Biological and chemical characterization of antimicrobial activity in Arthrobacter spp. isolated from disease-suppressive compost

Antagonistic bacteria can act as biocontrol agents against various phytopathogens. Recently, Arthrobacter spp. demonstrated antifungal activity, but were not further characterized. In this study, the antimicrobial activity of Arthrobacter humicola strains M9-1A, M9-2, and M9-8, and Arthrobacter psychrophenolicus strain M9-17 were evaluated against nine plant pathogens in vitro, and their cell-free filtrates were additionally assessed for inhibition of Alternaria alternata and suppression of black mold disease on tomato fruit. Results indicated that A. humicola M9-1A and A. psychrophenolicus M9-17 were the most inhibitory, reducing growth of seven of the pathogens studied. Cell-free filtrates of A. psychrophenolicus M9-17 reduced the growth of most pathogens. All cell-free bacterial filtrates, except those from A. humicola M9-2, suppressed black mold on tomato fruit. Disk diffusion assays with ethyl acetate soluble culture filtrate extracts of all bacteria reduced the mycelial growth of A. alternata. Clear inhibition zones were observed for A. psychrophenolicus M9-17 extracts using drop bioassays. The antifungal compound N-acetyltryptamine was purified and characterized from the A. psychrophenolicus M9-17 cell-free ethyl acetate soluble extract. This study suggests that antibiosis may play a key role in the antimicrobial activity of Arthrobacter spp.     

Volatile organic compounds produced by Aureobasidium pullulans induce electrolyte loss and oxidative stress in Botrytis cinerea and Alternaria alternata

Aureobasidium pullulans is a yeast-like fungus that produces volatile organic compounds (VOCs) with antifungal properties. VOCs have the potential to trigger the production of intracellular reactive oxygen species (ROS), lipid peroxidation and electrolyte loss in microorganisms. The relationship among A. pullulans VOCs, induced ROS accumulation and electrolyte leakage was investigated in Botrytis cinerea and Alternaria alternata in vitro. Exposure to a mixture of A. pullulans VOCs: ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol, resulted in electrolyte leakage in both B. cinerea and A. alternata. Fluorescence microscopy using 2′,7′-dichlorofluorescein diacetate indicated triggered ROS accumulation in exposed fungal mycelia and the presence of the superoxide radical was evident by intense red fluorescence with dihydroethidium. Partial inhibition of enzymes of the mitochondrial respiratory chain complex I of B. cinerea and A. alternata by pre-treatment with rotenone reduced ROS accumulation in hypha exposed to A. pullulans VOCs and reversed the VOCs inhibition of fungal growth. Scanning electron micrographs revealed that B. cinerea and A. alternata hypha exposed to A. pullulans VOCs had altered cell wall structures. Our findings give insights into the potential mechanisms involved in the antifungal properties of A. pullulans in the suppression of B. cinerea and A. alternata growth in vitro.     

In vitro comparison of cilofungin alone and in combination with other antifungal agents against clinical isolates ofCandida species

Cilofungin, a lipopeptide antifungal agent, was tested for in vitro activity alone and in combination with ketoconazole, itraconazole, flucytosine and amphotericin B against 102 clinical isolates ofCandida species. At 48 hours all isolates ofCandida albicans, Candida tropicalis, Candida paratropicalis andCandida glabrata were inhibited by 5 mcg/ml of cilofungin. In contrast, the MIC90 forCandida krusei was 10 mcg/ml and forCandida parapsilosis >40 mcg/ml. The interaction of combinations of cilofungin with amphotericin B, itraconazole, ketoconazole and flucytosine was additive or indifferent at 48 hours for 100 %, 88 %, 78 % and 70 % of allCandida species isolates, respectively. Overall, cilofungin demonstrated good activity in vitro against mostCandida species isolates.     

Activity of amphotericin B and itraconazole against intraphagocyticCandida albicans

The activity of amphotericin B and itraconazole against intracellularCandida albicans was determined in vitro using murine resident peritoneal macrophages. Amphotericin B at concentrations of 0.5 and 2 µg/ml produced significantly less rapid killing of intracellular than of extracellularCandida albicans as measured in macrophage-free medium. Amphotericin B at concentrations of 0.1 µg/ml or itraconazole concentrations of up to 3 µg/ml produced only fungistatic or limited fungicidal activity against both intracellular and extracellular organisms. Against intracellularCandida albicans amphotericin B acted by direct antifungal action rather than through stimulation of macrophage function, as demonstrated by the fact that (i) activity persisted when macrophages were successively exposed to amphotericin B, washed and disrupted by sonication, and (ii) no activity was seen when amphotericin B was tested against intracellular amphotericin B-resistantCandida tropicalis orSalmonella typhimurium. Pre-exposure of macrophages to itraconazole (0.4 µg/ml) inhibited subsequent killing activity of amphotericin B (2 µg/ml) against intracellular susceptibleCandida albicans. These experiments validate the conventional methods of susceptibility testing for determining the fungistatic activity of antifungal agents.     

Isolation,structure elucidation and biological activity of angucycline antibiotics from an epiphytic yew streptomycete

In the course of study of epiphytic microorganisms occurring on the surface of roots of Taxus baccata L. a new strain Streptomyces sp. AC113 was isolated. According to 16S ribosomal DNA‐based identification the new strain is 99% identical with Streptomyces flavidofuscus. This strain cultivated in an arginine glycerol medium produced three major metabolites identified as (–)‐8‐O ‐methyltetrangomycin ( 1 ), 8‐O ‐methyltetrangulol ( 2 ) and 8‐O ‐methyl‐7‐deoxo‐7‐hydroxytetrangomycin ( 3 ). The chemical structures of these angucyclines were elucidated by 1D and 2D NMR as well as by mass spectrometry. Isolated angucycline metabolites showed significant antimicrobial activity against Bacillus cereus and Listeria mocytogenes. Cytotoxic activities of compounds 1 , 2 and 3 against four cell lines (B16, HT‐29 and non – tumor V79, L929) were evaluated. Compound 3 was the most potent anticancer agents with IC₅₀ 0.054 μg/ml against cell line B16. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Proposal for a fourth aquabirnavirus serogroup

Four putative aquabirnaviruses, based on morphology, nucleic acid type and partial RNA-dependent RNA polymerase gene (VP1) sequence, isolated from three tropical freshwater fish species were not neutralised by antisera against type members of the Aquabirnavirus genus serogroups A, B or C. Antisera produced against two of the isolates neutralised the homologous and heterologous isolates, but not any type member of Aquabirnavirus serogroups A, B or C. The serological comparisons suggest that the four isolates should be regarded as members of a fourth Aquabirnavirus serogroup, D.     

Antimicrobial properties of fungal macrolide antibiotics

Antibacterial, antifungal and antiprotozoal effects of curvularin, cyanein, monorden, and cytochalasins A, B and D were studied. Curvularin and cytochalasin B showed no inhibition of the microorganisms tested. Cyanein and cytochalasin D, besides their cytotoxicity, are antifungal antibiotics. Cytochalasin A inhibits growth of Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Candida albicans, Mucor corymbifer, Aspergillus fumigatus, and Botrytis cinerea. Monorden was active against B. subtilis, S. cerevisiae, C. albicans, B. cinerea, and also against Euglena gracilis. Cyanein, monorden, and cytochalasins A and D induce an intensive ramification of B. cinerea hyphae. Biological activity of the studied compounds is discussed in terms of their chemical structures.     

Caspofungine et mycoses à champignons rares : activité in vitro et in vivo chez l’animal et chez l’homme

Caspofungine belongs to the echinocandin family. It is a large lipopeptide molecule that inihibits the β-(1,3)-glucan synthesis, a cell wall component. No drug target is present in mammalian cells. In vitro data and experimental studies have demonstrated that caspofungin displays antifungal activity against most Candida spp. (including isolates resistant to azoles and amphotericin B), several species of filamentous fungi, including Aspergillus and certain dimorphic fungi, such as Histoplasma, Blastomyces and Coccidioïdes. However, the molecule displays no activity in vitro against Fusarium spp., Zygomycetes (Mucor circinelloides, Absidia corymbifera, Rhizomucor pusillus, Cunninghamella bertholletiae, etc.), Rhizopus spp. and Pseudoallescheria spp. Caspofungin generated low Minimum Inhibitory Concentrations (MICs) and Minimum Effective Concentrations (MECs) against Aspergillus but not against Fusarium. Studies were conducted using several experimental models of fungal infection. They were not active at clinically relevant concentrations against Zygomycetes, Cryptococcus neoformans or Fusarium spp. Absence of antagonism in combination with other antifungal drugs suggests that combination antifungal therapy could become a general feature of echinocandins, particularly in the case of invasive aspergillosis. The results of these first clinical trials are promising but further studies are required to define the exact role of caspofungin in the arsenal of antifungal agents. New trials using caspofungin alone or in combination are necessary to demonstrate its efficacy in the treatment of mycoses due to uncommon fungi.     

In vitro activity of amphotericin B,fluconazole and voriconazole against 162 <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> isolates from Africa and Cambodia

In order to determine the potential role that various antifungal agents might have in the management of cryptococcosis in tropical areas, the in vitro susceptibility of Cryptococcus neoformans isolates from Africa (n=52) and Cambodia (n=110) to three antifungal agents (amphotericin B, fluconazole and voriconazole) were compared using the E-test method. The results of this study (i) confirm the value of the E-test for testing the in vitro susceptibility of C. neoformans towards voriconazole; (ii) provide the first evidence demonstrating good activity of amphotericin B, fluconazole and voriconazole against Cambodian isolates; and (iii) show there are differences in susceptibility between African and Asian C. neoformans isolates, with Cambodian isolates appearing less susceptible to the agents tested but with amphotericin B maintaining good activity.     

Antibiosis of vineyard ecosystem fungi against food-borne microorganisms

Fermentation extracts from fungi isolated from vineyard ecosystems were tested for antimicrobial activities against a set of test microorganisms, including five food-borne pathogens (Staphylococcus aureus EP167, Acinetobacter baumannii (clinically isolated), Pseudomonas aeruginosa PAO1, Escherichia coli O157:H7 (CECT 5947) and Candida albicans MY1055) and two probiotic bacteria (Lactobacillus plantarum LCH17 and Lactobacillus brevis LCH23). A total of 182 fungi was grown in eight different media, and the fermentation extracts were screened for antimicrobial activity. A total of 71 fungi produced extracts active against at least one pathogenic microorganism, but not against any probiotic bacteria. The Gram-positive bacterium S. aureus EP167 was more susceptible to antimicrobial fungi broth extracts than Gram-negative bacteria and pathogenic fungi. Identification of active fungi based on internal transcribed spacer rRNA sequence analysis revealed that species in the orders Pleosporales, Hypocreales and Xylariales dominated. Differences in antimicrobial selectivity were observed among isolates from the same species. Some compounds present in the active extracts were tentatively identified by liquid chromatography–mass spectrometry. Antimicrobial metabolites produced by vineyard ecosystem fungi may potentially limit colonization and spoilage of food products by food-borne pathogens, with minimal effect on probiotic bacteria.     

Rapid flow cytometric susceptibility testing of Candida albicans.

A rapid flow cytometric assay for in vitro antifungal drug susceptibility testing was developed by adapting the proposed reference method for broth macrodilution testing of yeasts. Membrane permeability changes caused by the antifungal agent were measured by flow cytometry using propidium iodide, a nucleic acid-binding fluorochrome largely excluded by the intact cell membrane. We determined the in vitro susceptibility of 31 Candida albicans isolates and two quality control strains (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) to amphotericin B and fluconazole. Amphotericin B MICs ranged from 0.03 to 2.0 microg/ml, while fluconazole MICs ranged from 0.125 to 128 microg/ml. This method results in clear-cut endpoints that were reproducible. Four-hour incubation was required for fluconazole, whereas a 2-h incubation was sufficient for amphotericin B to provide MICs comparable to the reference macrodilution method developed by the National Committee for Clinical Laboratory Standards Subcommittee on Antifungal Susceptibility Tests. Results of these studies show that flow cytometry provides a rapid and sensitive in vitro method for antifungal susceptibility testing of C. albicans.     

Phologeny of Alternaria fungi known to produce host-specific toxins on the basis of variation in internal transcribed spacers of ribosomal DNA

The internal transcribed spacer regions (ITS1 and ITS2) of ribosomal DNA from Alternaria species, including seven fungi known to produce host-specific toxins, were analyzed by polymerase chain reaction-amplification and direct sequencing. Phylogenetic analysis of the sequence data by the Neighbor-joining method showed that the seven toxin-producing fungi belong to a monophyletic group together with A. alternata. In contract, A. dianthi, A. panax, A. dauci, A. bataticola, A. porri, A. sesami and A. solani, species that can be morphologically distinguished from A. alternata, could be clearly separated from A. alternata by phylogenetic analysis of the ITS variation. These results suggest that Alternaria pathogens which produce host-specific toxins are pathogenic variants within a single variable species, A. alternata.This paper is dedicated to the late Syoyo Nishimura     

Molecular karyotypes for Alternaria plant pathogens known to produce host-specific toxins

There are at least ten plant diseases caused by Alternaria species in which host-specific toxins (HSTs) are responsible for fungal pathogenicity. Of these HST-producers, seven are considered distinct pathotypes of the species Alternaria alternata, and the remaining three are among other species of pathogenic Alternaria. Inter- and intra-specific variation among Alternaria taxa, including HST-producers, was determined by electrophoretic karyotyping using pulsed-field gel electrophoresis. A. alternata including seven pathotypes of A. alternata and eight non-pathogenic strains had 9–11 chromosomal bands with estimated sizes ranging from 0.4 to 5.7 Mb. In contrast, Alternaria species that are morphologically distinct from A. alternata had 8–10 bands with sizes between 0.9 and 5.7 Mb. Estimated genome sizes of A. alternata and other Alternaria species ranged from 28.8 to 33.6 Mb and 25.1 to 30.7 Mb, respectively. Other species of pathogenic Alternaria were difficult to differentiate from A. alternata on the basis of chromosome-size polymorphisms alone, but Southern analysis using rDNA as a probe could, in some cases, differentiate between them. These results were cytologically confirmed by 4′,6-diamidino-2-phenylindole (DAPI) staining and fluorescence in situ hybridization with a rDNA probe for mitotic metaphase chromosomes prepared by the germ-tube burst method. Received: 20 October 1998 / 5 February 1999     

Characterization of an antifungal compound produced by Bacillus sp. strain A5F that inhibits Sclerotinia sclerotiorum

A potential antagonist, Bacillus sp. strain A₅F was isolated from soybean rhizosphere following in vitro dual plate screening. The bacterium displayed strong inhibitory activity in vitro against soybean stem rot pathogen, Sclerotinia sclerotiorum. The culture supernatant of strain A₅F completely suppressed the mycelial growth of the pathogen, indicating that suppression was due to the presence of antifungal compounds in the culture filtrate. The culture filtrate also suppressed other phytopathogenic fungi including Fusarium oxysporum and Macrophomina phaseolina, in vitro suggesting a broad spectrum antagonistic activity against fungal pathogens. Chemical extraction followed by chromatographic analysis resulted in two antifungal fractions. The high resolution‐electron spin ionization‐mass spectrometry (HR‐ESI‐MS) and Nuclear Magnetic Resonance (1D and 2D¹H) spectra of these antifungal fractions revealed the presence of antifungal compounds, one of which showed similarity to bacillomycin D. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Alternaria alternata TCTP, a novel cross-reactive ascomycete allergen

Defining more comprehensively the allergen repertoire of the ascomycete Alternaria alternata is undoubtedly of immense medical significance since this mold represents one of the most important, worldwide occurring fungal species responsible for IgE-mediated hypersensitivity reactions ranging from rhinitis and ocular symptoms to severe involvement of the lower respiratory tract including asthma with its life-threatening complications. Performing a hybridization screening of an excised A. alternata cDNA library with a radioactively labeled Cladosporium herbarum TCTP probe, we were able to identify, clone and purify the respective A. alternata homologue of TCTP which again represents a multifunctional protein that has been evolutionarily conserved from unicellular eukaryotes like yeasts to humans and appears, summarizing current literature, to be involved in housekeeping processes such as cell growth as well as cell-cycle progression, the protection of cells against various stress conditions including for instance apoptosis, and in higher organisms even in the allergic response. In this context, our present study characterizes recombinant A. alternata TCTP as a novel minor allergen candidate that displays a prevalence of IgE reactivity of approximately 4% and interestingly shares common, cross-reactive IgE epitopes with its C. herbarum and human counterparts as determined via Western blotting and in vitro inhibition approaches.     

The ultrasound‐assisted extraction and identification of antifungal substances from B. amyloliquefaciens strain NJN‐6 suppressing Fusarium oxysporum

The primary mechanism underlying antagonism among microorganisms is the production of antagonistic substances called antibiotics that inhibit the growth of pathogens. In this study, the antagonistic substances produced by the Bacillus amyloliquefaciens strain NJN‐6 that had antifungal activity against Fusarium oxysporum were extracted and identified. The active antifungal substance was extracted from dried leavening with ultrasound‐assisted extraction (UAE), using n ‐butanol as the extractant. HPLC/ESI‐MS was performed to investigate the components of the extracts. The results of the study showed that the antimicrobial substances consisted of three homologues of the iturin A family with molecular weights of 1043, 1057 and 1071 Da and of two homologues of the fengycin family with molecular weights of 1477 and 1491 Da. The effects of ultrasonic treatment time, extraction time and extractant volume, three major methodological parameters, were also studied to determine the optimal conditions for extraction. Compared with traditional extraction techniques, UAE is a simple, cheap and environmentally friendly method that represents a new option for the isolation and identification of lipopeptides and other active compounds. These antifungal substances extracted and identified from Bacillus amyloliquefaciens NJN‐6 will help us to understand its biocontrol mechanism against Fusarium oxysporum. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Pradimicins: A novel class of broad-spectrum antifungal compounds

Pradimicins are a new class of antifungal compounds currently undergoing preclinical and early phase I clinical trials. The pradimicin structure is characterized by an aglycone of dihydrobenzo (alpha) naphthacenequinone with substitutions by a D-amino acid and hexose sugar. Pradimicins prossess a novel mechanism of action consisting of a specific binding recognition to terminal D-mannosides of the cell wall ofCandida albicans, resulting in the formation of a ternary complex consisting of D-mannoside, pradimicin, and calcium that leads to disruption of the integrity of the fungal cell membrane. Pradimicin in the form of BMS-181184 has broad-spectrum in vitro antifungal activity againstCandida spp.,Cryptococcus neoformans, Aspergillus spp., dematiaceous molds, and the Zygomycetes.Fusarium spp. are comparatively resistant to high concentrations of pradimicin. Initial in vivo studies indicate that pradimicins have antifungal activity against experimental murine disseminated candidiasis and disseminated aspergillosis. Early studies indicate an excellent therapeutic index with no major end-organ toxicity. Pradimicins warrant further investigation for treatment of opportunistic mycoses in immunocompromised hosts.     

Antifungal activity of an allicin derivative against Penicillium expansum via induction of oxidative stress

The application of natural preservatives has become an attractive method for controlling postharvest decay of fruits and vegetables. Allicin, the main active ingredient of allium plants, has broad-spectrum antifungal activity. However, the unstable properties of allicin limit its wide application. In this study, 1-[(R)-ethylsulfinyl]sulfanylethane, a structurally stable derivative of allicin, was used to explore its antifungal activity and potential mechanism on the expansion of Penicillium expansum. We demonstrated the antifungal activity of 1-[(R)-ethylsulfinyl]sulfanylethane through in vitro antifungal experiments. At the concentration is 2 mg/L, 1-[((R)-ethylsulfinyl]sulfanyl]ethane can completely inhibit spore germination and mycelial growth, whereas the concentration of allicin needs to reach 16 mg/L. Fungal Biochemical assay indicated that decrease of mitochondrial membrane potential, overgeneration of reactive oxygen species, decrease of adenosine triphosphate and glutathione content, increase of superoxide dismutase, catalase, and malondialdehyde content. The results revealed that 1-[(R)-ethylsulfinyl]sulfanylethane induced mitochondrial dysfunction and oxidative stress in P. expansum. Due to its excellent antifungal activity, 1-[((R)-ethylsulfinyl]sulfanyl]ethane might be developed as a substitute for fungicides against P. expansum in postharvest fruit storage.     

Hyper‐responsive Toll‐like receptor 7 and 9 activation in NADPH oxidase‐deficient B lymphoblasts

Chronic granulomatous disease (CGD) is an inherited immunodeficiency linked with mutations in the multi-subunit leucocyte NADPH oxidase. Myeloid-derived phagocytic cells deficient in NADPH oxidase fail to produce sufficient levels of reactive oxygen species to clear engulfed pathogens. In this study we show that oxidase also influences B-cell functions, including responses to single-stranded RNA or unmethylated DNA by endosomal Toll-like receptors (TLRs) 7 and 9. In response to TLR7/9 ligands, B-cell lines derived from patients with CGD with mutations in either the NADPH oxidase p40^phox or p47^phox subunits produced only low levels of reactive oxygen species. Remarkably, cytokine secretion and p38 mitogen-activated protein kinase activation by these oxidase-deficient B cells was significantly increased upon TLR7/9 activation when compared with oxidase-sufficient B cells. Increased TLR responsiveness was also detected in B cells from oxidase-deficient mice. NADPH oxidase-deficient patient-derived B cells also expressed enhanced levels of TLR7 and TLR9 mRNA and protein compared with the same cells reconstituted to restore oxidase activity. These data demonstrate that the loss of oxidase function associated with CGD can significantly impact B-cell TLR signalling in response to nucleic acids with potential repercussions for auto-reactivity in patients.     

Fungicidal activity of thymol and carvacrol by disrupting ergosterol biosynthesis and membrane integrity against Candida

Natural isopropyl cresols have been reported to have antifungal activity. This work is an attempt to examine thymol and carvacrol against 111 fluconazole-sensitive and -resistant Candida isolates. Insight into the mechanism of action was elucidated by flow cytometric analysis, confocal imaging and ergosterol biosynthesis studies. The susceptibility tests for the test compounds were carried out in terms of minimum inhibitory concentrations (MICs), disc diffusion assays and time–kill curves against all Candida isolates by employing standard protocols. Propidium iodide (PI) cell sorting has been investigated by flow cytometric analysis and confocal imaging. Haemolytic activity on human erythrocytes was studied to exclude the possibility of further associated cytotoxicity. Both compounds were found to be effective to varying extents against all isolates, including the resistant strains. In contrast to the fungistatic nature of fluconazole, our compounds were found to exhibit fungicidal nature. Significant impairment of ergosterol biosynthesis was pronouncedly induced by the test entities. Negligible cytoxicity was observed for the same compounds. Furthermore, it was observed that the positional difference of the hydroxyl group in carvacrol slightly changes its antifungal activity. Carvacrol and thymol show strong fungicidal effect against all of the Candida isolates. The mechanisms of action of these natural isopropyl cresols appear to originate from the inhibition of ergosterol biosynthesis and the disruption of membrane integrity.