Quantitative and qualitative analysis of the antifungal activity of allicin alone and in combination with antifungal drugs

The antifungal activity of allicin and its synergistic effects with the antifungal agents flucytosine and amphotericin B (AmB) were investigated in Candida albicans (C. albicans). C. albicans was treated with different conditions of compounds alone and in combination (allicin, AmB, flucytosine, allicin + AmB, allicin + flucytosine, allicin + AmB + flucytosine). After a 24-hour treatment, cells were examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM) to measure morphological and biophysical properties associated with cell death. The clearing assay was conducted to confirm the effects of allicin. The viability of C. albicans treated by allicin alone or with one antifungal drug (AmB, flucytosine) in addition was more than 40% after a 24-hr treatment, but the viability of groups treated with combinations of more than two drugs was less than 32%. When the cells were treated with allicin alone or one type of drug, the morphology of the cells did not change noticeably, but when cells were treated with combinations of drugs, there were noticeable morphological changes. In particular, cells treated with allicin + AmB had significant membrane damage (burst or collapsed membranes). Classification of cells according to their cell death phase (CDP) allowed us to determine the relationship between cell viability and treatment conditions in detail. The adhesive force was decreased by the treatment in all groups compare to the control. Cells treated with AmB + allicin had a greater adhesive force than cells treated with AmB alone because of the secretion of molecules due to collapsed membranes. All cells treated with allicin or drugs were softer than the control cells. These results suggest that allicin can reduce MIC of AmB while keeping the same efficacy.     

Effect of Pichia membranaefaciens in combination with salicylic acid on postharvest blue and green mold decay in citrus fruits

The separate or combined effects of Pichia membranaefaciens and salicylic acid (SA) on the control of blue and green mold decay in citrus fruits were investigated. Results indicate that combining P. membranaefaciens (1 × 10⁸ CFU ml⁻¹) with SA (10 μg ml⁻¹) either in a point-inoculated or dipped treatment provided a more effective control of blue and green mold than separately applying yeast or SA. SA (10 μg ml⁻¹) did not significantly affect P. membranaefaciens growth in vitro but slightly increased the yeast population in fruit wounds. P. membranaefaciens plus SA effectively enhanced the phenylalanine ammonia-lyase, peroxidase, polyphenoloxidase, chitinase, and β-1,3-glucanase activities and stimulated the synthesis of phenolic compounds. The combined treatment did not impair quality parameters such as weight loss or titratable acidity, but resulted in low average natural infection incidence and increased total soluble solids and ascorbic acid contents in citrus fruits after 14 d at 20 °C.     

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.

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.     

Screening and identification of epiphytic yeasts with potential for biological control of green mold of citrus fruits

Epiphytic yeasts isolated from the surface of citrus fruits, harvested in several orchards in the Souss-Massa-Draa Valley, Agadir, Morocco, were in vivo screened for antagonistic activity against Penicillium digitatum, the causal agent of green mold of citrus. From a total of 245 yeast strains assessed for their biocontrol activity against P. digitatum, fifteen reduced the incidence of disease to less than 50%. The effectiveness of the best selected yeast strains showed that Pichia anomala (YT73), Debaryomyces hansenii (YT22) and Hanseniaspora guilliermondii (YT13) were the most effective, with a reduction of green mold incidence from 65 to ~80%, compared to the control. The identification of the fifteen selected yeast strains was carried out through an integrated approach including phenotypic and genotypic (sequencing of D1/D2 domain of 26S rDNA encoding gene) methods. These 15 selected were identified as: H. guilliermondii, D. hansenii, H. uvarum and P. anomala. The study of the dynamics of two of the best strains, H. guilliermondii and D. hansenii, showed that these strains can grow rapidly, by approximately 2 log units, in citrus fruit wounds. Such rapid growth in wounds indicates that these antagonist yeasts are excellent colonizers of citrus wounds and can thrive on citrus fruits as a substrate.     

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.     

In vitro activity of the synthetic lipopeptide PAL-Lys-Lys-NH(2) alone and in combination with antifungal agents against clinical isolates of Cryptococcus neoformans

The in vitro activity of the lipopeptide PAL-Lys-Lys-NH(2) (PAL), alone or combined with either fluconazole (FLU) or amphotericin B (AMB), was investigated against 14 Cryptococcus neoformans isolates. PAL MICs ranged from 1.0 to 4.0 microg/ml. Fungicidal activity was observed. Synergy, defined as a fractional inhibitory concentration (FIC) index of < or =0.5, was observed in 21.4% of PAL/AMB interactions. Antagonism (FIC index>4) was never observed. The broad antifungal activity and the positive interactions with AMB suggest that PAL can represent a promising candidate in infections due to C. neoformans.     

Enhancement of biocontrol activity of yeasts by adding sodium bicarbonate or ammonium molybdate to control postharvest disease of jujube fruits

AIMS: To assess the potential of sodium bicarbonate and ammonium molybdate as additives in enhancing the biocontrol efficacy of Rhodotorula glutinis and Cryptococcus laurentii against blue mould in jujube fruits. METHODS AND RESULTS: Two yeasts at a concentration of 107 CFU ml-1, in combination with 238 mmol l-1 sodium bicarbonate or 15 mmol l-1 ammonium molybdate, showed a significant inhibition effect on blue mould of jujube fruits stored at 20 degrees C for 5 days. The colonizing ability of the yeasts in wounded sites was significantly decreased in the presence of ammonium molybdate. CONCLUSIONS: Combining R. glutinis or C. laurentii with sodium bicarbonate or ammonium molybdate provided a more effective control of postharvest disease than using the antagonistic yeasts or the chemicals alone. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of sodium bicarbonate or ammonium molybdate reduced the number of antagonists required to efficiently control disease of postharvest fruits, which could result in the reduction of costs.     

Antimicrobial activity of tigecycline alone or in combination with rifampin against Staphylococcus epidermidis in biofilm

Staphylococcus epidermidis is a commensal inhabitant of the healthy human skin, but in the recent years, it has been recognized as a nosocomial pathogen especially in immunocompromised patients. The pathogenesis of S. epidermidis is thought to be based on its capacity to form biofilms on the surface of medical devices, where bacterial cells may persist, protected from host defence and antimicrobial agents. Rifampin has been shown to be one of the most active antimicrobial agents in the eradication of the staphylococcal biofilm. However, this antibiotic should not be used in monotherapy. Therefore, one of the objectives of our research was to study the efficacy of the tigecycline/rifampin combination against methicillin-resistant S. epidermidis embedded in biofilms. Of the 80 clinically significant S. epidermidis isolates, 75 strains possess the ability to form a biofilm. These bacteria formed the biofilm via ica-dependent mechanisms. However, other biofilm-associated genes, including aap (encoding accumulation-associated protein) and bhp (coding cell wall-associated protein), were present in 85 and 29 % of isolates, respectively. The biofilm structures of S. epidermidis strains were also analyzed in confocal laser scanning microscopy (CLSM) and the obtained image demonstrated differences in their architecture. In vitro studies showed that the MIC value for tigecycline against S. epidermidis growing in the biofilm ranged from 0.125 to 2 μg/mL. Tigecycline in combination with rifampin demonstrated higher activity against bacteria embedded in biofilms than tigecycline alone.     

Inhibitory activity of natural antimicrobial compounds alone or in combination with nisin against Enterobacter sakazakii

Aim: To determine the antimicrobial activity of natural organic compounds alone and in combination with nisin on the growth of Enterobacter sakazakii in laboratory media. Methods and Results: The minimum inhibitory concentrations (MIC) of five natural organic compounds were determined, and their effects in combination with nisin were evaluated by comparing treatment with each natural organic compound alone and in combination with 25 mg ml^?1 nisin in tryptic soy broth. Among the tested natural organic compounds, the MIC of carvacrol and thymol was 1·25 mmol l^?1 and showed the strongest inhibitory activity against E. sakazakii, whereas the MIC of cinnamic acid was higher than 5 mmol l^?1, and therefore showed the weakest inhibitory activity. However, the combination of each compound with nisin did not result in the enhancement of their antimicrobial activities except when nisin was combined with diacetyl. Conclusions: The order of inhibition attributed to natural organic compounds was carvacrol = thymol > eugenol > diacetyl > cinnamic acid, and only the combination of diacetyl and nisin showed a synergistic effect of inhibiting the growth of E. sakazakii. Significance and Impact of the Study: This study shows the potential of natural organic compounds for controlling E. sakazakii.     

Efficiency of different application methods of biocontrol agents and biocides in control of Fusarium root rot on some citrus rootstocks

Abstract

The efficiency of two biocontrol agents (Trichoderma harzianum NB and Bacillus subtilis NB) and two commercial biocides (Plant Guard and Rhizo-N) in controlling Fusarium root rot disease on some citrus rootstocks was evaluated under artificially infested soil in green house.

Fusrium root rot on citrus rootstocks seedlings i.e. sour orange (SO), volkamer lime (VL), rangpur lime (RP) and cleopatra mandarin (CL) was successfully controlled by dipping the root system of such seedlings in water suspensions of each biological treatment i.e. Trichoderma harzianum (spore suspension 5×10⁶ spore/ml), Bacillus subtilis (cell suspension 8×10⁷ cell/ml), Plant Guard (3 g/l) and Rhizo-N (4 g/l), then transplanted into artificially infested soil with Fusarium solani and drenched with enough water suspension of such biological treatments. Plant Guard (3 g/l) and Rhizo-N (4 g/l) were highly effective treatments in decreasing infection and severity of the disease, Fusarium density in rhizosphere soil and colonization of Fusarium solani in the roots of all tested seedlings.

Meanwhile, root dipping or soil drenching with the same treatments individually gave the least effect in reducing root rot incidenceon all tested rootstocks compared with application of the two methods together.

It should be noted that using biocontrol agents and commercial biocides could be successfully used in controlling root rot pathogens on citrus in commercial greenhouses or under field conditions before transplanting in new reclaimed lands in the desert.     

Larvicidal activity of plant extracts used alone and in combination with known synthetic larvicidal agents against Aedes aegypti

Larvicidal activity of acetone and petroleum ether extracts of four plants Murraya koenigii, Coriandrum sativum, Ferula asafoetida, Trigonella foenum graceum and synthetic larvicides Fenthion and Temephos used alone and in combination was carried out against A. aegypti larvae under laboratory conditions. Optimum values were found out by using different concentrations of each plant (ranged 25 ppm-900 ppm) and both synthetic larvicides (ranged 0.01-7.5 ppm). The synergistic study was carried out by using 0.05 ppm of Temephos and Fenthion with 25ppm of M. koenigii, F. asafetida, T. foenum graceum and 100 ppm of C. sativum. All the plants showed potential synergistic activity although showed comparatively poor larvicidal activity when tested individually.     

Improving function of biocontrol agents incorporated in antifungal fruit coatings: a review

The in-field performance of microbial biocontrol agents (BCAs) against fungal pathogens in fruit is subject to considerable variability due to their sensitivity to both adverse environmental conditions and their fluctuations. Therefore, to achieve an adequate development and implementation of biological agent-based products, it is necessary to improve their resistance and ability to control fungal diseases under a wide range of conditions. In this review, an overview of the latest strategies for the enhancement of the action of BCAs is given. The combination of the antagonists with edible polymers able to form coatings is one of the approaches with the greatest potential and it is analysed in depth. This formulation approach of biocontrol products, including adequate microbial protectants, can yield stable products with high microbial viability, ready for field applications, with improved adherence and survival of the BCA once applied in plant. The most recent studies into this field are reviewed and summarised.     

Isolation of plasma membrane fractions from green wheat leaves by free-flow electrophoresis or two-phase partition alone or in combination

Plasma membrane vesicles were isolated from shoots of light-grown wheat seedlings by preparative free-flow electrophoresis, aqueous polymer two-phase partition or both. Plasma membrane vesicles were identified from staining of thin sections prepared for electron microscopy with phosphotungstic acid at low pH. The orientation of the plasma membrane vesicles was determined from latency and trypsin sensitivity of K⁺ Mg²⁺ATPase and of glucan synthase II, and concanavalin A-peroxidase binding and membrane asymmetry visualized by electron microscopy. The K⁺Mg²⁺ATPase and of glucan synthase II activities of plasma membrane fractions isolated by two-phase partition were latent and trypsin resistant. The vesicles bound concanavalin A-peroxidase strongly and exhibited a cytoplasmic side-in morphology. These fractions of cytoplasmic side-in vesicles were less than 10% contaminated by cytoplasmic side-out vesicles. By free-flow electrophoresis, two populations of vesicles which stained with phosphotungstic acid at low pH, designated D and E, were obtained. The vesicle population with the lower electrophoretic mobility, fraction E, contained plasma membrane vesicles with properties similar to those of the plasma membrane vesicles obtained after two-phase partition. The phosphotungstic-reactive vesicles with greater electrophoretic mobility, fraction D, were concanavalin A unreactive with the cytoplasmic membrane leaflet outwards. Less than 50% of the K⁺Mg²⁺-ATPase activity of this fraction was latent and trypsin sensitive. The vesicles of fraction D appeared to be preferentially cytoplasmic side-out. The electrophoretic mobilities of cytoplasmic side-out (non-latent glucan synthase II activity) and cytoplasmic side-in (latent glncan synthase II activity) plasma membrane vesicles isolated from a frozen and thawed wheat plasma membrane fraction, corresponded with the mobilities of fraction D and E, respectively, again showing that the plasma membrane vesicles with the lesser electrophoretic mobility were cytoplasmic side-in. The cytoplasmic side-in and cytoplasmic side-out vesicles therefore showed opposite eletrophoretic mobilities compared with a previous free-flow electrophoretic separation of soybean plasma membranes. The majorities of the plasma membrane vesicles of both fractions D and E entered the upper phase upon two-phase partition with the phase composition used for purification of wheat plasma membranes. Thus, neither electrophoretic mobility nor phase partitioning characteristics can be used as the only criteria for assignment of vesicle orientation.     

6种抗真菌药物对皮肤癣菌体外抗真菌活性评价

目的评价6种抗真菌药物对皮肤癣菌体外抗真菌活性。方法采用CLSI推荐的M-38P方案对分离自足癣和体、股癣的皮肤癣菌进行联苯苄唑、硝酸舍他康唑、硝酸异康唑、盐酸布替萘芬、阿莫洛芬、利拉萘酯6种抗真菌药物敏感性测定。结果联苯苄唑MIC范围为0．03—4mg／L,MIC50为1mg／L,MIC90为2mg／L。硝酸舍他康唑分别为0．06—16mg／L、0．5mg／L和2mg／L。硝酸异康唑分别为0．03～2mg／L、0．25mg／L和0．5mg／L。盐酸布替萘芬分别为0．0025～0．04mg／L、0．01mg／L和0．02mg／L。阿莫罗芬分别为0．01～〉0．08mg／L、0．02mg／L和0．04mg／L。利拉萘酯分别为0．004—0．625mg／L、0．039mg／L和0．312mg／L。结论6种抗真菌药物对皮肤癣菌均有强的抗菌活性,由强到弱依次为布替萘芬、阿莫罗芬、利拉萘酯和咪唑类药物。     

In vitro synergistic activity of diketopiperazines alone and in combination with amphotericin B or clotrimazole against Candida albicans

The synergistic anticandidal activity of three diketopiperazines [cyclo-(l-Pro-l-Leu) (1), cyclo-(d-Pro-l-Leu) (2), and cyclo-(d-Pro-l-Tyr) (3)] purified from a Bacillus sp. N strain associated with entomopathogenic nematode Rhabditis (Oscheius) in combination with amphotericin B and clotrimazole was investigated using the macrodilution method. The minimum inhibitory concentration and minimum fungicidal concentration of the diketopiperazines was compared with that of the standard antibiotics. The synergistic anticandidal activities of diketopiperazines with amphotericin B or clotrimazole were assessed using the checkerboard and time-kill methods. The results of the present study showed that the combined effects of diketopiperazines with amphotericin B or clotrimazole predominantly recorded synergistic (<0.5). Time-kill study showed that the growth of the Candida was completely attenuated after 12–24 h of treatment with 50:50 ratios of diketopiperazines and antibiotics. These results suggest that diketopiperazines combined with antibiotics may be microbiologically beneficial and not antagonistic. These findings have potential implications in delaying the development of resistance as the anticandidal effect is achieved with lower concentrations of both drugs (diketopiperazines and antibiotics). The cytotoxicity of diketopiperazines was also tested against two normal human cell lines (L231 lung epithelial and FS normal fibroblast) and no cytotoxicity was recorded for diketopiperazines up to 200 μg/mL. The in vitro synergistic activity of diketopiperazines with antibiotics against Candida albicans is reported here for the first time.     

Antitumor activity of hyperthermia alone or in combination with cisplatin and melphalan in primary cultures of human malignant melanoma

The effects of heat and the interaction between hyperthermia and alkylating agents, such as cisplatin (CDDP) and melphalan (L-PAM) in human malignant melanoma biopsies have been investigated by a short-term assay based upon the inhibition of 3H-thymidine incorporation. Cell suspensions from 50 cutaneous and lymph nodal metastases were heated at 40.5 degrees C or at 42 degrees C for 1 h. There were significant antiproliferative effects due to heat in 10% of the tumors exposed to 40.5 degrees C and 34% to 42 degrees C. Thermal resistance was evident in 73% (at 40.5 degrees C) and 54% (at 43 degrees C) of tumors, and there was significant enhancement of cell growth in 17% and 12% of tumors. The combined effects of hyperthermia and drugs were studied on 36 tumors. Cell suspensions were exposed to different concentrations of CDDP or L-PAM for 1 h at 40.5 degrees C and 42 degrees C. Synergy between heat and CDDP was observed in 7% of cases treated with the lowest drug dose and 38% of cases treated with the highest (40.5 degrees C), with only a slight increase in the frequency of synergy at 42 degrees C. Synergy between heat and L-PAM was also observed in 12% to 44% of tumors at 42 degrees C as a function of drug concentration.     

Antibacterial activity of 15-azasteroids alone and in combination with antibiotics.

A new class of 15-azasteroid analogues has been synthesized and tested for antimicrobial activity. The compounds 1, 10, 11, 11a-tetrahydro-7-methoxy-11a-methyl-2H-naphth (1,2-g) indol (methoxyimine) and 1,10,11,11a-tetrahydro-11a-methyl-2H-naphth (1,2-g) indol-7-ol (hydroxyimine) inhibit the growth of Bacillus subtillis and Escherichia coli at concentrations as low as 10-5 M. Addition of either compound to the growth medium casued a rapid inhibition in the transport of radioactive glucose, uracil and several amino acids. The inhibition of growth and substrate transport was reversed following removl of the steroid from the medium. The evidence is consistent with a site of steroid action at the cell periphery. Combining the methoxyimine with polyor circulin at subinhibitory concentrations produced greatly enhanced antimicrobial activity against Pseudomonas fluorescens. Similar action was observed against B. subtilis when the azasteroid was combined with vancomycin or chloramphenicol. The inhibitory action of other antibiotics such as penicillin or erythromycin was not affected by addition of the test compound. The results suggest formation of a molecular complex between the azasteroid and antibiotic which is responsible for the enhanced biological activity.