Rapid Identification Of Yeasts Research Articles

Application of MALDI-TOF analysis to reveal diversity and dynamics of winemaking yeast species in wild-fermented, organically produced, New Zealand Pinot Noir wine

Rapid yeast identification is of particular importance in monitoring wine fermentation and assessing strain application in winemaking. We used MALDI-TOF MS analysis supported by 26 S rRNA gene sequence analysis and Saccharomyces-specific PCR testing to differentiate reference and field strains recovered from organic wine production facilities in Waipara, New Zealand, in which Pinot Noir wine was produced by spontaneous fermentations in the vineyard and in the winery. Strains were isolated from each of four key stages of each ferment to evaluate changes in taxonomic diversity. MALDI-TOF MS analysis was confirmed as an excellent yeast identification method, with even closely related Saccharomyces species readily distinguished. A total of 13 indigenous species belonging to eight genera were identified from Pinot Noir ferments, with taxonomic diversity generally reducing as fermentation progressed. However, differences between the taxa recovered were observed between the vineyard and winery ferments, despite the grapes used being from the same batch. Furthermore, some consistent proteomic differences between strains of S. cerevisiae, Hanseniasporum uvarum, Candida californica, Pichia membranifaciens and Starmerella bacillaris correlated with the different fermentation systems used. The high speed, low cost, taxonomic resolution and ability to characterise subtle changes in phenotype that may result from variations in environmental conditions makes MALDI-TOF analysis an attractive tool for further and wider applications in the wine industry. Such applications may include monitoring wine fermentation to actively support the consistency of high-quality wine products, and potentially for the development of such products too.

Anew Medium for Isolation and Diagnosis of Cryptococcus neoformans

Melanin produce by Cryptococcus neoformans yeastwas used to characterize this yeast.In present study,we used red onion extract in prepared a new medium.The isolates of yeast were cultivation. After 48 hours results show,the isolates of C.neoformans were appear in dark brown colonies while the colonies on SDA not produce any pigment.Also analysis resultsof red onion extract by usedUV-Spectometerand FT-IR appear this extract content phenolic compounds which causes to produce brown pigment.thus, this medium Red Onion Agar (RO A) useful for rapid identification of yeast C.neoformans

Enumeration and rapid identification of yeasts during extraction processes of extra virgin olive oil in Tuscany.

The aim of this study was to evaluate the occurrence of yeast populations during different olive oil extraction processes, carried out in three consecutive years in Tuscany (Italy), by analysing crushed pastes, kneaded pastes, oil from decanter and pomaces. The results showed yeast concentrations ranging between 10(3) and 10(5)CFU/g or per mL. Seventeen dominant yeast species were identified by random amplified polymorphic DNA with primer M13 and their identification was confirmed by restriction fragments length polymorphism of ribosomal internal transcribed spacer and sequencing rRNA genes. The isolation frequencies of each species in the collected samples pointed out that the occurrence of the various yeast species in olive oil extraction process was dependent not only on the yeasts contaminating the olives but also on the yeasts colonizing the plant for oil extraction. In fact, eleven dominant yeast species were detected from the washed olives, but only three of them were also found in oil samples at significant isolation frequency. On the contrary, the most abundant species in oil samples, Yamadazyma terventina, did not occur in washed olive samples. These findings suggest a phenomenon of contamination of the plant for oil extraction that selects some yeast species that could affect the quality of olive oil.

Direct colony polymerase chain reaction for rapid identification of yeasts isolated from blood specimen

Context: Bloodstream infections (BSIs) caused by yeasts have an increasing frequency due to the growing population of immunosuppressed individuals. Among yeasts, Candida remains the most prevalent species with the increase in the incidence of non-albicans Candida species. Apart from Candida, other yeasts are also involved in causing BSI. High mortality associated with Candida and other yeast infection can be reduced by prompt and appropriate antifungal therapy. Hence, rapid identification and speciation of yeasts isolated from blood play a significant role in the management of the patients. Since conventional methods used for speciation of Candida and other yeasts are laborious, time-consuming and often unclear, rapid and accurate molecular techniques are required. Materials and Methods: Instead of using purified genomic DNA as template for polymerase chain reaction (PCR), we used yeast colony and cell suspensions in water and 0.10M potassium hydroxide as template for PCR. Candida albicans, Trichosporon and Cryptococcus neoformans were used as reference strains. Further, a total of 100 yeast isolates were also tested. All reactions were performed using the universal fungal primers ITS1 and ITS4; the PCR products were then digested with restriction enzyme (Msp1). Results: Direct colony PCR (DCPCR) produced sharp and distinct bands compared to the cell suspensions with the reference strains. All the 100 clinical isolates tested also produced distinct bands. Conclusion: DCPCR approach not only reduces the DNA template preparation time but is also easy, rapid and reduces the cost of PCR.

MOLECULAR IDENTIFICATION OF YEAST OF THE PULQUE BY PCR-DGGE, A TRADITIONAL MEXICANBEVERAGE

Currently it is well known that yeasts play an essential role in the production of different beverages. In this paper, were identified some of the yeasts involved in the fermentation process of the pulque, a Mexican traditional beverage. Samples were collected from different regions of Mexico and yeasts were detected directly from samples without cultivation. Identifying the yeasts was obtained using amplification the D1/D2 domain of the 26S rRNA gene and Denaturing Gradient Gel Electrophoresis (DGGE). The results of DGGE showed different profiles of bands in each of the analyzed samples, indicating the presence of several species of yeast, which was also confirmed by sequencing of the bands corresponding to the domain D1/D2, succeeded in identifying five species of yeasts. The results obtained in this work demonstrated that the technique used for identification of yeasts of pulque was efficient. Besides, the optimization of this method could also allow rapid identification of yeasts and help understand the role of these in the fermentation process of this beverage, as well as the isolation of strains of interest for biotechnological purposes such as production of ethanol or metabolites with nutraceutical activity.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the rapid identification of yeasts causing bloodstream infections

Few studies have systematically standardised and evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of yeasts from bloodstream infections. This is rapidly becoming pertinent for early identification of yeasts and appropriate antifungal therapy. We used 354 yeast strains identified by polymerase chain reaction (PCR) sequencing for standardisation and 367 blind clinical strains for validation of our MALDI-TOF MS protocols. We also evaluated different sample preparation methods and found the on-plate formic acid extraction method as most cost- and time-efficient. The MALDI-TOF assay correctly identified 98.9% of PCR-sequenced yeasts. Novel main spectrum projections (MSP) were developed for Candida auris, C. viswanathii and Kodamaea ohmeri, which were missing from the Bruker MALDI-TOF MS database. Spectral cut-offs computed by receiver operating characteristics (ROC) analysis showed 99.4% to 100% accuracy at a log score of ≥1.70 for C. tropicalis, C. parapsilosis, C. pelliculosa, C. orthopsilosis, C. albicans, C. rugosa, C. guilliermondii, C. lipolytica, C. metapsilosis, C. nivariensis. The differences in the species-specific scores of our standardisation and blind validation strains were not statistically significant, implying the optimal performance of our test protocol. The MSPs of the three new species also were validated. We conclude that MALDI-TOF MS is a rapid, accurate and reliable tool for identification of bloodstream yeasts. With proper standardisation, validation and regular database expansion, its efficiency can be further enhanced.

Prospective assessment of FilmArray® technology for the rapid identification of yeast isolated from blood cultures

We prospectively assessed the ability of FilmArray® device to identify fungal species involved in bloodstream infections. It succeeded in identifying 85.7% of isolates. The automated readout of results enabled the rapid initiation of appropriate antifungal therapy. Thus, FilmArray® appeared as a reliable alternative diagnostic method for the most common yeast-like species.

Comparison between MALDI-TOF MS and FilmArray Blood Culture Identification panel for rapid identification of yeast from positive blood culture

In this study we evaluated MALDI-TOF MS and FilmArray methods for the rapid identification of yeast from positive blood cultures. FilmArray correctly identified 20/22 of yeast species, while MALDI-TOF MS identified 9/22. FilmArray is a reliable and rapid identification system for the direct identification of yeasts from positive blood cultures.

Evaluation of Xpert MTB/RIF assay for the diagnosis of paediatric TB

Background: The incidence of invasive fungal infections has increased worldwide with Candida species causing the majority of these. Increasing resistance to antifungal agents has beendescribed and contributes to the difficulty in treating these infections. Hence, rapid yeast identification and susceptibility testing methods that will facilitate prompt initiation of appropriate therapy are required to optimize patient management. The Vitek® 2 antifungal susceptibility testing system (bioMerieux) is a fully automated commercial method that allows both fungal identification and MIC determination simultaneously within 24hours, compared to themanual API® and Etest® (bioMerieux) methods. Methods&Materials: 60 routine positive yeast culture isolates, from different sites that had been processed for fungal identification and susceptibility testing, using the standard laboratory method were tested on the Vitek 2 system according to the manufacturer’s instructions. The Vitek® 2 YST and Vitek® 2 AST-YS07 cardswereused for identificationand susceptibility testing, respectively. Results: All of the Vitek® 2 results were available within 24hours. There was a 98% concordance with the identification results and 100% concordance with susceptibility test results between the two methods. All discordant samples were retested using the Vitek® MS MALDI-TOF identification method, and these were found to be in agreement with the Vitek® 2. The instrument was found to be easy to use and required minimal maintenance. Conclusion: The adoption of this fully automated fungal identification and susceptibility testing system in the laboratory will significantly impact on patient management and outcomes as it reduces technologist hands-on time, turnaround times, and variability in patient results by using a standardized format.

Identification of candida species using HiChrome agar in HIV-seropositive patients with oral candidiasis

Objectives: Candidiasis is one of the most common fungal infections affecting the oral cavity in HIV‑seropositive patients. Various Candida species, (both albicans as well as non‑albicans) are identified as the causative organisms. These species are identified by various methods. Chromogenic media are frequently used in direct and rapid identification of yeasts as different Candida species produce different colors on these media. Difference in expression of putative virulence factors and antifungal susceptibility pattern among different Candida species has raised the need for species–level identification. This study aims at identifying various Candida species using HiChrom agar in HIV‑seropositive patients in different clinical variants of oral candidiasis. Materials and Methods: Swabs were collected from 30 HIV‑seropositive patients with oral candidiasis. HiChrome agar was used to identify the Candida species. Results: Based on the color of the colonies on HiChrom agar we found that 46.6% (n = 14) patients showed a combination of light green and purple color colonies of C. albicans and C. tropicalis, 16.6% (n = 5) cases showed light green colonies of C. albicans, 13.3% (n = 4) cases produced dark green colonies of C. dublienisis, 10% (n = 3) cases produced pink color colonies of C. glabrata, 6.6% (n = 2) produced off white colonies of C. parapsilosis, 3.3% (n = 1) produced deep blue colonies of C. tropicalis and 3.3% (n = 1) cases showed combination of off white and purple color colonies of C. parapsilosis and C. tropicalis. C. albicans as well as mixed Candida species were commonly seen in pseudomembranous oral candidiasis. Conclusions: Identification of these species is important to target the specific drug for treatment.

Modified Protocol for Yeast Identification Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Advances in Matrix-assisted Laser Desorption Ionization–time of Flight Mass Spectrometry (MALDI-ToF MS) identification methods have greatly facilitated the rapid identification of yeasts. We validate the use of a shorter on-plate extraction method as well as a lower score cutoff value on the Bruker Biotyper. Overall, when the on-plate extraction method was used together with a score cutoff of > 1.7, a total of 151 of 187 (81%) <i>Candida</i> isolates were correctly identified. At species level, almost 100% of <i>C. krusei</i> and <i>C. albicans</i> isolates were correctly identified. We find the revised protocol to be effective for routine identification of yeasts.

Evaluation of PNA-FISH Yeast Traffic Light for Rapid Identification of Yeast Directly from Positive Blood Cultures and Assessment of Clinical Impact

The PNA-FISH Yeast Traffic Light assay was performed on 54 clinical isolates of yeasts inoculated into blood culture bottles. The assay showed high sensitivity (Candida albicans/C. parapsilosis, 100%; C. glabrata/C. krusei, 92.3%; C. tropicalis, 100%) and specificity (C. albicans/C. parapsilosis, 100%; C. glabrata/C. krusei, 94.8%; C. tropicalis, 100%). Case note review estimated a change in therapy in 29% of cases had the PNA-FISH result been available to the clinician.

Rapid identification of yeast by fluorescence in situ hybridisation from broth and blood cultures

Candida species including species other than Candida albicans are of importance as causative agents of sepsis in intensive-care units, requiring prompt initiation of targeted therapy. While fluconazole is usually active against Candida albicans, non-Candida albicans species often require more sophisticated approaches. A rapid species diagnosis is therefore desirable and can be provided by fluorescence in situ hybridisation (FISH). However, broad evaluation studies of described probes are largely lacking and the probe panel that has been described is incomplete. As an addition to previously described C. albicans FISH probes, we evaluated published DNA probes for C. glabrata and C. krusei, as well as newly designed DNA probes for C. krusei, C. lusitaniae, C. parapsilosis, C. tropicalis, Crypotococcus neoformans and a group of intrinsically fluconazole-resistant Candida species for FISH with 22 reference strains, 23 well-characterised laboratory control strains, 169 isolates from clinical samples and 48 blood cultures. Sensitivity and specificity of >99% were demonstrated for all evaluated species-specific probes, whereas the probe that binds to a heterogeneous group of intrinsically fluconazole-resistant Candida species correctly identified eight of nine fluconazole-resistant clinical isolates. FISH yielded reliable results using the classical FISH procedure as well as a recently described slide chamber-based method. Given this good sensitivity and specificity, FISH may be applied for rapid identification of yeast in screening analyses, thus giving the opportunity for more precise targeting of antimycotic therapy.

Evaluation of the system Axima/Saramis MALDI-TOF MS bioMérieux in a clinical laboratory

Introduction. Mass Spectrometry technology “Matrix-assisted laser desorption Ionisation/Time-of-Flight (MALDI-TOF MS)” offers a promising new approach for identification of bacteria and fungi of clinical interest. The objective of this study is to evaluate the comparison of microbial identifications obtained with the system Axima/Saramis bioMérieux and identifications obtained by conventional manual or automatic (Vitek 2- bioMérieux).The work was divided into a pilot phase in which the staff has been trained and are set instrument parameters and an operational phase. Methods. MALDI-TOF is a system for the detection and identification of species-specific highly conserved proteins (ribosomal DNA and ligands). Part of a bacterial colony, is deposited on a polymeric substrate, extracted by the addition of the matrix (acid α-Cyano-4-hydroxycinnamic) and analyzed by MALDI-TOF.The mass spectra obtained are compared with the database Saramis (Anagnostec) containing the reference spectra of common pathogens. Results. During the pilot phase were analyzed 303 microorganisms (281 bacteria and 22 fungi), of which 207 (68.3%) correctly identified to species level, 35 (11.6%) identified as a species but not the main one and 49 (16.2%) did not identified. In the second phase of the study were analyzed microorganisms 1417 (1341 bacteria and 76 fungi) in a period of 60 days in 1240 (87.5%) correctly identified to species level, 121 (8.5%) identified as a species but not the main one and 41 (2.9%) unidentified. Conclusions. By reducing the response time (15 min/1 sample), the use of MALDI-TOF system, bioMérieux facilitates the workflow in the clinical laboratory, and positive impact on length of stay and costs. In addition, the rapid identification of yeasts at the species level is definitely a value added important and independent execution of test sensitivity. Finally, a good operator training translates into improved overall system performance.

Molecular Identification of Yeasts Isolated from Dadih by RFLP-PCR and Assessment on Their Ability in Utilizing Lactate

A wide variety of yeasts have been involved in traditional fermented foods, and potentially contributed to the development of product properties. The existence of indigenous yeasts in dadih, a traditionally fermented form of buffalo milk of West Sumatera, has been reported but an accurate identification is still required to be conducted. This study was aimed to identify yeasts isolated from dadih using a molecular approach, and to evaluate their lactate utilization. A total of 51 isolates were characterized and identified as Pichia jadinii and Candida stellimalicola using PCR amplification of the 5.8S - internal transcribed spacer region combined with restriction fragment length polymorphism (RFLP) analyses and gene sequencing. The former species were the dominant one in this tested product. Their ability to utilize lactate was demonstrated, indicating that they could modify the sensory characteristics of dadih, and hence interact with the indigenous lactic acid bacteria in dadih. The restriction profiles of the dadih yeasts can be used as a data base for rapid identification of yeasts in the future. Further work is still needed to elucidate the dadih yeast ecology.

Rapid Identification of Yeast Isolates from Clinical Specimens in Critically Ill Trauma ICU Patients

Purpose:The purpose was to evaluate the performance of a commercially available chromogenic Candida speciation media and the Vitek 2 ID system for the identification of medically important yeasts and yeast-like organisms in a routine clinical microbiology laboratory.Materials and Methods:A total of 429 non duplicate, consecutive yeast strains were included during the 3.5-year study period. The performance of the Vitek 2 ID system and a chromogenic agar medium was evaluated against the gold standard conventional phenotypic and biochemical identification method for speciation of yeast isolates from trauma patients.Results:Candida tropicalis (64%) was the most common Candida species, followed by Candida albicans (14%), Candida rugosa (7%), and Candida parapsilosis (6.5%). Of the 429 isolates, 183 could be identified to species level by all the three methods. Agreement between the chromogenic agar method and conventional methods was 80% for Candida tropicalis, 100% for Candida rugosa, 89% for Candida albicans, and 77% for Candida parapsilosis. Vitek 2 had lower sensitivity, with agreement of 49% for Candida tropicalis, 100% for Candida rugosa, 39% for Candida albicans, and 31% for Candida parapsilosis.Conclusion:Thus, in long-term ICU patients, an increasing trend of isolating nonalbicans Candida spp. continues. The chromogenic agar medium is a convenient and economic method to identify commonly isolated species in busy clinical microbiology laboratories.

Species-specific PCR primers for the rapid identification of yeasts of the genus Zygosaccharomyces

Species-specific primer pairs that produce a single band of known product size have been developed for members of the Zygosaccharomyces clade including Zygosaccharomyces bailii, Zygosaccharomyces bisporus, Zygosaccharomyces kombuchaensis, Zygosaccharomyces lentus, Zygosaccharomyces machadoi, Zygosaccharomyces mellis and Zygosaccharomyces rouxii. An existing primer pair for the provisional new species Zygosaccharomyces pseudorouxii has been confirmed as specific. The HIS3 gene, encoding imidazole-glycerolphosphate dehydratase, was used as the target gene. This housekeeping gene evolves slowly and is thus well conserved among different isolates, but shows a significant number of base pair changes between even closely related species, sufficient for species-specific primer design. The primers were tested on type and wild strains of the genus Zygosaccharomyces and on members of the Saccharomycetaceae. Sequencing of the D1/D2 region of rDNA was used to confirm the identification of all nonculture collection isolates. This approach used extracted genomic DNA, but in practice, it can be used efficiently with a rapid colony PCR protocol. The method also successfully detected known and new hybrid strains of Z. rouxii and Z. pseudorouxii. The method is rapid, robust and inexpensive. It requires little expertise by the user and is thus useful for preliminary, large-scale screens.

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.

Rapid identification of yeasts from positive blood culture bottles by pyrosequencing

We have developed a rapid protocol for the identification of Candida species from positive blood cultures by combining a simple method for nucleic acid extraction and preparation using microbial storage cardboards with polymerase chain reaction (PCR) and pyrosequencing of a small region of the 18S rRNA gene. The protocol is robust and easy to implement and can be performed in 4 h. The method was tested against a collection of clinical blood cultures. Agreement of sequence identifications with standard microbiological methods was 100%.

Hichrom candida agar for identification of candida species

Chromogenic media are frequently used in direct and rapid identification of yeasts because different Candida species produce unique colors on these media. We used 60 isolates of Candida species including 30 C. albicans, 10 C. parapsilosis, 11 C. glabrata, five C. tropicalis, and four C. dubliniensis, isolated from various clinical specimens, to evaluate the performance of HiChrome Candida agar. These strains had been identified by germ tube test, morphology on cornmeal agar, chlamydospore formation on tobacco agar and sugar assimilation tests. The sensitivity and specificity results were: C. albicans (96.55 and 96.42%); C. parapsilosis (80 and 98.03%), C. glabrata (90.90 and 88.23%), C. tropicalis (100 and 100%) and C. dubliniensis (60 and 96.55%) respectively. HiChrom Candida agaris medium has been useful and capable of presumptive, rapid identification of Candida species within 48 hours.