Common Yeast Species Research Articles

Molecular Identification of Clinically Common and Uncommon Yeast Species

Background: The incidence of fungal infections caused by the yeasts and yeast-like species increased dramatically in immunocompromised patients, during the past several decades. However, a few of yeast and yeast-like species may colonize in skin and mucous membranes of healthy individuals. Objectives: The current study aimed at accurately identifying yeast and yeast-like species from clinical samples by molecular methods. Methods: A total of 1200 clinical samples were collected from patients with suspected fungal infection and 110 (9.16 %) yeast and yeast-like strains isolated and identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), PCR amplification of hwp1 gene, and sequencing. Results: In total, Candida albicans (n = 46) was the most frequently isolated species followed by C. parapsilosis complex (n = 17), C. tropicalis (n = 13), C. guilliermondii (n = 12), C. glabrata (n = 4), C. krusei and C. famata each (n = 3), C. kefyr, C. haemulonii and Cutaneotrichosporon jirovecii each (n = 2), and C. stellatoidea, C. intermedia, C. sorbosivorans, C. africana, Pichia kudriavzevii, and Trichosporon asahii each (n = 1). Interestingly, C. haemulonii, a multidrug resistant fungus was isolated from cutaneous and sputum samples for the first time in Iran. Conclusions: Nowadays, with growing population at risk for fungal infections and the emergence of some less virulent or non-pathogenic and uncommon yeasts not readily distinguishable with phenotypic assays, the accurate identification using molecular methods are warranted.

Species distribution, virulence factors, and antifungal susceptibility among Candida parapsilosis complex isolates from clinical specimens at Siriraj Hospital, Thailand, from 2011 to 2015.

The Candida parapsilosis complex has been described as the second or third most common yeast species isolated from patients with bloodstream infections worldwide. This complex consists of three species: C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis. The distribution of species in this complex has never been studied in Thailand. Here we investigated the molecular epidemiology, in vitro on virulence factors, and antifungal susceptibility profiles of isolates of these three species collected from patients in Siriraj Hospital, Thailand, from 2011 to 2015. Of the 96 C. parapsilosis complex isolates analyzed, 66 (68.75%) were identified as C. parapsilosis sensu stricto, 28 (29.17%) as C. orthopsilosis, and two (2.08%) as C. metapsilosis. Most strains were isolated from blood (81.25%). Proteinase activity was only detected in four (6.06%) and two (7.14%) isolates of C. parapsilosis sensu stricto and C. orthopsilosis, respectively. Sixty (90.91%) isolates of C. parapsilosis sensu stricto, 12 (42.86%) isolates of C. orthopsilosis, and all C. metapsilosis isolates showed phospholipase activity. Psuedohyphae formation was only detected in 33 (50%) and 15 (53.57%) isolates of C. parapsilosis sensu stricto and C. orthopsilosis, respectively. All isolates were susceptible to caspofungin. Most (85-100%) isolates were susceptible to antifungal drugs, but 3.13 - 6.25% were resistant to voriconazole and fluconazole. In conclusion, our findings revealed that C. parapsilosis sensu stricto was the most common species among clinical isolates of the C. parapsilosis complex, and the most commonly used antifungal agents generally exhibited good in vitro activity against these strains.

Epidemiology and antifungal susceptibilities of yeast isolates causing invasive infections across urban Beijing, China.

To investigate the species distribution and antifungal susceptibility profiles of yeast isolates causing invasive infections across Beijing. A total of 1201 yeast isolates recovered from blood and other sterile body fluids were correctly identified by matrix-assisted laser desorption/ionization TOF MS supplemented by DNA sequencing. Antifungal susceptibility testing was performed according to the Clinical and Laboratory Standards Institute broth microdilution method. Candida (95.5%) remained the most common yeast species isolated; Candida albicans (38.8%) and Candida parapsilosis (22.6%) were the leading species of candidemia. Azole resistances were mainly observed in Candida glabrata and Candida tropicalis isolates. This study outlined the epidemiologic data of invasive yeast infections and highlighted the need for continuous monitoring of azole resistances among C. glabrata and C. tropicalis isolates in Beijing.

Differential attraction of drosophilids to banana baits inoculated with Saccharomyces cerevisiae and Hanseniaspora uvarum within a Neotropical forest remnant.

BackgroundYeasts are a necessary requisite in the diet of most Drosophila species that, in turn, may vector their dispersal in natural environments. Differential attractiveness experiments and the isolation of yeasts consumed by Drosophila may be informative for characterizing this association. Hanseniaspora uvarum is among the most common yeast species isolated from Drosophila crops, with high attractiveness to drosophilids. Saccharomyces cerevisiae has been widely used to collect flies, and it allows broad sampling of almost all local Drosophila species. Pronounced differences in the field concerning Drosophila attractivity to baits seeded with these yeast species have been previously reported. However, few explicit generalizations have been set. Since late fifties, no field experiments of Drosophila attractivity were carried out in the Neotropical region, which is facing shifts in abiotic and biotic factors. Our objective is to characterize preference behavior that mediates the interaction in the wild among Neotropical Drosophila species and yeasts associated with them. We want to set a broad generalization about drosophilids attracted to these yeasts. Here we present the results of a differential attractiveness experiment we carried out in a natural Atlantic Rainforest fragment to assess the preferences of Drosophila species groups to baits inoculated with H. uvarum and S. cerevisiae.MethodsBoth yeast species were cultured in GYMP broth and separately poured in autoclaved mashed banana that was left fermenting. In the field, we collected drosophilids over five arrays of three different baits: non-inoculated autoclaved banana and banana inoculated with each yeast. In the laboratory the drosophilids were sorted to five sets according to their external morphology and/or genitalia: tripunctata; guarani; willistoni; exotic; and the remaining flies pooled in others.Results and ConclusionsUninoculated banana baits attracted virtually no flies. We found significant departures from random distribution over the other two baits (1:1 proportion) for all sets, except the pooled others. Flies of the sets willistoni and exotic preferred H. uvarum over S. cerevisiae, while the remaining sets were more attracted to S. cerevisiae. Previously, various authors reported similar patterns in attraction experiments with S. cerevisiae and H. uvarum. It is also noteworthy that both yeast species have been isolated from natural substrates and crops of Drosophila species. Taken together, these results suggest that the preferences among Drosophila species groups may be reflecting deep and stable relations with yeast species in natural environments. They can be summarized as: forest dwelling species from subgenus Drosophila (such as tripunctata and guarani groups) are attracted to banana baits seeded with S. cerevisiae; while exotic (as D. melanogaster) and subgenus Sophophora species are preferentially attracted to baits seeded with H. uvarum.

Sequence-based Analysis of the Vitis vinifera L. cv Cabernet Sauvignon Grape Must Mycobiome in Three South African Vineyards Employing Distinct Agronomic Systems.

Recent microbiomic research of agricultural habitats has highlighted tremendous microbial biodiversity associated with such ecosystems. Data generated in vineyards have furthermore highlighted significant regional differences in vineyard biodiversity, hinting at the possibility that such differences might be responsible for regional differences in wine style and character, a hypothesis referred to as “microbial terroir.” The current study further contributes to this body of work by comparing the mycobiome associated with South African (SA) Cabernet Sauvignon grapes in three neighboring vineyards that employ different agronomic approaches, and comparing the outcome with similar data sets from Californian vineyards. The aim of this study was to fully characterize the mycobiomes associated with the grapes from these vineyards. The data revealed approximately 10 times more fungal diversity than what is typically retrieved from culture-based studies. The Biodynamic vineyard was found to harbor a more diverse fungal community (H = 2.6) than the conventional (H = 2.1) and integrated (H = 1.8) vineyards. The data show that ascomycota are the most abundant phylum in the three vineyards, with Aureobasidium pullulans and its close relative Kabatiella microsticta being the most dominant fungi. This is the first report to reveal a high incidence of K. microsticta in the grape/wine ecosystem. Different common wine yeast species, such as Metschnikowia pulcherrima and Starmerella bacillaris dominated the mycobiome in the three vineyards. The data show that the filamentous fungi are the most abundant community in grape must although they are not regarded as relevant during wine fermentation. Comparison of metagenomic datasets from the three SA vineyards and previously published data from Californian vineyards revealed only 25% of the fungi in the SA dataset was also present in the Californian dataset, with greater variation evident amongst ubiquitous epiphytic fungi.

Molecular identification and antifungal susceptibility profiles of Candida parapsilosis complex species isolated from culture collection of clinical samples.

Candida parapsilosis is a common yeast species found in cases of onychomycosis and candidemia associated with infected intravascular devices. In this study, we differentiated Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis from a culture collection containing blood and subungual scraping samples. Furthermore, we assessed the in vitro antifungal susceptibility of these species to fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin. Differentiation of C. parapsilosis complex species was performed by amplification of the secondary alcohol dehydrogenase (SADH) gene and digestion by the restriction enzyme Ban I. All isolates were evaluated for the determination of minimal inhibitory concentrations using Etest, a method for antifungal susceptibility testing. Among the 87 isolates, 78 (89.7%) were identified as C. parapsilosis sensu stricto , five (5.7%) were identified as C. orthopsilosis , and four (4.6%) were identified as C. metapsilosis . Analysis of antifungal susceptibility showed that C. parapsilosis sensu strictoisolates were less susceptible to amphotericin B and itraconazole. One C. parapsilosis sensu stricto isolate was resistant to amphotericin B and itraconazole. Moreover, 10.2% of C. parapsilosis sensu stricto isolates were resistant to caspofungin. Two C. parapsilosis sensu strictoisolates and one C. metapsilosis isolate were susceptible to fluconazole in a dose-dependent manner. We reported the first molecular identification of C. parapsilosiscomplex species in State of Goiás, Brazil. Additionally, we showed that although the three species exhibited differences in antifungal susceptibility profiles, the primary susceptibility of this species was to caspofungin.

Patterns of Human Oral Yeast Species Distribution on Hainan Island in China

Infections by yeast strains of the genus Candida are among the most prevalent fungal infections of humans. These yeasts are common residents of the oral mucosa and other body surfaces. Since most yeast infections are due to endogenous strains and that species of Candida differ in virulence properties and in intrinsic susceptibilities to antifungal drugs, understanding the human commensal yeast flora can help designing effective treatment and prevention strategies against yeast infections. Here, we report the patterns of yeast species distributions in the oral cavities of 1,799 people from Hainan Island in southern China. Based on sequence information at the fungal barcode locus ITS regions, 368 of the 415 obtained oral yeast strains were identified as belonging to 26 yeast species, while the remaining 47 strains all showed significant sequence divergence to the currently described species. The four most common yeast species were C. albicans (42%), C. tropicalis (20%), C. glabrata (5.5%), and C. parapsilosis (4.1%) and 10 of the 26 yeast species were represented by only one strain each. Our analyses identified that the gender of hosts and ethnical background showed no contribution to oral yeast species distributions. However, the health status, place of birth, current residency, and the age of hosts all showed significant contributions to the distributions of the four dominant yeast species. We compared our results with those reported previously and discussed the potential mechanisms for the observed differences in oral yeast species distributions.

Oral Candida carriage and species prevalence amongst habitual gutka‐chewers and non‐chewers

Oral Candida colonisation is higher in tobacco smokers as compared to non-smokers; however, it remains unknown whether smokeless tobacco chewers are susceptible to increased oral Candida colonisation. The aim was to determine the oral Candida carriage and species prevalence amongst habitual gutka-chewers and non-chewers in a cohort from Karachi, Pakistan. Forty-five gutka-chewers and 45 non-chewers were included. Information regarding age, sex, duration of gutka-chewing habit, daily frequency of gutka consumption, duration of holding gutka in the mouth, daily frequency of tooth-brushing and tongue brushing was collected using a questionnaire. Oral yeast samples were collected by scraping the dorsum of the tongue and bilateral buccal mucosa with a sterile cotton swab. Identification of yeast species was performed using standard techniques. Tongue lesions were identified and recorded. Unstimulated whole salivary flow rate (UWSFR) was also measured. There was no significant difference in the mean age, UWSFR and oral Candida carriage among gutka-chewers and non-chewers. Individuals were chewing gutka since 4·4 years and were consuming five gutka sachets daily. Candida albicans (C. albicans) was the most common yeast species isolated from 57·8% gutka-chewers and 64.4% non-chewers. In 24.4% gutka-chewers and 22·2% non-chewers, two candidal strains (C. albicans and Candida tropicalis) were isolated. In conclusion, the present results indicated no significant difference in oral Candida carriage in habitual gutka-chewers and non-chewers.

Evaluation of the Commercial Rapid Trehalose Test (GLABRATA RTT) for the Point of Isolation Identification of Candida glabrata Isolates in Primary Cultures

Candidaemias account for 10-20% of nosocomial bloodstream infections depending on the study. Whilst Candida albicans remains the most frequently isolated species, Candida glabrata may be responsible for as many as 10-25% of all candidaemias. Moreover, C. glabrata is generally less susceptible to the azole antifungals than the majority of other pathogenic yeast species. Thus, a rapid test for the specific identification of isolates of C. glabrata would be useful for patient management if it could be performed at point of isolation, on primary cultures grown on standard mycological media directly from patient specimens. Under certain conditions, C. glabrata rapidly hydrolyses trehalose into glucose. The GLABRATA RTT kit allows detection of the preformed enzyme responsible for this action. This study has assessed GLABRATA RTT as an identification tool specifically at point of isolation. Sixty test isolates were evaluated: 39 clinical isolates of C. glabrata identified at the UK Mycology Reference Laboratory, examples of the recently described genetic relatives of C. glabrata, Candida nivariensis (n=6) and Candida bracarensis (n=1), and a selection of other common pathogenic yeast species (n=14). The test provided results within 30min. Although 77% (30/39) of confirmed C. glabrata isolates were correctly identified by GLABRATA RTT (positive trehalase test), 23% (9/39) of isolates gave negative or equivocal results. All other yeast species gave negative results. The performance of GLABRATA RTT in this study is compared to previous evaluations of the test which employed isolates pre-cultured on specialised media and to other existing conventional identification methodologies.

Separation of phenotypically indistinguishable Candida species, C. orthopsilosis, C. metapsilosis and C. parapsilosis, by capillary electromigration techniques

At the current state of laboratory diagnostics, methods for fast identification of phenotypically indistinguishable species are difficult or inaccurate. An example is represented by Candida parapsilosis, which is the second most common yeast species isolated from bloodstream infections. C. parapsilosis comprises a complex of three genetically distinct groups. Genotypes II and III have been designated as the separate species Candida orthopsilosis and Candida metapsilosis, phenotypically indistinguishable. The considerable genetic variability of these newly described yeasts species has caused difficulties in the development of molecular techniques for their precise identification. Similarly, the detection of biofilm formation, which is considered as an important yeast virulence factor, is accompanied by difficulties. In this study we optimize the first precise and reproducible method for the separation and possible identification of C. orthopsilosis, C. metapsilosis and C. parapsilosis as well as the detection of their ability to form biofilm. The method is based on capillary isoelectric focusing and capillary electrophoresis with UV detection. In capillary isoelectric focusing, very narrow pH gradients were established. With such gradients, differences in isoelectric points of biofilm-negative and biofilm-positive species calculated from the migration times of the selected p I markers were below 0.03 p I units. In the capillary zone electrophoresis narrow zones of the cells of Candida species were detected with sufficient resolution. The values of the isoelectric point and the migration velocities of the examined species were independent on the origin of the tested strains. Capillary isoelectric focusing was examined also for the separation and detection of the cultivated biofilm-negative C. parapsilosis in the blood serum.

English

During the past two decades, there has been a significant increase in the prevalence of fungal infections caused by Candida species. Oral candidiasis is a common opportunistic infection of the oral cavity caused by yeast fungi of the genus Candida on the mucous membranes of the mouth. To isolate and determine the incidence rate of oral Candida species in periodentitis and gingivitis patients referred to school of dentistry, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran, this study was carried out in 172 patients with periodentitis and gingivitis aged 11 to 72 years. Swabs samples were taken from salivary secretion, the palate mucosa and dentine carious lesions and were cultured directly on Sabouraud dextrose agar medium. Isolated yeasts were identified by CHROMagarCandida, germ tube test and Clamidoconidia formation (corn meal agar plus Tween 80 medium). Results showed the prevalence of Candida albicans (n = 120, 75%),Candida glabrata (n = 20, 12.5%), Cadida tropicalis (n = 10, 6.5%), Candida dubliniensis (n = 6, 4.0%) and Cadida krusei (n = 3, 2.0%). In this investigation, germ tube-test and chlamydospore formation were positive in the isolates that produced dark-green colonies and were considered as C. dubliniensis and light-green colonies were identified as C. albicans. CHROMagar Candida is a satisfactory isolation medium for oral and dental specimens. It is a satisfactory method for correct and rapid identification of common yeast species and easy recognition of mixed cultures in clinical samples.   Key words: Periodentitis, gingivitis, oral candidiasis, CHROMagar

Candida albicans identification: comparison among nine phenotypic systems and a multiplex PCR.

Candida albicans is the most common fungal pathogen isolated from clinical samples and is also the most common yeast species carried as a commensal by healthy individuals although some non-C. albicans species account for an important number of infections. To compare nine phenotypic systems for C. albicans identification [API 20C AUX; RapID Yeast Identification panel (RYIP); Vitek2 ID-YST system; chromogenic media, CHRO-Magar, Oxoid Chromogenic Candida Agar (OCCA), Candida ID2, Candida Identification Agar, CandiSelect 4, and Chromalbicans Agar] with multiplex PCR. A collection of 390 yeast strains was obtained by routine isolation from oral and vaginal swabs. All of the yeasts isolated were tested for germ tube formation, and then submitted to a multiplex PCR protocol tested in previous studies, and to nine phenotypical commercial methods, together with the reference ATCC strains. Comparison was limited to the ability of the tests to identify C. albicans. 253 isolates were provisionally identified as C. albicans by germ tube, and their identities were further confirmed with the multiplex PCR. Sensitivity of phenotypical systems ranged from 81.9% (Vitek2) to 87.7% (Candida ID2 e CHROMagar). For specificity, the highest value was 96.8% for Candida ID2, and the lowest value (75.1%) was for Chromalbicans Agar. Although with differences in discriminatory power, the methods tested showed overall acceptable levels of sensitivity and specificity respect to the multiplex PCR; therefore, all could be useful for C. albicans identification where molecular differentiation is not available.

Microbial communities and chemical changes during fermentation of sugary Brazilian kefir

The microorganisms associated with sugary Brazilian kefir beverage were investigated using a combination of culture-dependent and -independent methods. A total of 289 bacteria and 129 yeasts were identified via phenotypic and genotypic methods. Lb. paracasei (23.8%) was the major bacterial isolate identified, followed by Acetobacter lovaniensis (16.31%), Lactobacillus parabuchneri (11.71%), Lactobacillus kefir (10.03%) and Lactococcus lactis (10.03%). Saccharomyces cerevisiae (54.26%) and Kluyveromyces lactis (20.15%) were the most common yeast species isolated. Scanning electron microscopy showed that the microbiota was dominated by lemon-shaped yeast cells growing in close association with Lactobacillus (long and curved). Some lactic acid bacteria detected by sequence analysis of DGGE (denaturing gradient gel electrophoresis) bands were not recovered at any time through fermentation by plating. Conversely, DGGE fingerprints did not reveal bands corresponding to some of the species isolated by culturing methods. The bacteria Acetobacter lovaniensis and the yeast Kazachstania aerobia are described for the first time in sugary kefir. During the 24h of fermentation, the concentration of lactic acid ranged from 0.2 to 1.80mg/ml, and that of acetic acid increased from 0.08 to 1.12mg/ml. The production of ethanol was limited, reaching a final mean value of 1.24mg/ml.

Isolation and characterization of Candida kefyr orotidine‐5′‐phosphate decarboxylase (URA3) gene

Candida kefyr is a common yeast species that can be found in fermented milk and cheeses. As a first step to developing a gene transfer system for C. kefyr, the orotidine-5'-phosphate decarboxylase (URA3) gene was cloned, using degenerate PCR and genome walking. The uninterrupted open reading frame of the C. kefyr URA3 gene spans 801 bp, corresponding to 267 amino acid residues. The functionality of the gene was confirmed by complementation of ura3 auxotrophs of C. albicans and Saccharomyces cerevisiae. Phylogenetic analysis of the deduced amino acid sequence indicated that it shares a high degree of homology with other Candida URA3 homologues.

Comparison of 24-Hour and 48-Hour Voriconazole MICs as Determined by the Clinical and Laboratory Standards Institute Broth Microdilution Method (M27-A3 Document) in Three Laboratories: Results Obtained with 2,162 Clinical Isolates of Candida spp. and Other Yeasts

We evaluated the performance of the 24-h broth microdilution voriconazole MIC by obtaining MICs for 2,162 clinical isolates of Candida spp. and other yeasts; the 24-h results were compared to 48-h reference MICs to assess essential, as well as categorical, agreement. Although the overall essential agreement was 88.6%, it ranged from 96.4 to 100% for 6 of the 11 species or groups of yeasts tested. The overall categorical agreement was 93.2%, and it was above 90% for eight species. However, unacceptable percentages of very major errors (false susceptibility) were observed for Candida albicans (2.7%), C. glabrata (4.1%), C. tropicalis (9.7%), and other less common yeast species (9.8%). Since it is essential to identify potentially resistant isolates and breakpoints are based on 48-h MICs, it appears that the 24-h MIC is not as clinically useful as the 48-h reference MIC. However, further characterization of these falsely susceptible MICs for three of the four common Candida spp. is needed to understand whether these errors are due to trailing misinterpretation or if the 48-h incubation is required to detect voriconazole resistance. Either in vivo versus in vitro correlations or the determination of resistance mechanisms should be investigated.

In Vitro Amphotericin B Susceptibility of Korean Bloodstream Yeast Isolates Assessed by the CLSI Broth Microdilution Method, Etest, and Minimum Fungicidal Concentration Test

Although amphotericin B (AMB) has a wide spectrum of activity that encompasses the majority of yeast isolates, there have been recent reports suggesting that some yeast isolates exhibit decreased susceptibility to AMB. However, in vitro AMB susceptibility of yeast species isolates from blood cultures in Korea has not been fully surveyed. A total of 92 bloodstream yeast isolates from four Korean hospitals, representing 10 Candida species (69 isolates) and 4 non-Candida yeast species (23 isolates) were evaluated. AMB minimum inhibitory concentrations (MICs) were determined by two methods: the CLSI method and Etest. AMB minimum fungicidal concentrations (MFCs) were also determined. For all 92 yeast isolates, the CLSI method generated a restricted range of MICs (0.125 to 4 microgram/mL) with 3.3% exhibiting MICs > or =2 microgram/mL, and the corresponding MFC values ranged from 0.25 to 8 microgram/mL with 26.1% showing MFCs > or =2 microgram/mL. Etest produced the widest distribution of MICs, ranging from 0.03 to 32 microgram/mL. High AMB MICs (> or =0.38 microgram/mL) by Etest was observed in 34.8% of the isolates: Candida krusei (100%), Candida rugosa (100%), Trichosporon asashii (100%), Candida glabrata (82%), and Yarrowia lipolytica (75%). Etest disclosed that all isolates of Candida guilliermondii, Candida lusitaniae, Candida pelliculosa and Kodamaea ohmeri were highly susceptible to AMB (MIC < or =0.19 microgram/mL). Our study showed that Etest may be more useful to discriminate yeast isolates with reduced susceptibility to AMB, and some isolates of less common yeast species from Korea may have decreased AMB susceptibilities.

Incidence and anatomic localization of oral candidiasis in patients with AIDS hospitalized in a public hospital in Belo Horizonte, MG, Brazil

The objective of this study was to evaluate the incidence, anatomic localization and yeast species isolated from each clinical type of oral candidiasis. The clinical samples were obtained from 67 patients with AIDS with CD4 cell counts below 200 cells/mm3 and hospitalized in a public hospital (Eduardo de Menezes Hospital) in the city of Belo Horizonte, MG, Brazil. Yeasts were isolated using Chromagar® Candida. The results show that 50.7% of these patients had oral candidiasis. The pseudomembranous form was the most frequent clinical manifestation of oral candidiasis, followed by the erythematous and angular cheilite forms. The most common site of these clinical forms of oral candidiasis was the tongue. Candida albicans was the most common yeast species isolated from the lesions. However, other species were also found to be associated with these forms of oral candidiasis.

Molecular identification of yeast species associated with ‘Hamei’ — A traditional starter used for rice wine production in Manipur, India

In Manipur state of North-Eastern India, wine from glutinous rice using traditional solid state starter called ‘Hamei’ is particularly interesting because of its unique flavour. A total of 163 yeast isolates were obtained from fifty four ‘Hamei’ samples collected from household rice wine preparations in tribal villages of Manipur. Molecular identification of yeast species was carried out by analysis of the restriction digestion pattern generated from PCR amplified internal transcribed spacer region along with 5.8S rRNA gene (ITS1-5.8S-ITS2). Seventeen different restriction profiles were obtained from the size of PCR products and the restriction analysis with three endonucleases (Hae III, Cfo I and Hinf I). Nine groups were identified as S. cerevisiae, Pichia anomala, Trichosporon sp ., Candida tropicalis, Pichia guilliermondi, Candida parapsilosis, Torulaspora delbrueckii, Pichia fabianii and Candida montana by comparing this ITS-RFLP profile with type strains of common wine yeasts, published data and insilico analysis of ITS sequence data available in CBS yeast database . ITS-RFLP profile of eight groups was not matching with available database of 288 common wine yeast species. The most frequent yeast species associated with ‘Hamei’ were S. cerevisiae (32.5%), P. anomala (41.7%) and Trichosporon sp. (8%). The identity of major groups was confirmed by additional restriction digestion of ITS region with Hind III, EcoRI, Dde I and Msp I. The genetic diversity of industrially important S. cerevisiae group was investigated using Pulsed Field Gel Electrophoresis (PFGE). Although most of the 53 strains of S. cerevisiae examined were exhibited a common species specific pattern, a distinct degree of chromosomal length polymorphism and variable number of chromosomal DNA fragments were observed with in the species. Cluster analysis showed seven major karyotypes (K1–K7) with more than 83% similarity. The karyotype pattern K1 was the most frequent (67.9%) among the strains from different samples. Other karyotypes K2–K7 were very unique with less than 80% similarity. Finally using mitochondrial DNA restriction analysis (mt-DNA RFLP), S. cerevisiae strains belonging to the major karyotype K1 were distinctly differentiated with highly polymorphic bands by Hinf I and Hae III endonucleases.

Molecular identification of pathogenic fungi

Systemic fungal infections represent a major cause of morbidity and mortality in immunocompromised patients. The ever-increasing number of yeast species associated with human infections that are not covered by conventional identification kits, and the fact that moulds isolated from deep infections are frequently impossible to identify using classical methods due to lack of sporulation, has driven the need for rapid, robust molecular identification techniques. We recently developed a rapid method of preparing fungal genomic DNAs using Whatman FTA filters, which has greatly facilitated molecular identification. Mould isolates cultured from dark grain mycetomas (destructive infections of skin/subcutaneous tissues that progress to involve muscle and bone) invariably fail to produce features by which they can be identified and were taxonomic mysteries. PCR amplification and sequencing of 250 bp of the internal transcribed spacer region 1 (ITS1) allowed us to distinguish between the known agents of mycetoma, to describe three new species associated with this disease and to define phylogenetic relationships. For yeasts, 153 isolates encompassing 47 species that had failed to be identified using classical methods were unambiguously identified by conventional sequencing of 350 bp of the 26S rRNA D1D2 region. These represented 5% of the isolates examined and included common species with atypical biochemical and phenotypic profiles, and rarer species infrequently associated with infection. Our recent studies indicate that FTA extraction coupled with pyrosequencing of 25 bp of ITS2 could potentially identify most common yeast species from pure culture in half a day. Together, these data underscore the importance of molecular techniques for fungal identification.

Yeast diversity in rice–cassava fermentations produced by the indigenous Tapirapé people of Brazil

The Tapirapé people of the Tapi'itãwa tribe of Brazil produce several fermented foods and beverages, one of which is called 'cauim'. This beverage usually makes up the main staple food for adults and children. Several substrates are used in its production, including cassava, rice, corn, maize and peanuts. A fermentation using rice and cassava was conducted, and samples were collected at 4-h intervals for microbial analysis. The yeast population was low at the beginning of the fermentation and reached 6.9 x 10(7) CFU mL(-1) after 48 h. During the fermentation process common yeast species were identified by sequencing of the D1/D2 domain of the large-subunit (26S) rRNA gene. The predominant yeast species found was Candida tropicalis. Candida intermedia, Candida parapsilosis, Pichia guilliermondii, Saccharomyces cerevisiae and Trichosporon asahii were also found in high numbers during the fermentation. Exophiala dermatidis, often associated with blastomycosis, was found in the mass before inoculation and during the initial stages of the fermentation. Examination of these indigenous fermented foods may provide clues as to how food production and preservation can be expanded and thereby contribute to improve nutrition in native tribes in the region.