Essential Oils,Silver Nanoparticles and Propolis as Alternative Agents Against Fluconazole Resistant Candida albicans,Candida glabrata and Candida krusei Clinical Isolates

Development of effective and safe therapeutic treatment of fungal infections remains one of the major challenge for modern medicine. The aim of presented investigation was to analyze the in vitro antifungal activity of selected essential oils, ethanolic extracts of propolis and silver nanoparticles dropped on TiO₂ against azole-resistant C. albicans (n = 20), C. glabrata (n = 14) and C. krusei (n = 10) clinical isolates. Among tested essential oils, the highest activity has definitely been found in the case of the oil isolated from the bark of Cinnamomum cassia, with MIC and MFC values for all tested strains in the range of 0.0006–0.0097 % (v/v) and 0.0012–0.019 % (v/v), respectively. High activity was also observed for the Lemon, Basil, Thyme, Geranium and Clove (from buds) essential oils. Significant differences in fungicidal activity have been observed in the case of four tested propolis samples. Only one of them revealed high activity, with MFC values in the range from 0.156 to 1.25 % (v/v). Satisfactory fungicidal activity, against C. albicans and C. glabrata isolates, was also observed in the case of silver nanoparticles, however C. krusei isolates were mostly resistant. We also revealed that constituents of most of essential oils and propolis as well as silver nanoparticles are not substrates for drug transporters, which belong to the most important factors affecting resistance of Candida spp. clinical isolates to many of conventional antimycotics. To conclude, the results of our investigation revealed that essential oils, propolis and silver nanoparticles represent high potential for controlling and prevention candidiasis.     

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.     

Antibacterial Activity of Green Synthesized Silver Nanoparticles Using Vasaka (Justicia adhatoda L.) Leaf Extract

There is an increasing demand for silver nanoparticles due to its wide applicability in various area of biological science such as in field of antimicrobial and therapeutics, biosensing, drug delivery etc. To use in bioprocess the silver nanoparticles should be biocompatible and free from toxic chemicals. In the present study we report a cost effective and environment friendly route for green synthesis of silver nanoparticles using Vasaka (Justicia adhatoda L.) leaf extract as reducing as well as capping agent. This plant has been opted for the present study for its known medicinal properties and it is easily available. The biosynthesized silver nanoparticles are characterized by UV–Vis spectroscopy and TEM analysis. It is observed the nanoparticles are well shaped and the average particle size is 20 nm in the range of 5–50 nm. The antibacterial activity of these nanoparticles against Pseudomonas aeruginosa MTCC 741 has been measured by disc diffusion method, agar cup assay and serial dilution turbidity measurement assay. The results show green synthesized silver nanoparticles, using Vasaka leaf extract, have a potential to inhibit the growth of bacteria.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0512-1) contains supplementary material, which is available to authorized users.     

Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin

Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9) isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.     

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies with Aspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellow peroba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium berghei in mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.     

Antibodies from Chicken Eggs as Probes for Antigens from Pasteuria penetrans Endospores

The bacteria Pasteuria spp. have been identified as among the most promising of several microbial organisms currently under investigation as biological control agents of plant-parasitic nematodes. As part of our goal to develop methods to discriminate isolates of Pasteuria penetrans with different host preferences, we investigated the potential of developing antibody probes to identify endospores of different isolates of P. penetrans. Polyclonal IgY antibodies were raised in chickens against endospores of P. penetrans isolates P20 and P100. Hens were injected with P20 or P100 endospore suspensions and boosted at 14 days. Anti-spore titers were determined with ELISA on yolk extracts of individual eggs as a function of time. The highest titers were found in eggs produced at 22 to 35 days after initial injections. Yolk extracts showing the highest titers were combined and processed to provide partially purified IgY preparations. SDS-PAGE and immunoblot analyses identified protein antigens with Mr values of 23-24, 46, and 57-59 KDa common to both P20 and P100 endospores. One protein antigen with an Mr value of 62 KDa was unique to the PI00 endospores. The IgY antibodies reduced the attachment of Pasteuria endospores to their nematode hosts, indicating antibody interaction with antigens on the endospore surface that are involved in the recognition and attachment processes.     

Antimicrobial Activities of Silver Nanoparticles Synthesized by Using Water Extract of Arnicae anthodium

Green synthesis of nanoparticles has gained significant importance in recent years and has become the one of the most preferred methods. Also, green synthesis of nanoparticles is valuable branch of nanotechnology. Plant extracts are eco-friendly and can be an economic option for synthesis of nanoparticles. This study presents method the synthesis of silver nanoparticles using water extract of Arnicae anthodium. Formation of silver nanoparticles was confirmed by UV–visble spectroscopy, Fourier transform infrared spectroscopy and total reflection X-ray fluorescence analysis. The morphology of the synthesized silver nanoparticles was verified by SEM–EDS. The obtained silver nanoparticles were used to study their antimicrobial activity.     

Suppression of Alfalfa Growth by Concommitant Populations of Pratylenchus penetrans and two Fusarium species

Growth of alfalfa (Medicago sativa cv. Vernal) seedlings was compared after inoculation with combinations of either Pratylenchus penetrans and Fusarium soloni or P. penetrans and F. oxysporum f. sp. medicaginis. A synergistic disease interaction occurred in alfalfa when F. oxysporum and P. penetrans were added simultaneously to the soil. Alfalfa growth was suppressed at all inoculum levels of P. penetrans and F. oxysporum, but not with F. solani. Seedlings inoculated with the nematode alone gave lower yields than when inoculated with either Fusarium species alone. Fusarium oxysporum, but not F. solani, was pathogenic to alfalfa under similar experimental conditions. Fusarium oxysporum did not alter the populations of P. penetrans in alfalfa roots, whereas the presence of F. solani was associated with a diminished number of P. penetrans in the roots.     

Antifungal activity of C3a and C3a-derived peptides against Candida

Antimicrobial peptides are generated during activation of the complement system [Nordahl et al. Proc. Natl. Acad. Sci. U. S. A. 2004, 101:16879-16884]. Here we show that the anaphylatoxin C3a exerts antimicrobial effects against the yeast Candida. Fluorescence microscopy and electron microscopy analysis demonstrated that C3a-derived peptides bound to the cell surface of Candida, and induced membrane perturbations and release of extracellular material. Various Candida isolates were found to induce complement degradation, leading to generation of C3a. Arginine residues were found to be critical for the antifungal and membrane breaking activity of a C3a-derived antimicrobial peptide, CNY21 (C3a; Cys⁵⁷-Arg⁷⁷). A CNY21 variant with increased positive net charge displayed enhanced antifungal activity. Thus, C3a-derived peptides can be utilized as templates in the development of peptide-based antifungal therapies.     

Investigation of aryl isonitrile compounds with potent,broad-spectrum antifungal activity

Invasive fungal infections present a formidable global public health challenge due to the limited number of approved antifungal agents and the emergence of resistance to the frontline treatment options, such as fluconazole. Three fungal pathogens of significant concern are Candida, Cryptococcus, and Aspergillus given their propensity to cause opportunistic infections in immunocompromised individuals. New antifungal agents composed of unique chemical scaffolds are needed to address this public health challenge. The present study examines the structure-activity relationship of a set of aryl isonitrile compounds that possess broad-spectrum antifungal activity primarily against species of Candida and Cryptococcus. The most potent derivatives are capable of inhibiting growth of these key pathogens at concentrations as low as 0.5 µM. Remarkably, the most active compounds exhibit an excellent safety profile and are non-toxic to mammalian cells even at concentrations up to 256 µM. The present study lays the foundation for further investigation of aryl isonitrile compounds as a new class of antifungal agents.     

The Influence of Trichoderma harzianum on the Root-knot Fusarium Wilt Complex in Cotton

Wilt-susceptible cultivar ''Rowden'' cotton was inoculated wilh Meloidogyne incognita (N), Trichoderma harzianum (T), and Fusarium oxysporum f. sp. vasinfectum (F) alone and in all combinations in various time sequences. Plants inoculated with F alone or in combination with T did not develop wilt, Simultaneous inoculation of 7-day-old seedlings with all three organisms (NTF) produced earliest wilt. However, plants receiving nematodes at 7 days and Fusarium and Trichoderma at 2 or 4 weeks later (N-T-F, N-TF) developed the greatest wilt between 49-84 days after initial nematode inoculation. During the same period, Fusarium added 4 weeks after initial nematode inoculation (N-F) and Fusarium added 4 weeks after initial simultaneous inoculation of nematode and Trichoderma (NT-F) produced the least wilt. The addition of Fusarium inhibited nematode reproduction. Simultaneous inoculation with nematodes and Trichoderma (NT-) resulted in the greatest root gall development, whereas nematodes alone produced the greatest number of larvae. In comparison with noninoculated controls (CK), treatments involving all three organisms inhibited plant growth, plants inoculated with the nematode alone (N-) or with nematodes and Trichoderma (NT-) simultaneously had greatest root weight. Any treatment involving the nematode resulted in fewer bolls per plant and greater necrosis on roots than the noninoculated checks.     

Ecdysteroids in Axenically Propagated Caenorhabditis elegans and Culture Medium

Ecdysteroids (insect molting hormones) from Caenorhabditis elegans were chromatographically purified and quantified by radioimmunoassay. Nematodes from semidefined medium contained the immunoreactive equivalent of 460 pg ecdysone per gram dry weight. Culture medium, however, contained the immunoreactive equivalent of 68 times the quantity within the nematodes. In a defined medium lacking immunoreactivity, C. elegans contained 520 pg ecdysone equivalents per gram dry weight but reproduced slowly. Reproduction of C. elegans in defined medium was enhanced by formulation in agar. Propagation of C. elegans in either agar-based or aqueous defined medium supplemented with [¹⁴C]cholesterol of high specific activity failed to result in production of radiolabeled free ecdysteroids or polar or apolar ecdysteroid conjugates. Failure to demonstrate ecdysteroid biosynthesis in C. elegans raises questions about the ecdysteroids identified previously in nematodes being products of endogenous biosynthesis, a necessary condition for these compounds to be nematode hormones.     

Antifungal potential of extracellular metabolites produced by Streptomyces hygroscopicus against phytopathogenic fungi

Indigenous actinomycetes isolated from rhizosphere soils were assessed for in vitro antagonism against Colletotrichum gloeosporioides and Sclerotium rolfsii. A potent antagonist against both plant pathogenic fungi, designated SRA14, was selected and identified as Streptomyces hygroscopicus. The strain SRA14 highly produced extracellular chitinase and β-1,3-glucanase during the exponential and late exponential phases, respectively. Culture filtrates collected from the exponential and stationary phases inhibited the growth of both the fungi tested, indicating that growth suppression was due to extracellular antifungal metabolites present in culture filtrates. The percentage of growth inhibition by the stationary culture filtrate was significantly higher than that of exponential culture filtrate. Morphological changes such as hyphal swelling and abnormal shapes were observed in fungi grown on potato dextrose agar that contained the culture filtrates. However, the antifungal activity of exponential culture filtrates against both the experimental fungi was significantly reduced after boiling or treatment with proteinase K. There was no significant decrease in the percentage of fungal growth inhibition by the stationary culture filtrate that was treated as above. These data indicated that the antifungal potential of the exponential culture filtrate was mainly due to the presence of extracellular chitinase enzyme, whereas the antifungal activity of the stationary culture filtrate involved the action of unknown thermostable antifungal compound(s).     

Predisposition of Broadleaf Tobacco to Fusarium Wilt by Early Infection with Globodera tabacum tabacum or Meloidogyne hapla

In greenhouse experiments, broadleaf tobacco plants were inoculated with tobacco cyst (Globodera tabacum tabacum) or root-knot (Meloidogyne hapla) nematodes 3, 2, or 1 week before or at the same time as Fusarium oxysporum. Plants infected with nematodes prior to fungal inoculation had greater Fusarium wilt incidence and severity than those simultaneously inoculated. G. t. tabacum increased wilt incidence and severity more than did M. hapla. Mechanical root wounding within 1 week of F. oxysporum inoculation increased wilt severity. In field experiments, early-season G. t. tabacum control by preplant soil application of oxamyl indirectly limited the incidence and severity of wilt. Wilt incidence was 48%, 23%, and 8% in 1989 and 64%, 60%, and 19% in 1990 for 0.0, 2.2, and 6.7 kg oxamyl/ha, respectively. Early infection of tobacco by G. t. tabacum predisposed broadleaf tobacco to wilt by F. oxysporum.     

Structure–activity relationship of cytochrome bc1 reductase inhibitor broad spectrum antifungal ilicicolin H

Ilicicolin H is a broad spectrum antifungal agent showing sub micro g/mL MICs against Candida spp., Aspergillus fumigatus and Cryptococcus spp. It is a potent inhibitor (C₅₀ 2–3 ng/mL) of the mitochondrial cytochrome bc1 reductase with over 1000-fold selectivity against rat liver cytochrome bc1 reductase. Structure–activity relationship of semisynthetic derivatives by chemical modification of ilicicolin H and its 19-hydroxy derivative produced by biotransformation have been described. Basic 4′-esters and moderately polar N- and O-alkyl derivatives retained antifungal and the cytochrome bc1 reductase activities. 4′,19-Diacetate and 19-cyclopropyl acetate retained antifungal and enzyme activity and selectivity with over 20-fold improvement of plasma protein binding.     

Ultrastructural Changes Caused by Fusarium oxysporum f. sp. lycopersici in Meloidogyne javanica Induced Giant Cells in Fusarium Resistant and Susceptible Tomato Cultivars

Tomato (Lycopersicon esculentum Mill.) seedlings, susceptible (cv. Pearson A-I Improved) and resistant (cv. Pearson Improved) to race 1 Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyd &Hans., were inoculated with Meloidogyne javanica (Trueb) Chitwood second-stage juveniles and 3 weeks later with race 1 F. oxysporum f. sp. lycopersici spores. One week after fungal inoculation, no fungus was visible in root tissue of the tomato cultivars and the giant cells were normal. Two weeks after fungal inoculation, abundant hyphae were visible in xylem tissues of Fusarium-susceptible but not of Fusarium-resistant plants. In susceptible plants, giant cell degeneration occurred, characterized by membrane and organelle disruption. In addition, where hyphae were in direct contact with the giant cell, dissolution of the giant cell wall occurred. Three weeks after fungal inoculation, fungal hyphae and spores were visible inside xylem tissues and giant cells in Fusarium-susceptible plants and in xylem tissue of the resistant plants. In susceptible and resistant plants, giant cell degeneration was apparent. Giant cell walls were completely broken down in Fusarium-susceptible tomato plants. In both cultivars infected by Fusarium, giant cell nuclei became spherical and dark inclusions occurred within the chromatin material which condensed adjacent to the fragmented nuclear membrane. No such ultrastructural changes were seen in the giant cells of control plants inoculated with nematode alone. Giant cell deterioration in both cultivars is probably caused by toxic fungal metabolites.     

Volatile Substances Produced by Fusarium oxysporum from Coffee Rhizosphere and Other Microbes affect Meloidogyne incognita and Arthrobotrys conoides

Microorganisms produce volatile organic compounds (VOCs) which mediate interactions with other organisms and may be the basis for the development of new methods to control plant-parasitic nematodes that damage coffee plants. In the present work, 35 fungal isolates were isolated from coffee plant rhizosphere, Meloidogyne exigua eggs and egg masses. Most of the fungal isolates belonged to the genus Fusarium and presented in vitro antagonism classified as mutual exclusion and parasitism against the nematode-predator fungus Arthrobotrys conoides (isolated from coffee roots). These results and the stronger activity of VOCs against this fungus by 12 endophytic bacteria may account for the failure of A. conoides to reduce plant-parasitic nematodes in coffee fields. VOCs from 13 fungal isolates caused more than 40% immobility to Meloidogyne incognita second stage juveniles (J₂), and those of three isolates (two Fusarium oxysporum isolates and an F. solani isolate) also led to 88-96% J₂ mortality. M. incognita J₂ infectivity decreased as a function of increased exposure time to F. oxysporum isolate 21 VOCs. Gas chromatography-mass spectrometry (GC-MS) analysis lead to the detection of 38 VOCs produced by F. oxysporum is. 21 culture. Only five were present in amounts above 1% of the total: dioctyl disulfide (it may also be 2-propyldecan-1-ol or 1-(2-hydroxyethoxy) tridecane); caryophyllene; 4-methyl-2,6-di-tert-butylphenol; and acoradiene. One of them was not identified. Volatiles toxic to nematodes make a difference among interacting microorganisms in coffee rhizosphere defining an additional attribute of a biocontrol agent against plant-parasitic nematodes.     

Fungal Parasites of the Potato Cyst Nematode Globodera rostochiensis: Isolation and Reinfection

Fungal parasitism of eggs of the potato cyst nematode Globodera rostochiensis was < 1, 3, and 17% at three sites in Sweden. The fungi isolated most frequently from infected eggs were a Septocylindrium-like fungus ( 19 %), Exophiala spp. (17 %), and Cylindrocarpon spp. (13 %). Verticillium suchtasporium was isolated from infected eggs at a low frequency (4%). In laboratory experiments V. suchlasporium infected 93% of the eggs within cysts after 10 days on dilute corn meal agar. This species showed chitinase and protease activity. Infection of eggs by the Septocylindrium-like fungus was moderate, whereas Cylindrocarpon destructans and Cladosporium cladosporoides did not infect eggs. No chitinase activity was found in these fungi, but protease activity was recorded in all. Growth of the fungi in cysts did not influence the number of physiologically disordered eggs.     

Biocontrol Potential of Siderophore Producing Heavy Metal Resistant <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> RZS3 vis-à-vis Organophosphorus Fungicide

In present study in vitro phytopathogen suppression activity of siderophoregenic preparations of Ni and Mn resistant Alcaligenes sp. STC1 and Pseudomonas aeruginosa RZS3 SH-94B isolated from soil were found superior over the chemical pesticide. Siderophore rich culture broth and siderophore rich supernatant exerted antifungal activity against Aspergillus niger NCIM 1025, Aspergillus flavus NCIM 650, Fusarium oxysporum NCIM 1281, Alternaria alternata ARI 715, Cercospora arachichola, Metarhizium anisopliae NCIM 1311 and Pseudomonas solanacerum NCIM 5103. Siderophore rich broth and supernatant exhibited potent antifungal activity vis-à-vis oraganophosphorus chemical fungicide; kitazine. The minimum fungicidal concentration required was 25 μl for Aspergillus niger, Aspergillus flavus, Fusarium oxysporum, Cercospora arachichola, Metarhizium anisopliae, Pseudomonas solanacerum and 75 μl for A. alternata.     

Benzimidazole tethered pyrrolo[3,4-b]quinoline with broad-spectrum activity against fungal pathogens

Fungal infections caused by Candida and Cryptococcus are particularly dangerous for immunocompromised individuals. In this study, we identified that benzimidazole fused pyrrolo[3,4-b]quinoline compounds have potent antifungal activity against several clinical isolates of pathogenic fungal strains. Specifically, the compound 6a did not show cytotoxicity against mammalian cells at a concentration that inhibits the growth of fungal strains. In addition, the compound 6a also significantly reduced the metabolic activity of fungal cells in the Candida albicans biofilms. Collectively, our results indicate that benzimidazole fused quinoline compounds have a potential to develop as an antifungal agents.