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PreprintCited by 48Antifungal resistance and susceptibilityFungal and yeast genetics researchYeasts and Rust Fungi Studies

Genotypic diversity and unrecognized antifungal resistance among populations of Candida glabrata from positive blood cultures

Open access
Badrane, Hassan, Cheng, Shaoji, Dupont, Chris, Hao, Binghua, Driscoll, Eileen, Morder, Kristen, Liu, Guojun, Newbrough, Anthony +5 more·2023·Nature Communications
DOI 10.21203/rs.3.rs-2706400/v1PMC PMC10104189
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Abstract

The longstanding paradigm is that most bloodstream infections (BSIs) are caused by a single organism. We performed whole genome sequencing of five-to-ten strains from blood culture (BC) bottles in each of ten patients with Candida glabrata BSI. We demonstrated that BCs contained mixed populations of clonal but genetically diverse strains. Genetically distinct strains from two patients exhibited phenotypes that were potentially important during BSIs, including differences in susceptibility to antifungal agents and phagocytosis. In both patients, the clinical microbiology lab recovered a fluconazole-susceptible index strain, but we identified mixed fluconazole-susceptible and –resistant populations. Diversity in drug susceptibility was likely clinically relevant, as fluconazole-resistant strains were subsequently recovered by the clinical laboratory during persistent or relapsing infections. In one patient, unrecognized respiration-deficient small colony variants were fluconazole-resistant and significantly attenuated for virulence during murine candidiasis. Our data suggest a new population-based paradigm of C. glabrata genotypic and phenotypic diversity during BSIs.

AI snapshot

Written from the paper’s full text.

TL;DR

Whole-genome sequencing of multiple *Candida glabrata* colonies from positive blood cultures in 10 patients showed that each culture contained clonal but genetically diverse strains. In two patients, this diversity included unrecognized fluconazole-resistant subpopulations, including respiration-deficient small colony variants, with differences in antifungal susceptibility, phagocytosis, and virulence.

Key findings

  • Across 10 patients, 94 *C. glabrata* blood-culture strains segregated into 4 sequence types and 3 clades; within-patient strains averaged 3,598 nucleotide differences, with the highest in patient J (7,507) and lowest in patient C (2,067).
  • In patient L, strain L4 had fluconazole, posaconazole, and isavuconazole MICs ≥8-fold, ≥16-fold, and ≥32-fold higher than other L strains, respectively; it carried a PDR1 G346C substitution, and engineered PDR1 G346C strains had higher azole MICs (256 and 64 μg/mL) than wild-type PDR1 controls (32 and 16 μg/mL).
  • In patient J, SCV strains J6–J10 had higher fluconazole and voriconazole MICs (≥64 μg/mL and 16 μg/mL) than NCV strains J1–J4 (4–8 μg/mL and 0.5 μg/mL); SCVs J5 and J9 were more resistant than J1 to neutrophil phagocytosis (12.6±2.7% and 10.9±2.6% vs. 31.8±1.8%) and killing (18.5±8.9% and 22.0±2.4% vs. 39.4±7.7%).
  • J9 caused significantly lower tissue burdens than J1 in kidneys and spleens at days 1, 3, and 7 and in livers at day 7 in mice; no mice died by day 21 after injection of either strain.

Methods

  • Positive blood culture bottles from 10 patients with *C. glabrata* BSI were streaked, and the clinical index strain plus 4–9 additional colonies per patient underwent Illumina NextSeq whole-genome sequencing; index strains also underwent Oxford Nanopore MinION sequencing.
  • Bioinformatic analyses used a Nextflow pipeline with BWA, GATK, SnpEff, BCFTOOLS, FastTree/RAxML, PubMLST, GENOME-STRip, FLYE, Pilon, and progressiveMauve to assess SNPs/indels, phylogeny, sequence types, gene copy-number variants, and mitochondrial copy number.
  • Phenotypic assays included CLSI broth dilution antifungal susceptibility testing, qRT-PCR, PDR1 mutant construction, adherence to Hep-2 cells, neutrophil phagocytosis/killing, paraquat killing, respiration/flow cytometry/electron microscopy, and a murine hematogenously disseminated candidiasis model.

Limitations

  • The paper states that only 10 strains per blood culture bottle were sequenced, which may not represent the entire *C. glabrata* population; it also states that only first BSI episodes were sequenced, results cannot be extrapolated to other *Candida* spp. or pathogens, and the patients were severely ill with extensive histories, polymicrobial BSIs, and recent antifungal exposure.
  • The paper notes that metagenomic sequencing directly from blood might give more comprehensive coverage but is currently limited by low microbial DNA concentrations and difficulty assigning variants to individual strains.
  • Evident from the text: detailed phenotypic characterization was performed for strains from only 2 of 10 patients, and the in vitro spiked blood culture experiment used only index strain J1, so the generality of the phenotypic findings remains uncertain.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.21203/rs.3.rs-2706400/v1

Cites

68 of 68 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 26 cited sources checked · 0 supported · 0 flagged

  • Findingcitation

    PDR1 and CDR1 were up-regulated by 14.6±4.0-fold and 257±11.8-fold, respectively, in strain J9 compared with strain J1.

    “As expected from these reports, PDR1 and CDR1 were up-regulated by 14.6±4.0-fold and 257±11.8-fold, respectively, in strain J9 compared with strain J1 (RT-PCR).”
    Quote in the paperSubstantial claim
  • Findingcitation

    L4 carried a mutation within PDR1 conferring a G346C substitution within the central regulatory domain of transcriptional regulator Pdr1.

    “L4 carried a mutation within PDR1, conferring a G346C substitution within the central regulatory domain of transcriptional regulator Pdr1.17”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [17] Simonicova L, Moye-Rowley WS. Functional information from clinically-derived drug resistant forms of the Candida glabrata Pdr1 transcription factor. PLoS Genet. 2020; 16(8): e1009005. 32841236 PMC7473514

      Not checked in the free run.

  • Findingreasoning

    Strain J5 was most consistent with a SCV on balance.

    “Therefore, on balance, strain J5 was most consistent with a SCV.”
    Quote in the paperNot a substantial claim
  • Findingown data

    PDR1 and CDR1 expression were significantly higher in L4 than in other L strains by RT-PCR.

    “Using RT-PCR, we showed that PDR1 and CDR1 expression were significantly higher in L4 than in other L strains [Figure 4].”
    Quote in the paperSubstantial claim
  • Findingown data

    Within-patient single nucleotide polymorphisms in C. glabrata were not distributed uniformly along the chromosomes but were predominantly clustered in hot spots, particularly in subtelomeric regions.

    “Single nucleotide polymorphisms (SNPs) within-patient were not distributed uniformly along the chromosomes, but rather were predominantly clustered in hot spots, particularly in subtelomeric regions [Supplemental Figure 1].”
    Quote in the paperNot a substantial claim
  • May combine two claims. 32
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 20

  • May combine two claims.

    Findingown data

    Adherence of strain L2 to Hep-2 cells was significantly higher (91.6±9.2%) than that of either L4 or L6 (66.3±3.4% and 78.0±1.7%, respectively; p=0.03), while L4 and L6 did not significantly differ from each other.

    “Adherence of strain L2 to Hep-2 cells was significantly higher (91.6±9.2%) than that of either L4 or L6 (66.3±3.4% and 78.0±1.7%, respectively; p=0.03, Brown-Forsythe ANOVA test); the latter strains did not significantly differ from each other in adherence.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    SCV strains J5 and J9 were more resistant than J1 to neutrophil phagocytosis (12.6±2.7% and 10.9±2.6%, respectively, vs. 31.8 ±1.8; p<0.0001) and killing (18.5±8.9% and 22.0±2.4% vs 39.4.8±7.7; p=0.0007 and 0.0001, respectively).

    “SCV strains J5 and J9 were more resistant than J1 to neutrophil phagocytosis (12.6±2.7% and 10.9±2.6%, respectively, vs. 31.8 ±1.8; p<0.0001) and killing (18.5±8.9% and 22.0±2.4% vs 39.4.8±7.7; p=0.0007 and 0.0001, respectively).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Fluconazole and voriconazole MICs were higher against strains J6-J10 (≥64 μg/mL and 16 μg/mL, respectively) than against J1-J4 (4-8 μg/mL and 0.5 μg/mL, respectively); MICs against J5 were intermediate.

    “Along these lines, fluconazole and voriconazole MICs were higher against strains J6-J10 (≥ 64 μg/mL and 16 μg/mL, respectively) than they were against J1-J4 (4–8 μg/mL and 0.5 μg/mL, respectively). Fluconazole and voriconazole MICs against J5 (32 μg/mL and 1 μg/mL, respectively) were intermediate to those against the other strains.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Oxygen consumption by strain J1 was significantly reduced upon exposure to antimycin A, sodium cyanide or salicylhydroxamic acid (SHAM), whereas oxygen consumption by strain J9 was already diminished in the absence of electron transport inhibition and was not reduced by these inhibitors.

    “As expected, oxygen consumption by strain J1 was significantly reduced upon exposure to antimycin A, sodium cyanide or salicylhydroxamic acid (SHAM) [Figure 5F]. In contrast, oxygen consumption by strain J9 was already diminished in the absence of electron transport inhibition, and it was not reduced upon exposure to antimycin A, sodium cyanide or SHAM.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After spiking a sterile blood culture bottle with index strain J1 and incubating for 3 days, sub-cultured colonies were homogenous and consistent with NCVs, with no evidence of SCVs after 5 days.

    “After 3 days of incubation, turbidity was observed. Aliquots sub-cultured on SDA plates revealed homogenous colonies consistent with NCVs. There was no evidence of SCVs after 5 days of incubation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There were no within-patient gene duplications or large-scale genetic differences such as aneuploidies among the C. glabrata strains.

    “There were no within-patient gene duplications or large-scale genetic differences such as aneuploidies.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    J9 caused significantly lower tissue burdens than J1 in kidneys and spleens at 1, 3 and 7 days, and in livers at 7 days, in mice with hematogenously disseminated candidiasis; none of the mice died at day 21 following injection of 10^7 CFU of either strain.

    “None of the mice died at day 21 following lateral tail vein injections of 107 CFU of either strain. J9 caused significantly lower tissues burdens than J1 in kidneys and spleens at 1, 3 and 7 days, and in livers at 7 days [Figure 6]”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    L8 did not carry a PDR1 mutation, but CDR1 expression was increased by >12-fold compared with the index strain L1.

    “L8 did not carry a PDR1 mutation, but CDR1 expression was increased by >12-fold compared with the index strain L1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Twelve genes were deleted among strains from patients EF, J, H, and K, 8 of which encoded adhesins.

    “Twelve genes were deleted among strains from these patients, 8 of which encoded adhesins [Table 2].”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The 94 C. glabrata strains represented 4 sequence types and segregated into three distinct clades: ST3 (patients J, K, P, ST), ST26 (patient H), and ST10 (patients C, D, I, L) plus ST179 (patient EF).

    “Strains represented 4 sequence types (STs), and they segregated into three distinct clades: 1) ST3 (patients J, K, P, ST), 2) ST26 (patient H), 3) ST10 (patients C, D, I, L), and ST179 (patient EF) [Figures 2A and B].”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Mitochondrial genome homology among strains from patient J was 96.71%−99.98%, while for other strains within-patient homology was higher (99.69%−100% DNA identity).

    “Mitochondrial genome homology (i.e., DNA identity) among strains from patient J was 96.71%−99.98%. For other strains, within-patient homology was higher (99.69%−100% DNA identity).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    On average, 70.4% and 29.6% of variants were synonymous and non-synonymous, respectively; 99.5% and 0.5% of non-synonymous SNPs were missense and nonsense, respectively.

    “On average, 70.4% and 29.6% of variants were synonymous and non-synonymous, respectively; 99.5% and 0.5% of the non-synonymous SNPs were missense and nonsense, respectively.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    On the mitochondrial phylogenetic tree, strain J11 clustered with SCV strains and was closest to strain J8.

    “On mitochondrial phylogenetic tree, strain J11 clustered with SCV strains, and was closest to strain J8 [Figure 7B].”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Estimated mitochondrial genome copy numbers of J strains ranged from 1 to 35; most SCV strains had ≤18 copies, while strains J1-4 and J6 had 30 and 35 copies, respectively.

    “Estimated mtN ranged from 1 to 35. Most SCV strains had ≤18 copies (J9, n=1; J5, n=8; J10, n=13; J7 and J8, n=18). In contrast, estimated mtN for strains J1–4 and J6 were 30 and 35, respectively.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Strain J11 was a SCV that demonstrated slow growth in YPD media, inability to grow in YP glycerol, small size on SDA plates, purple colonies on eosin plates, and resistance to fluconazole, voriconazole and posaconazole.

    “J11 was a SCV that demonstrated slow growth in YPD media, inability to grow in YP glycerol, small size on SDA plates, purple colonies on eosin plates, and resistance to fluconazole, voriconazole and posaconazole.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There was a within-patient average of 3,598 nucleotide differences across all 10 patients, with J and C strains showing the highest (7,507) and lowest (2,067) within-patient nucleotide differences, respectively.

    “There was a within-patient average of 3,598 nucleotide differences across all 10 patients. J and C strains showed the highest (7,507) and lowest (2,067) within-patient nucleotide differences, respectively.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Fluconazole-resistant L strains harbored the PDR1 G346C mutation.

    “Resistant strains harbored the PDR1 G346C mutation described above.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    J11 clustered with other J strains on the nuclear genome phylogenetic tree and was therefore highly related to J strains recovered from the initial blood culture.

    “J11 clustered with other J strains on nuclear genome phylogenetic tree [Figure 7A]. Therefore, J11 was highly related to J strains recovered from the initial blood culture.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Patient L's blood cultures remained positive for C. glabrata ~48 hours after IV fluconazole treatment, and the clinical microbiology laboratory then identified the isolate as azole-resistant.

    “Upon diagnosis of fluconazole-susceptible C. glabrata BSI, patient L was treated with IV fluconazole. Blood cultures ~48 hours later remained positive for C. glabrata, which was now identified by the clinical microbiology laboratory as azole-resistant [Table 1].”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In both patients with detailed phenotypic analysis, the clinical microbiology laboratory recovered a fluconazole-susceptible index strain, but mixed fluconazole-susceptible and fluconazole-resistant populations were identified by the study.

    “In both patients, the clinical microbiology lab recovered a fluconazole-susceptible index strain, but we identified mixed fluconazole-susceptible and –resistant populations.”
    • Also stated in: § DISCUSSION · plain
      “In both patients, blood cultures were comprised of mixed fluconazole-susceptible and -resistant populations, but the clinical microbiology laboratory only identified a fluconazole-susceptible index strain.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Strains J1-J5 carried fewer mitochondrial nucleotide differences than did J6-J10, and they clustered in a clade with P, K and ST strains, in keeping with nuclear phylogeny.

    “Strains J1-J5 carried fewer mitochondrial nucleotide differences than did J6-J10 [Figure 3B and 3C], and they clustered in a clade with P, K and ST strains, in keeping with nuclear phylogeny.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Using site-directed mutagenesis, G346C is a gain-of-function mutation that results in PDR1 hyperactivation and enhanced downstream CDR1 activation.

    “Using site-directed mutagenesis, we demonstrated that G346C is a gain-of-function mutation that results in PDR1 hyperactivation and enhanced downstream CDR1 activation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Compared to NCV strain J1, SCV strain J9 was relatively resistant to neutrophil phagocytosis and killing, but it was significantly attenuated for virulence in mice with hematogenously disseminated candidiasis.

    “Compared to NCV strain J1, SCV strain J9 was relatively resistant to neutrophil phagocytosis and killing, but it was significantly attenuated for virulence in mice with hematogenously disseminated candidiasis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    NCV strains J1-J4 had normal numbers of mitochondria and few mitochondrial gene mutations.

    “NCV strains J1-J4 had normal numbers of mitochondria and few mitochondrial gene mutations.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Patient J subsequently had a relapsing infection due to a fluconazole-resistant SCV strain (J11) that clustered phylogenetically with SCVs from the initial blood culture.

    “Our finding has clinical significance since patient J subsequently had a relapsing infection due to a fluconazole-resistant SCV strain (J11) that clustered phylogenetically with SCVs from the initial blood culture.”
    Quote in the paperSubstantial claim

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

    Findingown data

    Two patients had invasive candidiasis within the previous 90 days.

    “and 2 patients had invasive candidiasis within the previous 90 days.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Azole-resistant strains from patient J carried wild-type PDR1, but they exhibited SCV phenotypes including slow growth on enriched medium, attenuated proliferation in non-fermentable carbon sources, and mitochondrial and aerobic respiratory defects.

    “Azole-resistant strains from patient J carried wild-type PDR1, but they exhibited SCV phenotypes including slow growth on enriched medium, attenuated proliferation in non-fermentable carbon sources, and mitochondrial and aerobic respiratory defects.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The longstanding paradigm is that most bloodstream infections, including candidemia, are caused by a population of genetically identical strains derived from a single organism that passes through a bottleneck.

    “The longstanding paradigm is that most BSIs, including candidemia, are caused by a population of genetically identical strains derived from a single organism that passes through a bottleneck (“single organism” or “independent action” hypothesis).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [11] Rubin LG. Bacterial colonization and infection resulting from multiplication of a single organism. Rev Infect Dis. 1987; 9(3): 488– 493. 3299635

      Not checked in the free run.

    • cites [12] Moxon ER, Murphy PA. Haemophilus influenzae bacteremia and meningitis resulting from survival of a single organism. Proc Natl Acad Sci U S A. 1978; 75(3): 1534– 1536. 306628 PMC411507

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Positive blood cultures from individual patients with monomicrobial carbapenem-resistant Klebsiella pneumoniae bloodstream infection were comprised of mixed populations of genetically and phenotypically diverse strains, including differences in antibiotic susceptibility and virulence.

    “We recently demonstrated, for the first time, that positive blood cultures from individual patients with monomicrobial bacterial BSI (carbapenem-resistant Klebsiella pneumoniae) were comprised of mixed populations of genetically and phenotypically diverse strains, including those demonstrating differences in antibiotic susceptibility and virulence16.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] Cheng S, Fleres G, Chen L, Within-Host Genotypic and Phenotypic Diversity of Contemporaneous Carbapenem-Resistant Klebsiella pneumoniae from Blood Cultures of Patients with Bacteremia. mBio. 2022; 13(6): e0290622. 36445082 PMC9765435

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    PDR1 regulates multidrug transporter gene CDR1, which is linked to fluconazole resistance.

    “PDR1 regulates multidrug transporter gene CDR1, which is linked to fluconazole resistance18.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [18] Tsai HF, Krol AA, Sarti KE, Bennett JE. Candida glabrata PDR1, a transcriptional regulator of a pleiotropic drug resistance network, mediates azole resistance in clinical isolates and petite mutants. Antimicrob Agents Chemother. 2006; 50(4): 1384– 1392. 16569856 PMC1426987

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Azole exposure has been linked to mitochondrial damage, and patient J received 28 days of fluconazole prophylaxis prior to bloodstream infection.

    “Azole exposure has been linked to mitochondrial damage37–41, and patient J received 28 days of fluconazole prophylaxis prior to BSI.”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [37] Brun S, Berges T, Poupard P, Mechanisms of azole resistance in petite mutants of Candida glabrata. Antimicrob Agents Chemother. 2004; 48(5): 1788– 1796. 15105136 PMC400549

      Not checked in the free run.

    • cites [38] Ferrari S, Sanguinetti M, De Bernardis F, Loss of mitochondrial functions associated with azole resistance in Candida glabrata results in enhanced virulence in mice. Antimicrob Agents Chemother. 2011; 55(5): 1852– 1860. 21321146 PMC3088236

      Not checked in the free run.

    • cites [39] Sanglard D, Ischer F, Bille J. Role of ATP-binding-cassette transporter genes in high-frequency acquisition of resistance to azole antifungals in Candida glabrata. Antimicrob Agents Chemother. 2001; 45(4): 1174– 1183. 11257032 PMC90441

      Not checked in the free run.

    • cites [40] Zhang X, Moye-Rowley WS. Saccharomyces cerevisiae multidrug resistance gene expression inversely correlates with the status of the F(0) component of the mitochondrial ATPase. J Biol Chem. 2001; 276(51): 47844– 47852. 11602584

      Not checked in the free run.

    • cites [41] Kaur R, Castano I, Cormack BP. Functional genomic analysis of fluconazole susceptibility in the pathogenic yeast Candida glabrata: roles of calcium signaling and mitochondria. Antimicrob Agents Chemother. 2004; 48(5): 1600– 1613. 15105111 PMC400560

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Azole resistance in various C. glabrata strains from patients L and J was associated with increased expression of transcriptional regulator gene PDR1 and, in turn, up-regulation of efflux gene CDR1.

    “Azole resistance in various C. glabrata strains from patients L and J was associated with increased expression of transcriptional regulator gene PDR1 and, in turn, up-regulation of efflux gene CDR1.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    A strength of the study design is that C. glabrata were collected as blood cultures were being processed according to standard clinical microbiology lab practices.

    “A strength of our study design is that C. glabrata were collected as blood cultures were being processed according to standard clinical microbiology lab practices.”
    Quote in the paperSubstantial claim

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

    Interpretationnone given

    The results cannot be extrapolated to BSIs by other Candida spp. or other pathogens.

    “Our results cannot be extrapolated to BSIs by other Candida spp. or other pathogens.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    In a WGS analysis of C. glabrata from longitudinal cultures of various body sites over 0-90 days, inter-strain genetic variation was primarily due to standing, pre-existing diversity within the population rather than to accumulation of new mutations.

    “In a WGS analyses of C. glabrata from longitudinal cultures of various body sites over 0–90 days, investigators previously concluded that inter-strain genetic variation was primarily due to standing, pre-existing diversity within the population rather than to accumulation of new mutations7.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [7] Carrete L, Ksiezopolska E, Gomez-Molero E, Genome Comparisons of Candida glabrata Serial Clinical Isolates Reveal Patterns of Genetic Variation in Infecting Clonal Populations. Front Microbiol. 2019; 10: 112. 30809200 PMC6379656

      Not checked in the free run.

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

    Interpretationreasoning

    The extent of diversity detected may reflect particular features of the patients, who were severely ill and had extensive past medical histories.

    “The extent of diversity we detected may reflect particular features of our patients, who were severely ill and who had extensive past medical histories (Table 1).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    SCV strain J9 was less virulent than NCV J1 following intravenous inoculation in the mouse model, but SCVs were able to persist in patient J after resolution of candidemia and re-emerge to cause relapsing infection.

    “For example, SCV strain J9 was less virulent than NCV J1 following intravenous inoculation in our mouse model, but nevertheless SCVs were able to persist in patient J after resolution of candidemia and re-emerge to cause relapsing infection.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Correct integration of the deletion cassette was verified by PCR.

    “Correct integration was verified by PCR.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All qRT-PCR experiments were done in independent biological triplicates and are shown as mean with standard deviation for each time point.

    “All experiments were done in independent biological triplicates and are shown as mean with standard deviation (SD) for each time point.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The cloned PDR1 sequence was confirmed by Sanger sequencing.

    “The cloned PDR1 sequence was confirmed by Sanger sequencing.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Three independent experiments were performed for the respirometry assays.

    “Three independent experiments were performed.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The study only sequenced strains from the first episode of BSI.

    “We only sequenced strains from the first episode of BSI.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    C. glabrata were confirmed by matrix assisted laser desorption ionization-time of flight mass spectrometry.

    “C. glabrata were confirmed by matrix assisted laser desorption ionization-time of flight mass spectrometry.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Previous studies of virulence of C. glabrata SCVs have yielded conflicting results.

    “Previous studies of virulence of C. glabrata SCVs have yielded conflicting results.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Antifungal susceptibility testing was performed using the broth dilution technique according to the Clinical and Laboratory Standards Institute (CLSI) standardized method.

    “Antifungal susceptibility testing was performed using the broth dilution technique according to the Clinical and Laboratory Standards Institute (CLSI) standardized method62.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [62] Institute CaLS. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard. Fourteh Edition. M27– A3. Wayne, PA.

      Not checked in the free run.

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

    Study design and conductnone given

    C. glabrata ATCC 90030 was incorporated into each set of antifungal susceptibility experiments as quality control.

    “C. glabrata ATCC 90030 was incorporated into each set of experiments as quality control.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    A C. albicans SCV that emerged after hematogenous passage through mouse organs caused lower mortality and acute tissue burdens during disseminated candidiasis than its parent strain, but it persisted within tissue for a prolonged period.

    “We previously reported that a C. albicans SCV that emerged after hematogenous passage through mouse organs caused lower mortality and acute tissue burdens during disseminated candidiasis than its parent strain, but it persisted within tissue for a prolonged period19.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] Cheng S, Clancy CJ, Zhang Z, Uncoupling of oxidative phosphorylation enables Candida albicans to resist killing by phagocytes and persist in tissue. Cell Microbiol. 2007; 9(2): 492– 501. 16987332

      Not checked in the free run.

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

    Study design and conductnone given

    Phagocytosis and killing experiments were performed in triplicate and repeated at least twice.

    “The experiments were performed in triplicate and repeated at least twice.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    A fluconazole-resistant oropharyngeal C. glabrata SCV was more virulent than an antecedent fluconazole-susceptible strain.

    “In another study, however, a fluconazole-resistant oropharyngeal C. glabrata SCV was more virulent than an antecedent fluconazole-susceptible strain38.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] Ferrari S, Sanguinetti M, De Bernardis F, Loss of mitochondrial functions associated with azole resistance in Candida glabrata results in enhanced virulence in mice. Antimicrob Agents Chemother. 2011; 55(5): 1852– 1860. 21321146 PMC3088236

      Not checked in the free run.

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

    Premisecitation

    An SCV generated by ethidium bromide treatment exhibited reduced virulence during murine disseminated candidiasis.

    “In one report, an SCV generated by ethidium bromide treatment exhibited reduced virulence during murine disseminated candidiasis.43”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [43] Brun S, Dalle F, Saulnier P, Biological consequences of petite mutations in Candida glabrata. J Antimicrob Chemother. 2005; 56(2): 307– 314. 15958498

      Not checked in the free run.

50 of 50

  • The paper relies on this without support.

    At present, it is unknown whether bloodstream infections are commonly caused by genetically diverse populations rather than single organisms.

    “At present, it is unknown if BSIs are commonly caused by genetically diverse populations, rather than single organisms.”

  • The paper relies on this without support.

    Standard microbiology laboratory protocol processes positive clinical cultures by testing single microbial strains from morphologically distinct colonies.

    “Standard microbiology laboratory protocol in processing positive clinical cultures is to test single microbial strains from morphologically distinct colonies.”

  • The paper relies on this without support.

    The ten enrolled patients are representative of patients with C. glabrata bloodstream infection.

    “We enrolled 10 patients with C. glabrata BSIs [Table 1].”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Petite C. glabrata colonies isolated from blood samples were efficiently cleared in the spleen, liver, and kidney of a systemic infection mouse model.

    “petite C. glabrata colonies isolated from blood samples from a recent study were also efficiently cleared in the spleen, liver, and kidney of a systemic infection mouse model (40).”

    From Overlooked <i>Candida glabrata</i> petites are echinocandin tolerant, induce host inflammatory responses, and display poor <i>in vivo</i> fitness (2023) · cites this paper as [40]

    The Results (Phenotypic diversity of C. glabrata strains; page unavailable) report that the blood-culture-derived small-colony strain J9 produced significantly lower kidney and spleen burdens than J1 on days 1, 3, and 7, and a lower liver burden on day 7. Lower burdens support reduced persistence relative to J1, but the passage does not establish that J9 was efficiently cleared from all three organs. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Underestimation of petite resulted in fluconazole therapeutic failure and the emergence of pure petite isolates from later blood samples.

    “this underestimation of petite resulted in fluconazole therapeutic failure and the emergence of pure petite isolates from later blood samples (40).”

    From Overlooked <i>Candida glabrata</i> petites are echinocandin tolerant, induce host inflammatory responses, and display poor <i>in vivo</i> fitness (2023) · cites this paper as [40]

    The Discussion (page unavailable) says petite variants may be underestimated because they appear late in culture, and reports a later relapsing infection with a fluconazole-resistant petite variant. It does not say underestimation caused fluconazole treatment failure. The Results, “Patient J” (page unavailable), place the later isolate in an intra-abdominal infection, not a later blood sample; the patient received caspofungin followed by voriconazole prophylaxis. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • The prevalence of petite isolates appeared to be 10% (1/10) in a recent candidemia study exploring the genotypic diversity of C. glabrata colonies growing from the same blood sample.

    “The prevalence of petite isolates also appeared to be 10% (1/10) in a recent candidemia study exploring the genotypic diversity of C. glabrata colonies growing from the same blood sample (40).”

    From Overlooked <i>Candida glabrata</i> petites are echinocandin tolerant, induce host inflammatory responses, and display poor <i>in vivo</i> fitness (2023) · cites this paper as [40]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Ten-strain-per-bottle statement overlooks patient I

    Minor

    The limitation says 10 strains were sequenced per blood culture bottle, but the strain labels specify only five strains for patient I (the index strain and strains #2-5).

1 of 1

Cites

Flagged first, then by citations

1 of the 69 references couldn’t be matched to a known paper.

Cited by

Sorted by citations

Related

Authors
Badrane, H., Cheng, S., Dupont, C., Hao, B., Driscoll, E., Morder, K., Liu, G., Newbrough, A., Fleres, G., Kaul, D., Espinoza, J., Clancy, C., & Nguyen, M. H.
Affiliations
  • University of Pittsburgh (US)
  • J. Craig Venter Institute
  • University of Pittsburgh
  • J. Craig Venter Institute (US)
Corresponding author
Nguyen, Minh Hong
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2023-04-03
Volume
14
Issue
1
Pages
5918-5918
ISSN
2041-1723 (ISSN-L)
DOI
10.21203/rs.3.rs-2706400/v1
PMC
PMC10104189
OpenAlex
W4386968272
Cited by
48
References
69
Citations by year
2026: 9 · 2025: 17 · 2024: 17 · 2023: 5
FWCI
9.88
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • U.S. Department of Health and Human Services (1U54GH009824)
  • U.S. Department of Veterans Affairs (1I01BX001955, I01 BX001955)
  • Division of Intramural Research, National Institute of Allergy and Infectious Diseases (R21AI152018, R21AI160098, 1R01AI170111-01)
  • University of Pittsburgh (SCR 022735, S10OD028483)
  • York University
  • National Institutes of Health (1U54GH009824, 1s10od028483-01a1)
  • Center for Research Computing, University of Pittsburgh (SCR_022735)
  • National Human Genome Research Institute (U54HG009824)
  • National Institute of Allergy and Infectious Diseases (R21AI152018, R21AI160098, R01AI170111)
Topics
  • Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Yeasts and Rust Fungi Studies
Keywords
Candida glabrata, bloodstream infection, genotypic diversity, antifungal resistance, fluconazole resistance, clonal populations, phagocytosis, small colony variants, virulence attenuation

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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