Narrow mutational signatures drive acquisition of multidrug resistance in the fungal pathogen Candida glabrata

May combine two claims.

None of the tested strains presented improved growth on flucytosine or amphotericin B compared to wild-type strains, although a few strains presented higher susceptibility to amphotericin B.

“None of the tested strains presented improved growth on flucytosine (5-FC, pyrimidine analog) or amphotericin B (ampB, polyene) when compared to wild-type (WT) strains, although a few strains presented higher susceptibility to ampB.”

May combine two claims.

All surviving strains acquired stable resistance to the exposed agent(s), with the resistance phenotype kept for several generations in standard growth conditions after removal of the selective agent.

“All of the surviving strains acquired stable resistance to the exposed agent(s); that is, the resistance phenotype was kept for several generations in standard growth conditions after the removal of the selective agent”

May combine two claims.

Similar results were observed for the two tested echinocandins (ani and caspofungin), while the two tested azoles presented more disparate patterns, with few strains growing better on voriconazole than on fluconazole.

“Similar results were observed for the two tested echinocandins (ani and caspofungin), while the two tested azoles presented more disparate patterns, with few strains growing better on voriconazole (vrz) than on flz”

May combine two claims.

When simultaneously exposed to both fluconazole and anidulafungin, 21 populations (43.75%) died, including all replicates of each of 2 strains from clade I (CST109) and clade III (M12).

“However, when simultaneously exposed to both drugs, 21 populations (43.75%) died, including all replicates of each of 2 strains from clade I (CST109) and clade III (M12).”

May combine two claims.

Increased resistance to both drugs (MDR) was often achieved, including all surviving ANIFLZ samples, a majority of AinF (91.6%) and FinA (97.9%) samples, and 44.7% of ANI samples.

“Increased resistance to both drugs (MDR) was often achieved, including all surviving ANIFLZ samples, a majority of AinF (91.6%) and FinA (97.9%) samples, and, due to the mentioned cross-resistance, in 44.7% of ANI samples (Figure 2C; Data S1).”

May combine two claims.

All fluconazole-exposed samples showed a tendency to reduce fitness (p < 10−5, Kolmogorov-Smirnov test), while the mean fitness of ANI samples remained unaltered (p > 0.05).

“All flz-exposed samples showed a tendency to reduce fitness (p < 10−5, Kolmogorov-Smirnov test), while the mean fitness of ANI samples remained unaltered (p > 0.05).”

May combine two claims.

Both ANI parents carried only one FKS2 mutation and alterations in CNE1 ortholog.

“Interestingly, both of the ANI parents carried only one FKS2 mutation and alterations in CNE1 ortholog, encoding an endoplasmic reticulum (ER) protein involved in the quality control of misfolded proteins.28”

• cites [28] Molinari M. Eriksson K.K. Calanca V. Galli C. Cresswell P. Michalak M. Helenius A. Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control Mol. Cell 13 2004 125 135 14731400 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Aneuploidies were common in experiments involving exposure to fluconazole, but they were not detected in cells exposed only to anidulafungin.

“we found that aneuploidies were common in experiments involving exposure to flz, but they were not detected in cells exposed only to ani (Figure 5A).”

May combine two claims.

These five strains harbored mutations in ERG3 (3 AinF strains) and ERG11 (2 strains).

“These strains harbored mutations in ERG3 (3 AinF strains, discussed below) and ERG11 (2 strains).”

May combine two claims.

Among ERG11 mutations, K152 was the most altered amino acid (12/16 samples), followed by ERG11-Y141 (2/16 samples).

“Among ERG11 mutations, K152 was the most altered amino acid (12/16 samples), followed by ERG11-Y141 (2/16 samples).”

May combine two claims.

Many fluconazole-exposed samples retained fitness levels within 2 standard deviations of the mean of YPD-exposed strains (56% of ANIFLZ, 77% AinF, 81% FLZ, and 68% FinA), and only a few samples (2.9%, 5/8 of them ANIFLZ) had severely reduced fitness levels below 50% of the corresponding WT strain.

“Nevertheless, many of the flz-exposed samples retained fitness levels within 2 standard deviations of the mean of YPD-exposed strains (56% of ANIFLZ, 77% AinF, 81% FLZ, and 68% FinA), and only a few samples (2.9%, 5/8 of them ANIFLZ) had severely reduced fitness levels below 50% of the corresponding WT strain.”

May combine two claims.

Three resistant strains carried only non-HS FKS mutations (FKS1-R1422L and FKS1-F708S; FKS1-W681L and FKS2-K265∗; and FKS2-A651T), and whole-genome sequencing revealed no additional mutations outside FKS genes that could explain their resistance phenotype.

“Three resistant strains carried only such non-HS FKS mutations (FKS1-R1422L and FKS1-F708S; FKS1-W681L and FKS2-K265∗; and FKS2-A651T; Data S2), and whole-genome sequencing of these strains revealed no additional mutations outside FKS genes that could explain their resistance phenotype”

May combine two claims.

No newly acquired SVs were found in YPD samples, while drug-evolved strains accumulated a small number (<10) of variants.

“The analysis of genome-wide mutational patterns revealed no newly acquired SVs in YPD samples, while the drug-evolved strains accumulated a small number (<10) of variants (Figures 4A and 4B).”

May combine two claims.

The most common altered gene under exposure to fluconazole was PDR1, which was in many instances (14/37 strains) accompanied by alterations in ERG11.

“The most common altered gene under exposure to flz was PDR1, which was in many instances (14/37 strains) accompanied by alterations in ERG11 (Figure 6; Data S3).”

May combine two claims.

ERG11 mutations and aneuploidies in ChrE, bearing this gene, were strongly anticorrelated, with a single ANIFLZ sample carrying both alterations.

“Importantly, ERG11 mutations and aneuploidies in ChrE, bearing this gene, were strongly anticorrelated, with a single ANIFLZ sample carrying both alterations.”

May combine two claims.

Repeatability may be unique to each phenotype and condition, where AinF and ANIFLZ samples have particularly higher phenotypic variability.

“Our analysis (Figure S1E) indicates that repeatability may be unique to each phenotype and condition, where AinF and ANIFLZ samples have particularly higher phenotypic variability.”

May combine two claims.

Mutations preferentially occurred in FKS2 over FKS1 and in HS1 over HS2.

“Mutations preferentially occurred in FKS2 over FKS1 and in HS1 over HS2 (Figure 3)”

May combine two claims.

All resistant strains carried mutations or duplications in at least one of these nine genes, and the subset of mutated genes largely separated samples by treatment.

“Importantly, all of the resistant strains carried mutations or duplications in at least one of these genes, and the subset of mutated genes largely separated samples by treatment.”

May combine two claims.

The most frequently mutated site in ani-adapted samples was FKS2-F659 (63 samples, 52.1%), with the most prevalent alteration being F659del (52 samples, 43%), which was the only FKS mutation in 26 samples (21.5%).

“Overall, the most frequently mutated site in ani-adapted samples was FKS2-F659 (63 samples, 52.1%; Data S2), with the most prevalent alteration being F659del (52 samples, 43%), which was the only FKS mutation in 26 samples (21.5%).”

May combine two claims.

Three ERG3-mutated strains subsequently exposed to fluconazole (AinF) did not acquire additional mutations in PDR1 or ERG11, nor did they present ChrE duplications.

“When ERG3-mutated strains were subsequently exposed to flz (AinF), three of them did not acquire additional mutations in PDR1 or ERG11, nor did they present ChrE duplications”

May combine two claims.

The trailing effect occurred in most (8/10) ANI samples with ERG3 mutations, leading to high MIC values in the range of FLZ samples.

“We observed this effect occurring in most (8/10) ANI samples with ERG3 mutations, leading to high MIC values that were in the range of FLZ samples (Figure S7C).”

May combine two claims.

Competition assays showed a competitive disadvantage of ERG3 mutants in the absence of drug treatment, but an advantage in the presence of anidulafungin.

“Competition assays between strains carrying the WT and the mutated ERG3 allele showed a competitive disadvantage of ERG3 mutants in the absence of drug treatment, but an advantage in the presence of ani.”

May combine two claims.

One FinA sample carried a PDR1 premature termination codon, which resulted in susceptibility and reduced fluconazole resistance below the median rAUC value of YPD samples.

“One of them carried a PDR1 premature termination codon (∗), which resulted in susceptibility according to our MIC-based thresholding (STAR Methods) and reduced flz resistance below the median rAUC value of YPD samples.”

May combine two claims.

Sample BG2_11H_ANI bears an ERG3 premature termination codon and presents a mismatch between flz MIC and rAUC.

“Conversely, there is one sample (BG2_11H_ANI) bearing an ERG3 premature termination codon and presenting a mismatch between flz MIC and rAUC.”

May combine two claims.

The identified ERG3 mutants do not have deletions in mtDNA and do not have a petite phenotype.

“we can discard this effect in the identified ERG3 mutants due to the absence of deletions in the mtDNA (Figure 6; Data S3) and the absence of a petite phenotype (Figure S7E).”

Classified as probably not a substantial claim.

The threshold of MIC more than 2 times the WT MIC separated samples clearly into susceptible and resistant strains.

“This threshold separated our samples clearly into susceptible and resistant strains (Figures S1C and S1D).”

May combine two claims.

In ANI strains, equal numbers of cases (2 each) had either ERG3 or FKS mutations predating the other, and 5 cases had both mutations traced to the same intermediate generation.

“we traced the order of appearance of ERG3 and FKS mutations along intermediate generations in ANI strains and found equal numbers of cases (2 each) in which either ERG3 or FKS mutations predated the other one, and 5 cases in which both mutations are traced to the same intermediate generation (Table S3).”

May combine two claims.

Neither phylogenetic clade nor the presence of non-synonymous mutations in MSH2 are good predictors of the ability to develop MDR.

“Our results show that neither phylogenetic clade nor the presence of non-synonymous mutations in MSH2 are good predictors of the ability to develop MDR, which is pervasive in C. glabrata.”

May combine two claims.

Measurement errors are similar between MIC and rAUC, but MIC is more consistent across independently evolved strains of the same condition.

“In addition, although measurement errors are similar (Figures S1A and S1B) MIC is more consistent across independently evolved strains of the same condition (Figures 2B and S1C).”

May combine two claims.

Adaptation to anidulafungin often results in cross-resistance to fluconazole, but not the other way around.

“An important result from our experiment is the observation that adaptation to ani often results in cross-resistance to flz (but not the other way around).”

May combine two claims.

No acquisition of anidulafungin resistance was observed in FLZ samples but only as a result of anidulafungin (FinA).

“No acquisition of ani resistance was observed in FLZ samples but only as a result of ani (FinA).”

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

The total number of analyzed samples was 48 FLZ, 47 FinA, 47 ANI, 48 AinF, 21 ANIFLZ and 48 YPD.

“the total number of analyzed samples was as follows: 48 FLZ, 47 FinA, 47 ANI, 48 AinF, 21 ANIFLZ and 48 YPD.”

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

SLL2_glab was sequenced within this project and assigned to clade 7.

“SLL2 glab was sequenced within this project and assigned to clade 7.”

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

2 samples were found as susceptible but have rAUC values in the range of resistant samples.

“Note that 2 samples (marked with “?”) were found as susceptible but have rAUC values in the range of resistant samples.”

May combine two claims.

In the experiment, alterations in ERG3 often appeared under anidulafungin exposure and were retained in subsequent fluconazole exposure, sometimes without further mutation explaining fluconazole resistance.

“In our experiment, alterations in this gene often appeared under ani exposure and were retained in subsequent flz exposure, sometimes without any further mutation being acquired that would explain the acquisition of resistance to flz.”

May combine two claims.

One ANI sample showed high anidulafungin resistance but also low basal fitness, so the high resistance value may not be representative.

“One of the ANI samples showed high ani resistance (above 1.0, meaning the fitness was higher in ani than in no drug), but also showed low basal fitness, which means that the high resistance value may be not representative.”

May combine two claims.

Combined exposure to both drugs prevented adaptation in a significant fraction of the cases, with two strains from two different clades showing an inability to develop resistance.

“However, the combined exposure to both drugs prevented adaptation in a significant fraction of the cases, with two strains from two different clades showing an inability to develop resistance in this scenario.”

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

Three samples did not acquire any new mutation in the recurrent genes in AinF.

“the gray circles outline 3 samples that did not acquire any new mutation in the recurrent genes in AinF.”

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

In the CBS138 wild-type strain, the same trial of transformations was performed but selection of positive transformants was unsuccessful.

“The same trial of transformations was done on CBS138 WT strain but the selection of positive transformants was unsuccessful.”

May combine two claims.

Relative coverage was correlated with the distance to the telomere (smiley-pattern), which may be an artifact of library preparation and/or sequencing, with this effect varying across samples.

“As noted in other studies, we found that relative coverage was correlated with the distance to the telomere (hereafter referred as “smiley-pattern”), which may be an artifact of library preparation and/or sequencing, with this effect varying across samples.”

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

In Candida albicans, MIC data were found for 187/478 clinical isolates.

“In Candida albicans, we could find MIC data for 187/478 clinical isolates.”

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based). May combine two claims. The quote only partly states this claim.

In C. albicans, 39/186 and 9/150 isolates were resistant to all tested azoles or echinocandins, respectively.

“This yielded 39/186 and 9/150 isolates resistant to all tested azoles or echinocandins, respectively.”

May combine two claims.

EF1620_7B_ANI had low fitness (by both fAUC and DR) in the absence of the drug, with a small increase in the lower flz concentrations.

“We found that this sample had low fitness (by both fAUC and DR) in the absence of the drug, with a small increase in the lower flz concentrations.”

May combine two claims.

In Candida glabrata, MIC data were found in 126/393 clinical isolates, including resistance to fluconazole (126/126), posaconazole (84/126), voriconazole (91/126), isavuconazole (37/126), micafungin (42/126), anidulafungin (9/126) and caspofungin (91/126).

“In Candida glabrata, we could find these data in 126/393 clinical isolates, including resistance to fluconazole (flz 126/126), posaconazole (pos 84/126), voriconazole (vrz 91/126), isavuconazole (ivz 37/126), micafungin (mif 42/126), anidulafungin (ani 9/126) and caspofungin (cas 91/126).”

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

Two C. glabrata samples had resistance to both azole and echinocandin classes of drugs.

“We could find two samples with resistance to both classes of drugs.”

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based). The quote only partly states this claim.

One C. albicans sample had resistance to both azole and echinocandin classes of drugs.

“We could find one sample with resistance to both classes of drugs.”

May combine two claims.

In CST34_2A_AinF, only an inverted heterozygous breakpoint matching the start of the aneuploid region of ChrE was found, which was not enough to explain the source of the duplication.

“Conversely, we could only find an inverted heterozygous breakpoint matching the start of the aneuploid region of ChrE in CST34_2A_AinF, which was not enough to explain the source of the duplication.”

May combine two claims.

In C. glabrata, 41/126 and 19/91 isolates were resistant to all tested azoles or echinocandins, respectively.

“This yielded 41/126 and 19/91 isolates resistant to all tested azoles or echinocandins, respectively.”

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

No heterozygous breakend events were detected in the aneuploid chromosomes.

“Note that we did not detect any such heterozygous events in the aneuploid chromosomes.”

May combine two claims.

Two FinA samples had genomic rearrangements breaking the FKS1 coding region, including one deletion at the beginning of the coding sequence and one balanced inverted translocation between ChrG and ChrM; both samples carried FKS2 mutations, suggesting these are complementary FKS1 alterations.

“Finally, we found two FinA samples with GR breaking the FKS1 coding region, including one deletion at the beginning of the coding sequence (with relative coverage < 0.01) and one balanced inverted translocation between ChrG and ChrM (Figure S3C). Both samples carried FKS2 mutations (potentially conferring ani resistance), suggesting that these rearrangements are complementary FKS1 alterations with”

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

Strains containing NAT1 grew in lower abundance on the antibiotic than on YPD plates alone.

“Since we observed that strains containing NAT1 were growing in lower abundance on the antibiotic than on YPD plates alone, the total number of cells were accounted for this discrepancy.”

May combine two claims.

One ANIFLZ sample had a deletion at the beginning of the ERG3 CDS and one FinA sample had a deletion in the ERG3 5′ region, both associated with low relative coverage spanning the breakpoint.

“Regarding ERG3, we found one ANIFLZ sample with a deletion at the beginning of the CDS and a FinA sample with a deletion in the 5′ region (potentially spanning the promoter, and related to the loss of CAGL0F01815 g (see Results). Both of these were associated with low relative coverage (< 0.01) spanning the breakpoint, which further confirmed these deletions (Figure S3A).”

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

C. glabrata often is the second most prevalent cause of Candida infection.

“C. glabrata often being the second most prevalent cause of infection.6”

• cites [6] Pfaller M.A. Diekema D.J. Turnidge J.D. Castanheira M. Jones R.N. Twenty Years of the SENTRY Antifungal Surveillance Program: Results for Candida Species From 1997-2016 Open Forum Infect. Dis. 6 Suppl 1 2019 S79 S94 30895218 PMC6419901 sourceNot checked yet

  Not checked in the free run.

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

Echinocandin resistance has been linked to non-synonymous variations in two conserved hotspot regions of FKS genes.

“whereas echinocandin resistance has been linked to non-synonymous variations in two conserved hotspot (HS; i.e., frequently mutated) regions of FKS genes.16”

• cites [16] Perlin D.S. Mechanisms of echinocandin antifungal drug resistance Ann. N Y Acad. Sci. 1354 2015 1 11 26190298 PMC4626328 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Previous studies on in vitro-acquired drug resistance in C. glabrata have evaluated fitness costs of echinocandin resistance or used transcriptomics to unveil mechanisms contributing to azole resistance, but the genome-wide genetic alterations involved during this process remain elusive.

“Previous studies on in vitro-acquired drug resistance in C. glabrata have evaluated the fitness costs of echinocandin resistance23 or used transcriptomics to unveil the mechanisms contributing to azole resistance,24 but the genome-wide genetic alterations involved during this process remain elusive.”

• cites [23] Duxbury S.J.N. Bates S. Beardmore R.E. Gudelj I. Evolution of drug-resistant and virulent small colonies in phenotypically diverse populations of the human fungal pathogen Candida glabrata Proc. Biol. Sci. 287 2020 20200761 32673559 PMC7423662Not checked yet

  Not checked in the free run.

• cites [24] Cavalheiro M. Costa C. Silva-Dias A. Miranda I.M. Wang C. Pais P. Pinto S.N. Mil-Homens D. Sato-Okamoto M. Takahashi-Nakaguchi A. A Transcriptomics Approach To Unveiling the Mechanisms of Evolution towards Fluconazole Resistance of a Clinical Isolate Antimicrob. Agents Chemother. 63 2019 e00995-18 30348666 PMC6325195 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Low adaptation potential to simultaneous fluconazole and anidulafungin is strain-specific and not clade-specific.

“populations from other strains from these clades survived, indicating that low adaptation potential is strain- and not clade-specific.”

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

Known mechanisms of azole resistance in C. glabrata almost exclusively consist of gain-of-function mutations in PDR1.

“Known mechanisms of azole resistance in C. glabrata almost exclusively consist of gain-of-function mutations in PDR1, which encodes a transcriptional regulator of drug efflux pumps,15”

• cites [15] Sanglard D. Ischer F. Calabrese D. Majcherczyk P.A. Bille J. The ATP binding cassette transporter gene CgCDR1 from Candida glabrata is involved in the resistance of clinical isolates to azole antifungal agents Antimicrob. Agents Chemother. 43 1999 2753 2765 10543759 PMC89555 sourceNot checked yet

  Not checked in the free run.

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

C. glabrata is phylogenetically closer to Saccharomyces cerevisiae than to most other Candida pathogens, which may imply different routes for drug adaptation compared to other Candida species.

“C. glabrata belongs to the Nakaseomyces clade and is phylogenetically closer to Saccharomyces cerevisiae than to most other Candida pathogens,7 which may imply different routes for drug adaptation as compared to other Candida species.”

• cites [7] Gabaldón T. Carreté L. The birth of a deadly yeast: tracing the evolutionary emergence of virulence traits in Candida glabrata FEMS Yeast Res. 16 2016 fov110 26684722 PMC5815135 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

The evolutionary processes underpinning the acquisition of antifungal drug resistance are poorly understood.

“Despite the pressing challenge that the emergence of antifungal resistance represents for human health and food security, we have a limited understanding of the evolutionary processes leading to drug adaptation in fungi.5”

• cites [5] Ksiezopolska E. Gabaldón T. Evolutionary Emergence of Drug Resistance in Candida Opportunistic Pathogens Genes (Basel) 9 2018 461 PMC6162425 30235884 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Aneuploidies were adaptive per se and not because they allowed faster evolution of duplicated genes.

“indicating they have not accumulated new mutations since their duplication, and, therefore, that aneuploidies were adaptive per se and not because they allowed faster evolution of duplicated genes.”

May combine two claims.

Although the set of genes recurrently mutated during acquisition of resistance is rather limited (nine genes), the number of specific mutations is large and highly diverse and only partially covered by our experiment or clinical surveys.

“These results suggest that although the set of genes recurrently mutated during the acquisition of resistance is rather limited (nine genes), the number of specific mutations (i.e., which residue is mutated and what type of mutation occurs) is large and highly diverse and only partially covered by our experiment or clinical surveys.”

May combine two claims.

Fluconazole resistance or the presence of aneuploidies are compatible with virulence.

“suggesting that flz resistance or the presence of aneuploidies are compatible with virulence.”

May combine two claims.

The low overlap is expected from the large diversity of identified mutations in our experiments and is similarly low for mutations identified in actual clinical surveys.

“This low overlap is, however, expected from the actual large diversity of the identified mutations in our experiments (Figure S5B; Data S2 and S3) and is similarly low for mutations identified in actual clinical surveys (e.g., SENTRY6).”

• cites [6] Pfaller M.A. Diekema D.J. Turnidge J.D. Castanheira M. Jones R.N. Twenty Years of the SENTRY Antifungal Surveillance Program: Results for Candida Species From 1997-2016 Open Forum Infect. Dis. 6 Suppl 1 2019 S79 S94 30895218 PMC6419901 sourceNot checked yet

  Not checked in the free run.

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

Altered adhesion has been linked to azole resistance in C. glabrata.

“Altered adhesion has been linked to azole resistance in C. glabrata,35”

• cites [35] Vale-Silva L.A. Moeckli B. Torelli R. Posteraro B. Sanguinetti M. Sanglard D. Upregulation of the Adhesin Gene EPA1 Mediated by PDR1 in Candida glabrata Leads to Enhanced Host Colonization mSphere 1 2016 e00065-15 27303714 PMC4863579 sourceNot checked yet

  Not checked in the free run.

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

Alterations in the promoter region of the efflux pump CDR1 have already been reported in azole-resistant strains.

“Alterations in the promoter region of the efflux pump CDR1 have already been reported in azole-resistant strains,33,34”

• cites [33] Tsai H.-F. Krol A.A. Sarti K.E. Bennett J.E. Candida glabrata PDR1, a transcriptional regulator of a pleiotropic drug resistance network, mediates azole resistance in clinical isolates and petite mutants Antimicrob. Agents Chemother. 50 2006 1384 1392 16569856 PMC1426987 sourceNot checked yet

  Not checked in the free run.

• cites [34] Looi C.Y. D’ Silva E.C. Seow H.F. Rosli R. Ng K.P. Chong P.P. Increased expression and hotspot mutations of the multidrug efflux transporter, CDR1 in azole-resistant Candida albicans isolates from vaginitis patients FEMS Microbiol. Lett. 249 2005 283 289 16006060 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

ERG3 alterations have a dual role in adaptation to anidulafungin and in the development of cross-resistance to fluconazole in C. glabrata.

“Our results support a dual role of ERG3 alterations in the adaptation to ani and in the development of cross-resistance to flz in C. glabrata.”

Classified as probably not a substantial claim.

Voriconazole could be a promising therapeutic alternative.

“Importantly, many flz-resistant strains were susceptible to vrz, which could be a promising therapeutic alternative.”

• AssumptionRests on an assumption: The in vitro drug concentrations and exposure regimens mimic clinical antifungal exposure.

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

The scarcity of sequenced genomes for MDR clinical strains and the lack of information on the treatment regime they were exposed to prevented assessment of how commonly this cross-resistance mechanism occurs in clinics.

“The scarcity of sequenced genomes for MDR clinical strains and the lack of information of the treatment regime they were exposed to (STAR Methods) prevented us from assessing how commonly this cross-resistance mechanism occurs in the clinics”

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

ERG3 mutations leading to depletion of ergosterol and accumulation of less toxic sterols when ERG11 is inhibited have been implicated in cross-resistance between azoles and polyenes in S. cerevisiae and C. albicans and between echinocandins and azoles in C. parapsilosis.

“ERG3 mutations leading to the depletion of ergosterol and the accumulation of less toxic sterols when ERG11 is inhibited have been implicated in cross-resistance between azoles and polyenes in S. cerevisiae and C. albicans40, 41, 42, 43 and between echinocandins and azoles in C. parapsilosis.44,45”

• cites [40] Cowen L.E. Sanglard D. Howard S.J. Rogers P.D. Perlin D.S. Mechanisms of Antifungal Drug Resistance Cold Spring Harb. Perspect. Med. 5 2014 a019752 25384768 PMC4484955 sourceNot checked yet

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• cites [41] Kelly S.L. Lamb D.C. Kelly D.E. Manning N.J. Loeffler J. Hebart H. Schumacher U. Einsele H. Resistance to fluconazole and cross-resistance to amphotericin B in Candida albicans from AIDS patients caused by defective sterol delta5,6-desaturation FEBS Lett. 400 1997 80 82 9000517 sourceNot checked yet

  Not checked in the free run.

• cites [42] Martel C.M. Parker J.E. Bader O. Weig M. Gross U. Warrilow A.G.S. Rolley N. Kelly D.E. Kelly S.L. Identification and characterization of four azole-resistant erg3 mutants of Candida albicans Antimicrob. Agents Chemother. 54 2010 4527 4533 20733039 PMC2976150 sourceNot checked yet

  Not checked in the free run.

• cites [43] Morio F. Pagniez F. Lacroix C. Miegeville M. Le Pape P. Amino acid substitutions in the Candida albicans sterol Δ5,6-desaturase (Erg3p) confer azole resistance: characterization of two novel mutants with impaired virulence J. Antimicrob. Chemother. 67 2012 2131 2138 22678731 sourceNot checked yet

  Not checked in the free run.

• cites [44] Rybak J.M. Dickens C.M. Parker J.E. Caudle K.E. Manigaba K. Whaley S.G. Nishimoto A.T. Luna-Tapia A. Roy S. Zhang Q. Loss of C-5 Sterol Desaturase Activity Results in Increased Resistance to Azole and Echinocandin Antifungals in a Clinical Isolate of Candida parapsilosis Antimicrob. Agents Chemother. 61 2017 e00651-17 28630186 PMC5571332 sourceNot checked yet

  Not checked in the free run.

• cites [45] Papp C. Bohner F. Kocsis K. Varga M. Szekeres A. Bodai L. Willis J.R. Gabaldón T. Tóth R. Nosanchuk J.D. Triazole Evolution of Candida parapsilosis Results in Cross-Resistance to Other Antifungal Drugs, Influences Stress Responses, and Alters Virulence in an Antifungal Drug-Dependent Manner mSphere 5 2020 e00821-20 33115837 PMC7593601 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Fluconazole resistance may be common after the failure of anidulafungin therapy, so that fluconazole treatment following anidulafungin may also result in therapy failure.

“our data suggest that flz resistance may be common after the failure of ani therapy, so that flz treatment following ani may also result in therapy failure.”

• AssumptionRests on an assumption: The in vitro drug concentrations and exposure regimens mimic clinical antifungal exposure.

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

Resistance to fluconazole is often spontaneously acquired in C. glabrata by partial or total loss of mitochondrial DNA, rendering a petite phenotype.

“Resistance to flz is often spontaneously acquired in C. glabrata by partial or total loss of mitochondrial DNA, rendering a so-called petite phenotype.30”

• cites [30] Kaur R. Castaño I. Cormack B.P. Functional genomic analysis of fluconazole susceptibility in the pathogenic yeast Candida glabrata: roles of calcium signaling and mitochondria Antimicrob. Agents Chemother. 48 2004 1600 1613 15105111 PMC400560 sourceNot checked yet

  Not checked in the free run.

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

The 12 strains of C. glabrata used in this study include 11 clinical strains previously analyzed and one commensal strain SLL2_glab.

“The 12 strains of C. glabrata used in this study are listed in the key resources table. Eleven clinical strains had been previously analyzed for several phenotypic characteristics, including susceptibility to various drugs.20 In addition, they have been shown to belong to seven genetically distinct clades. The remaining strain (SLL2_glab) was isolated from an oral wash of a healthy individual from”

• cites [20] Carreté L. Ksiezopolska E. Pegueroles C. Gómez-Molero E. Saus E. Iraola-Guzmán S. Loska D. Bader O. Fairhead C. Gabaldón T. Patterns of genomic variation in the opportunistic pathogen Candida glabrata suggest the existence of mating and a secondary association to the human host Curr. Biol 28 2018 15 27.e7 29249661 PMC5772174 sourceNot checked yet

  Not checked in the free run.

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

These findings are consistent with a recent report of fluconazole cross-resistance in ani-adapted C. glabrata isolates.

“Importantly, these findings are consistent with a recent report of flz cross-resistance in ani-adapted C. glabrata isolates.38”

• cites [38] Hatwig C. Balbueno E.A. Bergamo V.Z. Pippi B. Fuentefria A.M. Silveira G.P. Multidrug-resistant Candida glabrata strains obtained by induction of anidulafungin resistance in planktonic and biofilm cells Braz. J. Pharm. Sci. 55 2019 10.1590/s2175-97902019000218025 sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim.

The trailing effect has been reported with azoles and Candida species.

“This effect has been reported with azoles and Candida species.65, 66, 67”

• cites [65] Zomorodian K. Bandegani A. Mirhendi H. Pakshir K. Alinejhad N. Poostforoush Fard A. In Vitro Susceptibility and Trailing Growth Effect of Clinical Isolates of Candida Species to Azole Drugs Jundishapur J. Microbiol. 9 2016 e28666 27127587 PMC4841996 sourceNot checked yet

  Not checked in the free run.

• cites [66] Rueda C. Puig-Asensio M. Guinea J. Almirante B. Cuenca-Estrella M. Zaragoza O. CANDIPOP Project from GEIH-GEMICOMED (SEIMC) and REIPI Evaluation of the possible influence of trailing and paradoxical effects on the clinical outcome of patients with candidemia Clin. Microbiol. Infect. 23 2017 49.e1 49.e8 27677697 sourceNot checked yet

  Not checked in the free run.

• cites [67] Marcos-Zambrano L.J. Escribano P. Sánchez-Carrillo C. Bouza E. Guinea J. Scope and frequency of fluconazole trailing assessed using EUCAST in invasive Candida spp. isolates Med. Mycol. 54 2016 733 739 27161788 sourceNot checked yet

  Not checked in the free run.

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

Acquisition of ERG3 mutations upon echinocandin exposure has been described in C. auris.

“In addition, acquisition of ERG3 mutations upon echinocandin exposure has also been described in C. auris.46”

• cites [46] Carolus H. Pierson S. Muñoz J.F. Subotić A. Cruz R.B. Cuomo C.A. Van Dijck P. Genome-wide analysis of experimentally evolved Candida auris reveals multiple novel mechanisms of multidrug-resistance mBio 12 2020 e03333-20 PMC8092288 33820824 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The observed CDR1 mutations increase azole efflux and thus decrease fluconazole susceptibility.

“We propose that the observed CDR1 mutations increase azole efflux and thus decrease flz susceptibility.”

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

rAUC values provided a continuous estimate of resistance, better suited for quantitative analyses.

“However, rAUC values provided a continuous estimate of resistance, which is better suited for quantitative analyses (such as those of Figures 2E and 7C).”

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

ERG4, similar to ERG3, may influence resistance to fluconazole.

“ERG4 is another gene involved in the ergosterol biosynthesis pathway, which, similar to ERG3, may influence resistance to flz.”

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

Susceptibility tests were performed in at least three replicates following the EUCAST E.DEF 7.3.1 protocol.

“Susceptibility tests were performed in at least three replicates following the EUCAST E.DEF 7.3.1. protocol.64”

• cites [64] Arendrup M.C. Cuenca-Estrella M. Lass-Flörl C. Hope W. EUCAST-AFST EUCAST technical note on the EUCAST definitive document EDef 7.2: method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts EDef 7.2 (EUCAST-AFST) Clin. Microbiol. Infect. 18 2012 E246 E247 22563750 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Although the set of genes altered during adaptation may be limited, the diversity of possible resistance-conferring mutations in each affected gene is very large.

“our results suggest that although the set of genes altered during the process of adaptation may be limited, the diversity of possible resistance-conferring mutations in each of the affected genes is very large.”

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

Cell membrane modifications related to changes in ergosterol production affect the structure and composition of the cell wall.

“it has been reported that cell membrane modifications related to changes in ergosterol production affect the structure and composition of the cell wall.47”

• cites [47] Lesage G. Bussey H. Cell wall assembly in Saccharomyces cerevisiae Microbiol. Mol. Biol. Rev. 70 2006 317 343 16760306 PMC1489534 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

These results support selection for ERG3 mutations only during anidulafungin treatment and point to a possible explanation for the lack of clinical cases showing this alteration.

“These results support the selection for ERG3 mutations only during ani treatment and point to a possible explanation for the lack of clinical cases showing this alteration.”

• AssumptionRests on an assumption: The fitness cost of ERG3 mutants measured in vitro and in G. mellonella reflects their fitness in human hosts.

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

ERG3 deletion in C. glabrata does not affect fluconazole susceptibility.

“earlier work showing that ERG3 deletion in C. glabrata does not affect flz susceptibility.29”

• cites [29] Geber A. Hitchcock C.A. Swartz J.E. Pullen F.S. Marsden K.E. Kwon-Chung K.J. Bennett J.E. Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility Antimicrob. Agents Chemother. 39 1995 2708 2717 8593007 PMC163017 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Sample P35_10E_FinA has two reciprocal inverted interchromosomal breakpoints between close positions of ChrG (breaking the FKS1 CDS) and ChrM, interpreted as an inverted balanced translocation.

“This approach yielded one sample (P35_10E_FinA) with two reciprocal inverted interchromosomal breakpoints between close positions (less than 200 bp apart) of chromosome (Chr) G (breaking the CDS of FKS1) and ChrM. These were called as two independent unbalanced translocations, but we interpret them as an inverted balanced translocation between the two chromosomes.”

May combine two claims.

Taken together, the results suggest that complex structural variation may contribute to drug resistance and that breakpoint calling can explain the precise events leading to CNV and aneuploidies.

“Taken together, these results suggest that complex structural variation may contribute to drug resistance. They also show how breakpoint calling can explain the precise events leading to CNV and aneuploidies.”

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

The mixed chromosome in EB0911 is much shorter than the reference ChrI, perhaps resulting in a lower fitness cost of this aneuploidy.

“Interestingly, this chromosome is much shorter than the reference ChrI, perhaps resulting in a lower fitness cost of this aneuploidy.”

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

The presence and absence of ERG3 mutations was confirmed by Sanger sequencing.

“Presence and absence of ERG3 mutations was confirmed by Sanger sequencing.”

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

All genomic rearrangements discussed in the text were positively confirmed with PCR.

“Presence of all the GRs discussed in the text was confirmed with PCR using primers specifically designed to provide amplicons only in the presence of the GR (translocations) or with a different size (deletions). Results are presented in Figure S3F. All events were positively confirmed.”

May combine two claims.

The low overlap between the mutations described here and clinical isolates is expected and comparable to other datasets of well-known resistance-conferring mutations.

“However, this low overlap is expected and comparable to other datasets of well-known resistance-conferring mutations.”

Classified as probably not a substantial claim.

The conservative approach of defining GRs as those where no breakend matched a breakend in any parent ensures high confidence in the final set of variants.

“This is an extremely conservative approach (as most called breakends in the parents may be false positives) to ensure high confidence in our final set of variants.”

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

All these rearrangements were confirmed through PCR.

“We confirmed all these rearrangements through PCR (see below).”

May combine two claims.

An unbalanced translocation explains the partial duplication of ChrE in CBS138_9F_FLZ, with the right arm of ChrE duplicated and attached to ChrJ, replacing the left-end at the breakpoint; the configuration is proposed to result from a pre-existing rearrangement in the WT strain.

“We found an unbalanced translocation explaining the partial duplication of ChrE in CBS138_9F_FLZ.”

• Also stated in: § Method details · hedged

  “We propose that this configuration is the result of a pre-existing rearrangement in the WT strain, which explains why the deleted region does not span the entire left-end of the chromosome.”

• Also stated in: § Method details · plain

  “Accordingly, the ChrE breakend was called heterozygous, while the ChrJ was haploid (Figure S3D).”

May combine two claims.

For BG2_11H_ANI, although MIC is in the WT range, visual inspection of the flz concentration-versus-fitness curve showed a trailing effect around 50% of growth, implying increased resistance.

“Although MIC is in the WT range, visual inspection of the flz concentration-versus-fitness curve showed a trailing effect around 50% of growth (Figure S7C), implying increased resistance.”