Whole-Genome and Targeted-Amplicon Sequencing of Fluconazole-Susceptible and -Resistant Candida parapsilosis Isolates from Kuwait Reveals a Previously Undescribed N1132D Polymorphism in CDR1.
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ABSTRACT:
SUBMITTER: Asadzadeh M
PROVIDER: S-EPMC7848974 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background and Purpose:Candida albicans is the most common Candida species (sp.) isolated from fungal infections. Azole resistance in Candida species has been considerably increased in the last decades. Given the toxicity of the antimicrobial drugs, resistance to antifungal agents, and drug interactions, the identification of new antifungal agents seems essential. In this study, we assessed the antifungal effects of biogenic selenium nanoparticles on C. albicans and determined the expression of ERG11 and CDR1 genes. Materials and Methods:Selenium nanoparticles were synthesized with Bacillus sp. MSH-1. The ultrastructure of selenium nanoparticles was evaluated with a transmission electron microscope. The antifungal susceptibility test was performed according to the modified Clinical and Laboratory Standards Institute M27-A3 standard protocol. The expression levels of the CDR1 and ERG11 genes were analyzed using the quantitative real-time polymerase chain reaction (PCR) assay. Results:The azole-resistant C. albicans and wild type C. albicans strains were inhibited by 100 and 70 µg/mL of selenium nanoparticle concentrations, respectively. The expression of CDR1 and ERG11 genes was significantly down-regulated in these selenium nanoparticle concentrations. Conclusion:As the findings indicated, selenium nanoparticles had an appropriate antifungal activity against fluconazole-resistant and -susceptible C.albicans strains. Accordingly, these nanoparticles reduced the expression of CDR1 and ERG11 genes associated with azole resistance. Further studies are needed to investigate the synergistic effects of selenium nanoparticles using other antifungal drugs.
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Project description:We investigated the azole resistance mechanisms and clinical features of fluconazole-nonsusceptible (FNS) isolates of Candida tropicalis recovered from Korean surveillance cultures in comparison with fluconazole-less-susceptible (FLS) isolates. Thirty-five clinical isolates of C. tropicalis, comprising 9 FNS (fluconazole MIC, 4 to 64 ?g/ml), 12 FLS (MIC, 1 to 2 ?g/ml), and 14 control (MIC, 0.125 to 0.5 ?g/ml) isolates, were assessed. CDR1, MDR1, and ERG11 expression was quantified, and the ERG11 and UPC2 genes were sequenced. Clinical features of 16 patients with FNS or FLS bloodstream isolates were analyzed. Both FNS and FLS isolates had >10-fold higher mean expression levels of CDR1, MDR1, and ERG11 genes than control isolates (P values of <0.02 for all). When FNS and FLS isolates were compared, FNS isolates had 3.4-fold higher mean ERG11 expression levels than FLS isolates (P = 0.004), but there were no differences in those of CDR1 or MDR1 Of all 35 isolates, 4 (2 FNS and 2 FLS) and 28 (8 FNS, 11 FLS, and 9 control) isolates exhibited amino acid substitutions in Erg11p and Upc2p, respectively. Both FNS and FLS bloodstream isolates were associated with azole therapeutic failure (3/4 versus 4/7) or uncleared fungemia (4/6 versus 4/10), but FNS isolates were identified more frequently from patients with previous azole exposure (6/6 versus 3/10; P = 0.011) and immunosuppression (6/6 versus 3/10; P = 0.011). These results reveal that the majority of FNS C. tropicalis isolates show overexpression of CDR1, MDR1, and ERG11 genes, and fungemia develops after azole exposure in patients with immunosuppression.
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Project description:We recently observed the emergence of fluconazole-resistant Candida parapsilosis bloodstream isolates harboring a Y132F substitution in Erg11p in South Korea. These Y132F isolates had a higher propensity to cause clonal transmission than other fluconazole-resistant isolates and persisted within hospitals for several years, as revealed by microsatellite typing.
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Project description:We used whole-genome sequencing to analyse a collection of 35 fluconazole resistant and 7 susceptible Candida parapsilosis isolates together with coverage analysis and GWAS techniques to identify new mechanisms of fluconazole resistance. Phylogenetic analysis shows that although the collection is diverse, two probable outbreak groups were identified. We identified copy number variation of two genes, ERG11 and CDR1B, in resistant isolates. Two strains have a CNV at the ERG11 locus; the entire ORF is amplified in one, and only the promoter region is amplified in the other. We show the annotated telomeric gene CDR1B is actually an artefactual in silico fusion of two highly similar neighbouring CDR genes due to an assembly error in the C. parapsilosis CDC317 reference genome. We report highly variable copy numbers of the CDR1B region across the collection. Several strains have increased expansion of the two genes into a tandem array of new chimeric genes. Other strains have experienced a deletion between the two genes creating a single gene with a reciprocal chimerism. We find translocations, duplications, and gene conversion across the CDR gene family in the C. parapsilosis species complex, showing that it is a highly dynamic family.
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Project description:Fluconazole-resistant Candida parapsilosis (FLZR-CP) outbreaks are a growing public health concern and have been reported in numerous countries. Patients infected with FLZR-CP isolates show fluconazole therapeutic failure and have a significantly increased mortality rate. Because fluconazole is the most widely used antifungal agent in most regions with outbreaks, it is paramount to restore its antifungal activity. Milbemycin oxim (MOX), a well-known canine endectocide, is a potent efflux pump inhibitor that significantly potentiates the activity of fluconazole against FLZR C. glabrata and C. albicans. However, the FLZ-MOX combination has not been tested against FLZR-CP isolates, nor is it known whether MOX may also potentiate the activity of echinocandins, a different class of antifungal drugs. Furthermore, the extent of involvement of efflux pumps CDR1 and MDR1 and ergosterol biosynthesis enzyme ERG11 and their link with gain-of-function (GOF) mutations in their transcription regulators (TAC1, MRR1, and UPC2) are poorly characterized among FLZR-CP isolates. We analyzed 25 C. parapsilosis isolates collected from outbreaks in Turkey and Brazil by determining the expression levels of CDR1, MDR1, and ERG11, examining the presence of potential GOF mutations in their transcriptional regulators, and assessing the antifungal activity of FLZ-MOX and micafungin-MOX against FLZR and multidrug-resistant (MDR) C. parapsilosis isolates. ERG11 was found to be universally induced by fluconazole in all isolates, while expression of MDR1 was unchanged. Whereas mutations in MRR1 and UPC2 were not detected, CDR1 was overexpressed in three Brazilian FLZR-CP isolates, which also carried a novel TAC1L518F mutation. Of these three isolates, one showed increased basal expression of CDR1, while the other two overexpressed CDR1 only in the presence of fluconazole. Interestingly, MOX showed promising antifungal activity against FLZR isolates, reducing the FLZ MIC 8- to 32-fold. However, the MOX and micafungin combination did not exert activity against an MDR C. parapsilosis isolate. Collectively, our study documents that the mechanisms underpinning FLZR are region specific, where ERG11 mutations were the sole mechanism of FLZR in Turkish FLZR-CP isolates, while simultaneous overexpression of CDR1 was observed in some Brazilian counterparts. Moreover, MOX and fluconazole showed potent synergistic activity, while the MOX-micafungin combination showed no synergy.
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Project description:Antimicrobial drug resistance can limit the ability to effectively treat patients. Numerous factors have been proposed to impact the development of antimicrobial resistance, including those specific to the drug and the dosing regimen. The field of investigation that examines the relationship between dosing regimen and outcome is termed antimicrobial pharmacokinetics and pharmacodynamics. Our prior in vivo investigations examined the relationship between fluconazole pharmacodynamics and the modulation of isogenic resistant and susceptible Candida albicans populations in a mixed-inoculum design (1). The goal of the current studies was to examine the impact of fluconazole pharmacodynamics on resistance emergence from a susceptible parent population over time using a murine systemic-candidiasis model. Both microbiologic and transcriptional endpoints were examined during the evolution of cell populations. As in our previous investigation, the more frequently administered dosing regimen prevented the emergence of a resistant cell phenotype. Conversely, dosing regimens that produced prolonged sub-MIC concentrations were associated with resistance development. The studies also demonstrated a striking relationship between fluconazole pharmacodynamic exposures and the mRNA abundance of drug resistance-associated efflux pumps. Global transcriptional profiling of cell populations during the progressive emergence of a resistance phenotype provides insight into the mechanisms underlying this complex physiologic process.
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Project description:Candida parapsilosis causes ~35% of all candidemia cases in neonates. High-resolution fingerprinting of C. parapsilosis isolates from neonatal intensive care unit (NICU) patients in Maternity Hospital (MH) was performed to identify epidemiologically related strains. Sixty-eight bloodstream/colonizing strains isolated from 59 NICU patients, two isolates from health care workers (HCWs) from MH and 18 bloodstream isolates from two other hospitals were used. Six microsatellite markers were employed, isolates were assigned a numerical microsatellite genotype (MSG), dendrogram was constructed and similarities between genotypes were visualized by minimum spanning tree. Fifty bloodstream isolates from MH yielded 37 MSGs with 20 isolates clustering in 7 MSGs. Duplicate isolates and colonizing strains yielded same/highly similar MSG as bloodstream isolates. Colonizing strains from two non-candidemia patients yielded unique MSGs while others belonged to a cluster. All isolates from HCWs and from two other hospitals belonged to unique MSGs. Cluster isolates came from patients in NICU-1 or from neonates in NICU-1 and other NICUs. Clonal complexes comprising closely related genotypes indicative of microevolution were also detected. Our data show that some C. parapsilosis strains have persisted in MH environment over several years and these endemic genotypes were transmitted to other patients in NICU-1 and/or other nearby NICUs.
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Project description:Candida dubliniensis and Candida albicans are two closely related species. Although C. dubliniensis is less pathogenic, it has a higher propensity to develop resistance to fluconazole and some strains exhibit intrinsic resistance to 5-flucytosine (5-FC). All 5-FC-resistant isolates from Kuwait were previously shown to belong to one of seven internal transcribed spacer (ITS) region of rDNA-based haplotypes. This study performed fingerprinting of C. dubliniensis isolates by multilocus sequence typing (MLST) to determine population structure of 5-FC-resistant and -susceptible strains and compared the results with data from a global collection of isolates. Fifty-two C. dubliniensis isolates previously analyzed and 58 additional isolates mostly collected during 2010-2013 and characterized by phenotypic and molecular methods were used. ITS-based haplotypes were identified by haplotype-specific PCR and/or by PCR-DNA sequencing of rDNA. Population structure was determined by 8-loci-based MLST. E-test was used to determine susceptibility to 5-FC, fluconazole, voriconazole and amphotericin B. Five ITS haplotypes (ITSH) were detected among 110 C. dubliniensis isolates. The ITSH1 was most common (n = 80 isolates) followed by ITSH4 (n = 25 isolates). Two isolates each belonged to ITSH5 and ITSH8 while one isolate belonged to ITSH7. MLST identified 16 diploid sequence types (DSTs) including six new DSTs. DST11 (n = 52) and DST14 (n = 25) were dominant genotypes and were confined (together with DST21) to Middle-Eastern countries. Other DSTs (excluding some new DSTs) had a wider global distribution as they were identified from various other countries. Only ITSH4 isolates (n = 25) belonged to DST14, were resistant to 5-FC and contained S29L mutation in CdFCA1. ITSH5, ITSH7 and ITSH8 isolates belonged to different DSTs. Thus, clinical C. dubliniensis isolates in Kuwait exhibited limited genotypic heterogeneity and most isolates belonged to region-specific DSTs. All 5-FC-resistant C. dubliniensis isolates belonged to ITSH4 and MLST-based DST14 genotype. Placement of some isolates into additional ITS haplotypes is also supported by MLST data.
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Project description:Candida tropicalis is the fourth leading cause of candidemia in Turkey. Although C. tropicalis isolates from 1997 to 2017 were characterized as fully susceptible to antifungals, the increasing global prevalence of azole-non-susceptible (ANS) C. tropicalis and the association between high fluconazole tolerance (HFT) and fluconazole therapeutic failure (FTF) prompted us to re-evaluate azole susceptibility of C. tropicalis in Turkey. In this study, 161 C. tropicalis blood isolates from seven clinical centers were identified by ITS rDNA sequencing, genotyped by multilocus microsatellite typing, and tested for susceptibility to five azoles, two echinocandins, and amphotericin B (AMB); antifungal resistance mechanisms were assessed by sequencing of ERG11 and FKS1 genes. The results indicated that C. tropicalis isolates, which belonged to 125 genotypes grouped into 11 clusters, were fully susceptible to echinocandins and AMB; however, 18.6% of them had the ANS phenotype but only two carried the ANS-conferring mutation (Y132F). HFT was recorded in 52 isolates, 10 of which were also ANS. Large proportions of patients infected with ANS and HFT isolates (89 and 40.7%, respectively) showed FTF. Patients infected with azole-susceptible or ANS isolates did not differ in mortality, which, however, was significantly lower for those infected with HFT isolates (P = 0.007). There were significant differences in mortality (P = 0.02), ANS (P = 0.012), and HFT (P = 0.007) among genotype clusters. The alarming increase in the prevalence of C. tropicalis blood isolates with ANS and HFT in Turkey and the notable FTF rate should be a matter of public health concern.
| S-EPMC7573116 | biostudies-literature
Project description:Candida parapsilosis is a common cause of invasive candidiasis worldwide and is the most commonly is7olated Candida species among pediatric and neonatal populations. Previous work has demonstrated that nonsynonymous mutations in the gene encoding the putative transcription factor CpMrr1 can influence fluconazole susceptibility. However, the direct contribution of these mutations and how they influence fluconazole resistance in clinical isolates are poorly understood. We identified 7 nonsynonymous CpMRR1 mutations in 12 isolates from within a collection of 35 fluconazole-resistant clinical isolates. The mutations leading to the A854V, R479K, and I283R substitutions were further examined and found to be activating mutations leading to increased fluconazole resistance. In addition to CpMDR1, we identified two other genes, one encoding a major facilitator superfamily (MFS) transporter (CpMDR1B, CPAR2_603010) and one encoding an ATP-binding cassette (ABC) transporter (CpCDR1B, CPAR2_304370), as being upregulated in isolates carrying CpMRR1-activating mutations. Overexpression of CpMDR1 in a susceptible strain and disruption in resistant clinical isolates that overexpress CpMDR1 had little to no effect on fluconazole susceptibility. Conversely, overexpression of either CpMDR1B or CpCDR1B increased resistance, and disruption in clinical isolates overexpressing these genes decreased fluconazole resistance. Our findings suggest that activating mutations in CpMRR1 represent important genetic determinants of fluconazole resistance in clinical isolates of C. parapsilosis, and unlike what is observed in Candida albicans, this is primarily driven by upregulation of both MFS (CpMdr1B) and ABC (CpCdr1B) transporters.
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