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      Molecular mechanisms of azole resistance in Candida bloodstream isolates

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          Abstract

          Background

          Antifungal resistance rates are increasing. We investigated the mechanisms of azole resistance of Candida spp. bloodstream isolates obtained from a surveillance study conducted between 2012 and 2015.

          Methods

          Twenty-six azole non-susceptible Candida spp. clinical isolates were investigated. Antifungal susceptibilities were determined using the Sensititre YeastOne® YO10 panel. The ERG11 gene was amplified and sequenced to identify amino acid polymorphisms, while real-time PCR was utilised to investigate the expression levels of ERG11, CDR1, CDR2 and MDR1.

          Results

          Azole cross-resistance was detected in all except two isolates. Amino acid substitutions (A114S, Y257H, E266D, and V488I) were observed in all four C. albicans tested. Of the 17 C. tropicalis isolates, eight (47%) had ERG11 substitutions, of which concurrent observation of Y132F and S154F was the most common. A novel substitution (I166S) was detected in two of the five C. glabrata isolates. Expression levels of the various genes differed between the species but CDR1 and CDR2 overexpression appeared to be more prominent in C. glabrata.

          Conclusions

          There was interplay of various different mechanisms, including mechanisms which were not studied here, responsible for azole resistance in Candida spp in our study.

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          Most cited references14

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          Mechanisms of azole resistance in clinical isolates of Candida glabrata collected during a hospital survey of antifungal resistance.

          The increasing use of azole antifungals for the treatment of mucosal and systemic Candida glabrata infections has resulted in the selection and/or emergence of resistant strains. The main mechanisms of azole resistance include alterations in the C. glabrata ERG11 gene (CgERG11), which encodes the azole target enzyme, and upregulation of the CgCDR1 and CgCDR2 genes, which encode efflux pumps. In the present study, we evaluated these molecular mechanisms in 29 unmatched clinical isolates of C. glabrata, of which 20 isolates were resistant and 9 were susceptible dose dependent (S-DD) to fluconazole. These isolates were recovered from separate patients during a 3-year hospital survey for antifungal resistance. Four of the 20 fluconazole-resistant isolates were analyzed together with matched susceptible isolates previously taken from the same patients. Twenty other azole-susceptible clinical C. glabrata isolates were included as controls. MIC data for all the fluconazole-resistant isolates revealed extensive cross-resistance to the other azoles tested, i.e., itraconazole, ketoconazole, and voriconazole. Quantitative real-time PCR analyses showed that CgCDR1 and CgCDR2, alone or in combination, were upregulated at high levels in all but two fluconazole-resistant isolates and, to a lesser extent, in the fluconazole-S-DD isolates. In addition, slight increases in the relative level of expression of CgSNQ2 (which encodes an ATP-binding cassette [ABC] transporter and which has not yet been shown to be associated with azole resistance) were seen in some of the 29 isolates studied. Interestingly, the two fluconazole-resistant isolates expressing normal levels of CgCDR1 and CgCDR2 exhibited increased levels of expression of CgSNQ2. Conversely, sequencing of CgERG11 and analysis of its expression showed no mutation or upregulation in any C. glabrata isolate, suggesting that CgERG11 is not involved in azole resistance. When the isolates were grown in the presence of fluconazole, the profiles of expression of all genes, including CgERG11, were not changed or were only minimally changed in the resistant isolates, whereas marked increases in the levels of gene expression, particularly for CgCDR1 and CgCDR2, were observed in either the fluconazole-susceptible or the fluconazole-S-DD isolates. Finally, known ABC transporter inhibitors, such as FK506, were able to reverse the azole resistance of all the isolates. Together, these results provide evidence that the upregulation of the CgCDR1-, CgCDR2-, and CgSNQ2-encoded efflux pumps might explain the azole resistance in our set of isolates.
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            The presence of an FKS mutation rather than MIC is an independent risk factor for failure of echinocandin therapy among patients with invasive candidiasis due to Candida glabrata.

            Echinocandins are frontline agents against invasive candidiasis (IC), but predictors for echinocandin therapeutic failure have not been well defined. Mutations in Candida FKS genes, which encode the enzyme targeted by echinocandins, result in elevated MICs and have been linked to therapeutic failures. In this study, echinocandin MICs by broth microdilution and FKS1 and FKS2 mutations among C. glabrata isolates recovered from patients with IC at our center were correlated retrospectively with echinocandin therapeutic responses. Thirty-five patients with candidemia and 4 with intra-abdominal abscesses were included, 92% (36/39) of whom received caspofungin. Twenty-six percent (10) and 74% (29) failed and responded to echinocandin therapy, respectively. Caspofungin, anidulafungin, and micafungin MICs ranged from 0.5 to 8, 0.03 to 1, and 0.015 to 0.5 μg/ml, respectively. FKS mutations were detected in 18% (7/39) of C. glabrata isolates (FKS1, n = 2; FKS2, n = 5). Median caspofungin and anidulafungin MICs were higher for patients who failed therapy (P = 0.04 and 0.006, respectively). By receiver operating characteristic (ROC) analyses, MIC cutoffs that best predicted failure were >0.5 (caspofungin), >0.06 (anidulafungin), and >0.03 μg/ml (micafungin), for which sensitivity/specificity were 60%/86%, 50%/97%, and 40%/90%, respectively. Sensitivity/specificity of an FKS mutation in predicting failure were 60%/97%. By univariate analysis, recent gastrointestinal surgery, prior echinocandin exposure, anidulafungin MIC of >0.06 μg/ml, caspofungin MIC of >0.5 μg/ml, and an FKS mutation were significantly associated with failure. The presence of an FKS mutation was the only independent risk factor by multivariate analysis (P = 0.002). In conclusion, detection of C. glabrata FKS mutations was superior to MICs in predicting echinocandin therapeutic responses among patients with IC.
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              Mechanisms of azole resistance in 52 clinical isolates of Candida tropicalis in China.

              To explore the mechanisms underlying azole resistance in clinical isolates of Candida tropicalis collected in China by focusing on their efflux pumps, respiratory status and azole antifungal target enzyme.
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                Author and article information

                Contributors
                jocelyn.teo.q.m@sgh.com.sg
                shannon.lee.j.y@sgh.com.sg
                tan.ai.ling@singhealth.com.sg
                robyn.lim@duke-nus.edu.sg
                cai.yiying@sgh.com.sg
                lim.tze.peng@sgh.com.sg
                +65 63213401 , andrea.kwa.l.h@sgh.com.sg
                Journal
                BMC Infect Dis
                BMC Infect. Dis
                BMC Infectious Diseases
                BioMed Central (London )
                1471-2334
                17 January 2019
                17 January 2019
                2019
                : 19
                : 63
                Affiliations
                [1 ]ISNI 0000 0000 9486 5048, GRID grid.163555.1, Department of Pharmacy, , Singapore General Hospital, ; Blk 8 Level 2, Outram Road, Singapore, 169608 Singapore
                [2 ]ISNI 0000 0001 2180 6431, GRID grid.4280.e, Saw Swee Hock School of Public Health, , National University of Singapore, ; 12 Science Drive 2, #10-01, Singapore, 117549 Singapore
                [3 ]ISNI 0000 0000 9486 5048, GRID grid.163555.1, Department of Microbiology, , Singapore General Hospital, ; Outram Road, Singapore, 169608 Singapore
                [4 ]ISNI 0000 0001 2180 6431, GRID grid.4280.e, Department of Pharmacy, , National University of Singapore, ; 18 Science Drive 4, Singapore, 117543 Singapore
                [5 ]ISNI 0000 0001 2180 6431, GRID grid.4280.e, Singhealth Duke-NUS Pathology Academic Clinical Programme, ; 8 College Road, Level 4, Singapore, 169857 Singapore
                [6 ]ISNI 0000 0001 2180 6431, GRID grid.4280.e, Singhealth Duke-NUS Medicine Academic Clinical Programme, ; 8 College Road, Level 4, Singapore, 169857 Singapore
                [7 ]ISNI 0000 0004 0385 0924, GRID grid.428397.3, Emerging Infectious Diseases, , Duke-National University of Singapore Medical School, ; 8 College Rd, Singapore, 169857 Singapore
                [8 ]ISNI 0000 0004 0385 0924, GRID grid.428397.3, Present address: Program in Health Services and Systems Research, , Duke-National University of Singapore Medical School, ; 8 College Rd, Singapore, 169857 Singapore
                Author information
                http://orcid.org/0000-0001-8981-4411
                Article
                3672
                10.1186/s12879-019-3672-5
                6337757
                30654757
                37b16718-2a7e-4c80-9cc4-7f13089ffc46
                © The Author(s). 2019

                Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 5 March 2018
                : 2 January 2019
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001349, National Medical Research Council;
                Award ID: NMRC/TA/0025/2013
                Award ID: NMRC/CG/016/2013
                Funded by: FundRef http://dx.doi.org/10.13039/100004319, Pfizer;
                Award ID: WS2347894
                Funded by: FundRef http://dx.doi.org/10.13039/100004324, Astellas Pharma US;
                Award ID: NA
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2019

                Infectious disease & Microbiology
                candida,antifungal resistance,genomics
                Infectious disease & Microbiology
                candida, antifungal resistance, genomics

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