Isolation of proteolytic psychrotrophic yeasts from fresh raw seafoods

A total of 103 cultures of yeasts were isolated from seven kinds of fresh raw seafoods. The isolates comprised six genera, Candida, Cryptococcus, Debaryomyces, Rhodotorula, Sterigmatomyces and Trichosporon , and included 21 different species. All the isolates were psychrotrophic yeasts. Proteolytic activities of 50 psychrotrophic strains were studied by use of skim milk within the temperature range of 0–42°C. All the strains showed various degrees of proteolysis. In particular, Candida lipolytica. Trichosporon pullulans and Candida scottii were active species at low temperatures. Sensory spoilage due to the proteolytic yeasts were observed in mackerel homogenates stored at 10°C. C. lipolytica -inoculated homogenates caused spoilage with ammoniacal odours after 1 week of storage. Values of total volatile basic nitrogen at 10°C were highest with C. lipolytica among 35 strains tested, followed by Tr. pullans. Trichosporon cutaneum, C. scottii, Rhodotorula glutinis and Cryptococcus luteolus. Proteolytic psychrotrophic yeasts were widely distributed in raw seafoods.     

Antifungal activity of propolis extract against yeasts isolated from onychomycosis lesions

The aim of this study was to determine the in vitro activity of propolis extract against 67 yeasts isolated from onychomycosis in patients attending at the Teaching and Research Laboratory of Clinical Analysis of the State University of Maringá. The method used was an adaptation made from the protocol approved by the National Committee for Clinical Laboratory Standards. The yeasts tested were: Candida parapsilosis 35%, C. tropicalis 23%, C. albicans 13%, and other species 29%. The propolis extract showed excellent performance regarding its antifungal activity: the concentration capable of inhibiting the all of the yeasts was 5 x 10(-2) mg/ml of flavonoids and 2 x 10(-2) mg/ml of flavonoids stimulated their cellular death. Trichosporon sp. were the most sensitive species, showing MIC50 and MIC90 of 1.25 x 10(-2) mg/ml of flavonoids, and C. tropicalis was the most resistant, with CFM50 of 5 x 10(-2) mg/ml of flavonoids and MFC90 of 10 x 10(-2) mg/ml. In view of the fact that propolis is a natural, low cost, nontoxic product with proven antifungal activity, it should be considered as another option in the onychomycosis treatment.     

Identification of medically important yeasts by sequence analysis of the internal transcribed spacer and D1/D2 region of the large ribosomal subunit

BackgroundThe prevalence of opportunistic yeast infections has increased in recent decades as the result of an increasing immunocompromised patient population.AimsTo evaluate ribosomal RNA (rRNA) gene sequence to identify medically important yeast species, to investigate the performance of both the rRNA gene internal transcribed spacer (ITS) and D1/D2 region in identifying clinically relevant yeasts, and to compare these results with those of a standard phenotypic method.MethodsBoth regions from 50 yeast strains, comprising 45 clinical isolates and 5 reference strains, were amplified using PCR and then sequenced. The sequences were compared to reference data available from the GenBank database of the National Center for Biotechnology Information using the BLASTn tool.ResultsUsing ID32C, 88% (44/50) of all strains were identified accurately at the species level, although 6% were misidentified; two Candida eremophila isolates were identified as Candida glabrata and Candida tropicalis, and one Saprochaete clavata isolate was identified as Saprochaete capitata. Two of the four isolates identified by phenotypic methods as Trichosporon asahii were defined so by analyzing the ITS region, but the remaining two were not distinguishable from closely related species. Based on the D1/D2 region, these four isolates had 100% sequence identity with T. asahii, Trichosporon japonicum, and Trichosporon asteroides. The isolate identified as Trichosporon inkin using ID32C could not be distinguished from Trichosporon ovoides by analyzing the ITS and D1/D2 regions.ConclusionsIdentifying medically important yeasts by sequencing the ITS and D1/D2 region is a rapid and reliable alternative to conventional identification methods. For a diagnostic algorithm, we suggest a two-step procedure integrating conventional methods (e.g. microscopic morphology on corn meal agar with Tween® 80 and API ID32C®) and sequence analysis of the ITS and D1/D2 region.     

A survey of mycotic otitis externa of dogs in Lisbon

One hundred and thirteen dogs of different breeds and with different clinical forms of external otitis were mycologically and bacteriologically examined. Forty six of those dogs showed abnormal cerumen with a high yeasts contamination. These yeasts belong to four species: Malassezia pachydermatis (80.4%), Cryptococcus laurentii (13.1%), Candida parapsilosis (4.3%) and Trichosporon cutaneum (2.1%). All strains, excepting C. laurentii were highly lipolytic. Most of the clinical cases associated with those yeasts were chronic, with hyperkeratosis and lichenification, and most of them were relapsed otitis (91.3%). The most affected dogs were a pendulous ears breeding (65.7%) and males (86.8%). Some dogs had other cutaneous disorders (seborrhoeic dermatitis, pemphigus). In vitro tests, using seven different antifungal drugs were systematically performed. All strains revealed to be 5-fluorocytosine-resistant and 32% of them were also resistant to nystatin. One M. pachydermatis isolated was resistant to all of the tested antifungal drugs.     

Carnitine acetyltransferase activity in oleaginous yeasts

Abstract The highest activities of carnitine acetyltransferase (CAT) were found in non-oleaginous yeasts ( Candida utilis and Saccharomyces cerevisiae ); lower activities, ranging from 50% down to 3% of the highest values, were found in various strains of oleaginous yeasts ( Candida curvata, Lipomyces starkeyi, Rhodosporidium toruloides and Trichosporon cutaneum ). Supply of acetyl units into the cytosol of the latter, but not of the former yeasts, was therefore necessarily reliant on the action of ATP: citrate lyase (ACL), which was present in all oleaginous yeasts. There was no correlation, however, between the amount of lipid in the oleaginous yeasts and the specific activities of either CAT or ACL. Activity of CAT was increased up to 30-fold by growing yeasts on a triacylglycerol.     

Identification of yeasts of clinical interest on CHROMagar Candida culture medium.

The efficiency of CHROMagar Candida was evaluated as a medium for the presumptive identification of yeasts. We tested 36 different yeast species, pertaining to 9 genera: one Blastoschizomyces, 20 Candida, five Cryptococcus, two Geotrichum, one Kloeckera, two Pichia, three Rhodotorula, one Saccharomyces and one Trichosporon, to determine the colony colors and characteristics on this medium. Afterwards, we identified 2,230 strains isolated directly on CHROMagar Candida from clinical samples by specific colouration and morphology of the colonies after 72 hours. Their results were compared with standard methods for the identification of yeasts. The sensitivity and specificity were both superior to 97% for all strains, 100% and 100% for Candida albicans, 97.3% and 99.9% for Candida glabrata, 92.3% and 99.6% for Candida krusei, 90.3% and 99.6% for Candida parapsilosis, and 100% and 100% for Candida tropicalis. CHROMagar Candida is a very useful medium for the culture of clinical samples; its use for identification of yeasts has an accuracy of 97.5%, close to 100% of conventional methods.     

Yeasts associated with fresh and frozen pulps of Brazilian tropical fruits

The occurrence of yeasts on ripe fruits and frozen pulps of pitanga (Eugenia uniflora L), mangaba (Hancornia speciosa Gom.), umbu (Spondias tuberosa Avr. Cam.), and acerola (Malpighia glaba L) was verified. The incidence of proteolytic, pectinolytic, and mycocinogenic yeasts on these communities was also determined. A total of 480 colonies was isolated and grouped in 405 different strains. These corresponded to 42 ascomycetous and 28 basidiomycetous species. Candida sorbosivorans, Pseudozyma antarctica, C. spandovensis-like, C. spandovensis, Kloeckera apis, C. parapsilosis, Rhodotorula graminis, Kluyveromyces marxianus, Cryptococcus laurentii, Metchnikowia sp (isolated only from pitanga ripe fruits), Issatchenkia occidentalis and C. krusei (isolated only from mangaba frozen pulps), were the most frequent species. The yeast communities from pitanga ripe fruits exhibited the highest frequency of species, followed by communities from acerola ripe fruits and mangaba frozen pulps. Yeast communities from frozen pulp and ripe fruits of umbu had the lowest number of species. Except the yeasts from pitanga, yeast communities from frozen pulp exhibited higher number of yeasts than ripe fruit communities. Mycocinogenic yeasts were found in all of the substrates studied except in communities from umbu ripe fruits and pitanga frozen pulps. Most of the yeasts found to produce mycocins were basidiomycetes and included P. antarctica, Cryptococcus albidus, C. bhutanensis-like, R. graminis and R. mucilaginosa-like from pitanga ripe fruits as well as black yeasts from pitanga and acerola ripe fruits. The umbu frozen pulps community had the highest frequency of proteolytic species. Yeasts able to hydrolyse casein at pH 5.0 represented 38.5% of the species isolated. Thirty-seven percent of yeast isolates were able to hydrolyse casein at pH 7.0. Pectinolytic yeasts were found in all of the communities studied, excepted for those of umbu frozen pulps. The highest frequency of pectinolytic activity was found in mangaba frozen pulp communities. Around 30% of all isolates produced pectinases. The ability to split arbutin was observed in all communities ranging from 8% in yeasts from pitanga frozen pulps to 40.6% in acerola ripe fruit communities. Among 432 species tested, 125 were active for beta-glucosidase production, and Kloeckera apis, P. antarctica, C. sorbosivorans, and C. spandovensis-like were the most active species.     

Ascorbic acid specific utilization by some yeasts

One hundred and eighty strains of yeasts belonging to 17 genus and 53 species were screened for their ability to grow on ascorbic acid and iso-ascorbic acid as the sole carbon source. Most of the tested strains (157) were unable to grow on either compound. Strains of seven species of the genus Cryptococcus, of two Candida species, of Filobasidiella neoformans, Trichosporon cutaneum, Lipomyces starkeyi, Hansenula capsulata, and one strain of Aureobasidium pullulans were able to grow on ascorbic as well as on iso-ascorbic acid. Conversely, four strains of Aureobasidium pullulans, Candida blankii, and Cryptococcus dimennae could use only ascorbic acid for growth.     

Significance of Molecular Identification and Antifungal Susceptibility of Clinically Significant Yeasts and Moulds in a Global Antifungal Surveillance Programme

The increasing diversity of opportunistic fungi causing serious invasive fungal infections (IFI) has been documented. Accurate identification (ID) is important in guiding therapy, determining prognosis for IFIs and in epidemiological surveys. We assessed the utility of PCR-based methods for the ID of yeasts and moulds that either were uncommon, failed conventional ID, or represented unusual biochemical or phenotypic profiles of common species. Among 1,790 viable fungal clinical isolates received during the SENTRY Program in 2010, 322 strains from 40 study sites had ID confirmed by molecular methods. Isolates were previously identified in participant institutions. Yeasts that were not confirmed by morphology on CHROMagar, growth at 45?°C (Candida albicans/dubliniensis), or assimilation of trehalose (C. glabrata) as well as non-Candida yeasts and all moulds were amplified and sequenced using primers amplifying one or more of the following genes: ITS, 28S, β-tubulin (Aspergillus spp.), TEF (Fusarium spp.), IGS (Trichosporon spp.). The isolates selected for molecular ID included 149 isolates of Candida species, 77 of Aspergillus species, 73 non-Candida yeasts, and 23 other moulds (a total of 41 different species). Overall, the ID determined by the submitting site was confirmed for 189 isolates (58.7?%): Aspergillus spp. (64.1?% correct); Candida spp. (60.1?% correct); non-Candida yeasts (58.9?% correct); non-Aspergillus moulds (30.4?% correct). Species with high levels of concordance between conventional and molecular ID included A. fumigatus (95.0 %), C. lusitaniae (100?%), C. dubliniensis (92.3?%), C. kefyr (100?%), and C. neoformans (90.2?%). Only 50.0?% of isolates of C. albicans and 59.1?% of C. glabrata selected due to unusual phenotypic or biochemical features were found to be correctly identified by the submitting site. Molecular methods for the identification of fungal pathogens are an important adjunct to the conventional identification of many less common clinically relevant yeasts and moulds including species of Candida with unusual or erroneous phenotypic or biochemical profiles. Molecular confirmation of fungal identification is essential in epidemiological surveys such as SENTRY.     

Characterization of oleaginous yeasts revealed two novel species: Trichosporon cacaoliposimilis sp. nov. and Trichosporon oleaginosus sp. nov

Two new species in the anamorphic basidiomycetous genus Trichosporon (Tremellomycetes, Agaricomycotina) were uncovered in a DNA sequence-based molecular analysis of oleaginous yeasts maintained in the ATCC Mycology Collection. One yeast is named as Trichosporon cacaoliposimilis sp. nov. for its capability of synthesizing and accumulating a large amount of lipids having a composition equivalent to that of natural cacao butter. The type strain is ATCC 20505(T), originally deposited as Trichosporon sp. The other can use food industry wastes and agricultural byproducts as the substrate for growth and accumulation of a high level of oil and accordingly is named Trichosporon oleaginosus sp. nov. The type strain is ATCC 20509(T), previously identified as Cryptococcus curvatus. Molecular phylogenetic analyses indicate that T. cacaoliposimilis is a novel taxon in the Gracile clade of the genus, close to T. gracile and T. dulcitum, and that T. oleaginosus belongs to the Cutaneum clade, with T. jirovecii as the closest sister taxon. Other oleaginous yeasts were identified as new strains of known taxa, T. insectorum, Candida orthopsilosis and C. palmioleophila.     

Phenotypic switching and beta-N-acetylhexosaminidase activity of the pathogenic yeast Trichosporon asahii

The pathogenic yeast Trichosporon asahii is the major causative agent of deep-seated trichosporonosis in immunocompromised patients. Although infection by this microorganism is becoming increasingly frequent, information related to its pathogenicity and virulence factors is still limited. Therefore, we investigated phenotypic switching in colony morphology, and the production of extracellular enzymes as a virulence factor. Sixty-one clinical isolates of T. asahii produced four different morphological types on Sabouraud dextrose agar (SDA): 69% WF (white farinose), 18% WP (white pustular), 10% Y (yellowish white), and 3% WC (white cerebriform). Strains of the three major types (WF, WP, and Y) produced two to five colony types when cultured on SDA at 37 C. The frequency of switching between colony types was 10(-2) to 10(-4), as in Candida albicans and Cryptococcus neoformans. Notably, most of the colonies switched to the smooth (S) type irreversibly, at frequencies of 10(-2) to 10(-3). No secreted aspartic proteinase or phospholipase activity was detected in T. asahii, while beta-N-acetylhexosaminidase activity, which catalyzes the hydrolysis of terminal nonreducing N-acetyl-D-hexosamine residues in N-acetyl-beta-D-hexosaminides, was found. Furthermore, enzymatic activity of the S type was significantly greater than that of the parent type in all strains. No other clinically relevant Trichosporon species (T. mucoides, T. inkin, and T. ovoides ) produced this enzyme. These results provide basal information for understanding the pathogenic potential of T. asahii.     

Conversion of pentoses by yeasts

The utilization and conversion of D-xylose, D-xylulose, L-arabinose, and xylitol by yeast strains have been investigated with the following results: (1) The majority of yeasts tested utilize D-xylose and produce polyols, ethanol, and organic acids. The type and amount of products formed varies with the yeast strains used. The most commonly detected product is xylitol. (2)The majority of yeasts tested utilize D-xylulose aerobically and fermentatively to produce ethanol, xylitol, D-arabitol, and organic acids. The type and amount of products varies depending upon the yeast strains used. (3) Xylitol is a poor carbon and energy source for most yeasts tested. Some yeast strains produce small amounts of ethanol from xylitol. (4) Most yeast strains utilize L-arabinose, and L-arabitol is the common product. Small amounts of ethanol are also produced by some yeast strains. (5) Of the four substrates examined, D-xylulose was the perferred substrate, followed by D-xylose, L-arabinose, and xylitol. (6) Mutant yeast strains that exhibit different metabolic product patterns can be induced and isolated from Candida sp. Saccharomyces cerevisiae, and other yeasts. These mutant strains can be used for ethanol production from D-xylose as well as for the study of metabolic regulation of pentose utilization in yeasts.     

Randomly amplified polymorphic DNA (RAPD) PCR for the identification of yeasts isolated from dairy products

In the present work randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) with primers M13 and RF2 was applied to the identification at species level of yeast strains isolated from cheeses. RAPD-PCR analysis of the type strains of different yeast species gave distinctive band profiles that allowed a clear differentiation of all the considered species. Forty-two of the 48 dairy associated yeasts were clearly assigned to the species Saccharomyces cerevisiae, Kluyveromyces marxianus (anamorph Candida kefyr), Kluyveromyces lactis (anamorph Candida sphaerica), Debaryomyces hansenii (anamorph Candida famata), Yarrowia lipolytica and Torulaspora delbrueckii (anamorph Candida colliculosa). The method, which is rapid and easy to perform, could be a useful tool for the identification of yeasts present in dairy products.     

Enzyme activities in oleaginous yeasts accumulating and utilizing exogenous or endogenous lipids

The activities of ATP: citrate lyase (ACL; EC 4. 1. 3. 8), carnitine acetyltransferase (CAT; EC 2. 3. 1. 7), NADP+-dependent isocitrate dehydrogenase (ICDH; EC 1. 1. 1. 42), isocitrate lyase (ICL; EC 4. 1. 3. 1) and malic enzyme (malate dehydrogenase; EC 1. 1. 1. 40) were measured in four oleaginous yeasts, Candida curvata D, Trichosporon cutaneum and two strains of Rhodosporidium toruloides, grown either to accumulate lipid, or to utilize their own lipid reserves or an exogenous supply of lipid. During lipid utilization, activities of ACL and malic enzyme diminished to low levels; CAT and ICL increased considerably in activity and ICDH activity was slightly increased but catalase (EC 1. 11. 1. 6) diminished in activity in both strains of R. toruloides. In all cases, yeasts utilizing exogenous lipid showed greater changes in enzyme activities than cells utilizing their endogenous reserves. Electron micrographs of Candida curvata D showed proliferation of peroxisomes in starved cells utilizing their own lipid reserve though peroxisomes were more in evidence when the yeast had been grown on exogenous lipid. In Lipomyces starkeyi, which shows only minimal utilization of its stored lipid and furthermore cannot grow on exogenous lipid, only the occasional peroxisome was seen when cells were starved of carbon.     

临床相关毛孢子菌的鉴定及体外药物敏感性研究

目的探讨临床相关毛孢子菌的鉴定方法及对常见抗真菌药物的体外敏感性。方法对48株临床分离的毛孢子菌分别通过形态学、API20C AUX、Vitek 2 Compact及核糖体rDNA ITS序列分析等方法鉴定到种;采用浓度梯度法（E-test）测定氟康唑、伏立康唑、伊曲康唑、两性霉素B及卡泊芬净对48株毛孢子菌的最低抑菌浓度。结果形态学和API20C AUX、Vitek 2 Compact不能准确区分不同种的毛孢子菌,以核糖体rDNA ITS序列分析将48株毛孢子菌鉴定为8个种：阿萨希毛孢子菌,星型毛孢子菌,皮瘤毛孢子菌,真皮毛孢子菌,皮肤毛孢子菌,赖巴克毛孢子菌,T.domesticum,T.jirovecii。体外药敏结果显示：卡泊芬净对毛孢子菌无体外活性,MIC〉32μg/mL;氟康唑和两性霉素B对毛孢子菌活性差,体外活性最好的药物是伏立康唑和伊曲康唑。结论常规方法不易将毛孢子菌准确鉴定到种的水平,ITS序列分析准确快速,可以辅助临床区分难鉴定毛孢子菌。毛孢子菌药敏谱不同于临床常见其他酵母菌,氟康唑和两性霉素B对其活性差,伏立康唑具有良好的体外抗菌活性。     

Yeasts Associated with Various Amazonian Native Fruits

Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.Key words: yeast diversity, fruit, Amazonia, PCR-RFLP, 5.8S-ITS     

Identification and susceptibility against fluconazole and albaconazole of 100 yeasts' strains isolated from vaginal discharge

Vulvovaginal candidiasis is a condition that affects a great number of fertile women. It is considered the second cause of genital infection after vaginosis due to GAM complex. Candida albicans is the most frequent isolated species from vaginal discharge. However, sometimes more than one yeast species could be found in the same clinical sample that are more resistant to antifungal drugs. Nowadays, it is necessary to identify properly up to species level the isolated microorganism and to determine the antifungal susceptibility profile. One hundred strains obtained from vaginal discharge of 94 patients suffering acute vulvovaginal candidiasis were studied. The identification of the isolates showed: C. albicans 86%, Candida glabrata 6%, Candida inconspicua 3%, Candida krusei 2% and Candida intermedia, Candida holmii and Trichosporon asahii one case each. Minimal inhibitory concentrations (MIC) of all the yeasts against fluconazole and albaconazole were performed. C. glabrata, C. krusei and C. inconspicua were the most resistant against fluconazole, on the other hand albicans was susceptible to this drug. All the isolates presented MIC against albaconazole much lower than fluconazole.     

Yeast flora of plant rhizosphere and phyllosphere]

The species belonging to the genera Cryptococcus and Hansenula with saturnian spores predominate in the rhizosphere of agricultural plants. The sporiferous strains of Debaryomyces, Hanseniaspora apiculata, Metschnikowia pulcherrima, and asporogenic Candida krusei and Trichosporon cutaneum prevail in the rhizosphere of wild plants. Candida krusei and Trichosporon cutaneum prevail in the rhizosphere of wild plants. The cultures of Rhodotorula, Candida krusei and Metschnikowia pulcherrima are typical of the phyllosphere of both cultural and wild plants. The phyllosphere of cultural plants contains also the asporogenic strains of Cryptococcus, Candida tropicalis, Trichosporon pullulans, Tr. cutaneum, and Hansenula, while Hanseniaspora apiculata and Saccharomyces cerevisiae predominate in the phyllosphere of wild plants. The yeast flora of the majority of studied plants is diverse and comprises 10--20 species (in cabbage, potato, linden, aspen, and pear trees). The rhizophere and phyllosphere of some plants contain only 2 to 4 yeast species (onion, hop, wild apple).     

Oxidation of amines by yeasts grown on 1-aminoalkanes or putrescine as the sole source of carbon,nitrogen and energy

The maximum growth rate of Trichosporon cutaneum CBS 8111 in chemostat cultures was 0.185 h^-1 on ethylamine and 0.21 h^-1 on butylamine, that of Candida famata CBS 8109 was 0.32 h^-1 on putrescine.The amine oxidation pattern of the ascomycetous strains studied, viz. Candida famata CBS 8109, Stephanoascus ciferrii CBS 4856 and Trichosporon adeninovorans CBS 8244 was independent of the amine that had been used as the growth substrate. It resembled that of benzylamine/putrescine oxidase found in other ascomycetous yeasts. However, differences in pH optimum and substrate specificity were observed between the amine-oxidizing systems of these three species.The amine oxidation pattern of cell-free extracts of Trichosporon cutaneum CBS 8111 varied with the amine that was used as growth substrate. The enzyme system produced by Cryptococcus laurentii CBS 7140 failed to oxidize isobutylamine and benzylamine, and showed a high pH optimum.The synthesis of amine oxidase in the four yeast strains studied was not repressed by ammonium chloride and was weakly repressed by glucose but was strongly repressed if both compounds were present in the growth medium.