (AmB) activity against Candida albicans using the superoxide (O2-) generator paraquat (PQ). The effects of PQ on AmB activities against growth, viability, membrane permeability and respiration were examined in a wild-type parent strain (K) and a respiration-deficient mutant (KRD-19) since PQ-induced superoxide generation depends on respiration. In the parent strain, the minimal inhibitory concentration (MIC) of AmB, 0.25 microg/ml, tested with a liquid culture was lowered to 0.025 microg/ml by 1 mM PQ. Such a PQ-induced decrease in the MIC value of AmB was minimal in the mutant. Similar PQ-induced enhancement of AmB activity toward the parent strain was also observed with growth on an agar medium. In viability tests, when candidal cells were exposed to AmB (0.1 microg/ml) for I h, the lethality of AmB was enhanced by 1 mM PQ only in the parent strain. Exogenous superoxide dismutase and catalase failed to diminish the enhancing effect of PQ on the growth inhibitory activity of AmB in the parent strain, suggesting an interaction between superoxide and AmB in candidal cells. The enhancement of AmB activity by PQ, observed preferentially in the wild-type strain, can be explained by extensive superoxide generation depending on respiration. These results suggest that oxidative damage induced by superoxide is involved in AmB activity against C. albicans.     

Amphotericin B susceptibility testing of Candida species by flow cytometry.

We have developed an 8 hr flow cytometry (FCM) method for assessing susceptibility of yeasts to amphotericin B (AmpB). The method detects both high-level and relative-resistance to the drug. Variables found to affect fluorescence of control and AmpB treated cells included pH, presence of glucose, incubation conditions, concentration and length of exposure to both AmpB and ethidium bromide (ETBR), and the degree of resistance to AmpB. The FCM method was optimized based on increased red fluorescence intensity (RF), decreased forward angle light scatter (FALS), and a negative gating technique. A dose response was seen between 0.1 and 10 micrograms AmpB/ml for the susceptible control strain. Greater than 50% of cells from all susceptible strains tested transfer into the negative gate when exposed to 2.5 micrograms Amp B/ml while fewer than 5% of cells of the highly resistant C. tropicalis (ATCC 28707) are affected at concentrations up to 20 micrograms/ml. This method may provide a more accurate assessment of Amp B susceptibility than conventional tube dilution methods.     

Antimicrobial activity of [2-(methacryloyloxy)ethyl]trimethylammonium chloride against Candida spp

BackgroundCandida-associated denture stomatitis is the most common manifestation of oral candidal infection, caused mainly by Candida albicans. Several authors have attempted to add antifungal agents or antiseptics to denture temporary soft lining materials or to denture acrylic resins, without relevant results. Therefore, the investigation of a quaternary ammonium functionalized compound [2-(methacryloyloxy)ethyl]trimethylammonium chloride (MADQUAT), which copolymerizes with methacrylates and which could act as a fungal inhibitor, is of paramount importance.AimsTo evaluate the in vitro activity of MADQUAT against Candida species.MethodsThirty-one Candida strains were used to determine the in vitro antifungal activity of this compound. The minimum inhibitory concentrations and minimum fungicidal concentrations of MADQUAT and nystatin were determined.ResultsMADQUAT showed antifungal properties at concentrations of 6.25 to > 100 mg/ml, and fungicidal activity between 25 and > 100 mg/ml. The quantitative determinations of the fungistatic and fungicidal activity of MADQUAT showed fungistatic activity against all Candida albicans, Candida krusei and Candida parapsilosis strains, revealing fungicidal activity against some strains of the other species.ConclusionsMADQUAT has antifungal activity against Candida spp. Moreover, the sensitivity to this substance varies across the different species in terms of MIC values and fungicidal or fungistatic activity.     

棘白菌素对氟康唑耐药的念珠菌体外药物敏感性的研究

目的评价3种棘白菌素类药物（卡泊芬净、米卡芬净、阿尼多芬净）体外对氟康唑耐药念珠菌的药物敏感性。方法采用微量液体稀释法和琼脂稀释法测定最小抑制浓度（MIC）。结果微量液体稀释法：59株耐药白念珠菌3种药物MIC50均为0.06μg/mL,米卡芬净、阿尼多芬净的MIC范围均为0.015～0.125μg/mL,卡泊芬净为0.015～0.25μg/mL;8株耐药光滑念珠菌MIC值均为0.063μg/mL。琼脂稀释法：59株耐药白念珠菌和8株耐药光滑念珠菌3种药物MIC值均为0.063μg/mL。结论3种棘白菌素类药物可能具有治疗氟康唑耐药的念珠菌感染的临床价值。     

Activity of antifungal agents alone and in combination against echinocandin-susceptible and -resistant Candida parapsilosis strains

Background

Candida parapsilosis may acquire resistance to echinocandins, a fact that prompts the search for new therapeutic options.

In vitro activity of propyl gallate-azole drug combination against fluconazole- and itraconazole-resistant Candida albicans strains

AIMS: The influence of an antioxidant, propyl gallate (PG), on the in vitro antifungal activity of itraconazole and fluconazole, was investigated to determine whether PG could increase the antifungal activity and reduce strain resistance. METHODS AND RESULTS: Susceptibility tests were performed against azole-resistant isolates of Candida albicans by the microbroth dilution method in the presence of PG at 400 microg ml-1. PG-triazole combination brought about a marked reduction of inhibitory azole concentration. In particular, the MIC90 for itraconazole and fluconazole dropped from 1 microg ml-1 to 0.125 microg ml-1 and from > 64 microg ml-1-8 microg ml-1, respectively. CONCLUSION: It is likely that more than one mechanism is involved in the above synergistic interaction, including effects of PG on ATP synthesis, thus reducing the ABC transporters activity, or an effect on the target of azole, i.e. the P-450 cytochrome. SIGNIFICANCE AND IMPACT OF THE STUDY: The PG-triazole combination may have a role in future topical antifungal strategies but other studies are warranted.