Polymorphic internal transcribed spacer region 1 DNA sequences identify medically important yeasts

Abstract

Species-specific polymorphisms in the noncoding internal transcribed spacer 2 (ITS2) region of the rRNA operon provide accurate identification of clinically significant yeasts. In this study, we tested the hypothesis that ITS1 noncoding regions contain diagnostically useful alleles. The length of ITS1 region PCR products amplified from 40 species (106 clinical strains, 5 reference strains, and 30 type strains) was rapidly determined with single-base precision by automated capillary electrophoresis. Polymorphisms in the PCR product length permitted 19 species to be distinguished by ITS1 alone, compared with 16 species distinguished by using only ITS2. However, combination of both ITS alleles permitted identification of 30 species (98% of clinical isolates). The remaining 10 species with PCR products of similar sizes contained unique ITS alleles distinguishable by restriction enzyme analysis. DNA sequence analysis of amplified ITS1 region DNA from 79 isolates revealed species-specific ITS1 alleles for each of the 40 pathogenic species examined. This provided identification of unusual clinical isolates, and 53 diagnostic ITS1 sequences were deposited in GenBank. Phylogenetic analyses based on ITS sequences showed a similar overall topology to 26S rRNA gene-based trees. However, different species with identical 26S sequences contained distinct ITS alleles that provided species identification with strong statistical support. Together, these data indicate that the analysis of ITS polymorphisms can reliably identify 40 species of clinically significant yeasts and that the capacity for identifying potentially new pathogenic species by using this database holds significant promise.

FIG. 1

(A) ITS1 sequence-based phylogenetic tree of 41 clinically significant yeast species. A consensus neighbor-joining dendrogram with 1,000 bootstrap replicates was based on 419 aligned positions of complete ITS1 sequences with adjacent partial sequences of 18S and 5.8S rRNA genes. GenBank accession numbers of sequences generated in this study are presented in Table 2. Numbers at the nodes indicate the bootstrap values. Lower bars indicate relative genetic distance. (B) ITS2 sequence-based phylogenetic tree of 42 clinically significant yeast species. Consensus neighbor-joining dendrogram with 1,000 bootstrap replicates was based on 432 aligned positions of complete ITS2 sequences with adjacent partial sequences of 5.8S and 26S rRNA genes. The sequences of C. albicans (no. L28817) and C. parapsilosis (no. U10988), as well as our sequences used for tree building, were previously published (2). Accession numbers of additional ITS2 sequences generated in this study are shown in Table 2 (footnote d). (C) 26S sequence-based phylogenetic tree of 41 clinically significant yeast species. Consensus neighbor-joining dendrogram with 1,000 bootstrap replicates was based on 565 aligned positions of the D1/D2 region of the 26S rRNA genes. Sequences of the following organisms (accession numbers in parentheses) were retrieved from GenBank: C. albicans (no. U45776), C. dubliniensis (no. U57685), C. famata (no. U45808), C. freyschussii (no. AF017242), C. glabrata (no. U44808), C. guilliermondii (no. U45709), C. intermedia (no. U44809), C. kefyr (no. U94924), C. krusei (no. U76347), C. lambica (no. AF020435), C. lusitaniae (no. U44817), C. parapsilosis (no. U45754), C. pararugosa (no. U62306), C. pelliculosa (no. U74592), C. rugosa (no. U45727), C. tropicalis (no. AB034689), C. utilis (no. U73570), C. zeylanoides (no. U45832), C. diffluens (no. AF075502), C. humicolus (no. AF189836), C. laurentii (no. AF075469), C. liquefaciens (no. AF181515), C. uniguttulatus (no. AF075468), E. fibuliger (no. U40088), P. fabianii (no. U73573), P. veronae (no. U73576), S. cerevisiae (no. U44806), S. salmonicolor (no. AF070439), T. asahii (no. AF105393), T. cutaneum (no. AF075483), T. inkin (no. AF105396), T. jirovecii (no. AF105398), and T. mucoides (no. AF075515). Sequences of the following organisms (accession numbers in parentheses) are from this study: C. albidus (no. AF335982), C. neoformans (no. AF335984), P. farinosa (no. AF335974), P. ohmeri (no. AF335976), P. ohmeri sequevar 1 (no. AF335975), R. glutinis (no. AF335985), R. rubra (no. AF335986), and Y. lipolytica (no. AF335977).

FIG. 1

(A) ITS1 sequence-based phylogenetic tree of 41 clinically significant yeast species. A consensus neighbor-joining dendrogram with 1,000 bootstrap replicates was based on 419 aligned positions of complete ITS1 sequences with adjacent partial sequences of 18S and 5.8S rRNA genes. GenBank accession numbers of sequences generated in this study are presented in Table 2. Numbers at the nodes indicate the bootstrap values. Lower bars indicate relative genetic distance. (B) ITS2 sequence-based phylogenetic tree of 42 clinically significant yeast species. Consensus neighbor-joining dendrogram with 1,000 bootstrap replicates was based on 432 aligned positions of complete ITS2 sequences with adjacent partial sequences of 5.8S and 26S rRNA genes. The sequences of C. albicans (no. L28817) and C. parapsilosis (no. U10988), as well as our sequences used for tree building, were previously published (2). Accession numbers of additional ITS2 sequences generated in this study are shown in Table 2 (footnote d). (C) 26S sequence-based phylogenetic tree of 41 clinically significant yeast species. Consensus neighbor-joining dendrogram with 1,000 bootstrap replicates was based on 565 aligned positions of the D1/D2 region of the 26S rRNA genes. Sequences of the following organisms (accession numbers in parentheses) were retrieved from GenBank: C. albicans (no. U45776), C. dubliniensis (no. U57685), C. famata (no. U45808), C. freyschussii (no. AF017242), C. glabrata (no. U44808), C. guilliermondii (no. U45709), C. intermedia (no. U44809), C. kefyr (no. U94924), C. krusei (no. U76347), C. lambica (no. AF020435), C. lusitaniae (no. U44817), C. parapsilosis (no. U45754), C. pararugosa (no. U62306), C. pelliculosa (no. U74592), C. rugosa (no. U45727), C. tropicalis (no. AB034689), C. utilis (no. U73570), C. zeylanoides (no. U45832), C. diffluens (no. AF075502), C. humicolus (no. AF189836), C. laurentii (no. AF075469), C. liquefaciens (no. AF181515), C. uniguttulatus (no. AF075468), E. fibuliger (no. U40088), P. fabianii (no. U73573), P. veronae (no. U73576), S. cerevisiae (no. U44806), S. salmonicolor (no. AF070439), T. asahii (no. AF105393), T. cutaneum (no. AF075483), T. inkin (no. AF105396), T. jirovecii (no. AF105398), and T. mucoides (no. AF075515). Sequences of the following organisms (accession numbers in parentheses) are from this study: C. albidus (no. AF335982), C. neoformans (no. AF335984), P. farinosa (no. AF335974), P. ohmeri (no. AF335976), P. ohmeri sequevar 1 (no. AF335975), R. glutinis (no. AF335985), R. rubra (no. AF335986), and Y. lipolytica (no. AF335977).

FIG. 1

(A) ITS1 sequence-based phylogenetic tree of 41 clinically significant yeast species. A consensus neighbor-joining dendrogram with 1,000 bootstrap replicates was based on 419 aligned positions of complete ITS1 sequences with adjacent partial sequences of 18S and 5.8S rRNA genes. GenBank accession numbers of sequences generated in this study are presented in Table 2. Numbers at the nodes indicate the bootstrap values. Lower bars indicate relative genetic distance. (B) ITS2 sequence-based phylogenetic tree of 42 clinically significant yeast species. Consensus neighbor-joining dendrogram with 1,000 bootstrap replicates was based on 432 aligned positions of complete ITS2 sequences with adjacent partial sequences of 5.8S and 26S rRNA genes. The sequences of C. albicans (no. L28817) and C. parapsilosis (no. U10988), as well as our sequences used for tree building, were previously published (2). Accession numbers of additional ITS2 sequences generated in this study are shown in Table 2 (footnote d). (C) 26S sequence-based phylogenetic tree of 41 clinically significant yeast species. Consensus neighbor-joining dendrogram with 1,000 bootstrap replicates was based on 565 aligned positions of the D1/D2 region of the 26S rRNA genes. Sequences of the following organisms (accession numbers in parentheses) were retrieved from GenBank: C. albicans (no. U45776), C. dubliniensis (no. U57685), C. famata (no. U45808), C. freyschussii (no. AF017242), C. glabrata (no. U44808), C. guilliermondii (no. U45709), C. intermedia (no. U44809), C. kefyr (no. U94924), C. krusei (no. U76347), C. lambica (no. AF020435), C. lusitaniae (no. U44817), C. parapsilosis (no. U45754), C. pararugosa (no. U62306), C. pelliculosa (no. U74592), C. rugosa (no. U45727), C. tropicalis (no. AB034689), C. utilis (no. U73570), C. zeylanoides (no. U45832), C. diffluens (no. AF075502), C. humicolus (no. AF189836), C. laurentii (no. AF075469), C. liquefaciens (no. AF181515), C. uniguttulatus (no. AF075468), E. fibuliger (no. U40088), P. fabianii (no. U73573), P. veronae (no. U73576), S. cerevisiae (no. U44806), S. salmonicolor (no. AF070439), T. asahii (no. AF105393), T. cutaneum (no. AF075483), T. inkin (no. AF105396), T. jirovecii (no. AF105398), and T. mucoides (no. AF075515). Sequences of the following organisms (accession numbers in parentheses) are from this study: C. albidus (no. AF335982), C. neoformans (no. AF335984), P. farinosa (no. AF335974), P. ohmeri (no. AF335976), P. ohmeri sequevar 1 (no. AF335975), R. glutinis (no. AF335985), R. rubra (no. AF335986), and Y. lipolytica (no. AF335977).