Assessment of
            <i>Candida glabrata</i>
            Strain Relatedness by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing

Lin, Chi-Yang; Chen, Yee‐Chun; Lo, Hsiu‐Jung; Chen, Kuo-Wei; Li, Shuying

doi:10.1128/jcm.00699-07

Cited by 61 publications

(53 citation statements)

References 41 publications

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“…Another finding of our study is that no microsatellite genotypes were associated with any of the clinical data recorded, as previously reported (8,12,19). Additionally, in the eight infected patients, we have shown that C. glabrata candidemia originated from colonizing isolates, as previously described for both C. glabrata (12) and C. albicans (4,22).…”

supporting

confidence: 62%

Multiple-Locus Variable-Number Tandem-Repeat Analysis for Rapid Typing of Candida glabrata

et al. 2007

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A multiple-locus variable-number tandem-repeat analysis (MLVA) using six microsatellite markers was assessed in 127 Candida glabrata isolates. Thirty-seven different genotypes, stable both in vitro and in vivo, were observed. The highest discriminatory power (D ‫؍‬ 0.902) was reached by using only four markers. MLVA seems to be relevant for C. glabrata typing.Candida glabrata has recently emerged as a major pathogen, causing mucosal and systemic infections (10,17). Several methods, such as electrophoretic karyotyping, restriction enzyme analysis, Southern blotting with probes, randomly amplified polymorphic DNA, and multilocus sequence typing (MLST), have been used to distinguish and type C. glabrata isolates (2,7,8,14,21,28). Microsatellite polymorphism analysis has been widely used for typing fungi (3,4,11,15,18,25), and this could be an alternative, easy-to-perform, reproducible method suitable for large-scale studies of C. glabrata epidemiology. Recently, Foulet et al. described three polymorphic microsatellite markers to investigate the delineation of clinical C. glabrata isolates (12). The discriminatory power of this method was good but not optimal. The aim of our study was to assess a microsatellite-based multiple-locus variable-number tandemrepeat analysis (MLVA) using new markers for C. glabrata typing.One hundred twenty-seven C. glabrata strains were analyzed, including four reference strains, 98 independent clinical isolates, and 25 epidemiologically related isolates. These 25 were from the blood cultures and peripheral site isolates of eight patients with C. glabrata candidemia. Genomic DNA was extracted by boiling with Chelex resin as previously described (6, 23). Six microsatellite markers were selected from the C. glabrata DNA sequences available in the GenBank database (29). Primer sequences were designed with Primer3 software (24), and locations in the C. glabrata genome were determined with the Genolevures database (http://cbi.labri.fr/Genolevures/) ( Table 1). For each primer set, PCRs were performed with a 20-l final volume containing 1 l of DNA, each deoxynucleoside triphosphate at 200 M, a forward primer and a 5Ј-dyelabeled reverse primer at 0.25 M each, and 1 U of Taq DNA polymerase (Promega, Madison, WI). The amplification conditions were 5 min at 95°C, followed by 35 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 45 s and then a final step of 72°C for 10 min. For any given isolate, amplicons of each PCR were pooled before multiplex fragment sizing with a Ceq 8000 Genetic Analyzer (Beckman Coulter, Fullerton, CA). Strain IHEM 9670 was run as a control in each experiment. As allele sizing depends on dyes and the analyzer used for electrophoresis, results were expressed as the exact size of the sequence (determined by sequencing of each representative allele for each locus) to allow further interlaboratory comparisons of MLVA results (13,20). The reproducibility and stability of the method were assessed as described elsewhere (25) . Primer specificity was also tested against the sequ...

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confidence: 62%

Multiple-Locus Variable-Number Tandem-Repeat Analysis for Rapid Typing of Candida glabrata

et al. 2007

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“…Interestingly, CC64, which was not associated with a given MLST clade, corresponds in a recent paper by Lott et al (33) to an MLST group clustering as many as 18.3% of the tested strains, supported by a 99% bootstrap value (100% in the Dodgson study), each with a synapomorphic NMT1 allele (NMT1-5). Conversely, we considered genotype RT60 to be included in a potential CC since the single strain we had exhibited allele 12 for the NMT1 locus (data not shown), characteristic of clade VII (15,31,33).…”

Section: Resultsmentioning

confidence: 99%

Multilocus Microsatellite Markers for Molecular Typing of Candida glabrata : Application to Analysis of Genetic Relationships between Bloodstream and Digestive System Isolates

et al. 2010

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Candida glabrata has emerged as the second most common etiologic agent, after Candida albicans, of superficial and invasive candidiasis in adults. Strain typing is essential for epidemiological investigation, but easy-to-use and reliable typing methods are still lacking. We report the use of a multilocus microsatellite typing method with a set of eight markers on a panel of 180 strains, including 136 blood isolates from hospitalized patients and 34 digestive tract isolates from nonhospitalized patients. A total of 44 different alleles were observed, generating 87 distinct genotypes. In addition to perfect reproducibility, typing ability, and stability, the method had a discriminatory power calculated at 0.97 when all 8 markers were associated, making it suitable for tracing strains. In addition, it is shown that digestive tract isolates differed from blood culture isolates by exhibiting a higher genotypic diversity associated with different allelic frequencies and preferentially did not group in clonal complexes (CCs). The demonstration of the occurrence of microevolution in digestive strains supports the idea that C. glabrata can be a persistent commensal of the human gut.

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“…Fragments of the C. tropicalis genes ICL1, MDR1, SAPT2, SAPT4, XYR1, and ZWF1a have already been analyzed (32,33). Previous reports have indicated that MLST can provide additional information on the mechanisms of genetic variability, such as the persistence, replacement, and microevolution of strains in the hosts (30,34,35).…”

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confidence: 99%

Multilocus Sequence Typing of Candida tropicalis Shows the Presence of Different Clonal Clusters and Fluconazole Susceptibility Profiles in Sequential Isolates from Candidemia Patients in São Paulo, Brazil

et al. 2013

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fThe profiles of 61 Candida tropicalis isolates from 43 patients (28 adults and 15 children) diagnosed with candidemia at two teaching hospitals in São Paulo, Brazil, were characterized by multilocus sequence typing (MLST). For the 14 patients who had bloodstream infections, 32 isolates were serially collected from their blood and/or catheters. Thirty-nine diploid sequence types (DSTs) were differentiated. According to the C. tropicalis MLST database (http://pubmlst.org/ctropicalis/), 36 DSTs and 23 genotypes identified from the 61 isolates had not previously been described. This report represents the first study to characterize sequential isolates of C. tropicalis from candidemia cases in South America. Microvariation in a single gene was found in the sequential isolates from 7 patients. The main polymorphisms occurred in the alleles of the XYR1 gene, specifically at nucleotide positions 215, 242, and 344. Macrovariation in six gene fragments was detected in the isolates from 3 patients. eBURST analysis added two new groups to this study (groups 6 and 18). Additionally, susceptibility tests indicate that 3 isolates were resistant to fluconazole. No correlation was found between the DSTs and susceptibility to fluconazole and/or selective antifungal pressure. Two patients were sequentially infected with resistant and susceptible strains. MLST is an important tool for studying the genetic diversity of multiple/sequential isolates of patients with candidemia, allowing the comparison of our data with those from other regions of the world, as well as allowing an analysis of the genetic relationship among several clones in sequential isolates from the same or different candidemia patient sites (blood or catheter).

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Assessment of Candida glabrata Strain Relatedness by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing

Cited by 61 publications

References 41 publications

Multiple-Locus Variable-Number Tandem-Repeat Analysis for Rapid Typing of Candida glabrata

Multiple-Locus Variable-Number Tandem-Repeat Analysis for Rapid Typing of Candida glabrata

Multilocus Microsatellite Markers for Molecular Typing of Candida glabrata : Application to Analysis of Genetic Relationships between Bloodstream and Digestive System Isolates

Multilocus Sequence Typing of Candida tropicalis Shows the Presence of Different Clonal Clusters and Fluconazole Susceptibility Profiles in Sequential Isolates from Candidemia Patients in São Paulo, Brazil

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