Antifungal Drug Concentration Impacts the Spectrum of Adaptive Mutations in Candida albicans

May combine two claims.

Lineages evolved to drug concentrations above their ancestral MIC50 tended to acquire a different suite of mutational changes and increased in drug tolerance.

“By contrast, lineages evolved to drug concentrations above their ancestral MIC50 tended to acquire a different suite of mutational changes and increased in drug tolerance (the ability of a subpopulation of cells to grow slowly above their MIC50).”

May combine two claims.

Lineages evolved to drug concentrations close to their MIC50 tended to rapidly evolve an increased MIC50 and acquired distinct segmental aneuploidies and copy number variations.

“In general, lineages evolved to drug concentrations close to their MIC50 (the level of drug that reduces growth by 50% in the ancestor) tended to rapidly evolve an increased MIC50 and acquired distinct segmental aneuploidies and copy number variations.”

May combine two claims.

Higher drug concentrations (supra-MIC) selected for increases in drug tolerance, but only rarely increased resistance.

“Higher drug concentrations (supra-MIC) selected for increases in drug tolerance, but only rarely increased resistance.”

May combine two claims.

All lineages derived from the third genetic background (FH1, starting MIC 8 μg/ml) acquired trisomy of chromosome 5 and a concomitant increase in MIC during adaptation to the 8 μg/ml environment, but not during adaptation to lower or higher drug concentrations.

“Strikingly, all lineages derived from this genetic background acquired trisomy of chromosome 5 and a concomitant increase in MIC during adaptation to the 8 μg/ml environment, but not during adaptation to lower or higher drug concentrations.”

May combine two claims.

Drug concentrations near the starting MIC selected for increases in the frequency of segmental aneuploidies and increases in resistance (MIC).

“We first identified the spectrum and effect of mutations acquired in two different genetic backgrounds with the same starting MIC (0.5 μg/ml) and found that drug concentrations near the starting MIC selected for increases in the frequency of segmental aneuploidies and increases in resistance (MIC).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The copy number breakpoints of all segmental aneuploidies occurred at long repeat sequences.

“The copy number breakpoints of all segmental aneuploidies occurred at long repeat sequences as described previously (Todd et al. 2019; Todd and Selmecki 2020).”

• cites Todd et al. 2019 sourceNot checked yet

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• cites Todd and Selmecki 2020 sourceNot checked yet

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No identical SNVs arose independently in different lineages, but several genes acquired SNVs in different lineages (RIM101, DAL5, RGD3, PXP2, TERT, orf19.3604, orf19.6457, and orf19.6970).

“Although no identical SNVs arose independently in different lineages, several genes acquired SNVs in different lineages (RIM101, DAL5, RGD3, PXP2, TERT, orf19.3604, orf19.6457, and orf19.6970).”

May combine two claims.

Ten LOH events were detected in seven of the 48 lineages, including whole-chromosome LOH events (5/10) and segmental chromosome LOH events (5/10) ranging from ∼8 kb to ∼716 kb.

“Ten LOH events were detected in seven of the 48 lineages, including whole-chromosome LOH events (5/10) and segmental chromosome LOH events (5/10) ranging from ∼8 kb to ∼716 kb (Table 3).”

May combine two claims.

Only one segmental aneuploidy was identified above 1 μg/ml FLC (Lineage J64) and occurred at the rDNA array on ChrR.

“Only one segmental aneuploidy was identified above 1 μg/ml FLC (Lineage J64) and occurred at the rDNA array on ChrR, a common copy number breakpoint in clinical isolates.”

May combine two claims.

The greatest number of SNVs in SC5314-derived lineages was in the no-drug-evolved lineages (33/57), and in P75063-derived lineages was in the 1 μg/ml FLC-evolved lineages (28/69).

“The greatest number of SNVs were in the SC5314 no drug-evolved lineages (33/57, fig. 3E) and the P75063 1 μg/ml FLC-evolved lineages (28/69, fig. 3F).”

The quote only partly states this claim.

The greatest number of SNVs per treatment in FH1 lineages was in the 8 μg/ml FLC lineages.

“The greatest number of SNVs per treatment was in the 8 μg/ml FLC lineages (fig. 4C).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Similar GO Term Finder analysis of genes with SNVs from drug-treated (1, 8, 64 μg/ml FLC) lineages with progenitors SC5314, P75063, and FH1 identified no significantly enriched GO terms.

“Similar analyses were conducted with the set of all genes with SNVs from drug-treated (1, 8, 64 μg/ml FLC) lineages with progenitors SC5314, P75063, and FH1, but no significant terms were identified.”

May combine two claims.

FH1 remains diploid at all drug concentrations tested, whereas polyploidization occurred more frequently in P75063 lineages than in SC5314 lineages.

“Here we found that FH1 remains diploid at all drug concentrations tested, whereas polyploidization occurred more frequently in P75063 lineages than in SC5314 lineages (fig. 2).”

May combine two claims.

Polyploidy was frequently associated with chromosome aneuploidy, and all the polyploid isolates randomly selected for sequencing contained amplification of ChrR.

“Using whole-genome sequencing we also found polyploidy was frequently associated with chromosome aneuploidy, and interestingly, all the polyploid isolates that we randomly selected for sequencing contained amplification of ChrR (fig. 3A and B).”

May combine two claims.

Adaptation to FLC did not occur in either the absence of drug or in drug concentrations below that of the progenitor's starting MIC.

“Adaptation to FLC did not occur in either the absence of drug or in drug concentrations below that of the progenitor's starting MIC.”

Classified as probably not a substantial claim. May combine two claims.

Fluconazole concentrations vary between different tissues in the body (1 μg/ml to over 22 μg/ml), with the highest concentration detected in the spleen.

“Additionally, FLC concentrations vary between different tissues in the body (1 μg/ml to over 22 μg/ml), with the highest concentration of FLC being detected in the spleen (Fischman et al. 1993).”

• cites Fischman et al. 1993 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Up to ∼20% of cells in a population can become polyploid in as little as 8 h when cultured in 10 μg/ml fluconazole due to multipolar spindle formation and/or cytokinesis failure.

“Up to ∼20% of cells in a population can become polyploid in as little as 8 h when cultured in 10 μg/ml FLC due to multipolar spindle formation and/or cytokinesis failure (Harrison et al. 2014).”

• cites Harrison et al. 2014 sourceNot checked yet

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Classified as probably not a substantial claim. May combine two claims.

Little is known about how prolonged exposure to a constant drug concentration impacts evolutionary trajectories and what distinguishes selection for azole resistance from azole tolerance.

“The impact of drug concentration on the spectrum and effect of mutations acquired during adaptation is not known for any fungal pathogen, and how the specific level of a given stress influences the distribution of beneficial mutations has been poorly explored in general.”

• cites Cowen et al. 2000 sourceNot checked yet

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• cites Anderson et al. 2003 sourceNot checked yet

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Classified as probably not a substantial claim.

The impact of drug concentration on the frequency of polyploidization events and the competitive fitness of polyploid cells over short and long timescales in fluconazole remains poorly understood.

“However, the impact of drug concentration on frequency of polyploidization events and the competitive fitness of polyploid cells over short and long timescales in FLC, remain poorly understood (Gerstein and Sharp 2021).”

• cites Gerstein and Sharp 2021Not checked yet

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Drug-tolerant fungal cells are often present as a distinct subpopulation and can evolve independently of drug resistance, existing alongside drug-resistant or susceptible cells.

“Drug-tolerant fungal cells are often present as a distinct subpopulation and can evolve independently of drug resistance, existing alongside drug-resistant or susceptible cells (Gerstein and Sethi 2022).”

• cites Gerstein and Sethi 2022Not checked yet

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Antifungal drug tolerance occurs via enhanced signaling of cellular stress response pathways including modulation of the calcium signaling pathway, nutrient detection, and HSP90 activation.

“Currently, it is thought that antifungal drug tolerance occurs via enhanced signaling of cellular stress response pathways including modulation of the calcium signaling pathway, nutrient detection, and HSP90 activation (Cowen and Steinbach 2008; Rosenberg et al. 2018; Murphy and Bicanic 2021).”

• cites Cowen and Steinbach 2008Not checked yet

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• cites Rosenberg et al. 2018 sourceNot checked yet

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• cites Murphy and Bicanic 2021Not checked yet

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May combine two claims.

Azole tolerance and cross-tolerance to different azole drugs can evolve rapidly in vitro.

“Intriguingly, our recent experiments demonstrate that azole tolerance and cross-tolerance to different azole drugs can evolve rapidly in vitro (Todd and Selmecki 2020; Kukurudz et al. 2022), yet the mechanisms that distinguish drug-resistant and drug-tolerant phenotypes in fungi are not fully characterized.”

• cites Todd and Selmecki 2020 sourceNot checked yet

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• cites Kukurudz et al. 2022 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

About 50% of all fluconazole-resistant Candida albicans isolates contain at least one aneuploid chromosome, and chromosomal instability can increase the frequency of fluconazole-resistant cells.

“Additionally, ∼50% of all FLC-resistant C. albicans isolates contain at least one aneuploid chromosome (Selmecki et al. 2006) and chromosomal instability, in general, can increase the frequency of FLC-resistant cells (Brimacombe et al. 2019; Yang, Todd, et al. 2021).”

• cites Selmecki et al. 2006 sourceNot checked yet

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• cites Brimacombe et al. 2019 sourceNot checked yet

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• cites Yang, Todd, et al. 2021Not checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In one example, six lineages from one Candida albicans strain were maintained at increasing concentrations of fluconazole for 300-400 generations, with MIC50 quantified every 10-20 generations and drug concentration increased to twice the MIC50, to a maximum of 128 μg/ml fluconazole.

“In one example, six lineages from one C. albicans strain were maintained at increasing concentrations of FLC for 300–400 generations, where the MIC50 was quantified every 10–20 generations and the drug concentration was increased to twice the MIC50, to a maximum concentration of 128 μg/ml FLC (Cowen et al. 2000).”

• cites Cowen et al. 2000 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Tolerance in fungi seems to be a stable characteristic and isolates that evolve increased tolerance do not change in the prolonged absence of fluconazole.

“Tolerance in fungi seems to be a stable characteristic and isolates that evolve increased tolerance do not change in the prolonged absence of FLC (Rosenberg et al. 2018).”

• cites Rosenberg et al. 2018 sourceNot checked yet

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May combine two claims.

i(5L) can arise rapidly under fluconazole selection, occurs in diverse genetic backgrounds of Candida albicans, and frequently results in multi-azole resistance.

“i(5L) can arise rapidly under FLC selection, occurs in diverse genetic backgrounds of C. albicans, and frequently results in multi-azole resistance (Selmecki et al. 2008, 2009).”

• cites Selmecki et al. 2008 sourceNot checked yet

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• cites Selmecki et al. 2009 sourceNot checked yet

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Isochromosome 5L (i(5L)) causes drug resistance via an increase in the copy number of ERG11 and TAC1.

“One specific segmental aneuploidy, isochromosome 5L (i(5L)), causes drug resistance via an increase in the copy number of ERG11 and TAC1 (Selmecki et al. 2006, 2008).”

• cites Selmecki et al. 2006 sourceNot checked yet

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• cites Selmecki et al. 2008 sourceNot checked yet

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Fungal drug tolerance is linked to azole treatment failure and the inability to clear an infection, despite isolates being considered drug-sensitive by typical MIC50 assays.

“Fungal drug tolerance is linked to azole treatment failure and the inability to clear an infection, despite these isolates being considered drug-sensitive by the typical MIC50 assays (Sanglard et al. 2003; Delarze and Sanglard 2015; Rosenberg et al. 2018; Berman and Krysan 2020).”

• cites Sanglard et al. 2003 sourceNot checked yet

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• cites Rosenberg et al. 2018 sourceNot checked yet

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• cites Berman and Krysan 2020 sourceNot checked yet

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Different concentrations of drug can select for different genotypic and phenotypic outcomes in vitro and may explain observed in vivo drug response variation.

“This is the first evidence that different concentrations of drug can select for different genotypic and phenotypic outcomes in vitro and may explain observed in vivo drug response variation.”

• AssumptionRests on an assumption: In vitro evolution under constant fluconazole concentrations reflects the selective pressures and adaptive outcomes occurring during clinical antifungal therapy.

Probably not a substantial claim: may not pass S3 (load-bearing).

In a similar example, three lineages from one genetic background of haploid Saccharomyces cerevisiae were exposed to increasing concentrations of fluconazole over 400 generations, starting at 16 μg/ml and increasing every 100 generations to 32, 64, and 128 μg/ml fluconazole.

“In a similar example, three lineages from one genetic background of haploid S. cerevisiae were exposed to increasing concentrations of FLC over the span of 400 generations, starting at 16 μg/ml FLC and increasing every 100 generations to 32 μg/ml, 64 μg/ml, and 128 μg/ml FLC (Anderson et al. 2003).”

• cites Anderson et al. 2003 sourceNot checked yet

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Classified as probably not a substantial claim.

The impact of genetic and environmental factors, including fluconazole concentration, on the acquisition of drug resistance and tolerance phenotypes is not known.

“Overall, the impact of genetic and environmental factors, including FLC concentration, on the acquisition of drug resistance and tolerance phenotypes is not known.”

• cites Cowen et al. 2000 sourceNot checked yet

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• cites Anderson et al. 2003 sourceNot checked yet

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May combine two claims.

Drug concentration dramatically impacts both the phenotypic and genotypic basis of adaptation of lineages from two diverse genetic backgrounds of C. albicans evolved for 100 generations.

“In summary, drug concentration dramatically impacts both the phenotypic and genotypic basis of adaptation of lineages from two diverse genetic backgrounds of C. albicans evolved for 100 generations.”

May combine two claims.

Polyploidy, aneuploidy, and segmental aneuploidy are likely a faster route to adapting to FLC in diploid C. albicans isolates, and these karyotypic mutations are more likely to repeatedly arise and be selected independently in different lineages than rare adaptive point mutations, at least in the early stages of FLC exposure.

“The overall pattern is that polyploidy, aneuploidy, and segmental aneuploidy is likely a faster route to adapting to FLC in diploid C. albicans isolates and these karyotypic mutations are more likely to repeatedly arise and be selected independently in different lineages than rare adaptive point mutations, at least in the early stages of FLC exposure (Yang, Todd, et al. 2021; Gerstein and Sethi 2”

• cites Yang, Todd, et al. 2021Not checked yet

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• cites Gerstein and Sethi 2022Not checked yet

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May combine two claims.

Drug concentration can impact selection for, and possibly formation of, the recurrent segmental aneuploidy i(5L).

“This is the first report of i(5L) formation in the reference strain SC5314 and the first indication that drug concentration can impact selection for, and possibly formation of, this recurrent segmental aneuploidy.”

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Ploidy analysis by flow cytometry may be underestimating the frequency of chromosome copy number changes, which are more common than polyploidization, in the SC5314 background.

“This suggests that ploidy analysis by flow cytometry may be underestimating the frequency of chromosome copy number changes, that are more common than polyploidization, in the SC5314 background.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Twelve single colonies from each progenitor isolate were selected at random.

“Twelve single colonies from each progenitor isolate were selected at random”

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Strain background can significantly influence adaptation, with the initial MIC relative to the environment impacting both mutational spectrum and evolutionary trajectory during FLC treatment.

“Together, these findings indicate that strain background can significantly influence adaptation, with the initial MIC relative to the environment impacting both mutational spectrum and evolutionary trajectory during FLC treatment.”

May combine two claims.

Segmental aneuploidies recurrently and predominantly form only at FLC concentrations near the MIC of the original progenitor isolate, and the propensity to acquire segmental aneuploidy is background-dependent.

“The experiments here are the first to clarify that segmental aneuploidies recurrently and predominantly form only at FLC concentrations that are near the MIC of the original progenitor isolate, and furthermore suggest that the propensity to acquire segmental aneuploidy is background-dependent.”

Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

These findings have broad implications for antifungal drug therapies and clinical best practices and could underlie why some treatment regimens fail.

“These findings have broad implications for antifungal drug therapies and clinical best practices and could underlie why some treatment regimens fail.”

• AssumptionRests on an assumption: In vitro evolution of C. albicans under fluconazole reflects the adaptation and treatment failure that occur in clinical host niches.

Probably not a substantial claim: may not pass S3 (load-bearing).

Read depth was corrected for both chromosome-end bias and GC-content.

“Read depth was corrected for both chromosome-end bias and GC-content.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In general, aneuploidy alters cell physiology in ways that may promote antifungal tolerance or resistance.

“In general, aneuploidy alters cell physiology in ways that may promote antifungal tolerance or resistance.”

May combine two claims.

Cells exposed to supra-MIC concentrations of antifungal drug may be able to slow growth and division enough to preserve viability, whereas lower concentrations may still allow cells to progress through the cell cycle, leading to acquisition of segmental aneuploidy or point mutations that result in bona fide resistance.

“Cells exposed to supra-MIC concentrations of antifungal drug may be able to slow growth and division enough to preserve viability, whereas lower concentrations of antifungal drugs may still allow cells to progress through the cell cycle leading to the acquisition of segmental aneuploidy or point mutations that result in bona fide resistance.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Approximate LOH boundaries were confirmed by visual inspection in IGV (v2.8.2).

“Approximate LOH boundaries were identified from YMAP GBrowse allele ratio tracks, and confirmed by visual inspection in IGV (IGV, v2.8.2) (Thorvaldsdottir et al. 2013).”

• cites Thorvaldsdottir et al. 2013 sourceNot checked yet

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Near-MIC concentrations of FLC (at or two times above the progenitor MIC) selected for lineages with significantly increased drug resistance, whereas supra-MIC concentrations selected for lineages with significantly increased drug tolerance.

“In general, concentrations of FLC that were at or two times above the MIC (near-MIC) of the progenitor selected for lineages that have significantly increased drug resistance, whereas higher concentrations of FLC (supra-MIC) selected for lineages with significantly increased drug tolerance.”

May combine two claims.

The critical distinction is not drug concentration per se, but a physiological breakpoint where cells experience one degree of stress near their initial MIC, and a different degree of stress below and above their initial MIC.

“Our results suggest that the critical distinction is not drug concentration per se, but a physiological breakpoint where cells experience one degree of stress near their initial MIC, and a different degree of stress below and above their initial MIC.”

May combine two claims.

Recurrent and distinct whole-chromosome and segmental aneuploidy was extremely prevalent in drug concentrations near the initial MIC, with the specific evolved karyotype linked to both progenitor strain and drug environment.

“The acquisition of recurrent and distinct whole-chromosome and segmental aneuploidy was extremely prevalent in drug concentrations near the initial MIC, with the specific evolved karyotype linked to both progenitor strain and drug environment.”

May combine two claims.

Selection seems to act either to increase FLC resistance or FLC tolerance, suggesting phenotypic improvement in these traits represents distinct peaks on the fitness landscape.

“Specifically, we observed that selection seems to act either to increase FLC resistance or FLC tolerance, suggesting phenotypic improvement in these traits represents distinct peaks on the fitness landscape.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All growth curves were performed in biological triplicate.

“All growth curves were performed in biological triplicate.”

Classified as probably not a substantial claim.

All variants were verified visually using the Integrative Genomics Viewer (IGV, v2.8.2).

“All variants were verified visually using the Integrative Genomics Viewer (IGV, v2.8.2) (Thorvaldsdottir et al. 2013).”

• cites Thorvaldsdottir et al. 2013 sourceNot checked yet

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