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

Abstract

Background: Meyerozyma 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.

Results: We 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.

Conclusions: We 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.

Figure 1

Differentiation of M. guilliermondii and M. caribbica by TaqI digestion of ITS1-5.8S-ITS2. A: Multiple sequence alignment of representative ITS1-5.8S-ITS2 sequences of the two species obtained from NCBI GenBank and CBS yeast database showing position of TaqI recognition site (highlighted) which distinctly differentiated the two species. B: TaqI restriction digestion profile of ITS1-5.8S-ITS2 amplicons obtained from some of the representative isolates. Lane 1: C. guilliermondii ATCC 6260; Lane 2 − 12: isolates of M. guilliermondii genotype group MG (A1S10Y1, A2S10Y1, A3S9Y1, A2S9Y1, A3S11Y1, A3S2Y1, A3S6Y1, A2S6Y1, A1S9Y1, Kw3S3Y1 and Kw2S11Y2); Lane 13 – 20: isolates of M. caribbica genotype group MC (A1S10Y2a, A1S10Y3, A1S10Y5, Kw3S2Y1, Kw2S3Y1, Kw3S3Y3, Kw3S3Y4 and Kw1S7Y2); Lane M: PCR 100 bp Low DNA ladder (Sigma-Aldrich).

Figure 2

Neighbour-joining (NJ) phylogenetic tree showing taxa-specific separation of M. guilliermondii from M. caribbica. The tree was constructed based on the evolutionary distance calculated using Kimura-2 parameter from the nucleotide sequence of ITS1-5.8S-ITS2. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches for values >40%. The bar represents 1% sequence divergence. GenBank accession numbers are mentioned within the parentheses. S. cerevisiae was the outgroup in the analysis. ^T = Type strain.

Figure 3

mtDNA-RFLP based dendrogram showing distinct clustering of M. guilliermondii and M. caribbica. The dendrogram was constructed using UPGMA algorithm on Jaccard similarity coefficients generated from HaeIII and HinfI restriction digestion profile of mtDNA of some of the representative isolates. Value at each branch node indicates the branch quality with 1000 bootstrap replications. The scale represents the similarity.

Figure 4

PFGE karyotype patterns of isolates belonging to M. guilliermondii and M. caribbica genotype groups. Lane 1: C. guilliermondii ATCC 6260; Lane 2 − 3: M. guilliermondii isolates A1S10Y1 and Kw2S11Y2; Lane 4 − 11: M. caribbica isolates A1S10Y2a, A1S10Y3, A1S10Y5, Kw3S2Y1, Kw2S3Y1, Kw3S3Y3, Kw3S3Y4 and Kw1S7Y2; Lane M: S. cerevisiae PFGE marker (Sigma-Aldrich). Right arrow indicates the co-migrating chromosomal doublets showing strain level diversity.