The fungicidal activity of novel nanoemulsion (X8W60PC) against clinically important yeast and filamentous fungi

Surfactant nanoemulsions are water in oil preparations that proved to have a broad spectrum biocidal activity against a variety of microorganisms including Gram-positive and Gram-negative bacteria, spores and enveloped viruses. These preparations are non-toxic to the skin, mucous membrane and gastrointestinal tissues at biocidal concentrations. In this study, 0.1% of the nanoemulsion designated X8W60PC has shown fungicidal activity against yeast including Candida albicans and C. tropicalis in 15 minutes. C. tropicalis was more sensitive than C. albicans, which required a longer time or a higher concentration of the nanoemulsion to achieve killing. Neutral to slightly alkaline pH was more effective in killing the yeast cells than acidic pH. Using the minimum inhibitory concentration assay, 0.08% of the nanoemulsion was inhibitory to C. albicans, and parapsilosis and filamentous fungi including Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum, Aspergillus fumigatus and Fusarium oxysporum. None of the individual ingredients was as effective a fungicidal as the nanoemulsion at equivalent concentration. This shows that the nanoemulsion structure is an important factor in the anti-fungal activity. The X8W60PC has great potential as a topical anti-fungal agent and further investigation into the mechanism of fungicidal action is warranted.     

The fungicidal activity of novel nanoemulsion (X8W60PC) against clinically important yeast and filamentous fungi

Surfactant nanoemulsions are water in oil preparations that proved to have a broad spectrum biocidal activity against a variety of microorganisms including Gram-positive and Gram-negative bacteria, spores and enveloped viruses. These preparations are non-toxic to the skin, mucous membrane and gastrointestinal tissues at biocidal concentrations. In this study, 0.1% of the nanoemulsion designated X8W₆₀PC has shown fungicidal activity against yeast including Candida albicans and C. tropicalis in 15 minutes. C. tropicalis was more sensitive than C. albicans, which required a longer time or a higher concentration of the nanoemulsion to achieve killing. Neutral to slightly alkaline pH was more effective in killing the yeast cells than acidic pH. Using the minimum inhibitory concentration assay, 0.08% of the nanoemulsion was inhibitory to C. albicans, and parapsilosis and filamentous fungi including Microsporum gypseum,Trichophyton mentagrophytes,Trichophyton rubrum,Aspergillus fumigatus andFusarium oxysporum.None of the individual ingredients was as effective a fungicidal as the nanoemulsion at equivalent concentration. This shows that the nanoemulsion structure is an important factor in the anti-fungal activity. The X8W₆₀PC has great potential as a topical anti-fungal agent and further investigation into the mechanism of fungicidal action is warranted.This revised version was published online in October 2005 with corrections to the Cover Date.     

Antibiotic activity of the pyrenocines

Pyrenocine A, a phytotoxin produced by Pyrenochaeta terrestris (Hansen) Gorenz, Walker and Larson, possesses general antibiotic activity against plants, fungi, and bacteria. Effective doses for 50% inhibition (ED50s) are 4 micrograms/mL for onion seedling elongation; 14, 20, 20, and 25 micrograms/mL for the germination of asexual spores of Fusarium oxysporum f. sp. cepae, Fusarium solani f. sp. pisi, Mucor hiemalis, and Rhizopus stolonifer, respectively. Pyrenocine A also inhibits the linear mycelial growth of both P. terrestris and F. oxysporum with ED50s calculated as 77 and 54 micrograms/mL, respectively. Gram-positive bacteria are more susceptible to pyrenocine A than Gram-negative bacteria. ED50s are estimated as 30, 45, and 200 micrograms/mL for the inhibition of growth of Bacillus subtilis, Staphylococcus aureus, and Escherichia coli, respectively, with Pseudomonas aeruginosa resistant to those concentrations tested. Pyrenocine A acts primarily as a biostatic rather than a biocidal agent with all organisms tested showing some degree of recovery when released from pyrenocine A. Pyrenocines B and C show little antibiotic activity in the bioassays performed.     

Phylogenetic analysis on the bacteria producing non-volatile fungistatic substances

This study characterized the soil bacteria producing non-volatile fungistatic substances. Among the 2,100 colonies of soil bacteria randomly isolated from seven agricultural soil samples, 518 isolates (24.67% of total) showed fungistatic activity toward nematophagous fungi Paecilomyces lilacinus and Trichoderma viride by producing non-volatile substances. A phylogenetic analysis based on amplified ribosomal DNA restriction analysis (ARDRA) and 16S rDNA sequence placed the 518 bacteria in three groups of the domain Bacteria: Actinomycetales, Bacillales, and Gammaproteobacteria. Three genera, Arthrobacter, Bacillus, and Pseudomonas, were the most frequently encountered groups.     

Resistance of vaccinia virus to inactivation by solvent/detergent treatment of blood products.

The inactivation of enveloped viruses by two different solvent/detergent combinations, i.e. tri-n-butyl phosphate (TNBP)/Triton X-100 or TNBP/Tween 80, has been investigated using a high purity factor VIII (Replenate) and factor IX (Replenine) respectively. Treatment with TNBP/Triton X-100 rapidly inactivated all the typical enveloped viruses tested, i.e. Sindbis, semliki forest virus (SFV), herpes simplex virus type-1 (HSV-1) and vesicular stomatitis virus (VSV), by 3.7-5.8 log within 15 seconds. While virus inactivation with TNBP/Tween 80 was slower, effective inactivation of Sindbis, HSV-1, VSV and human immunodeficiency virus type-1, i.e. 4.1-->6.3 log, occurred within 30 minutes. In contrast, vaccinia virus was relatively resistant to inactivation in either of these solvent/detergent combinations. Incubation times of 10 minutes for TNBP/Triton X-100 or 6-24 hours for TNBP/Tween 80, were required to reach inactivation levels of about 4 log.     

Assignment of defensin gene(s) to human chromosome 8p23

A relatively abundant component of the polymorphonuclear leukocyte granulocytes has been recently isolated and called defensin. Defensins have antimicrobial activity against gram-positive and gram-negative bacteria and enveloped viruses. A cDNA insert for defensin HNP-1 (DEF1) has been used to map the gene(s) to human chromosome 8p23 using a mouse/human somatic cell hybrid panel and in situ hybridization to normal human metaphase chromosomes. Because of the similarity of HNP-1 defensin to other defensins, it is likely that two of these genes map to this region.     

Chemotactic assay for biological effects of silver nanoparticles

A method is proposed for assessing the biocidal efficacy of water-dispersed nanoparticles of silver. It is based on negative chemotaxis of the plasmodia of the slime mold Physarum polycephalum. Biocidal and repellent effects were compared for silver nanoparticles, Ag⁺ ions, and AOT in solution and in the agar gel. In such characteristics as increasing the period of auto-oscillations of contractile activity, decreasing the area of spreading on substrate, and substrate preference in spatial tests, silver nanoparticles proved to be substantially more effective than Ag⁺ and AOT. The lethal concentrations of the nanoparticles were close to those found earlier for bacteria and viruses. The chemotactic tests allow quantitative assessment of the biological reaction and monitoring its dynamics; in resolution, they are superior to the tests based on the lethal action of biocidal agents.     

Inhibition of Influenza A Virus Infection In Vitro by Peptides Designed In Silico

Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H⁺ ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.     

Structure and Leukemogenic Activity of a Murine Leukemia Virus

Purified Friend viruses obtained from chronically infected tissue cultures were studied under the electron microscope in an effort to correlate the fine structure of the particles to their leukemogenic activity under varied experimental conditions, i.e., temperature treatments and exposure to Tween 80, amyl acetate, or ether. It was observed that an intact viral envelope was a prerequisite to leukemogenic activity as tested by intraperitoneal inoculation of newborn mice. It was also noted that the percentage of C particles was not increased after heating for 1 hr at 45 C (treatment which, however, completely inactivated the viruses). Digestion with ribonuclease indicated the presence of ribonucleic acid within the nucleoids of "enveloped A particles," which shows that these are not immature particles. The significance of the simultaneous presence of "enveloped A" and C particles is discussed.     

Inactivation of lipid enveloped viruses by octanoic Acid treatment of immunoglobulin solution.

Inactivation of lipid enveloped viruses by treatment with octanoic acid has been investigated for three intravenous immunoglobulin preparations, using Human Immunodeficiency Virus, Bovine Viral Diarrhoea Virus, Sindbis Virus and Pseudorabies Virus as test viruses. At a concentration of 7.45 g octanoic acid per kg solution complete inactivation of lipid enveloped viruses to below detectable level (>5.36, >4.68, >6.25 and >5.55 log(10), respectively) was achieved within the first minutes of treatment. Octanoic acid treatment as described here, has been demonstrated as an effective and rapid virus inactivation procedure, which shows high robustness at the tested ranges of temperature, pH and protein content of the test material. However, pH must be considered as a critical parameter of treatment, as octanoic acid fails to inactivate lipid coated viruses at basic pH. At suitable conditions, e.g. pH<6.0 and a concentration of >3.7 g/kg, octanoic acid treatment gives reliable and highly effective inactivation of lipid enveloped viruses.     

Antimicrobial mechanism of action of surfactant lipid preparations in enteric Gram-negative bacilli

Two surfactant lipid preparations (SLPs) were investigated to determine their mechanism of antimicrobial action. 8N8, a water-in-oil emulsion, and W60C, a liposome, both have bactericidal activity against Gram-positive bacteria and non-enteric Gram-negative bacteria. Additionally, W60C is bactericidal for enteric Gram-negative bacilli when suspended in deionized water. Zeta potential measurements suggested that the resistance of Gram-negative bacilli to 8N8 might be caused by ionic repulsion. Addition of 50 micromol 1(-1) ethylene diamine tetra acetic acid in 100 mmol 1(-1) Tris buffer to either SLPs yielded efficient bactericidal activity against Gram-negative bacilli. This appeared to be due to disruption of the outer membrane and the chelation of divalent cations, as the addition of excess calcium inhibited the antimicrobial effect. Electron microscopy studies documented that 8N8 disrupts the bacterial cell wall, lysing the bacteria, while W60C fuses and internalizes within the cell, causing damage without immediate cell lysis. Understanding the mechanisms of action of these biocidal formulations will help to produce improved formulations with broader spectra of activity.     

芽胞杆菌及其代谢物抑制包膜病毒感染的研究进展

包膜病毒指具有一层脂质双层膜的病毒,如流感病毒、冠状病毒等,这些包膜病毒每年在世界范围内导致许多严重的疾病,严重威胁人类健康。使用抗病毒药物是预防与治疗病毒感染的主要策略,芽胞杆菌(Bacillus)及其代谢物能够抑制多种包膜病毒的感染。本文综述了芽胞杆菌代谢的粗提物、肽、酶、胞外聚合物、小双链RNA和热灭活的枯草芽胞杆菌孢子在抗包膜病毒感染中发挥的重要作用,其机制是通过直接破坏病毒包膜、阻止膜融合、与病毒基因组RNA直接配对、催化裂解病毒RNA、激活天然免疫反应等对抗病毒,期望为包膜病毒的持续预防和治疗提供参考。     

Antifungal activity displayed by cereulide, the emetic toxin produced by Bacillus cereus

In this study, the fungistatic activity of Bacillus cereus cereulide-producing strains was demonstrated against nine fungal species. The role of cereulide was confirmed using plasmid-cured derivatives and ces knockout mutants. The fungistatic spectra of cereulide and valinomycin, a chemically related cyclododecadepsipeptide, were also compared and found to be similar but distinct.     

Inhibition of enveloped virus infection of cultured cells by valproic acid

Valproic acid (VPA) is a short-chain fatty acid commonly used for treatment of neurological disorders. As VPA can interfere with cellular lipid metabolism, its effect on the infection of cultured cells by viruses of seven viral families relevant to human and animal health, including eight enveloped and four nonenveloped viruses, was analyzed. VPA drastically inhibited multiplication of all the enveloped viruses tested, including the zoonotic lymphocytic choriomeningitis virus and West Nile virus (WNV), while it did not affect infection by the nonenveloped viruses assayed. VPA reduced vesicular stomatitis virus infection yield without causing a major blockage of either viral RNA or protein synthesis. In contrast, VPA drastically abolished WNV RNA and protein synthesis, indicating that this drug can interfere the viral cycle at different steps of enveloped virus infection. Thus, VPA can contribute to an understanding of the crucial steps of viral maturation and to the development of future strategies against infections associated with enveloped viruses.     

Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses

Enveloped viruses often enter cells via endocytosis; however, specific endocytic trafficking pathway(s) for many viruses have not been determined. Here we demonstrate, through the use of dominant-negative Rab5 and Rab7, that influenza virus (Influenza A/WSN/33 (H1N1) and A/X-31 (H3N2)) requires both early and late endosomes for entry and subsequent infection in HeLa cells. Time-course experiments, monitoring viral ribonucleoprotein colocalization with endosomal markers, indicated that influenza exhibits a conventional endocytic uptake pattern – reaching early endosomes after approximately 10 min, and late endosomes after 40 min. Detection with conformation-specific hemagglutinin antibodies indicated that hemagglutinin did not reach a fusion-competent form until the virus had trafficked beyond early endosomes. We also examined two other enveloped viruses that are also pH-dependent for entry – Semliki Forest virus and vesicular stomatitis virus. In contrast to influenza virus, infection with both Semliki Forest virus and vesicular stomatitis virus was inhibited only by the expression of dominant negative Rab5 and not by dominant negative Rab7, indicating an independence of late endosome function for infection by these viruses. As a whole, these data provide a definitive characterization of influenza virus endocytic trafficking and show differential requirements for endocytic trafficking between pH-dependent enveloped viruses .     

抗菌肽及其临床应用研究进展

抗菌肽是生物体在抵抗病原微生物的防御反应过程中产生的一类具有抗微生物活性的小分子多肽。抗菌肽是机体天然免疫系统的重要组成部分,具有广谱的抗革兰氏阳性、阴性菌活性,对真菌、某些有包膜的病毒、寄生虫以及肿瘤细胞也有抑制活性。抗菌肽具有不同于传统抗生素的独特抗菌机制,病原菌不宜对其产生耐药性,有可能成为一种新的抗生素替代品。介绍了抗菌肽的来源与分类、理化特性与生物学活性,并重点阐述其最新的临床应用进展。     

Lipid-specific membrane activity of human beta-defensin-3

Defensins represent a major component of innate host defense against bacteria, fungi, and enveloped viruses. One potent defensin found, e.g., in epithelia, is the polycationic human beta-defensin-3 (hBD3). We investigated the role of the lipid matrix composition, and in particular the presence of negatively charged lipopolysaccharides (LPS) from sensitive (Escherichia coli, Salmonella enterica serovar Minnesota) or resistant (Proteus mirabilis) Gram-negative bacteria or of the zwitterionic phospholipids of human cells, in determining the action of polycationic hBD3 on the different membranes, and related to their biological activity. The main focus was directed on data derived from electrical measurements on a reconstitution system of the OM as a planar asymmetric bilayer composed on one side of LPS and on the other of a phospholipid mixture. Our results demonstrate that the antimicrobial activity and the absence of cytotoxicity can be explained by the lipid-specificity of the peptide. A clear correlation between these aspects of the biological activity of hBD3 and its interaction with lipid matrices could be found. In particular, hBD3 could only induce lesions in those membranes resembling the lipid composition of the OM of sensitive bacterial strains. The permeation through the membrane is a decisive first step for the biological activity of many antimicrobial peptides. Therefore, we propose that the lipid-specificity of hBD3 as well as some other membrane-active antimicrobial peptides is important for their activity against bacteria or mammalian cells.     

Composition and antimicrobial activity of fatty acids detected in the hygroscopic secretion collected from the secretory setae of larvae of the biting midge Forcipomyia nigra (Diptera: Ceratopogonidae)

The hygroscopic secretion produced by the secretory setae of terrestrial larvae of the biting midge Forcipomyia nigra (Winnertz) was analysed using gas chromatography coupled with mass spectrometry (GC-MS). The viscous secretion is stored at the top of each seta and absorbs water from moist air. GC-MS analyses (four independent tests) showed that the secretion contained 12 free fatty acids, the most abundant of which were oleic (18:1), palmitic (16:0), palmitoleic (16:1) and linoleic (18:2). Other acids identified were valeric (5:0), enanthic (7:0), caprylic (8:0), pelargonic (9:0), capric (10:0), lauric (12:0), myristic (14:0) and stearic (18:0). Two other compounds, glycerol and pyroglutamic acid, were also found. The antibacterial activity of the fatty acids and pyroglutamic acid was tested using the agar disc diffusion method and targeted Gram positive (Bacillus cereus, Bacillus subtilis, Enterococcus faecalis) and Gram negative bacterial strains (Citrobacter freundii, Pseudomonas aeruginosa, Pseudomonas fluorescens). The antifungal activity was tested by determining minimal inhibitory concentration (MIC) of examined compounds. Fatty acids were tested against enthomopathogenic fungi (Paecilomyces lilacinus, Paecilomyces fumosoroseus, Lecanicillium lecanii, Metarhizium anisopliae, Beauveria bassiana (Tve-N39), Beauveria bassiana (Dv-1/07)). The most effective acids against bacterial and fungal growth were C(9:0), C(10:0) and C(16:1), whereas C(14:0), C(16:0,) C(18:0) and C(18:1) demonstrated rather poor antifungal activity and did not inhibit the growth of bacteria. The antimicrobial assay investigated mixtures of fatty and pyroglutamic acids (corresponding to the results of each GC-MS test): they were found to be active against almost all the bacteria except P. fluorescens and also demonstrated certain fungistatic activity against enthomopathogenic fungi. The hygroscopic secretion facilitates cuticular respiration and plays an important role in the antimicrobial protection of F. nigra larvae living in moist terrestrial habitats.     

黄颡鱼肠道病原拮抗性芽孢杆菌的筛选与特性研究

采用80℃水浴法从黄颡鱼肠道分离到65 株芽孢杆菌, 牛津杯法拮抗试验筛选到3 株抑菌活性很强的菌株, API 50CHB 试剂盒鉴定它们为枯草芽孢杆菌。选取抑菌活性最强的F14 进行特性研究。结果显示: F14在80℃、90℃和沸水浴中处理5min 后的存活率分别为96.7%、95.3%、52.9%。随时间的延长, F14 在90℃和沸水浴中的存活率均出现了较大幅度的下降。沸水浴处理10min 时, 其存活率减少至8.3%。在pH 3.0—5.0的范围内, 处理F14 1h、2h 后的存活率分别在98%、87%以上。F14 代谢粗提物具有较强的抑菌作用, 病原性嗜水气单胞菌在粗提物中培养2h, 细菌数量就开始急剧下降, 至 24h 时已检测不到活的细菌。此外, 喷洒在饲料中的F14 在室温、4℃和?20℃条件下储存, 细菌数量在4℃和?20℃存储20d 均无显著变化, 仅在室温下存储至第20 天时显著减少。研究表明, F14 抗菌谱较广, 抑菌作用强, 耐高温和酸性环境, 在饲料中稳定性好, 具有作为水产益生菌应用的潜力。       

Inactivation of laboratory animal RNA-viruses by physicochemical treatment

Eight commonly used chemical disinfectants and physical treatments (UV irradiation and heating) were applied to both enveloped RNA viruses (Sendai virus, canine distemper virus) and unenveloped RNA viruses (Theiler's murine encephalomyelitis virus, reo virus type 3) to inactivate infectious virus particles. According to the results, alcohols (70% ethanol, 50% isopropanol), formaldehyde (2% formalin), halogen compounds (52ppm iodophor, 100ppm sodium hypochlorite), quaternary ammonium chloride (0.05% benzalkonium chloride) and 1% saponated cresol showed virucidal effects giving more than 99.95% reduction in the infectivity of virus samples of Sendai virus and canine distemper after 10 minutes exposure. There was no significant difference in the effects on the two enveloped RNA viruses. The susceptibility of unenveloped RNA viruses to chemical disinfectants and physical treatments differed greatly from the enveloped viruses. The two unenveloped viruses showed distinct resistance to 50% isopropanol, 2% formalin, 1% saponated cresol and to physical treatments (heating at 45, 56, 60 degrees C, and UV irradiation). These results indicate that using physicochemical methods to inactivate RNA viruses in laboratory animal facilities should be considered in accordance with the characteristics of the target virus. For practical purposes in disinfecting enveloped RNA viruses, 70% ethanol, 0.05% quaternary ammonium chloride and 1% saponated cresol diluted in hot water (greater than 60 degrees C) are considered as effective as UV irradiation. For unenveloped RNA viruses, halogen compounds, more than 1,000 ppm sodium hypochlorite or 260 ppm iodophor are recommended over a period of 10 minutes for disinfecting particles, although these compounds result in an oxidation problem with many metals.