Nucleic acid sequence-based amplification (NASBA) detection of medically important Candida species.

Nucleic Acid Sequence Based Amplification (iNASBA), an isothermal amplification technique for nucleic acids, was evaluated for the identification of medically important Candida species using primers selected from 18S rRNA sequences conserved in fungi. An RNA fragment of 257 nucleotides was amplified for Candida albicans. Nineteen different fungi were tested for rRNA amplification with the NASBA. All were positive when analyzed on agarose gel, whereas human RNA was negative. For the identification of Candida species, NASBA amplification products were analyzed in an enzyme bead-based detection format, using species-specific biotinylated probes and a generic Candida HRPO probe or a membrane-based system using biotinylated probes and avidin-HPRO. Discrimination of the major human pathogenic Candida spp. was based on a panel of biotinylated probes for C. krusei, C. tropicalis, C. albicans, C. glabrata, and C. lusitaniae. Using rRNA dilutions obtained from pure cultures of C. albicans, the combination of NASBA and the enzymatic bead-based detection yielded a sensitivity equivalent to 0.01 CFU. In a model system using 1 ml of artificially contaminated blood as few as 1-10 CFU of C. albicans could be detected. Testing of 68 clinical blood samples from patients suspected of candidemia showed that eight samples were positive for C. albicans and one for C. glabrata. Testing of 13 clinical plasma samples from patients suspected of fungemia identified the presence of C. albicans in two specimens. The whole procedure of sample preparation, amplification and identification by hybridization can be performed in 1 day. This speed and the observed sensitivity of the assay make the NASBA a good alternative to PCR for the detection of candidemia.     

Evaluation of a digoxigenin-labelled probe for detection of Aeromonas spp.

An outer membrane protein-based Digoxigenin (DIG)-labelled DNA probe was developed for the specific detection of Aeromonas sp. from food/environmental/clinical samples. Dot blot reaction answered for all the Aeromonas isolates and was negative for Escherichia coli , Pseudomonas sp., Klebsiella sp., Vibrio parahaemolyticus , V. harveyi , V. alginolyticus , V. vulnificus . Edwardsiella tarda and Staphylococcus sp. As this protein is highly conserved in various Aeromonas species, the probe has the potential for use as a rapid and reliable diagnostic tool.     

内蒙古西部区酸粥中酵母菌的分离鉴定及优势菌分析

从内蒙古地区采集28份酸粥样品,从中分离出40株酵母菌,并对其进行了分子生物学鉴定和生物多样性分析。26S rDNA D1/D2区域 (600bp左右)碱基序列分析结果表明,酸粥中的酵母菌有Issatchenkia orientalis、Saccharomyces cerevisiae、Geotrichum sp.、Candida pararugosa、 Candida parapsilosis、Trichosporon asahii、Trichosporon coremiiforme、Clavispora lusitaniae和Candida tropicalis。经过分析,Issatchenkia orientalis(75%,Frequency percentage)为酸粥中的优势菌。     

Five novel species of the anamorphic genus Candida in the Cyberlindnera clade isolated from natural substrates in Taiwan

Twelve strains representing five novel yeast species were isolated from natural samples distributed in mountain areas in Taiwan during 2007 and 2009. Sequence analysis of the D1/D2 domain of the large subunit (LSU) rRNA gene revealed that these species are members of the Cyberlindnera clade. These five new species have a greater than 1% difference from their closest relatives in the sequences of the D1/D2 domain of the LSU rRNA gene and were well separated from their closest relatives in terms of physiological characteristics. Moreover, a sexual state could not be found in these five novel yeast species. Therefore, the scientific names of Candida maesa sp. nov. (type strain GJ8L01^T), Candida takata sp. nov. (type strain EN25S01^T), Candida taoyuanica sp. nov. (type strain GY15S07^T), Candida hungchunana sp. nov. (type strain NC3W71^T) and Candida stauntonica sp. nov. (type strain GY13L05^T) were proposed to accommodate these yeasts.     

DNA probes for the rapid identification of medically important Candida species using a multianalyte profiling system

Recently, a new flow cytometric technology to detect multiple DNA target sequences in a single microtiter well plate was developed [multianalyte profiling (MAP) System, Luminex Corp., Austin, TX]. DNA probes, directed to the internal transcribed spacer 2 region of ribosomal DNA, were therefore designed to detect and differentiate PCR amplicons from six medically important Candida species using this system. Each probe was covalently linked to one of 100 available microsphere (bead) sets. Biotinylated PCR amplicons were then hybridized to the complementary probe on each bead set. Bound amplicons were detected fluorometrically using a streptavidin-linked reporter dye, R-phycoerythrin. Specific hybridization was noted for all six Candida species probes (mean sample-to-background ratio+/-standard error: Candida albicans, 58.7+/-1.2; Candida tropicalis, 53.2+/-3.8; Candida glabrata, 46.9+/-2.1; Candida parapsilosis, 59.9+/-1.6; Candida krusei, 54.7+/-3.7 vs. 0.9+/-0.03 for all heterologous Candida species DNA targets and vs. 1.0+/-0.1 for samples containing water instead of DNA; P < 0.001). The limit of test sensitivity was 0.5 pg of DNA. A sample could be processed and analyzed within 1 h post-PCR amplification. Therefore, the multianalyte profiling system was rapid, sensitive and specific for the detection and differentiation of the most medically important species of Candida.     

Modulation of chromium(VI) toxicity by organic and inorganic sulfur species in yeasts from industrial wastes

Two chromium(VI) resistant yeast strains (Candida sp. and Rhodosporidium sp.) were isolated from industrial wastes. Four different yeasts, three from the Industrial Yeast Collection and one of pharmaceutical origin, were also studied in relation to chromate toxicity and its alleviation by sulfur species. The growth of yeasts from industrial wastes was inhibited by 50% by high concentrations of Cr(VI): Candida sp. by 4 mM Cr(VI) and Rhodosporidium sp. by 10 mM Cr(VI) in Sabouraud Broth medium. The other Cr(VI)-sensitive yeasts were inhibited by 0.1 mM Cr(VI). The general mechanism of chromium resistance in Candida sp. and Rhodosporidium sp. was due to reduced uptake of chromium, but not to biological reduction from Cr(VI) to Cr(III). In Cr(VI)-sensitive yeasts, chromium was accumulated as much as 10-fold, as in Saccharomyces cerevisiae. Cr(VI) toxicity in Candida sp. was modulated from Cr(VI)-resistance to Cr(VI)-hypersensitivity depending on the addition of methionine, cysteine, sulfate and djenkolic acid. If Candida sp. was grown in the presence of S-amino acids, especially methionine, it was more resistant than if the sulfur source was sulfate. When sulfate transport was enhanced by addition of djenkolic acid, Candida sp. became hypersensitive. Rhosporidium sp. was always resistant to Cr(VI) because sulfate transport was inefficient and it assimilated sulfur as S-amino acids. Cr(VI)-sensitive yeasts required larger amounts of S-amino acids, especially methionine, to tolerate Cr(VI) toxicity. Cysteine was toxic for C.famata 6016 above 50 microM.     

Characterisation of the diversity and physiology of cellobiose-fermenting yeasts isolated from rotting wood in Brazilian ecosystems

We investigated the yeast species associated with rotting wood samples obtained from Brazilian ecosystems, with a special focus on cellobiose-fermenting species. About 647 yeast strains were isolated from rotting wood samples collected from the areas of Atlantic rainforest, Cerrado, and Amazonian forest. Eighty-six known species and 47 novel species of yeasts were isolated. Candida boidinii, Cyberlindnera subsufficiens, Meyerozyma guilliermondii, Schwanniomyces polymorphus, Candida natalensis, and Debaryomyces hansenii were the most frequently isolated species. Among the cellobiose-fermenting yeasts, 14 known and three novel yeast species were identified. Scheffersomyces queiroziae, Sc. amazonensis, Yamadazyma sp.1, Hanseniaspora opuntiae, C. jaroonii, and Candida tammaniensis were the main ethanol-producing yeasts. These species also produced an intracellular β-glucosidase responsible for cellobiose hydrolysis. In fermentation assays using a culture medium containing 50 g L^?1 cellobiose, ethanol production was observed in all cases; Sc. queiroziae and Sc. amazonensis showed the highest yield, efficiency, and productivity. Candida jaroonii and Yamadazyma sp.1 strains also showed high efficiency in cellobiose fermentation, while C. tammaniensis and H. opuntiae strains produced low amounts of ethanol. This study shows the potential of rotting wood samples from Brazilian ecosystems as a source of yeasts, including new species as well as those with promising biotechnological properties.     

Three novel species of the anamorphic yeast genus Candida in the Candida intermedia clade found in Japan, Thailand and Taiwan

Four strains of yeasts isolated in Japan, Thailand and Taiwan were found to represent three novel species of the genus Candida. The three species are located in a clade including Candida tsuchiyae, Candida thailandica and Candida akabanensis in a tree based on the D1/D2 domain sequences of the large subunit rRNA genes but clearly differentiated from these relative species. Three novel species are proposed for these strains, i. e., Candida berkhoutiae sp. nov., for strains ST-49(T) (=BCC 7749(T)=NBRC 106733(T)=CBS 11722(T)) isolated from insect frass in Thailand and SA13S01 (=NBRC 106053) isolated from soil in Taiwan, Candida ezoensis sp. nov., for strain Y07-1601-2(T) (=NBRC 105019(T)=CBS 11753(T)) isolated from forest soil in Japan, and Candida inulinophila sp. nov., for ST-369(T) (=BCC 15081(T)=NBRC 106735(T)=CBS 11725(T)) isolated from an unidentified wild mushroom from Thailand.     

Three anamorphic yeast species Candida sanitii sp. nov., Candida sekii sp. nov. and Candida suwanaritii, three novel yeasts in the Saturnispora clade isolated in Thailand

Nine strains of three novel anamorphic yeast species were obtained from samples collected in Thailand including six strains (RV96, RV152, R14, RS9, RS58 and EA1) obtained from estuarine waters collected from two mangrove forests, one strain (ST84) from insect frass and two strains (SR16 and UB13) from forest soils. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and the sequence analysis of the D1/D2 domain of the large subunit rRNA gene, the nine strains were found to represent three novel Candida species in the Saturnispora clade. Five strains (RV96, RV152, R14, RS9 and RS58) were assigned as a single novel species, which was named Candida sanitii sp. nov. The type strain is RV152^T (BCC 25967^T=NBRC 103864^T=CBS 10864^T). Strain EA1 was named as Candida suwanaritii sp. nov. The type strain is EA1^T (BCC 29900^T=NBRC 104877^T=CBS 11021^T). Three strains (ST84, SR16 and UB13) represented another novel species, for which Candida sekii sp. nov. is proposed. The type strain is ST84^T (BCC 8320^T=NBRC 105671^T=CBS 10931^T).     

Glycosphingolipids as a possible signature of microbial communities in activated sludge and the potential contribution of fungi to wastewater treatment under cold conditions

Seven strains of fungi were isolated from activated sludge and identified as Mucor sp., Geotrichum sp., Trichosporon sp., Candida sp., and Trichoderma sp. by 28S rDNA D2 region sequences analysis. The structures of the main ceramide monosaccharides (CMSs) from these fungi were identified as glucosylceramide (GlcCer) consisting of ceramide moieties of 9-methyl-octadeca-sphingadienine (9-Me d18:2), with 2-hydroxyhexadecanoate (h16:0) (Mucor sp. and Geotrichum sp.), 2-hydroxyoctadecanoate (h18:0) (Trichosporon sp. and Candida sp.), and 2-hydroxyoctadecenoate (h18:1) (Trichoderma sp.). Seasonal changes in glycosphingolipids in activated sludge suggest the possibility that microbial flora in activated sludge changes with the seasons, and that fungi adaptable to low temperatures dominate in the cold period, resulting in the maintenance of stable effluent quality. Mucor sp., Geotrichum sp., and Candida sp. satisfactorily reduced the BOD of synthetic sewage at 10 degrees C. These results indicate that fungi in activated sludge can contribute to wastewater treatment in cold conditions.     

Four novel species of the anamorphic yeast genus Candida found in Thailand and Taiwan

Four strains of yeasts isolated in Thailand and Taiwan were found to represent four distinct novel species of the ascomycetous anamorphic yeast genus Candida. These strains are located in the Clavispora-Metschnikowia clade in a phylogenetic tree based on the D1/D2 domain sequences of the large subunit rRNA genes. Together with Candida picinguabensis and Candida saopaulonensis, the four novel species constitute a well-separated subclade from other species of the Clavispora-Metschnikowia clade. Three species from Thailand are described as Candida bambusicola sp. nov. (type strain, ST-50(T) = BCC 7750(T) = NBRC 106734(T) = CBS 11723(T)), Candida nongkhaiensis sp. nov. (type strain, ST-95(T) = BCC 8331(T) = NBRC 105874(T) =CBS 11724(T)) and Candida succicola sp. nov. (type strain, ST-631(T) = BCC 15314(T) = NBRC 106736(T) = CBS 11726(T)), respectively, and the species from Taiwan is described as Candida touchengensis sp. nov. (type strain, SY4S03(T) = NBRC 102647(T) = BCRC 23097(T) = CBS 10585(T)).     

Stability of microbial communities in goat milk during a lactation year: molecular approaches

The microbial communities in milks from one herd were evaluated during 1-year of lactation, using molecular methods to evaluate their stability and the effect of breeding conditions on their composition. The diversity of microbial communities was measured using two approaches: molecular identification by 16S and 18S rDNA sequencing of isolates from counting media (two milks), and direct identification using 16S rDNA from clone libraries (six milks). The stability of these communities was evaluated by counting on selective media and by Single Strand Conformation Polymorphism (SSCP) analysis of variable region V3 of the 16S rRNA gene and variable region V4 of the 18S rRNA gene. One hundred and eighteen milk samples taken throughout the year were analyzed. Wide diversity among bacteria and yeasts in the milk was revealed. In addition to species commonly encountered in milk, such as Lactococcus lactis, Lactococcus garvieae, Enterococcus faecalis, Lactobacillus casei, Leuconostoc mesenteroides, Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus caprae, Staphylococcus equorum, Micrococcus sp., Kocuria sp., Pantoea agglomerans and Pseudomonas putida, sequences were affiliated to other species only described in cheeses, such as Corynebacterium variabile, Arthrobacter sp., Brachybacterium paraconglomeratum, Clostridium sp. and Rothia sp. Several halophilic species atypical in milk were found, belonging to Jeotgalicoccus psychrophilus, Salinicoccus sp., Dietza maris, Exiguobacterium, Ornithinicoccus sp. and Hahella chejuensis. The yeast community was composed of Debaryomyces hansenii, Kluyveromyces lactis, Trichosporon beigelii, Rhodotorula glutinis, Rhodotorula minuta, Candida pararugosa, Candida intermedia, Candida inconspicua, Cryptococcus curvatus and Cryptococcus magnus. The analyses of microbial counts and microbial SSCP profiles both distinguished four groups of milks corresponding to four periods defined by season and feeding regime. The microbial community was stable within each period. Milks from winter were characterized by Lactococcus and Pseudomonas, those from summer by P. agglomerans and Klebsiella and those from autumn by Chryseobacterium indologenes, Acinetobacter baumanii, Staphylococcus, Corynebacteria and yeasts. However, the composition of the community can vary according to factors other than feeding. This study opens new investigation fields in the field of raw milk microbial ecology.     

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 from U.S.A. soils

Summary Investigations of yeasts from 38 U.S.A. soils samples show the occurrence of 22 different species (16 sporogenous and 6 asporogenous).The most widespread species were Pichia fermentans and Hansenula anomala. The other isolated species were Saccharomyces ellipsoideus, Torulaspora delbrueckii, Pichia membranefaciens, Saccharomyces smittii, Saccharomyces carlsbergensis, Saccharomyces uvarum, Torulaspora rosei, Zygosaccharomyces rouxii, Zygosaccharomyces n. sp., Hansenula saturnus, Hansenula californica, Hansenula n. sp., Hansenula suaveolens, Debaryomyces n. sp., Torulopsis glabrata, Torulopsis n. sp. Candida tropicalis, Candida robusta, Rhodotorula glutinis, Trichosporon cutaneum and Trichosporon cutaneum var. multisporus.     

Susceptibility to antifungal agents of Candida sp. and biofilm formation

In recent years the increase in frequency of fungal infections with Candida sp. was noticed. These infections are connected with ability of Candida sp. to form biofilm on surfaces of biomaterials used in medicine. Furthermore fungal infections make serious therapeutic problems because ofbiofilm resistance to antifungal agents actually. The aim of the study was to evaluate the susceptibility to antifungal agents of Candida sp. and their ability to form biofilm on different biomaterials. 50 strains of Candida sp. isolated from patients of University Hospital No. 1 of dr A. Jurasz in Bydgoszcz were examined. API Candida (bioMérieux) tests were used to identify Candida sp. strains. The susceptibility of the yeast strains to antifungal agents was evaluated by ATB FUNGUS 2 INT (bioMérieux) tests. The susceptibility of examined strains to voriconazole, posaconazole, caspofungin and anidulafungin was assessed by means ofEtests (AB BIODISK) method employing drug concentrations from 0,002 to 32 microg/ml. All analysed strains were susceptible to amphotericin B and caspofungin. Biofilm formation on different biomaterials (silicon, latex, polychloride vinyl, polypropylene, nylon) was measured after 72 hour incubation at 37 degrees C. All examined yeasts formed biofilm on all analysed biomaterials. The highest number of strains formed biofilm on surface of polychloride vinyl: 23 (92,0%) by C. albicans strains and 24 (96,0%) Candida non-albicans strains. The lowest number of the strains formed biofilm on the surface of nylon: 12 (48,0%) of C. albicans strains and 9 (36,0%) of Candida non-albicans strains. The studied strains resistant to azoles and anidulafungin display stronger ability to form biofilm on surfaces of all analysed biomaterials.     

Enrichment of butteroil with conjugated linoleic acid via enzymatic interesterification (acidolysis) reactions

Microbial lipases from Candida cylindracea, Pseudomonas sp., Mucor miehei and Candida antarctica were screened for their ability to incorporate conjugated linoleic acid into butteroil triacylglycerides. The lipase from Candida antarctica was employed in a substrates-only medium to increase the conjugated linoleic acid content of the acylglycerides from 0.6 to 15 g/100 g fat.     

Sensitivity of aerobic bacteria and Candida species to chlormidazole hydrochloride in vitro

Over 100 bacterial stains, including Candida sp., gram-positive cocci and some gram-negative bacilli, were tested. The majority of microorganisms was isolated from man. Only one bacterial strain (5 micrograms/mL) and 76% of Candida strains were sensitive to chlormidazole HCl (10 micrograms/mL). It may be assumed that most infections with Candida sp. will respond to therapy with drugs containing chlormidazole HCl. Relatively simple and inexpensive tube test may serve to evaluate microbial sensitivity to this agent, especially in case of isolates from patients.     

Lytic Enzymes for Gram-negative Bacteria Produced by Bacillus subtilis YT-25

The available energy, gross protein value, phosphorus availability and palatability of 16 samples of single cell protein were evaluated in 20 bioassays using total 2,136 depleted chicks.

Four protein samples were products from Aspergillus tamarii grown on waste water of a fish processing factory, three were from Aspergillus oryzae grown on either acetic acid medium or cooked soybean waste, three were from Candida sp. grown on citrus molasses extracted from peel wastes of citrus processing plants, four were from Candida utitis grown on wood molasses produced from various wood wastes, and two were from Pseudomonas sp. and Alteromonas thlasomethanolica grown on methanol.

Five of 16 samples had excellent nutritive value, comparable to single cell proteins available commercially in Europe. All samples were palatable to the chicks, and no sign of acute toxicity was observed.     

Microflora associated with the skin of the bowhead whale (Balaena mysticetus)

A study of the microbiological flora isolated from cultures of normal and lesional skin tissue samples collected from 19 bowhead whales (Balaena mysticetus) over a 4 yr period is presented. These cultures were obtained from 30 tissue samples (17 normal, 13 lesion) and 248 swab samples (157 normal, 91 lesion). Seven hundred-thirty bacterial and yeast isolations were made (285 normal, 445 lesion). Distribution revealed that 56% of the gram positive bacterial isolates, 75% of the gram negative bacterial isolates and 64% of the yeast isolates recovered were associated with lesional skin. It was found that 80% of one group of Corynebacterium sp. isolates, 90% of the Acinetobacter sp. isolates and 94% of the Moraxella sp. isolates were associated with lesional skin. Although the primary yeasts recovered were Candida spp., they were found on both normal and lesional skin. Enzymatic assays of isolates from normal and lesional skin demonstrated production of enzymes capable of causing necrosis. The majority of the microorganisms recovered were facultative anaerobes and many of them could be considered potential pathogens of mammalian hosts.