Purification of Candida guilliermondii and Pichia ohmeri killer toxin as an active agent against Penicillium expansum

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Molecular identification of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular identification of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila

Traditional phenotypic methods and commercial kits based on carbohydrate assimilation patterns are unable to consistently distinguish among isolates of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila. As result, these species are often misidentified. In this work, we established a reliable method for the identification/differentiation of these species. Our assay was validated by DNA sequencing of the polymorphic region used in a real-time PCR assay driven by species-specific probes targeted to the fungal ITS 1 region. This assay provides a new tool for pathogen identification and for epidemiological, drug resistance and virulence studies of these organisms.

CTAB, Triton X-100 and freezing-thawing treatments of Candida guilliermondii: Effects on permeability and accessibility of the glucose-6-phosphate dehydrogenase, xylose reductase and xylitol dehydrogenase enzymes

Cells of Candida guilliermondii (ATCC 201935) were permeabilised with surfactant treatment (CTAB or Triton X-100) or a freezing-thawing procedure. Treatments were monitored by in situ activities of the key enzymes involved in xylose metabolism, that is, glucose-6-phosphate dehydrogenase (G6PD), xylose reductase (XR) and xylitol dehydrogenase (XD). The permeabilising ability of the surfactants was dependent on its concentration and incubation time. The optimum operation conditions for the permeabilisation of C. guilliermondii with surfactants were 0.41 mM (CTAB) or 2.78 mM (Triton X-100), 30 degrees C, and pH 7 at 200 rpm for 50 min. The maximum permeabilisation measured in terms of the in situ G6PD activity observed was, in order, as follows: CTAB (122.4 +/- 15.7 U/g(cells)) > freezing-thawing, , (54.3 +/- 1.9 U/g(cells)) > Triton X-100 (23.5 +/- 0.0 U/g(cells)). These results suggest that CTAB surfactant is more effective in the permeabilisation of C. guilliermondii cells in comparison to the freezing-thawing and Triton X-100 treatments. Nevertheless, freezing-thawing was the only treatment that allowed measurable in situ XR activity. Therefore, freezing-thawing permeabilised yeast cells could be used as a source of xylose reductase for analytical purposes or for use in biotransformation process such as xylitol preparation from xylose. The level of in situ xylose reductase was found to be 13.2 +/- 0.1 U/g(cells).

Candida proteinases in the degradation of oral mucosal tissue components associated with Candida invasion

The aim of this thesis was to compare the degradation of human oral epithelial proteins by proteinases of different Candida yeast species. We focused on proteins associated with Candida invasion in the cell-to-cell junction, the basement membrane zone, the extracellular matrix, and local tissue inflammatory regulators. Another main objective was to evaluate the effect of the yeast/hyphal transition and pH on the degradative capability of Candida. The enzymatic activity of the Candida proteinases was verified by gelatin zymography. Laminins-332 (Lm-322) and -511(Lm-511) produced by human oral keratinocytes were gathered from the growth media, and E-cadherin (E-Cad) was isolated from the cell membrane of the keratinocytes by immunoprecipitation. The proteins were incubated with Candida cells and cell-free fractions, and degradation was detected by fluorography. Fibronectin degradation was visualised by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Matrix metalloproteinase-9 (MMP-9) activation and tissue inhibitor of metalloproteinase-1 (TIMP-1) fragmentation was detected by using the Western blot and enhanced chemoluminescence (ECL) techniques. Residual activity of TIMP-1 was evaluated by a casein degradation assay. A fluorimetric assay was used to detect and compare Candida proteinase activities with MMP-9. These studies showed that the ability of the different Candida yeast species to degrade human Lm-332, fibronectin, and E-Cad vary from strain to strain and that this degradation is pH-dependent. This indicates that local acidic pH in tissue may play a role in tissue destruction by activating Candida proteinases and aid invasion of Candida into deeper tissue. A potential correlation exists between the morphological form of the yeasts and the degradative ability; the C. albicans yeast form seems to be related to superficial infections, and hyphal forms can apparently invade deeper tissues between the epithelial cells by degradation of E-Cad. Basement membrane degradation is possible, especially in the junctional epithelium, which contains only Lm-332 as a structural component. Local tissue host inflammatory mediators, such as MMP-9, were activated, and TIMP-1 was degraded by certain Candida species, thus indicating the possibility of a weakened host tissue defence mechanism in vivo.

果实采后病害生物防治及其机理研究

　　分离和筛选了5种能有效防治采后果实病害的拮抗菌。其中，季也蒙假丝酵母(Candida guiliermondii(Cast) Langeroret Guerra)从引种拮抗菌中筛选获得，枯草芽孢杆菌（Bacillus subtilis）B-912从土壤中分离筛选获得，膜醭毕赤酵母（Pichia membranefaciens hansen）从桃果实伤口上分离获得，隐球酵母(Cryptococcus albidus (Saito) Skinner)和丝孢酵母（Trichosporon sp.）从桃果实表面分离获得。本文主要研究了这些拮抗菌对桃、油桃、苹果、梨和柑桔等我国主要水果采后病害的防治效果，并对其可能的抑菌机理进行了初步研究。结果如下： 1. Sx108 CFU/mL的C．guiliermondii和P．membranefaciens悬浮液可完全抑制病菌孢子浓度为5x104个/mL时桃和油桃果实软腐病(Rhizopus stolonifer(Ehrenb.ex Fr.) Vuill.)在25℃，15℃和3℃下的发生。lx108 CFU/mL的C．albidus和Trichosporon sp.悬浮液可完全抑制孢子浓度分别为lx105个/mL和5x104个/mL时苹果灰霉病(Botrytis cinerea)和青霉病（Penicillum expansum）在23℃-25℃和1℃下的发生。C．albidus和Trichosporon sp.对梨灰、青霉病也有一定抑制效果。B-912对柑桔果实青霉病(Penicillium italicum)、绿霉病(Penicillium digitatum)和核果类果实褐腐病(Monilinia fructicola)也有极好的抑制效果。生物防治效果与拮抗菌的浓度成正比，与病菌孢子浓度成反比。 2．拮抗酵母菌在室温和冷藏条件下都能迅速在果实伤口定殖，接种酵母菌48 h后，数量可增加20倍以上。拮抗菌和病菌孢子的接种时间与生物防治效果有关，先接种拈抗菌的抑菌效果显著地好于同时或后于病菌接种的效果。 3．温度对拮抗酵母菌的抑菌活力没有明显影响，无论是在室温还是在冷藏条件下，拮抗酵母菌都有同样的抑菌效果。但拮抗细菌B-912的抑菌效果与温度有一定关系。较高的温度有利于细菌拮抗作用的发挥。 4．拮抗菌能与常规的果实采后处理措施如钙处理、化学杀菌剂、冷藏和气调贮藏相结合。酵母菌与2% CaC12配合能明显地增强其抑菌能力;拮抗菌与低浓度的杀菌剂如扑海因混合使用，可达到高浓度杀菌剂的抑病效果;C．albidus和Trichosporon sp.对果实采后气调贮藏环境有良好的适应性，它们在气调下对采后苹果、梨的灰霉病和青霉病的抑制效果比冷藏条件下的好。 5．细菌B-912的抑菌机理与其产生抗菌素有关，B-912的滤液在in vitro上能有效地抑制病菌孢子的萌发，在invivo上也能明显地抑制果实采后病害的发生。拮抗酵母菌的抑菌机理则较复杂，但主要与病菌竞争营养有关，同时，C．guilliermondii和P．membranefaciens对软腐菌的抑制还通过产生水解酶如β-1，3一葡聚糖酶和几丁酶与病原菌直接作用，并参与诱导寄主产生抗性等

外源物质对酵母拮抗菌生活力的影响

　　近年来，酵母拮抗菌在水果采后病害防治中展示了良好的应用前景。然而，在实际应用中,酵母拮抗菌在逆境条件下会因为发生凋亡或细胞损伤而引起生活力的下降，最终导致拮抗菌抑病能力降低。研究酵母拮抗菌生活力下降的规律，提高酵母拮抗菌的生产效率，减少剂型加工过程中的细胞损伤，增强其对逆境条件的耐受力是增加或稳定生防制剂防治效果的有效途径。本文主要研究酵母拮抗菌正常培养过程中生活力下降的规律，筛选剂型加工过程中对酵母拮抗菌具有保护作用的化学物质，并对酵母拮抗菌的培养条件进行了优化。主要研究结果如下： 　　1. 在正常培养过程中，酵母拮抗菌Rhodotorula glutinis和Cryptococcus laurentii中细胞染色质凝集或细胞膜破损的发生一般在6天以后。外源加入的N-乙酰半胱氨酸及硅酸钠等物质在超过一定浓度时会加速酵母菌的死亡。 　　2. 在不同的液体悬浮制剂中，对R. glutinis而言，使用磷酸缓冲液（PBS）悬浮时保护效果最好；而C. laurentii悬浮在NYDB培养基中或海藻糖、乳糖溶液中时的生活力最高。 　　3. 以10 %葡萄糖 + 5 %脱脂牛奶作保护剂，可以有效地保持酵母拮抗菌C. laurentii冻干制剂的生活力，配合使用的保护效果高于它们单独使用时的保护效果。添加1 mM N-乙酰半胱氨酸能更好地保持拮抗菌制剂在常温保存过程中的生活力，这可能与这种还原性物质缓解了细胞内活性氧的积累有关。 　　4. 不同酵母拮抗菌对不同碳、氮源的利用能力有明显差异。在9种不同的碳源和10种不同的氮源中，Pichia membranefaciens能够最有效利用的碳、氮源是葡萄糖、果糖和多价胨，而Candida guilliermondii的最佳碳源和氮源分别是果糖和肉蛋白胨。

酵母拮抗菌和外源水杨酸对核果类果实抗病相关基因的诱导表达

在果实采后贮藏过程中，病原真菌的侵染会引起果实腐烂，造成巨大的经济损失。利用生物和非生物因子诱导果实抗病性，已经成为采后病害防治领域的一个研究热点。本文主要利用RT-PCR和RACE技术克隆果实抗病相关基因，通过分子杂交和蛋白羰基化免疫检测技术，研究了外源SA和酵母拮抗菌诱导果实抗病性机理，结果表明： 1. 通过优化RNA提取方法，能从含有多糖的冬枣、葡萄、甜樱桃、桃、番茄等果实中提取到质量较好的RNA，用于RT-PCR和Northern杂交。 2. 采用RT-PCR和RACE方法，从甜樱桃果实克隆了两个抗氧化相关基因CAT2（Genbank：EF165590）和GPX（Genbank：EF165591）和两个PR基因GLU-1（Genbank：EF177487）和GLU-3（Genbank：EF177488)。其中CAT2全长cDNA序列为1479 bp，编码492个氨基酸；GPX全长cDNA序列为513 bp，编码170个氨基酸；GLU-1全长cDNA序列为1050 bp，编码349个氨基酸；GLU-3部分cDNA序列为454 bp，编码141个氨基酸。 3. 酵母拮抗菌Pichia membranaefaciens处理不同成熟度的甜樱桃果实，能显著降低果实贮藏期间青霉病（Penicillium expansum）的发生，并且对低成熟度果实的病害防治效果更为明显。酵母拮抗菌的抑病机理与减轻了甜樱桃果实蛋白羰基化程度，诱导了果实抗氧化酶基因（CAT和GPX）和PR基因（GLU-1）的表达和提高了抗氧化酶（CAT和GPX）和β-1,3-葡聚糖酶的活性有关。 4. 四种酵母拮抗菌P. membranaefaciens, Cryptococcus laurentii, Candida guilliermondii和Rhodotorula glutinis处理桃果实，可显著降低贮藏期间的褐腐病（Monilinia fructicola）。这是由于酵母拮抗菌能抑制病原菌侵染造成的氧化胁迫和蛋白羰基化。此外，酵母拮抗菌处理还能显著诱导CAT、POD、几丁质酶、β-1,3-葡聚糖酶活性及相应基因的表达。 5. 水杨酸（SA，2 mM）处理采后不同成熟度的甜樱桃果实，能显著降低青霉病的危害。其抑病机理与SA处理能减轻P. expansum侵染引起的果实蛋白羰基化程度，显著提高CAT、GPX和β-1,3-葡聚糖酶基因的表达和相关的酶的活性有关。而2 mM的SA处理对P. expansum的生长没有直接抑制作用。 6. 水杨酸（SA，2 mM）与P. membranaefaciens（1×108 CFU/ml）配合处理能显著降低低温贮藏期间桃果实的褐腐病，并能提高几丁质酶、β-1,3-葡聚糖酶和POD的活性和相关基因的表达。另外，2 mM的SA对拮抗菌P. membranaefaciens的生长没有影响，但能够抑制病原菌M. fructicola的孢子萌发和菌丝扩展。

Development of a novel PCR assay for the identification of the black yeast, Exophiala (Wangiella) dermatitidis from adult patients with cystic fibrosis (CF)

Cystic fibrosis (CF) patients may suffer increased morbidity and mortality through colonisation, allergy and invasive infection from fungi. The black yeast, Exophiala dermatitidis (synonym Wangiella dermatitidis) has been found with increasing frequency in sputum specimens of CF patients, with reported isolation rates ranging from 1.1 to 15.7%. At present, no diagnostic PCR exists to aid with the clinical laboratory detection and identification of this organism. A novel species-specific PCR-based assay was developed for the detection of E. dermatitidis, based on employment of rDNA operons and interspacer (ITS) regions between these rDNA operons. Two novel primers, (designated ExdF & ExdR) were designed in silico with the aid of computer-aided alignment software and with the alignment of multiple species of Exophiala, as well as with other commonly described yeasts and filamentous fungi within CF sputum, including Candida. Aspergillus and Scedosporium. An amplicon of approximately 455 by was generated, spanning the partial ITS I region - the complete 5.8S rDNA region - partial ITS2 region, employing ExdF (forward primer [16-mer], 5'-CCG CCT ATT CAG GTC C-3' and ExdR (reverse primer [16-mer], 5'-TCT CTC CCA CTC CCG C-3', was employed and optimised on extracted genomic DNA from a well characterised culture of E. dermatitidis, as well as with high quality genomic DNA template from a further 16 unrelated fungi, including Candida albicans, C. dubliniensis, C. parapsilosis, C. glabrata, Scedosporium apiospermum, Penicillium sp., Aspergillus fumigatus, Aspergillus versicolor, Pichia guilliermondii, Rhodotorula sp., Trichosporon sp., Aureobasidium pullulans, Fusarium sp., Mucor hiemalis, Bionectria ochroleuca, Gibberella pulicaris. Results demonstrated that only DNA from E. dermatitidis gave an amplification product of the expected sire, whilst none of the other fungi were amplifiable. Subsequent employment of this primer pair detected this yeast from mycological cultures from 2/50 (4%) adult CF patients. These two patients were the only patients who were previously shown to have a cultural history of E. dermatitidis from their sputum. E. dermatitidis is a slow-growing fungus, which usually takes up to two weeks to culture in the microbiology laboratory and therefore is slow to detect conventionally, with the risk of bacterial overgrowth from common co-habiting pan- and multiresistant bacterial pathogens from sputum. namely Pseudomonas aeruginosa and Burkholderia cepacia complex organisms, hence this species-specific PCR assay may help detect this organism from CF sputum more specifically and rapidly. Overall, employment of this novel assay nay help in the understanding of the occurrence. aetiology and epidemiology of E. dermatitidis, as an emerging fungal agent in patients with CF. (C) 2008 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Trends in candidemia and antifungal susceptibility in a university hospital in Northern Ireland 2001-2006

Objectives: To describe the species distribution and antifungal susceptibility trends for documented episodes of candidemia at the Royal Hospitals, Belfast, 2001-2006. Methods: Laboratory-based retrospective observational study of all episodes of candidemia. Results: There were 151 episodes of candidemia. The species recovered were: 96 C. albicans; 26 C. glabrata; 18 C. parapsilosis; five C. tropicalis; four C. guilliermondii; one C. famata and one C. dubliniensis. We separated the data into two periods 2001-2003 and 2004-2006; contrary to the findings of other investigators, there was a notable trends toward increasing frequency of C. albicans and decreasing frequency of non-albicans species over time. Although the proportion of C. albicans, C. parapsilosis and C. tropicalis isolates susceptible to fluconazole was unchanged over time, a trend of decreased susceptibility of C. glabrata to fluconazole was noted over the six-year period. Overall, 73% and 7.7% of C. glabrata isolates had susceptible-dose-dependent and resistant phenotypes, respectively. The percentage of C. glabrata isolates susceptible to fluconazole (MIC

Estudo da evolução da microbiota em fermentações naturais de azeitona verde do Algarve

Disssertação de mest., Tecnologia de Alimentos, Instituto Superior de Engenharia, Univ. do Algarve, 2011

Marine Yeasts from the Slope Sediments of Arabian Sea and Bay of Bengal

The present study provides an account of the occurrence and diversity of marine yeasts in the slope sediments of Arabian Sea and Bay of Bengal. It also gives a clear idea about the role of yeasts in the benthic realm of marine ecosystem. The lipolytic potential of the organisms indicate the presence of rich lipid moieties in the study area. The isolates, Candida sp. SD 302 and Pichia guilliermondii SD 337 were proved to have potential oil degrading property and can be employed as bioremediators of oil spill after further characterization. The black yeasts isolated during the study area were found to have high commercial value by virtue of the by-products obtained from them. The melanin and the melanin degrading enzyme extracted from these organisms are potential bioactive materials for application in cosmetology.

Avaliação da atividade biológica de óleos essenciais sobre candida não albicans de origem clínica

Candidíase é um problema de importância crescente, devido o aumento do número de indivíduos imunocomprometidos e o surgimento de cepas resistentes aos antifúngicos convencionais. É de fundamental importância a busca por novos agentes antifúngicos mais eficazes, menos tóxicos, sendo os óleos essenciais (OEs) excelentes alternativas para esse propósito. Esse estudo investigou a atividade biológica do OE de Mentha spicata L. sobre Candida guilliermondii de origem anal e vaginal. Para tanto foram determinadas a Concentração Inibitória Mínima (CIM), Concentração Fungicida Mínima (CFM), cinética do crescimento das leveduras (Time-Kill), alterações micromorfológicas (técnica do microcultivo em câmara úmida) e investigação do mecanismo de ação antifúngico, utilizando o bioensaio do sorbitol. O OE de M. spicata foi obtido pelo processo de extração por destilação a vapor. Na análise fitoquímica desse óleo foi observada a presença de carvona com 84,32%, seguida pelo limoneno (13,70%) e traços de iso-dihidrocarvona (0,82%). Os resultados da análise da CIM variou entre 32 e 128 μg/mL. A CFM variou entre 64 e 1024 μg/mL. Na avaliação da ação de OE e da nistatina 100UI/mL, o antifúngico padrão apresentou o efeito fungicida a partir de 4 horas e para OE de M. spicata foi observado efeito fungistático na CIM, CIMX2 e CIMX4 frente às cepas avaliadas. O OE de M. spicata apresentou forte atividade antifúngica contra as cepas de C. guilliermondii, promovendo alterações micromorfológicas visíveis por microscopia óptica, nas concentrações testadas (CIM, CIMx2), resultado semelhante ao que se observou com a nistatina (100UI/mL). Na investigação do mecanismo de ação antifúngico foi constatado que houve alteração da CIM na presença de sorbitol, com elevação dos valores quatro vezes maior que a concentração inicial, o que indica que os componentes desse OE apresentam ação direta sobre a parede celular das leveduras. Conclui-se que o OE de Mentha spicata é um potencial agente terapêutico no tratamento de candidíase

Avaliação do sistema conservante em formulação com extrato hidroalcóolico de schinus terebinthifolius raddi nacardiaceae

Schinus terebinthifolius Raddi is used in the treatment of skin and mucosal injuries, infections of respiratory, digestive and genitourinary systems. Currently one of the biggest problems faced for the industry of phytopharmaceuticals with regard to the quality of raw materials is the microbial contamination. The aim this study was to evaluate the antimicrobial action of the hidroalcoholic extract of aroeira, beyond testing the effectiveness of the preservative system in hidrogel to the base of this extract. The extracts were prepared by maceration in the ratio of 1:10 of solvent plant/with alcohol 40%. The methods for microbial count were pour plate and test for specific microorganisms, analyzing in third copy each one of the samples. The antimicrobial activity of aroeira extracts was performed using an agar diffusion method, using strains of S. aureus, P. aeruginosa, E. coli, B. subtilis, C. albicans, C. tropicalis, C. krusei, C. guilliermondii, T. rubrum, M. gypseum, A. flavus and A. niger. The formula with aroeira was evaluated by the challenge test. This method consisted of artificial contamination the sample with separate inóculos of A. niger, C. albicans, E. coli e S. aureus aeruginosa and determinations of survivors for the method of counting for pour plate , during times 0, 24h, 48h, 7 days, 14 days, 21 days and 28 days. How much to the results, one verified that the extract of aroeira in the 13,5 concentration mg/mL presented antimicrobial activity for cepas of E. coli, B. subtilis, P. aeruginosa e S. aureus, producing inhibition zone, on average with 13 mm of diameter. However it did not present no fungi activity. The formula with aroeira containig both methylparaben and propylparaben showed a good efficacy in challenge test front to strains of A. niger, C. albicans, E. coli, S. aureus. The A criteria of European Pharmacopoeia, adopted in this work, was verified that this product revealed the good preservative efficacy for the challenge test, time interval of the 28 days. However, it is interesting to extend this study, in order to carry through the sped up stability and the test of shelf, to establish the validity of this formularization