Multilocus sequence typing of pathogenic Candida species.

Typing strains within a microbial species on the basis of DNA sequences at multiple loci has greatly advanced study of the epidemiology and evolutionary phylogenetics of many bacterial (9, 40, 60, 72) and fungal (26, 68) pathogens. Multilocus sequence typing (MLST) schemes have now been published for five of the opportunistically pathogenic Candida species: C. albicans (6, 7), C. dubliniensis (42), C. glabrata (19), C. krusei (30), and C. tropicalis (65). A sixth species, C. parapsilosis, shows too little sequence diversity to be typeable by MLST (64). This review discusses technical aspects and implications of findings from MLST studies of the genus Candida. There will inevitably be an emphasis on studies with C. albicans, since many more isolates of this species than of other Candida species have been subjected to MLST. The ability to discriminate between microbial strain types has been, for very many years, a topic of great importance to epidemiologists. Strain typing facilitates tracing of sources and routes of transmission of infection outbreaks. However, a paradox exists at the heart of strain typing. The question most often posed by an epidemiologist is whether two microbial isolates are the same type, but the methodology used for typing is designed to reveal differences between strain types and is usually assessed statistically for its success in differentiation. Identity is inferred by implication when two isolates cannot be distinguished; however, increasing technological finesse means that two isolates classed as identical types in one system may be shown to differ in another. One possible resolution to the paradox of proving strain identity would be to compare wholegenome sequences for two isolates, something that is already a technical possibility for selected isolates and which seems likely to become a more common event in the near future. However, because sequence differences may arise rapidly in highly variable regions of the genome, evidence for strain identity may never achieve its theoretical perfection. MLST, while a long way from providing complete genome sequences, generates evidence for similarities and differences between isolates from sequences determined, typically, for six to eight independent chromosomal genes. The MLST approach, originally devised by Maiden and colleagues (41), offers a number of advantages over most earlier phenotypic and genotypic typing methods. Sequencing data offer a higher level of reproducibility and minimal subjectivity in analysis compared with other technologies, and the sequences can be stored in Internet databases, offering an unprecedented degree of portability and accessibility to all interested users. (MLST databases can be accessed at two main host sites, http://pubmlst .org/ and http://www.mlst.net/.) Moreover, MLST is based on the same conceptual premise as multilocus enzyme electrophoresis, allelic variation, which means that MLST provides information relevant to population genetic studies as well as to epidemiology. The field of Candida epidemiology has already been substantially explored by a range of technologies. MLST data confirm the many previous studies in the field and offer an increment of refinement in the phylogenetic detail with which epidemiology can be explored experimentally. Strain typing as a routine clinical laboratory procedure is more familiar to bacteriologists than to mycologists, which is perhaps why, with the exception of Aspergillus fumigatus (2), MLST data only for Candida species and Cryptococcus neoformans have been made available on the central MLST databases. In fact, the MLST approach to strain delineation was used with Coccidioides immitis (31) at least as early as the year when bacteriologists first set up their Web-based MLST systems (41). MLST has been applied to epidemiological and phylogenetic research with other fungal opportunist pathogens, including Cryptococcus gattii (8, 26), Fusarium solani (74), and Batrachochytrium dendrobatidis (43), but the authors of these publications have not utilized the central MLST Web repository for their data, which makes it less easy for others to add or reanalyze the data. The opposite applies to MLST for Cryptococcus neoformans var. grubii (35), the latest fungal addition to the central MLST database (http://www.mlst.net/).