Computational protocol: Heteroresistance to Fluconazole Is a Continuously Distributed Phenotype among Candida glabrata Clinical Strains Associated with In Vivo Persistence

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Protocol publication

[…] Phylogenetic relationships between different C. glabrata strains were resolved by sequencing and analysis of the rapidly evolving IGS region between nuclear genes CDH1 and ERP6 on chromosome A as described previously (). Phylogenetic analysis included 48 fluconazole-susceptible C. glabrata isolates, 6 fluconazole-resistant clinical isolates, and reference C. glabrata strains CBS138, CBS15126, and BG2. After genomic DNA extraction, an ~660-bp fragment of the IGS was amplified with primers 00605 (5′ C TCA CAA ATG GAT TCC TTA AAG AGT TCG 3′) and 00627 (5′ GT C ACC AGA GTT GGA GTA CAT GTA G 3′). PCR was performed with 50-µl tubes containing 400 nM each primer under the following conditions: initial denaturation at 95°C (4.5 min) and then 35 cycles of 95°C (45 s), 52°C (1 min), and 72°C (1 min), followed by 72°C (7 min). The PCR products were sequenced with primers 00605 and 00627. Sequences were aligned with Clustal Omega (), and phylogenetic trees were constructed in MrBayes 3.2 () by Bayesian Markov chain Monte Carlo (MCMC) methodology. The average standard deviation of split frequencies (ASDSF) was calculated by comparing split and clade frequencies across 1 × 106 MCMC runs started from different randomly chosen trees. The ASDSF cutoff was set at <0.05. The final output is presented as a cladogram with the posterior probabilities for all of the splits. […]

Pipeline specifications

Software tools Clustal Omega, MrBayes
Application Phylogenetics
Organisms [Candida] glabrata, Mus musculus, Homo sapiens
Diseases Infection
Chemicals Adenosine Triphosphate, Fluconazole