Dataset Information

Genetic Basis of Azole and Echinocandin Resistance in Clinical Candida glabrata in Japan.

ABSTRACT: Infections caused by Candida glabrata have caused worldwide concern, especially when they are associated with increasing echinocandin and azole resistance. In this study, we analyzed the molecular mechanisms of azole and echinocandin resistance in C. glabrata isolates obtained from hospitalized patients in Japan from 1997 to 2019. All isolates were checked phenotypically for resistance and genotypically for mutations in PDR1, ERG11, hot spot 1 (HS1), HS2, and HS3 of FKS1, and HS1 and HS2 of FKS2, and all isolates were genotyped by multilocus sequence typing (MLST). Interestingly, 32.6% of the isolates were resistant to caspofungin, and 4.7% were resistant to micafungin. The isolates showed low rates of resistance to azoles, ranging from 2.3% to 9.3%, and only 4.7% of the isolates were non-wild type for flucytosine susceptibility. For the first time in Japan, 4.7% of the isolates were identified as multidrug-resistant strains. Nonsynonymous mutations in PDR1, including two novel mutations associated with azole resistance, were identified in 39.5% of the isolates, and a single nonsynonymous mutation was identified in ERG11 Nine isolates from the same patient harbored nonsynonymous mutations in HS1 of FKS2, and a single isolate harbored a single nonsynonymous mutation in HS1 of FKS1 MLST genotyping revealed 13 different sequence types (STs), with 3 new STs, and ST7 was the most prevalent among the patients (35%) and was associated with high resistance rates. Our results are of crucial clinical concern, since understanding the molecular mechanisms underlying fungal resistance is imperative for guiding specific therapy for efficient patient treatment and promoting strategies to prevent epidemic spread.

SUBMITTER: Khalifa HO

PROVIDER: S-EPMC7449192 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Genetic Basis of Azole and Echinocandin Resistance in Clinical Candida glabrata in Japan.

Khalifa Hazim O HO Arai Teppei T Majima Hidetaka H Watanabe Akira A Kamei Katsuhiko K

Antimicrobial agents and chemotherapy 20200820 9

Infections caused by Candida glabrata have caused worldwide concern, especially when they are associated with increasing echinocandin and azole resistance. In this study, we analyzed the molecular mechanisms of azole and echinocandin resistance in C. glabrata isolates obtained from hospitalized patients in Japan from 1997 to 2019. All isolates were checked phenotypically for resistance and genotypically for mutations in PDR1, ERG11, hot spot 1 (HS1), HS2, and HS3 of < ...[more]

PMID: 32571826

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Project description:Microarray was used to analyze azole resistance of Candida glabrata oropharyngeal isolates from 7 hematopoietic stem cell transplant recipients receiving fluconazole prophylaxis. Transcriptional profiling of the sequential-paired clinical isolates by microarray revealed 19 genes upregulated in the majority of resistant isolates compared to their paired-susceptible isolates. All seven resistant isolates had greater than two fold upregulation of CgPDR1, a master transcriptional regulator of PDR network, and all 7 resistant isolates showed upregulation of known CgPDR1-target genes. The altered transcriptome can be explained in part by the observation that all 7 resistant isolates had acquired a single nonsynonymous mutation in their CgPDR1 ORF. Four mutations occurred in the regulatory domain (L280P, L344S, G348A, S391L) and one in the activation domain (G943S) while two mutations (N764I, R772I) occurred in an undefined region. Association of azole resistance and the CgPDR1 mutations was investigated in the same genetic background by introducing the CgPDR1 sequences from one sensitive and five resistant isolates into a laboratory azole-sensitive strain (cgpdr1) via integrative transformation. The cgpdr1 strain was restored to wild-type fluconazole susceptibility when transformed with CgPDR1 from the susceptible isolate but became resistant when transformed with CgPDR1 from the resistant isolates. However, despite the identical genetic background, upregulation of CgPDR1 and CgPDR1-target genes varied between the 5 transformants, independent of the domain locations in which the mutations occurred. In sum, gain-of-function mutations in CgPDR1 not only contributed to the clinical azole resistance but different mutations had varying degrees of impact on the CgPDR1-target genes. Two groups (C1S/84 and C16R/84) consisted of 4 biological replicates in which a complemented strain sample was paired with the laboratory wild type strain 84 sample. Each group included 1 reciprocally labeled sample.

Project description:Purpose: Candida glabrata has emerged as the second or third most common non-albicans species responsible for an increasing number of systemic infections. Moreover, its high-level of resistance to azole is associated with a high mortality rate. This study aimed to evaluate nosocomial infections and resistance characteristics of C. glabrata and to explore the mechanism of azole resistance in C. glabrata. Patients and methods: Fifty-nine clinical C. glabrata isolates were collected from two hospitals in China. The susceptibility of the strains to antifungal agents was determined by both the ATB Fungus 3 strip and CLSI M27 broth microdilution method. Efflux of rhodamine 6G was examined to evaluate the effects of efflux pumps. The expression levels of CgCDR1, CgCDR2, CgSNQ2, CgERG11, and CgPDR1 were examined by real-time PCR. The sequences of CgERG11 and CgPDR1 were determined by PCR-based DNA sequencing. Results: All 59 isolates of C. glabrata were susceptible to flucytosine and amphotericin B. Twelve (20.3%) isolates were determined to be fluconazole-resistant, whereas 13 (22.0%) and 27 (45.7%) isolates were categorized as non-wild-type for itraconazole and voriconazole, respectively. Efflux pumps in azole-resistant isolates showed stronger effects than those in azole-susceptible-dose dependent isolates, which is consistent with the significant upregulation of CgCDR1 and CgCDR2 (P<0.05), whereas no obvious differences were found for CgSNQ2, CgERG11, and CgPDR1 (P>0.05). Sequencing of CgERG11 showed no alteration favoring the hypothesis that CgERG11 is not involved in the azole resistance of C. glabrata. Four CgPDR1 missense mutations were found in azole-resistant isolates, of which the high frequency of the CgPDR1 mutation, A848V, has not been reported previously. Conclusion: Efflux pump function is the main mechanism of resistance to fluconazole in our collected clinical isolates of C. glabrata, and further studies of the related gene disruption and genome-wide expression are needed to verify the function.

Project description: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.

Dataset Information

Genetic Basis of Azole and Echinocandin Resistance in Clinical Candida glabrata in Japan.

Publications

Genetic Basis of Azole and Echinocandin Resistance in Clinical Candida glabrata in Japan.

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