Collaborative Consensus for Optimized Multilocus Sequence Typing of Candida albicans

Abstract

A panel of 86 different Candida albicans isolates was subjected to multilocus sequence typing (MLST) in two laboratories to obtain sequence data for 10 published housekeeping gene fragments. Analysis of data for all possible combinations of five, six, seven, eight, and nine of the fragments showed that a set comprising the fragments AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b was the smallest that yielded 86 unique diploid sequence types for the 86 isolates. This set is recommended for future MLST with C. albicans.Multilocus sequence typing (MLST) is becoming a widely used approach to microbial isolate differentiation for epidemiological purposes (6). For Candida albicans, the species most often involved in deep-organ fungal diseases, MLST was introduced in 2002 (1), and a central internet database has been set up for deposition and analysis of C. albicans MLST data from any global source (http://calbicans.mlst.net). This MLST system is based on fragments of six C. albicans genes (in alphabetical order): ACC1, ADP1, GLN4, RPN2, SYA1, and VPS13.A second study of C. albicans MLST involved four of these genes plus four other gene fragments, AAT1a, AAT1b, MPIb, and ZWF1b (5). Both sets of gene fragments provided highly discriminatory typing systems that gave stable, reproducible results and could distinguish even closely related strains. While in principle the use of as many gene sequences as possible should enhance the discriminatory power of an MLST scheme, in practical and technical terms a compromise is required to provide the maximum level of isolate differentiation with the minimum set of fragments. Our two groups therefore agreed to collaborate by exchanging C. albicans isolates that had already been described in the published MLST papers and compiling a data set of sequences based on all 10 fragments that have been used for MLST. Analysis of these data allows us to propose an optimized gene set for routine use in C. albicans MLST research.A total of 92 C. albicans isolates (1, 5) were shared between the laboratories. These included duplicate cultures of isolate SC5314 and one culture of CAF2, derived from SC5314. As expected, identical MLST results were obtained for these three isolates, so two of the three data sets were excluded from analysis. Among the remaining 90 unique isolates, incomplete sequence data were obtained for 4, so the results for 86 isolates were analyzed to determine the optimal set of gene fragments for MLST.The method used for MLST was as previously described (1, 5). Both DNA strands in this diploid fungus were sequenced for each of 10 fragments (Table 1), and the sequences were recorded by the one-letter code for nucleotides from the International Union of Pure and Applied Chemistry nomenclature. For each fragment, each different genotype was assigned a unique number. Diploid sequence types (DSTs) are the numbers assigned to each unique combination of genotypes. Table 1 summarizes the characteristics of the 10 DNA fragments used for MLST with the 86 C. albicans isolates. The sizes ...

Multilocus sequence typing of Candida albicans isolates from a burn intensive care unit in Iran

“…Some DSTs (656, 124 and 172) of C. albicans were isolated from more than one patient in the same BICU during one month; therefore, it seems that colonized patients are a major reservoir of C. albicans in hospitals, and the isolation of C. albicans with the same DST in more than one patient may provide evidence for cross-contamination between patients, which might be a crucial factor for nosocomial infections (Bougnoux et al, 2003(Bougnoux et al, , 2004(Bougnoux et al, , 2006.…”

“…Briefly, genomic DNA was extracted using glass beads and the phenol/chloroform method (Yamada et al, 2002) and stored at 220 uC prior to use. MLST used seven housekeeping genes, AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13 and ZWF1b (Bougnoux et al, 2003). PCR amplification was performed for seven loci using a thermal cycler (Bio-Rad-C1000) and previously described primers (Bougnoux et al, 2003), followed by sequencing using an ABI 3730XL automatic sequencer (Applied Biosystems, Foster City, CA, U.S.A.).…”

“…MLST used seven housekeeping genes, AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13 and ZWF1b (Bougnoux et al, 2003). PCR amplification was performed for seven loci using a thermal cycler (Bio-Rad-C1000) and previously described primers (Bougnoux et al, 2003), followed by sequencing using an ABI 3730XL automatic sequencer (Applied Biosystems, Foster City, CA, U.S.A.). Sequence data were aligned manually using MEGA 5.05 (http:// www.megasoftware.net/) (Tamura et al, 2011) and BioEdit version 7.0.9 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) (Alignment, BioEdit Sequence 2011) software packages.…”

“…Five patients were sampled at different body sites and different times: skin burns (hand), blood and urine of patient 2 (P2) were sampled within a 7 day period; skin burns (hand and abdomen), blood and urine of P3 were sampled within a 10 day period; skin burns (foot) and urine of P7 were sampled within a 7 day period; skin burns of P13 including chest and scapula were sampled within a 5 day period; skin burns (chest) and two blood samples of P17 were sampled within a 21 day period. MLST was used to type the 30 clinical isolates as described previously (Bougnoux et al, 2003). Briefly, genomic DNA was extracted using glass beads and the phenol/chloroform method (Yamada et al, 2002) and stored at 220 uC prior to use.…”

“…Moreover, MLST sequences can be stored and characterized in multiple electronic formats, thus offering an unprecedented degree of portability and accessibility to other users (website http://calbicans.mlst. net) (Bougnoux et al, 2003(Bougnoux et al, , 2004. The purpose of this study was to investigate the MLST analysis of clinical isolates of C. albicans recovered from patients with burns in a burn intensive care unit (BICU) of a hospital at Sari city, Iran.…”