Global Analysis of the Evolution and Mechanism of Echinocandin Resistance in Candida glabrata

The evolution of drug resistance has a profound impact on human health. Candida glabrata is a leading human fungal pathogen that can rapidly evolve resistance to echinocandins, which target cell wall biosynthesis and are front-line therapeutics for Candida infections. Here, we provide the first global analysis of mutations accompanying the evolution of fungal drug resistance in a human host utilizing a series of C. glabrata isolates that evolved echinocandin resistance in a patient treated with the echinocandin caspofungin for recurring bloodstream candidemia. Whole genome sequencing identified a mutation in the drug target, FKS2, accompanying a major resistance increase, and 8 additional non-synonymous mutations. The FKS2-T1987C mutation was sufficient for echinocandin resistance, and associated with a fitness cost that was mitigated with further evolution, observed in vitro and in a murine model of systemic candidemia. A CDC6-A511G(K171E) mutation acquired before FKS2-T1987C(S663P), conferred a small resistance increase. Elevated dosage of CDC55, which acquired a C463T(P155S) mutation after FKS2-T1987C(S663P), ameliorated fitness. To discover strategies to abrogate echinocandin resistance, we focused on the molecular chaperone Hsp90 and downstream effector calcineurin. Genetic or pharmacological compromise of Hsp90 or calcineurin function reduced basal tolerance and resistance. Hsp90 and calcineurin were required for caspofungin-dependent FKS2 induction, providing a mechanism governing echinocandin resistance. A mitochondrial respiration-defective petite mutant in the series revealed that the petite phenotype does not confer echinocandin resistance, but renders strains refractory to synergy between echinocandins and Hsp90 or calcineurin inhibitors. The kidneys of mice infected with the petite mutant were sterile, while those infected with the HSP90-repressible strain had reduced fungal burden. We provide the first global view of mutations accompanying the evolution of fungal drug resistance in a human host, implicate the premier compensatory mutation mitigating the cost of echinocandin resistance, and suggest a new mechanism of echinocandin resistance with broad therapeutic potential.     

Green synthesis of selenium nanoparticles and evaluate their effect on the expression of ERG3, ERG11 and FKS1 antifungal resistance genes in Candida albicans and Candida glabrata

Drug resistance in Candida species has been considerably increased in the last decades. Given the opposition to antifungal agents, toxicity and interactions of the antimicrobial drugs, identifying new antifungal agents seems essential. This study assessed the antifungal effects of nanoparticles (NPs) on the standard strains of Candida albicans and Candida glabrata and determined the expression genes, including ERG3, ERG11 and FKS1. Selenium nanoparticles (Se-NPs) were biosynthesized with a standard strain of C. albicans and approved by several methods including, ultraviolet-visible spectrophotometer, X-ray diffraction technique, Fourier-transform infrared analysis, field-emission scanning electron microscopy and EDX diagram. The antifungal susceptibility testing performed the minimum inhibitory concentrations (MICs) using the CLSI M27-A3 and M27-S4 broth microdilution method. The expression of the desired genes was examined by the real-time PCR assay between untreated and treated by antifungal drugs and Se-NPs. The MICs of itraconazole, amphotericin B and anidulafungin against C. albicans and C. glabrata were 64, 16 and 4 µg ml⁻¹. In comparison, reduced the MIC values for samples treated with Se-NPs to 1 and 0·5 µg ml⁻¹. The results obtained from real-time PCR and analysis of the ∆∆C_q values showed that the expression of ERG3, ERG11 and FKS1 genes was significantly down-regulated in Se-NPs concentrations (P < 0·05). This study's evidence implies biosafety Se-NPs have favourable effects on the reducing expression of ERG3, ERG11 and FKS1 antifungal resistance genes in C. albicans and C. glabrata.     

The new mutation L321F in Candida albicans ERG11 gene may be associated with fluconazole resistance

BackgroundFor many years fluconazole has been commonly used to treat Candida infections. However, the indiscriminate use of this antimycotic therapy has favored the emergence of resistant isolates. Mutations in the ERG11 gene have been described as one of the primary mechanisms of resistance in Candida species.AimsIn this study we investigated missense mutations in ERG11 genes of Candida albicans, Candida glabrata and Candida tropicalis isolates previously evaluated by susceptibility testing to fluconazole.MethodsScreening for these mutations was performed on 19 Candida clinical isolates (eight C. albicans, five C. glabrata and six C. tropicalis) resistant and susceptible to fluconazole. The ERG11 gene was amplified by PCR with specific primers for each Candida species and analyzed by automated sequencing.ResultsWe identified 14 different missense mutations, five of which had not been described previously. Among them, a new mutation L321F was identified in a fluconazole resistant C. albicans isolate and it was analyzed by a theoretical three-dimensional structure of the ERG11p.ConclusionThe L321F mutation in C. albicans ERG11 gene may be associated with fluconazole resistance.     

Effect of temperature and water activity on the production of fumonisins by Aspergillus niger and different Fusariumspecies

Background

Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing.

Results

The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate.

Conclusion

The sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.     

SSCP analysis (ITS2, ERG11) in clinical Candida isolates from oral cavity in oncology patients

Antifungal prophylactic therapy in oncology patients has favors the emergence of diverse species of Candida. In the present study 32 clinic isolates of Candida spp., recovered from oral cavity, were evaluated testing their susceptibility to diverse antifungals by means of the microdilution method according with the document M-27A of the National Committee for Clinical Laboratory Standards (NCCLS). The response to antifungals was then compared with SSCP patterns of the gene ERG11, hoping to find mutations in this gene linked to resistance to the azole antifungals from samples of clinical origin. Three SSCP genotypes with diverse response to different antifungals were found. This study suggested that a link of mutations in the ERG11 gene of Candida albicans and antifungal resistance was not supported by this data. In addition, the isolates were also classified by conventional methods and their genetic diversity evaluated by means of SSCP analysis and evaluation of their ITS2 regions, identifying eight SSCP genotypes. This technique has the potential to be a more sensitive method for taxonomic classification.     

热带假丝酵母唑类耐药相关基因的研究进展

临床上热带假丝酵母(又称热带念珠菌)的分离率越来越高,唑类抗真菌药物因较低的细胞毒性且大多可口服给药,是治疗热带念珠菌感染的常用药物。我国耐唑类药物热带念珠菌的分离率较高,因此有必要了解其具体机制,为寻求新的药物作用靶点提供依据。目前认为,与热带念珠菌唑类耐药有关的主要机制有靶基因ERG11过度表达和突变、编码转录因子的upc2基因过度表达和突变、外排泵基因过度表达及其他相关基因过度表达等。本文就目前热带念珠菌唑类耐药机制的基因水平研究进展进行综述。     

Correlation between broth microdilution and disk diffusion methods for antifungal susceptibility testing of caspofungin, voriconazole, amphotericin B, itraconazole and fluconazole against Candida glabrata

Candida glabrata is one of the most frequent organisms isolated from superficial and invasive fungal infections, after Candida albicans. This organism also exhibits intrinsically low susceptibility to azole antifungals and treatment often fails. The microdilution method is not very practical for use in routine susceptibility testing in the clinical laboratory, thus necessitating the use of other methods. In this study, we compared the in vitro activity of five antifungal agents in three different groups (echinocandin, polyene and azole) against 50 C. glabrata isolates by broth microdilution and disk diffusion methods recommended by Clinical Laboratory Standards Institute CLSI M27-A3 and CLSI M44-A, respectively. All the isolates were susceptible to amphotericin B (100%) and 98% of the isolates were susceptible to caspofungin by the broth microdilution method. Within the azole group drugs, voriconazole was the most active followed by fluconazole and itraconazole in vitro. The highest rate of resistance was obtained against itraconazole with a high number of isolates defined as susceptible-dose dependent or resistant. Although the disk diffusion method is easy to use in clinical laboratories, it shows very poor agreement with the reference method for fluconazole and itraconazole against C. glabrata (8% and 14%, respectively).     

CRS–MIS in Candida glabrata: sphingolipids modulate echinocandin–Fks interaction

Infections with the azole‐refractory yeast Candida glabrata are now commonly treated with the echinocandins caspofungin (CSF) or micafungin (MCF). True resistance (> 32‐fold decreased susceptibility) to these lipopeptide inhibitors of cell wall synthesis is rare and strictly associated with mutations in integral membrane proteins Fks1 or Fks2. In contrast, mutants exhibiting 4‐ to 32‐fold CSF reduced susceptibility (CRS) were readily selected in vitro, and surprisingly demonstrated 4‐ to 32‐fold MCF increased susceptibility (MIS). Sequencing and gene deletion demonstrated that CRS–MIS is Fks‐independent. To explore alternative mechanisms, we initially employed Saccharomyces cerevisiae, and observed that CRS was conferred by multiple mutations (fen1Δ, sur4Δ, cka2Δ and tsc10‐ts) disrupting sphingolipid biosynthesis. Following this lead, C. glabrata fen1Δ and cka2Δ deletants were constructed, and shown to exhibit CRS–MIS. Sphingolipid analysis of CRS–MIS laboratory mutants and clinical isolates demonstrated elevated dihydrosphingosine (DHS) and phytosphingosine (PHS) levels, and consistent with this sequencing revealed fen1, sur4, ifa38 and sur2 mutations. Moreover, exogenous DHS or PHS conferred a CRS–MIS phenotype on wild‐type C. glabrata. Exogenous PHS failed, however, to suppress CRS–MIS in a sur2 mutant blocked in conversion of DHS to PHS, implying that accumulation of these intermediates confers CRS–MIS. We conclude that membrane sphingolipids modulate echinocandin–Fks interaction.     

Novel point mutations in the ERG11 gene in clinical isolates of azole resistant Candida species

The azoles are the class of medications most commonly used to fight infections caused by Candida sp. Typically, resistance can be attributed to mutations in ERG11 gene (CYP51) which encodes the cytochrome P450 14α-demethylase, the primary target for the activity of azoles. The objective of this study was to identify mutations in the coding region of theERG11 gene in clinical isolates of Candidaspecies known to be resistant to azoles. We identified three new synonymous mutations in the ERG11 gene in the isolates of Candida glabrata (C108G, C423T and A1581G) and two new nonsynonymous mutations in the isolates of Candida krusei - A497C (Y166S) and G1570A (G524R). The functional consequence of these nonsynonymous mutations was predicted using evolutionary conservation scores. The G524R mutation did not have effect on 14α-demethylase functionality, while the Y166S mutation was found to affect the enzyme. This observation suggests a possible link between the mutation and dose-dependent sensitivity to voriconazole in the clinical isolate of C. krusei. Although the presence of the Y166S in phenotype of reduced azole sensitivity observed in isolate C. kruseidemands investigation, it might contribute to the search of new therapeutic agents against resistant Candida isolates.     

Effect of antifungals on itraconazole resistant <Emphasis Type="Italic">Candida glabrata</Emphasis>

In the last decade, infections caused by Candida glabrata have become more serious, particularly due to its decreased susceptibility to azole derivatives and its ability to form biofilm. Here we studied the resistance profile of 42 C. glabrata clinical isolates to different azoles, amphotericin B and echinocandins. This work was also focused on the ability to form biofilm which plays a role in the development of antifungal resistance. The minimal inhibitory concentration testing to antifungal agents was performed according to the CLSI (Clinical and Laboratory Standards Institute) M27-A3 protocol. Quantification of biofilm was done by XTT reduction assay. All C. glabrata clinical isolates were resistant to itraconazole and sixteen also showed resistance to fluconazole. All isolates remained susceptible to voriconazole. Amphotericin B was efficient in a concentration range of 0.125–1 mg/L. The most effective antifungal agents were micafungin and caspofungin with the MIC₁₀₀ values of ≤0.0313–0.125 mg/L. Low concentrations of these agents reduced biofilm formation as well. Our results show that resistance of different C. glabrata strains is azole specific and therefore a single azole resistance cannot be assumed to indicate general azole resistance. Echinocandins proved to have very high efficacy against clinical C. glabrata strains including those with ability to form biofilm.     

Antifungal Susceptibility Testing of <Emphasis Type="Italic">Candida</Emphasis> and <Emphasis Type="Italic">Cryptococcus</Emphasis> Species and Mechanisms of Resistance: Implications for Clinical Laboratories

Purpose of Review

Resistance to antifungal drugs amongst Candida species is a growing concern, and azole resistance may be emerging in Cryptococcus species. This review provides a contemporary perspective, relevant to the clinical mycology laboratory, of antifungal susceptibility testing of these fungi, focussing on the challenges of phenotypic and genotypic methodologies to detect drug resistance.

Recent Findings

Standardised CLSI and EUCAST broth microdilution (BMD) susceptibility testing methods are the benchmark to determine clinical breakpoints (CBPs) and/or epidemiological cut-off values (ECVs) MICs for Candida and Cryptococcus spp. Commercial methods may be used but caution is required when employing BMD CBPs/ECVs to interpret results. Species-specific CBPs/ECVs for Candida spp. generally correlate well with predicting likelihood of therapeutic failure or of presence of a drug resistance mechanism with the exception of the echinocandins where the presence of specific FKS gene mutations and not the MIC correlates most accurately with clinical outcome. The relationship of presence of one or more mechanisms of azole resistance and drug MICs is uncertain. Next generation sequencing technology is offering insights into the relationships between susceptibility results obtained by phenotypic and genotypic methods. For Cryptococcus spp., CBPs are not established but species- and genetic type-specific EVCs are useful for guiding therapy where clinically indicated. Isolates of genotype VGII appear to exhibit the highest MICs.

Summary

Antifungal susceptibility testing of yeasts is important to detect drug resistance. For Candida spp., MICs have clinical utility for the azoles but detecting echinocandin resistance by genotypic methods is preferred. For Cryptococcus spp., ECVs are useful in guiding therapy.

     

Asexual sporulation facilitates adaptation: The emergence of azole resistance in Aspergillus fumigatus

Understanding the occurrence and spread of azole resistance in Aspergillus fumigatus is crucial for public health. It has been hypothesized that asexual sporulation, which is abundant in nature, is essential for phenotypic expression of azole resistance mutations in A. fumigatus facilitating subsequent spread through natural selection. Furthermore, the disease aspergilloma is associated with asexual sporulation within the lungs of patients and the emergence of azole resistance. This study assessed the evolutionary advantage of asexual sporulation by growing the fungus under pressure of one of five different azole fungicides over seven weeks and by comparing the rate of adaptation between scenarios of culturing with and without asexual sporulation. Results unequivocally show that asexual sporulation facilitates adaptation. This can be explained by the combination of more effective selection because of the transition from a multicellular to a unicellular stage, and by increased mutation supply due to the production of spores, which involves numerous mitotic divisions. Insights from this study are essential to unravel the resistance mechanisms of sporulating pathogens to chemical compounds and disease agents in general, and for designing strategies that prevent or overcome the emerging threat of azole resistance in particular.     

牛源近平滑念珠菌对氟康唑耐药性研究

以牛源近平滑念珠菌（Candida parapsilosis）为试验菌株,采用微量稀释法进行药物敏感性试验,PCR扩增测序检测ERG11基因突变,Realtime PCR检测ERG11、CDR1、MDR1、MRR1基因的mRNA表达量,探讨耐药相关基因在牛源近平滑念珠菌耐唑类药物中的作用,为牛源近平滑念珠菌的耐药研究提供参考。结果表明,近平滑念珠菌对5-氟胞嘧啶、两性霉素Ｂ的敏感率均高于75%,对唑类药物的耐药率均高于50%,其中对氟康唑的耐药率最高,达58.3%;所有菌株的ERG11基因中均检测出错义突变A395T,耐氟康唑和剂量依赖菌株的ERG11基因中检测出同义突变T591C;氟康唑耐药组ERG11、CDR1、 MDR1、MRR1基因表达水平均显著高于敏感组（P<0.05）。牛源近平滑念珠菌对唑类抗真菌药物的耐药率较高且具有多重耐药性。牛源近平滑念珠菌ERG11基因中的T591C突变以及ERG11、CDR1、MDR1、MRR1基因的高表达都可能在其对氟康唑耐药性的产生中起到一定的作用。     

Genome‐wide association and epistasis studies unravel the genetic architecture of sudden death syndrome resistance in soybean

Soybean [Glycine max (L.) Merr.] is an economically important crop that is grown worldwide. Sudden death syndrome (SDS), caused by Fusarium virguliforme, is one of the top yield‐limiting diseases in soybean. However, the genetic basis of SDS resistance, especially with respect to epistatic interactions, is still unclear. To better understand the genetic architecture of soybean SDS resistance, genome‐wide association and epistasis studies were performed using a population of 214 germplasm accessions and 31 914 SNPs from the SoySNP50K Illumina Infinium BeadChip. Twelve loci and 12 SNP–SNP interactions associated with SDS resistance were identified at various time points after inoculation. These additive and epistatic loci together explained 24–52% of the phenotypic variance. Disease‐resistant, pathogenesis‐related and chitin‐ and wound‐responsive genes were identified in the proximity of peak SNPs, including stress‐induced receptor‐like kinase gene 1 (SIK1), which is pinpointed by a trait‐associated SNP and encodes a leucine‐rich repeat‐containing protein. We report that the proportion of phenotypic variance explained by identified loci may be considerably improved by taking epistatic effects into account. This study shows the necessity of considering epistatic effects in soybean SDS resistance breeding using marker‐assisted and genomic selection approaches. Based on our findings, we propose a model for soybean root defense against the SDS pathogen. Our results facilitate identification of the molecular mechanism underlying SDS resistance in soybean, and provide a genetic basis for improvement of soybean SDS resistance through breeding strategies based on additive and epistatic effects.