Whole Genome Sequencing of Australian Candida glabrata Isolates Reveals Genetic Diversity and Novel Sequence Types

Biswas, Chayanika; Marcelino, Vanessa R.; Hal, Sebastiaan J. van; Halliday, Catriona; Martínez, Elena; Wang, Qinning; Kidd, Sarah; Kennedy, Karina; Marriott, Deborah; Morrissey, C. Orla; Arthur, Ian; Weeks, Kerry; Slavin, Monica; Sorrell, Tania C.; Sintchenko, Vitali; Meyer, Wieland; Chen, Sharon C.-A.

doi:10.3389/fmicb.2018.02946

Cited by 38 publications

(38 citation statements)

References 48 publications

(76 reference statements)

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“…Altogether, this supports that echinocandin resistance cannot be explained by MSH2 mutator phenotype, as previously reported (Delliere et al, 2016; Healey et al, 2016a; Biswas et al, 2018; Byun et al, 2018; Hou et al, 2018; Singh et al, 2018; Bordallo-Cardona et al, 2019). Likewise, no clear association between MSH2 sequence and increased fluconazole resistance or genotypes was detected on these isolates either (Biswas et al, 2018; Bordallo-Cardona et al, 2019), although a correlation with specific genetic types was previously described (Delliere et al, 2016; Byun et al, 2018; Hou et al, 2018). These results confirm that MSH2 substitutions may be constitutive variations from the gene rather than resistance-related or genotype-related mutations (Carrete et al, 2019).…”

Section: Discussionsupporting

confidence: 92%

“…Isolates from the same patient seemed to have a clonal origin by using these two typing techniques, although the use of next generation sequencing in order to compare their genomes would be necessary to prove if they are isogenic. The most frequent genotype among these patients was ST3, which has been reported as one of the most prevalent STs worldwide (Dodgson et al, 2003; Lott et al, 2012; Hou et al, 2017; Biswas et al, 2018; Byun et al, 2018; Mushi et al, 2018). No association between echinocandin resistance development and genetic type was found, which was in agreement with other reports (Dodgson et al, 2003; Abbes et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

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Clinical and Laboratory Development of Echinocandin Resistance in Candida glabrata: Molecular Characterization

et al. 2019

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The pathogenic yeast Candida glabrata has become a public health issue due to the increasing number of echinocandin resistant clinical strains reported. In this study, acquisition and development of resistance to this antifungal class were studied in serial C. glabrata isolates from five patients admitted in two Spanish hospitals with a resistant profile against echinocandins associated with different mutations in hot-spot 1 of FKS2 gene. For two of these patients susceptible FKS wild-type isolates obtained prior to resistant ones were also investigated. Isolates were genotyped using multilocus sequence typing and microsatellite length polymorphism techniques, which yielded comparable results. Susceptible and resistant isolates from the same patient had the same genotype, being sequence type (ST) 3 the most prevalent among them. Isolates with different FKS mutations but the same ST were present in the same patient. MSH2 gene alterations were also studied to investigate their correlation with antifungal resistance acquisition but no association was found with antifungal resistance nor with specific genotypes. In vitro exposure to increasing concentrations of micafungin to susceptible isolates developed colonies carrying FKS mutations in agar plates containing a minimum concentration of 0.06 mg/L of micafungin after less than 48 h of exposure. We investigated the correlation between development of resistance and genotype in a set of susceptible strains after being in vitro exposed to micafungin and anidulafungin but no correlation was found. Mutant prevention concentration values and spontaneous growth frequencies after selection with both echinocandins were statistically similar, although FKS mutant colonies were more abundant after micafungin exposure ( p < 0.001). Mutation S663P and F659 deletion were the most common ones found after selection with both echinocandins.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Clinical and Laboratory Development of Echinocandin Resistance in Candida glabrata: Molecular Characterization

et al. 2019

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“…This is consistent with several recent clinical studies that did not see an association between MSH2 genotype and prevalence of drug resistance in C. glabrata (27–29, 61, 62). However, in our previous study, we found that several MSH2 variants, including MSH2 E231G/L269F , when introduced on a plasmid into an ATCC 2001-derived msh2Δ mutant, did not fully rescue that strain’s elevated mutation rate (24).…”

Section: Discussionsupporting

confidence: 93%

“…Indeed, in Cryptococcus , naturally occurring mutations in MSH2 have been shown to contribute to microevolution and population diversity (59, 60). Yet, recent clinical studies have not found an association between specific MSH2 alleles and drug resistance (27–29, 61, 62), raising the question of whether clinical isolates carrying these alleles are true mutators. To answer this question, it is necessary to measure and directly compare mutation rates between clinical isolates of C.…”

Section: Introductionmentioning

confidence: 99%

A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens

Shor

Schuyler

Perlin

2019

mBio

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All evolutionary processes are underpinned by a cellular capacity to mutate DNA. To identify factors affecting mutagenesis, it is necessary to compare mutation rates between different strains and conditions. Drug resistance-based mutation reporters are used extensively to measure mutation rates, but they are suitable only when the compared strains have identical drug tolerance levels—a condition that is not satisfied under many “real-world” circumstances, e.g., when comparing mutation rates among a series of environmental or clinical isolates. Candida glabrata is a fungal pathogen that shows a high degree of genetic diversity and fast emergence of antifungal drug resistance. To enable meaningful comparisons of mutation rates among C. glabrata clinical isolates, we developed a novel fluorescence-activated cell sorting-based approach to measure the mutation rate of a chromosomally integrated GFP gene. We found that in Saccharomyces cerevisiae this approach recapitulated the reported mutation rate of a wild-type strain and the mutator phenotype of a shu1Δ mutant. In C. glabrata, the GFP reporter captured the mutation rate increases caused either by a genotoxic agent or by deletion of DNA mismatch repair gene MSH2, as well as the specific mutational signature associated with msh2Δ. Finally, the reporter was used to measure the mutation rates of C. glabrata clinical isolates carrying different alleles of MSH2. Together, these results show that fluorescence-based mutation reporters can be used to measure mutation rates in microbes under conditions of unequal drug susceptibility to reveal new insights about drivers of mutagenesis. IMPORTANCE Measurements of mutation rates—i.e., how often proliferating cells acquire mutations in their DNA—are essential for understanding cellular processes that maintain genome stability. Many traditional mutation rate measurement assays are based on detecting mutations that cause resistance to a particular drug. Such assays typically work well for laboratory strains but have significant limitations when comparing clinical or environmental isolates that have various intrinsic levels of drug tolerance, which confounds the interpretation of results. Here we report the development and validation of a novel method of measuring mutation rates, which detects mutations that cause loss of fluorescence rather than acquisition of drug resistance. Using this method, we measured the mutation rates of clinical isolates of fungal pathogen Candida glabrata. This assay can be adapted to other organisms and used to compare mutation rates in contexts where unequal drug sensitivity is anticipated.

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“…NGS further has potential to detect novel mutations implicated in phenotypic resistance that may otherwise be missed by targeted DNA sequencing. In C. glabrata, NGS has detected multiple SNPs in CgPDR1 and CgCDR1 in azoleresistant isolates; although none were definitively associated with resistance, azole-resistant isolates tended to have amino substitutions in pdr1 beyond the first 250 amino acid positions, unlike the susceptible isolates (Biswas et al, 2018). The cost of NGS is currently ∼AUD 80/sample (∼USD 57) but this is likely to fall with technological advances allowing ease of use for routine applications.…”

Section: Molecular Methods To Detect Azole Resistance In Candida Speciesmentioning

confidence: 99%

A New Age in Molecular Diagnostics for Invasive Fungal Disease: Are We Ready?

et al. 2020

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Whole Genome Sequencing of Australian Candida glabrata Isolates Reveals Genetic Diversity and Novel Sequence Types

Cited by 38 publications

References 48 publications

Clinical and Laboratory Development of Echinocandin Resistance in Candida glabrata: Molecular Characterization

Clinical and Laboratory Development of Echinocandin Resistance in Candida glabrata: Molecular Characterization

A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens

A New Age in Molecular Diagnostics for Invasive Fungal Disease: Are We Ready?

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