Reliable differentiation of Meyerozyma guilliermondii from Meyerozyma caribbica by internal transcribed spacer restriction fingerprinting

Abstract

BackgroundMeyerozyma guilliermondii (anamorph Candida guilliermondii) and Meyerozyma caribbica (anamorph Candida fermentati) are closely related species of the genetically heterogenous M. guilliermondii complex. Conventional phenotypic methods frequently misidentify the species within this complex and also with other species of the Saccharomycotina CTG clade. Even the long-established sequencing of large subunit (LSU) rRNA gene remains ambiguous. We also faced similar problem during identification of yeast isolates of M. guilliermondii complex from indigenous bamboo shoot fermentation in North East India. There is a need for development of reliable and accurate identification methods for these closely related species because of their increasing importance as emerging infectious yeasts and associated biotechnological attributes.ResultsWe targeted the highly variable internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) and identified seven restriction enzymes through in silico analysis for differentiating M. guilliermondii from M. caribbica. Fifty five isolates of M. guilliermondii complex which could not be delineated into species-specific taxonomic ranks by API 20 C AUX and LSU rRNA gene D1/D2 sequencing were subjected to ITS-restriction fragment length polymorphism (ITS-RFLP) analysis. TaqI ITS-RFLP distinctly differentiated the isolates into M. guilliermondii (47 isolates) and M. caribbica (08 isolates) with reproducible species-specific patterns similar to the in silico prediction. The reliability of this method was validated by ITS1-5.8S-ITS2 sequencing, mitochondrial DNA RFLP and electrophoretic karyotyping.ConclusionsWe herein described a reliable ITS-RFLP method for distinct differentiation of frequently misidentified M. guilliermondii from M. caribbica. Even though in silico analysis differentiated other closely related species of M. guilliermondii complex from the above two species, it is yet to be confirmed by in vitro analysis using reference strains. This method can be used as a reliable tool for rapid and accurate identification of closely related species of M. guilliermondii complex and for differentiating emerging infectious yeasts of the Saccharomycotina CTG clade.

Preliminary Analysis of Sugar Supplementation on Alcoholic Fermentation by <i>Meyerozyma guilliermondii</i>

“…Total yield of ethanol from M. guilliermondii and S. cerevisiae using molasses medium (initial reducing sugar concentration of 18.3 w/v%) revealed that M. guilliermondii produced higher volumes (10 ± 0.2 mg/ml) than that of S. cerevisiae (9 ± 0.2 mg/ml) at 96 hours (figure 7). The demand curve for assortment of low, medium to high alcohol content wines has informed the decision to study various tactics towards the production of low-medium [27]. Data from this study suggests that the non-Saccharomyces yeast; Meyerozyma guilliermondi, can be applied towards the production of various categories of wine.…”

“…Kluyveromyces marxianus [19], [20], Pichia kudriavzevii [21], [22], Dekkera bruxellensis [23], Zygosaccharomyces bailii [24]. Meyerozyma guilliermondii, a telemorph of Candida guilliermondii or wine yeast is one of such promising non Saccharomyces yeasts obtained from environmental samples with unique biotechnological applications and biological control potential [25], [26], [27], [28]. This yeast specie is known for its ease of consumption of xylose, naturally converting it to xylitol [29], [30].…”

Development of Two Molecular Approaches for Differentiation of Clinically Relevant Yeast Species Closely Related to Candida guilliermondii and Candida famata

“…Because these genetically related species are difficult to identify by conventional methods, molecular techniques are more suitable for their differentiation (24,30). ITS sequence analysis has been widely used as the gold standard for yeast species identification and for phylogenetic analysis (24,25,38).…”

“…Molecular methods such as PCR-restriction fragment length polymorphism (RFLP), real-time PCR, Luminex techniques, electrophoretic karyotyping, and PCR with type-specific primers have been developed to distinguish some of these species, but other species were not involved in those studies (30)(31)(32)(33). Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a promising technique that works well to distinguish yeast species, although a few closely related species (such as C. fermentati) may still be misidentified (34)(35)(36).…”

Diversidade genética e funcional de leveduras presentes em solos de mineração e áreas do entorno

“…São positivas para os testes de urease e azul de diazônio B (Boekhout & Fell, 1998 Espécies do gênero Meyerozyma são geneticamente próximas formando um complexo de espécies, possuem importância médica, biotecnológica e potencial para uso em controle biológico. M. guilliermondi e M. caribbica são extremamente difíceis para diferenciar por métodos fenotípicos, e as sequências provenientes do domínio D1/D2 falha ao discriminar essas duas espécies, sendo necessário uma análise concatenada com a região ITS para separação das espécies (Romi et al, 2014), fato observado nos isolados identificados no clado Meyerozyma.…”