Species Distribution, Azole Resistance and Related Molecular Mechanisms in Invasive Candida parapsilosis Complex Isolates: Increase of Fluconazole Resistance in 21 Years

ABSTRACTCandida parapsilosisis the second most prevalent fungal agent causing bloodstream infections. Nevertheless, there is little information about the molecular mechanisms underlying azole resistance in this species. Mutations (G1747A, A2619C, and A3191C) in theMRR1transcription factor gene were identified in fluconazole- and voriconazole-resistant strains. Independent expression ofMRR1genes harboring these mutations showed that G1747A (G583R) and A2619C (K873N) are gain-of-function mutations responsible for azole resistance, the first described inC. parapsilosis.

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Prevalence of Molecular Mechanisms of Resistance to Azole Antifungal Agents in Candida albicans Strains Displaying High-Level Fluconazole Resistance Isolated from Human Immunodeficiency Virus-Infected Patients

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.10.2676-2684.2001 ◽

2001 ◽

Vol 45 (10) ◽

pp. 2676-2684 ◽

Cited By ~ 314

Author(s):

Sofia Perea ◽

José L. López-Ribot ◽

William R. Kirkpatrick ◽

Robert K. McAtee ◽

Rebecca A. Santillán ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Candida Albicans ◽

Molecular Mechanisms ◽

Azole Resistance ◽

Mechanisms Of Resistance ◽

Fluconazole Resistance ◽

Immunodeficiency Virus ◽

Genes Encoding ◽

High Level ◽

Target Enzyme

ABSTRACT Molecular mechanisms of azole resistance in Candida albicans, including alterations in the target enzyme and increased efflux of drug, have been described, but the epidemiology of the resistance mechanisms has not been established. We have investigated the molecular mechanisms of resistance to azoles inC. albicans strains displaying high-level fluconazole resistance (MICs, ≥64 μg/ml) isolated from human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis. The levels of expression of genes encoding lanosterol 14α-demethylase (ERG11) and efflux transporters (MDR1 and CDR) implicated in azole resistance were monitored in matched sets of susceptible and resistant isolates. In addition,ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. The analysis confirmed the multifactorial nature of azole resistance and the prevalence of these mechanisms of resistance in C. albicans clinical isolates exhibiting frank fluconazole resistance, with a predominance of overexpression of genes encoding efflux pumps, detected in 85% of all resistant isolates, being found. Alterations in the target enzyme, including functional amino acid substitutions and overexpression of the gene that encodes the enzyme, were detected in 65 and 35% of the isolates, respectively. Overall, multiple mechanisms of resistance were combined in 75% of the isolates displaying high-level fluconazole resistance. These results may help in the development of new strategies to overcome the problem of resistance as well as new treatments for this condition.

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Molecular Mechanisms of Fluconazole Resistance in Candida parapsilosis Isolates from a U.S. Surveillance System

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04613-14 ◽

2014 ◽

Vol 59 (2) ◽

pp. 1030-1037 ◽

Cited By ~ 44

Author(s):

Nina T. Grossman ◽

Cau D. Pham ◽

Angela A. Cleveland ◽

Shawn R. Lockhart

Keyword(s):

Molecular Mechanisms ◽

Candida Parapsilosis ◽

Efflux Pump ◽

Point Mutations ◽

Resistance Rate ◽

Primary Therapy ◽

Fluconazole Resistance ◽

Content Type ◽

Fluconazole Resistant ◽

Dose Dependent

ABSTRACTCandida parapsilosisis the second or third most common cause of candidemia in many countries. The Infectious Diseases Society of America recommends fluconazole as the primary therapy forC. parapsilosiscandidemia. Although the rate of fluconazole resistance amongC. parapsilosisisolates is low in most U.S. institutions, the resistance rate can be as high as 7.5%. This study was designed to assess the mechanisms of fluconazole resistance in 706 incident bloodstream isolates from U.S. hospitals. We sequenced theERG11andMRR1genes of 122C. parapsilosisisolates with resistant (30 isolates; 4.2%), susceptible dose-dependent (37 isolates; 5.2%), and susceptible (55 isolates) fluconazole MIC values and used real-time PCR of RNA from 17 isolates to investigate the regulation ofMDR1. By comparing these isolates to fully fluconazole-susceptible isolates, we detected at least two mechanisms of fluconazole resistance: an amino acid substitution in the 14-α-demethylase geneERG11and overexpression of the efflux pumpMDR1, possibly due to point mutations in theMRR1transcription factor that regulatesMDR1. TheERG11single nucleotide polymorphism (SNP) was found in 57% of the fluconazole-resistant isolates and in no susceptible isolates. TheMRR1SNPs were more difficult to characterize, as not all resulted in overexpression ofMDR1and not allMDR1overexpression was associated with an SNP inMRR1. Further work to characterize theMRR1SNPs and search for overexpression of other efflux pumps is needed.

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Candida parapsilosis Resistance to Fluconazole: Molecular Mechanisms andIn VivoImpact in Infected Galleria mellonella Larvae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01177-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 6581-6587 ◽

Cited By ~ 56

Author(s):

Ana Carolina R. Souza ◽

Beth Burgwyn Fuchs ◽

Henrique M. S. Pinhati ◽

Ricardo A. Siqueira ◽

Ferry Hagen ◽

...

Keyword(s):

Molecular Mechanisms ◽

Candida Parapsilosis ◽

Galleria Mellonella ◽

Common Mutation ◽

Fluconazole Resistance ◽

Content Type ◽

Clonal Origin ◽

Fluconazole Resistant ◽

Resistant Strains

ABSTRACTCandida parapsilosisis the main non-albicans Candidaspecies isolated from patients in Latin America. Mutations in theERG11gene and overexpression of membrane transporter proteins have been linked to fluconazole resistance. The aim of this study was to evaluate the molecular mechanisms in fluconazole-resistant strains ofC. parapsilosisisolated from critically ill patients. The identities of the nine collectedC. parapsilosisisolates at the species level were confirmed through molecular identification with a TaqMan qPCR assay. The clonal origin of the strains was checked by microsatellite typing. TheGalleria mellonellainfection model was used to confirmin vitroresistance. We assessed the presence ofERG11mutations, as well as the expression ofERG11and two additional genes that contribute to antifungal resistance (CDR1andMDR1), by using real-time quantitative PCR. All of theC. parapsilosis(sensu stricto) isolates tested exhibited fluconazole MICs between 8 and 16 μg/ml. Thein vitrodata were confirmed by the failure of fluconazole in the treatment ofG. mellonellainfected with fluconazole-resistant strains ofC. parapsilosis. Sequencing of theERG11gene revealed a common mutation leading to a Y132F amino acid substitution in all of the isolates, a finding consistent with their clonal origin. After fluconazole exposure, overexpression was noted forERG11,CDR1, andMDR1in 9/9, 9/9, and 2/9 strains, respectively. We demonstrated that a combination of molecular mechanisms, including the presence of point mutations in theERG11gene, overexpression ofERG11, and genes encoding efflux pumps, are involved in fluconazole resistance inC. parapsilosis.

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A Transcriptomics Approach To Unveiling the Mechanisms ofIn VitroEvolution towards Fluconazole Resistance of aCandida glabrataClinical Isolate

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00995-18 ◽

2018 ◽

Vol 63 (1) ◽

Cited By ~ 8

Author(s):

Mafalda Cavalheiro ◽

Catarina Costa ◽

Ana Silva-Dias ◽

Isabel M. Miranda ◽

Can Wang ◽

...

Keyword(s):

Fungal Pathogen ◽

Biofilm Formation ◽

Molecular Mechanisms ◽

Antifungal Drugs ◽

Azole Resistance ◽

Fluconazole Resistance ◽

Content Type ◽

Antifungal Drug Resistance ◽

Genes Encoding ◽

Multidrug Resistance Transporters

ABSTRACTCandida glabratais an emerging fungal pathogen. Its increased prevalence is associated with its ability to rapidly develop antifungal drug resistance, particularly to azoles. In order to unravel new molecular mechanisms behind azole resistance, a transcriptomics analysis of the evolution of aC. glabrataclinical isolate (isolate 044) from azole susceptibility to posaconazole resistance (21st day), clotrimazole resistance (31st day), and fluconazole and voriconazole resistance (45th day), induced by longstanding incubation with fluconazole, was carried out. All the evolved strains were found to accumulate lower concentrations of azole drugs than the parental strain, while the ergosterol concentration remained mostly constant. However, only the population displaying resistance to all azoles was found to have a gain-of-function mutation in theC. glabrataPDR1gene, leading to the upregulation of genes encoding multidrug resistance transporters. Intermediate strains, exhibiting posaconazole/clotrimazole resistance and increased fluconazole/voriconazole MIC levels, were found to display alternative ways to resist azole drugs. Particularly, posaconazole/clotrimazole resistance after 31 days was correlated with increased expression of adhesin genes. This finding led us to identify the Epa3 adhesin as a new determinant of azole resistance. Besides being required for biofilm formation, Epa3 expression was found to decrease the intracellular accumulation of azole antifungal drugs. Altogether, this work provides a glimpse of the transcriptomics evolution of aC. glabratapopulation toward multiazole resistance, highlighting the multifactorial nature of the acquisition of azole resistance and pointing out a new player in azole resistance.

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