老鼠簕(Acanthus illicifolius L.)根际内生菌分离与活性评价

从老鼠簕(Acanthus illicifolius L.)根际土壤和植物根、叶、茎中共分离得到97株微生物,其中包括27株放线菌,44株细菌以及26株真菌,并且土壤中的分离得到的微生物最多。对获得的菌株进行细胞毒活性、抗金黄色葡萄球菌和抗白色念珠菌的测定,结果发现细菌的活性百分比最高,而放线菌和真菌活性百分比相对较小。     

厦门杏林湾红树林根际真菌分离及活性分析

【背景】海洋微生物是天然药物资源宝库,海洋环境中的微生物能够产生很多区别于陆生微生物的天然产物。红树林生长于陆地与海洋交界带的滩涂浅滩,是陆地向海洋过渡的特殊生态系统,可能蕴含着丰富的微生物资源和潜在的大量结构新颖的代谢产物。【目的】以厦门杏林湾红树林根际海泥和海水为研究对象,探索其中可培养海洋真菌的多样性及其发酵提取物的抗细菌和抗真菌活性,以期为发现新的药物先导化合物提供菌株资源。【方法】样品预处理后涂布于GPY、PDA、Martin和MEA这4种培养基,28℃倒置培养,待培养基上长出合适大小的菌落后,挑取单菌落接种于PDA培养基纯化。利用纸片扩散法对发酵提取物进行抗菌活性筛选,通过高效液相色谱分析提取物的化学多样性,并采用形态观察结合rDNAITS序列分析对活性较好的4株真菌进行菌种鉴定。【结果】共分离出71株真菌,其中49株真菌对金黄色葡萄球菌有抗性,6株真菌对白色念珠菌有抗性,2株真菌对大肠杆菌有抗性,菌株HS5-MEA-4和HS6-MEA-10初步鉴定为土曲霉,菌株HS5-GPY-7和HS6-GPY-15分别为棘孢曲霉和Aspergillus templicola。【结论】初步认知了厦门杏林湾红树林环境真菌多样性及其发酵提取物的活性和化学多样性特征,为后续海洋真菌次级代谢产物研究提供了依据。     

具抗真菌活性的海洋霉菌的筛选和初步研究

从中国南海海底沉积物与海水样品中分离出100多株海洋霉菌;以条件性致病真菌白色假丝酵母(Candida albieans),植物病原真菌镰刀菌(Fursarium sp．)等作为敏感测试菌株,初筛分离到30多株对上述测试真菌具有明显抑制作用的海洋霉菌;对其发酵液进行复筛,发现其中编号分别为B4^#-6、B4^#-3、1-B6-6、1-B6-10-5、1-B6-22、C^2#-5、A2—9和1-B6-10的海洋霉菌菌株其代谢产物粗提物有明显抗真菌活性;其抗真菌谱的实验结果表明菌株1-B6-10—5和B4^#-3的代谢产物还能抑制多种其他真菌的生长,这二个菌株具有良好的开发前景。     

蜚蠊肠道抗真菌活性放线菌的筛选与鉴定

对美洲大蠊肠道具有抗真菌活性的内生放线菌进行筛选和分类鉴定。以白色念珠菌、红色毛癣菌、黑曲霉和烟曲霉4种人体致病真菌为研究对象,采用牛津杯法和菌株对峙共培养法对159株蜚蠊肠道内生放线菌进行体外抗真菌活性筛选,采用16S rDNA PCR、Blast同源性比对和构建系统发育树等方法对菌株进行分子生物学鉴定。研究结果表明,45株内生放线菌具有抗真菌活性,诸多菌株抗真菌谱较广,有10株内生放线菌对4种受试真菌均有拮抗作用。其中,链霉菌属为优势菌属有32株,占71.11%;戈登氏菌属有6株,微杆菌属有2株,分枝杆菌属、无色杆菌属、小单孢菌属、纤维微菌属和壤霉菌属各有1株。说明蜚蠊肠道含有丰富的抗真菌活性放线菌资源。     

中药蛴螬多肽Probrelin抗白色念珠菌活性研究

[目的] 研究蛴螬多肽Probrelin对白色念珠菌的抗菌活性。[方法] 采用肉汤稀释法测定蛴螬多肽Probrelin对正常菌株及临床耐药菌株的最小抑菌浓度,同时结合平板计数法测定最小杀真菌浓度;通过不同浓度多肽处理后经平板计数绘制时间-杀菌动力学曲线;通过PI吸收实验检测多肽对白色念珠菌细胞膜完整性的影响;通过核酸阻滞实验检测多肽与核酸间是否具有结合作用;通过扫描电子显微镜检测多肽对白色念珠菌形态的影响;通过结晶紫染色法检测多肽对生物膜生成及成熟生物膜的影响;通过显微镜观察多肽对白色念珠菌菌丝形成的影响;通过棋盘法检测多肽与抗真菌药物间的相互效应;通过小鼠皮下感染模型检测多肽在生理条件下的抗白色念珠菌活性。[结果] 蛴螬多肽Probrelin对正常菌株及临床耐药菌株的最小抑菌浓度均为100 μg/mL,最小杀真菌浓度为100-200 μg/mL,且对白色念珠菌的杀菌动力学具有时间和浓度依赖性;该多肽以浓度依赖性的方式影响白色念珠菌细胞膜的完整性,并通过破坏白色念珠菌细胞壁的结构影响其形态,但与核酸间不具有结合作用;该多肽既可抑制白色念珠菌生物膜的形成,又可清除成熟生物膜,同时还可抑制白色念珠菌菌丝的形成;该多肽与抗真菌药物Clotrimazole间具有协同效应;在小鼠皮下感染模型中,该多肽可以有效杀灭白色念珠菌,进而抑制感染。[结论] 蛴螬多肽Probrelin对白色念珠菌具有良好的抑制杀灭活性,可以作为新的药物分子或模板分子用于抗白色念珠菌药物的研发。     

1株土壤放线菌的分类鉴定及其抗菌活性的研究

对从土壤微生物中筛选到的放线菌菌株1356进行分类学和抗菌活性的研究。采用多相分类法,对菌株的形态特征、培养特征、生理生化特性及16 SrRNA基因序列进行了研究。结果表明:该菌株的形态特征、培养特征、生理生化特性为链霉菌属的特征;16S rDNA序列分析及系统进化树分析表明其序列与灰色产色链霉菌的同源性最高;该菌株的发酵产物对番茄叶霉、白色念珠菌、小麦根腐菌等17种真菌均有不同程度的抑制作用。放线菌1356菌株具有广谱抗真菌活性而对细菌无作用;初步确定其为链霉菌属灰色产色链霉菌的一个亚种。     

酵母样真菌感染的菌型分布及药敏试验分析

目的:调查酵母样真菌在临床标本中的菌型分布情况;了解临床常见真菌对常用抗真菌药物的敏感性,为临床使用抗真菌药提供依据.方法:按<临床检验操作规程>进行菌株分离和鉴定;对分离的菌株用法国生物梅里埃公司生产的ATBFUNGUS抗真菌药物敏感性试剂盒进行药敏试验.结果:共检出酵母样真菌256株,其构成比:白色念珠菌161株(62.88%)、热带念珠菌58株(22.8%)、高里念珠菌12株(4.69%)、酵母菌10株(3.91%),未定型7株(2.73%);91株酵母样真菌药敏结果:5-FC敏感率95.6%、AmB 100%、NYS 93.4%、MIZ 80.2%、ECO 81.3%、KET 84.6%.11株MIZ耐药株,2株同时耐ECO与KET,9株为ECO中介、8株为KET中介,15株耐药株有10株为热带念珠菌.结论:引起深部真菌感染的酵母样真菌种类日渐增多,其中以白色念珠菌为主;大部分酵母样真菌对抗真菌药敏感,咪唑类药物有交叉耐药,多数热带念珠菌有耐药性.因此,酵母样真菌的菌种鉴定及药敏试验对临床深部真菌感染的防治具有重要的意义.     

来源于大兴安岭多年冻土可培养真菌及其发酵物的生物活性

本研究目的是分离大兴安岭多年冻土中的可培养真菌并测试它们产生的天然产物的生物活性。首先利用颗粒法在冻土中得到66株可培养真菌,通过内部转录间隔区序列对它们进行分子鉴定,发现它们隶属于55个分类单元,有8株真菌的ITS相似性≤97%。其中5株真菌属于耐冷真菌。研究结果发现6株真菌的提取物具有抗菌活性,其中外担子菌FSF028号菌株活性最强,同时具有抗真菌与抗细菌活性;柄孢壳菌FSF042号菌株具有较好的抗氧化活性;镰刀菌FSF029号菌株有较好的降解纤维素能力;此外还首次发现了Seltsamia属真菌具有抗氧化与产纤维素酶活性。     

湛江沿海潮间带产胞外抗菌活性海洋细菌的分离鉴定及生物多样性分析

从硇洲岛和徐闻珊瑚礁自然保护区潮间带采集海水和沉积物标本,采用纯培养的方法分离其中的海洋细菌;以金黄色葡萄球菌、枯草芽抱杆菌和大肠埃希菌等为指示菌,以氨苄青霉素、青霉素-链霉素为阳性对照,采用琼脂扩散法筛选抗菌活性菌株;采用基于16S rDNA序列比对及其系统发育分析对分离培养的阳性海洋细菌进行分类鉴定和生物多样性分析;为进一步从海洋细菌资源中发掘新型抗菌药物奠定基础。结果从106株海洋细菌中筛选出了44株抗细菌活性菌株,阳性率为41.5%。其中,11株具有抗金黄色葡萄球菌作用,31株有抗枯草芽孢杆菌作用,13株有抗大肠埃希菌作用。抗菌活性菌株分布于31属,优势属为芽孢杆菌属(Bacillus)和弧菌属(Vibrio)。这表明分离自硇洲岛和徐闻珊瑚礁自然保护区潮间带的海洋细菌中的抗菌活性菌株具有丰富的生物多样性。     

抗灰葡萄孢霉菌乳酸菌的筛选

从80株乳酸菌中筛选出45株具有抗灰葡萄孢霉菌活性的乳酸菌菌株,其中10株具有较强抗灰葡萄孢霉菌活性。对这10株乳酸菌菌株的抗植物致病真菌谱进行了研究,这10株乳酸菌对番茄早疫病菌,甜瓜疫霉菌,甜瓜枯萎病菌,苹果炭疽病菌的生长均有较强的抑制作用。其中1株具有广谱抗植物致病真菌活性的乳酸菌菌株BX6-4为植物乳杆菌。经番茄离体叶片接种试验发现,植物乳杆菌BX6-4的发酵液能够在体外强烈地抑制灰葡萄孢霉菌的生长。     

Evaluation of the antimicrobial activity of the acetone extract of the lichen Ramalina farinacea and its (+)-usnic acid, norstictic acid, and protocetraric acid constituents

The acetone extract of the lichen Ramalina farinacea and its (+)-usnic acid constituent showed antimicrobial activity against Bacillus subtilis, Listeria monocytogenes, Proteus vulgaris, Staphylococcus aureus, Streptococcus faecalis, Yersinia enterocolitica, Candida albicans, and Candida glabrata. Norstictic acid was active against Aeromonas hydrophila as well as the above microorganisms except Yersinia enterocolitica. Protocetraric acid showed activity only against the tested yeasts Candida albicans and Candida glabrata. The MIC values of the extract as well as of the three substances were determined. No antifungal activity of the acetone extract has been observed against ten filamentous fungi.     

Antifungal activity of coumarins

The antifungal activity of 40 coumarins was tested against the fungal strains: Candida albicans (ATCC 14053), Aspergillus fumigatus (ATCC 16913) and Fusarium solani (ATCC 36031), using the broth microdilution method. Osthenol showed the most effective antifungal activity among all the compounds tested, with a MIC value of 125 microg/ml for Fusarium solani and 250 micro/ml for Candida albicans and Aspergillus fumigatus. The antifungal potential of this prenylated coumarin can be related to the presence of an alkyl group at C-8 position.     

Fungicidal effect of prenylated flavonol, papyriflavonol A, isolated from Broussonetia papyrifera (L.) vent. against Candida albicans

Papyriflavonol A (PapA), a prenylated flavonoid (5,7,3',4'-tetrahydroxy-6,5'-di-(r,r-dimethylallyl)-flavonol), was isolated from the root barks of Broussonetia papyriferra. Our previous study showed that PapA has a broad-spectrum antimicrobial activity against pathogenic bacteria and fungi. In this study, the mode of action of PapA against Candida albicans was investigated to evaluate PapA as antifungal agent. The minimal inhibitory concentration (MIC) values were 10~25 microgram/ml for C. albicans and Saccharomyces cerevisiae, gram-negative bacteria (Escherichia coli and Salmonella typhimurium) and gram-positive bacteria (Staphylococcus epidermidis and Staphylococcus aureus). The kinetics of cell growth inhibition, scanning electron microscopy, and measurement of plasma membrane florescence anisotrophy revealed that the antifungal activity of PapA against C. albicans and S. cerevisiae is mediated by its ability to disrupt the cell membrane integrity. Compared with amphotericin B, a cell membrane disrupting polyene antibiotic, the hemolytic toxicity of PapA was negligible. At 10~25 microgram/ml of MIC levels for the tested strains, the hemolysis ratio of human erythrocytes was less than 5%. Our results suggest that PapA could be a therapeutic fungicidal agent having a broad spectrum antimicrobial agent.     

Antifungal activity of synthetic peptide derived from halocidin, antimicrobial peptide from the tunicate, Halocynthia aurantium

Halocidin is an antimicrobial peptide isolated from the hemocytes of the tunicate. Among the several known synthetic halocidin analogues, di-K19Hc has been previously confirmed to have the most profound antibacterial activity against antibiotic-resistant bacteria. This peptide has been considered to be an effective candidate for the development of a new type of antibiotic. In this study, we have assessed the antifungal activity of di-K19Hc, against a panel of fungi including several strains of Aspergillus and Candida. As a result, we determined that the MICs of di-K19Hc against six Candida albicans and two Aspergillus species were below 4 and 16 microg/ml, respectively, thereby indicating that di-K19Hc may be appropriate for the treatment of several fungal diseases. We also conducted an investigation into di-K19Hc's mode of action against Candida albicans. Our colony count assay showed that di-K19Hc killed C. albicans within 30s. Di-K19Hc bound to the surface of C. albicans via a specific interaction with beta-1,3-glucan, which is one of fungal cell wall components. Di-K19Hc also induced the formation of ion channels within the membrane of C. albicans, and eventually observed cell death, which was confirmed via measurements of the K+ released from C. albicans cells which had been treated with di-K19Hc, as well as by monitoring of the uptake of propidium iodide into the C. albicans cells. This membrane-attacking quality of di-K19Hc was also visualized via confocal laser and scanning electron microscopy.     

Antimicrobial activity of some Salvia species essential oils from Iran

The aerial parts of Salvia multicaulis, S. sclarea and S. verticillata were collected at full flowering stage. The essential oils were isolated by hydrodistillation and analyzed by combination of capillary GC and GC-MS. The in vitro antimicrobial activity of the essential oils were studied against eight Gram-positive and Gram-negative bacteria (Bacillus subtilis, Bacillus pumulis, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) and three fungi (Candida albicans, Saccharomyces cerevisiae and Aspergillus niger). The results of antibacterial activity tests of the essential oils according to the disc diffusion method and MIC values indicated that all the samples have moderate to high inhibitory activity against the tested bacteria except for P. aeruginosa which was totally resistant. In contrast to antibacterial activity, the oils exhibited no or slight antifungal property, in which only the oil of S. multicaulis showed weak activity against two tested yeasts, C. albicans and S. cerevisiae.     

Design,synthesis, and structure-activity relationship studies of novel triazole agents with strong antifungal activity against Aspergillus fumigatus

The incidence of invasive fungal infections has dramatically increased for several decades. In order to discover novel antifungal agents with broad spectrum and anti-Aspergillus efficacy, a series of novel triazole derivatives containing 1,2,3-benzotriazin-4-one was designed and synthesized. Most of the compounds exhibited stronger in vitro antifungal activities against tested fungi than fluconazole. Moreover, 6m showed comparable antifungal activity against seven pathogenic strains as voriconazole and albaconazole, especially against Aspergillus fumigatus (MIC = 0.25 μg/ml), and displayed moderate antifungal activity against fluconazole-resistant strains of Candida albicans. A clear SAR study indicated that compounds with groups at the 7-position resulted in novel antifungal triazoles with more effectiveness and a broader-spectrum.     

Design and synthesis of spiro[indole-thiazolidine]spiro[indole-pyrans] as antimicrobial agents

A series of novel spiro[indole-thiazolidine]spiro[indole-pyran] derivatives were synthesized from N-(bromoalkyl)indol-2,3-diones via monospiro-bisindole intermediates; the two indole nuclei being connected via N-(CH(2))(n)-N linker. Synthesized compounds were evaluated for their antimicrobial activities in vitro against three Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, and Staphylococcus epidermis), four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumonia) as well as four fungi (Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, and Candida albicans) using Cup plate method. Bis spiro-indoles exhibited stronger antibacterial and antifungal efficiency than their corresponding mono spiro-indoles. Compound 10e, the most active derivative was shown to inhibit the growth of all bacterial strains and two fungal strains (A. niger and C. albicans).     

Cross-Species Induction of Antimicrobial Compounds, Biosurfactants and Quorum-Sensing Inhibitors in Tropical Marine Epibiotic Bacteria by Pathogens and Biofouling Microorganisms

Enhancement or induction of antimicrobial, biosurfactant, and quorum-sensing inhibition property in marine bacteria due to cross-species and cross-genera interactions was investigated. Four marine epibiotic bacteria (Bacillus sp. S3, B. pumilus S8, B. licheniformis D1, and Serratia marcescens V1) displaying antimicrobial activity against pathogenic or biofouling fungi (Candida albicans CA and Yarrowia lipolytica YL), and bacteria (Pseudomonas aeruginosa PA and Bacillus pumilus BP) were chosen for this study. The marine epibiotic bacteria when co-cultivated with the aforementioned fungi or bacteria showed induction or enhancement in antimicrobial activity, biosurfactant production, and quorum-sensing inhibition. Antifungal activity against Y. lipolytica YL was induced by co-cultivation of the pathogens or biofouling strains with the marine Bacillus sp. S3, B. pumilus S8, or B. licheniformis D1. Antibacterial activity against Ps. aeruginosa PA or B. pumilus BP was enhanced in most of the marine isolates after co-cultivation. Biosurfactant activity was significantly increased when cells of B. pumilus BP were co-cultivated with S. marcescens V1, B. pumilus S8, or B. licheniformis D1. Pigment reduction in the quorum-sensing inhibition indicator strain Chromobacterium violaceum 12472 was evident when the marine strain of Bacillus sp. S3 was grown in the presence of the inducer strain Ps. aeruginosa PA, suggesting quorum-sensing inhibition. The study has important ecological and biotechnological implications in terms of microbial competition in natural environments and enhancement of secondary metabolite production.     

Screening for antibacterial and antifungal activities in marine benthic invertebrates from northern Norway

Benthic marine invertebrates collected from sub-Arctic regions of northern Norway, were found to be a promising source of novel bioactive compounds against human and fish pathogenic bacteria and fungi. Lyophilized material from seven species of ascidians, six sponges and one soft alcyonid coral were extracted with 60% acidified acetonitrile (ACN). After separation into an ACN-rich phase (ACN-extract) and an aqueous phase, and subsequent solid-phase extraction of the aqueous phase, fractions differing in polarity were obtained and screened for antibacterial and antifungal activities, along with the more lipophilic ACN-extracts. Antimicrobial activity was determined against two Gram-negative, two Gram-positive bacteria, and two strains of fungi. Notably, all the invertebrate species in the study showed activity against all four strains of bacteria and the two strains of fungi. In general, the aqueous fractions displayed highest antimicrobial activity, and the most potent extracts were obtained from the colonial ascidian Synoicum pulmonaria which displayed activity against bacteria and fungi at a concentration of 0.02 mg/ml; the lowest concentration tested.     

Synthesis and SAR studies of biaryloxy-substituted triazoles as antifungal agents

A series of 1-(substituted biaryloxy)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl) propan-2-ol were synthesized and their antifungal activities were evaluated against eight human pathogenic fungi in vitro. Seventeen compounds showed activity 4- to 64-fold higher than voriconazole against Candida albicans. SAR clearly suggested that introduction of a biaryloxy side chain greatly enhanced the antifungal activity of triazole analogs against Candida species.