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

2015 ◽  
Vol 59 (10) ◽  
pp. 6581-6587 ◽  
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.

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

2014 ◽  
Vol 59 (2) ◽  
pp. 1030-1037 ◽  
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.

scholarly journals Fluconazole-Resistant Candida auris Is Susceptible to Salivary Histatin 5 Killing and to Intrinsic Host Defenses

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
Ruvini U. Pathirana ◽  
Justin Friedman ◽  
Hannah L. Norris ◽  
Ornella Salvatori ◽  
Andrew D. McCall ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Candida Auris ◽  
Fluconazole Resistance ◽  
Content Type ◽  
Histatin 5 ◽  
Cellular Targets ◽  
Highly Sensitive ◽  
Fluconazole Resistant ◽  
Resistant Strains

ABSTRACT Candida auris is a newly identified species causing invasive candidemia and candidiasis. It has broad multidrug resistance (MDR) not observed for other pathogenic Candida species. Histatin 5 (Hst 5) is a well-studied salivary cationic peptide with significant antifungal activity against Candida albicans and is an attractive candidate for treating MDR fungi, since antimicrobial peptides induce minimal drug resistance. We investigated the susceptibility of C. auris to Hst 5 and neutrophils, two first-line innate defenses in the human host. The majority of C. auris clinical isolates, including fluconazole-resistant strains, were highly sensitive to Hst 5: 55 to 90% of cells were killed by use of 7.5 μM Hst 5. Hst 5 was translocated to the cytosol and vacuole in C. auris cells; such translocation is required for the killing of C. albicans by Hst 5. The inverse relationship between fluconazole resistance and Hst 5 killing suggests different cellular targets for Hst 5 than for fluconazole. C. auris showed higher tolerance to oxidative stress than C. albicans, and higher survival within neutrophils, which correlated with resistance to oxidative stress in vitro. Thus, resistance to reactive oxygen species (ROS) is likely one, though not the only, important factor in the killing of C. auris by neutrophils. Hst 5 has broad and potent candidacidal activity, enabling it to combat MDR C. auris strains effectively.

scholarly journals Transcriptional Profiling of Azole-Resistant Candida parapsilosis Strains

2011 ◽  
Vol 55 (7) ◽  
pp. 3546-3556 ◽  
Author(s):  
A. P. Silva ◽  
I. M. Miranda ◽  
A. Guida ◽  
J. Synnott ◽  
R. Rocha ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Candida Parapsilosis ◽  
Transcriptional Profiling ◽  
Point Mutations ◽  
Antifungal Drugs ◽  
Ergosterol Biosynthesis ◽  
Content Type ◽  
Resistant Strains ◽  
Major Facilitator

ABSTRACTHerein we describe the changes in the gene expression profile ofCandida parapsilosisassociated with the acquisition of experimentally induced resistance to azole antifungal drugs. Three resistant strains ofC. parapsilosiswere obtained following prolongedin vitroexposure of a susceptible clinical isolate to constant concentrations of fluconazole, voriconazole, or posaconazole. We found that after incubation with fluconazole or voriconazole, strains became resistant to both azoles but not to posaconazole, although susceptibility to this azole decreased, whereas the strain incubated with posaconazole displayed resistance to the three azoles. The resistant strains obtained after exposure to fluconazole and to voriconazole have increased expression of the transcription factorMRR1, the major facilitator transporterMDR1, and several reductases and oxidoreductases. Interestingly, and similarly to what has been described inC. albicans, upregulation ofMRR1andMDR1is correlated with point mutations inMRR1in the resistant strains. The resistant strain obtained after exposure to posaconazole shows upregulation of two transcription factors (UPC2andNDT80) and increased expression of 13 genes involved in ergosterol biosynthesis. This is the first study addressing global molecular mechanisms underlying azole resistance inC. parapsilosis; the results suggest that similarly toC. albicans, tolerance to azoles involves the activation of efflux pumps and/or increased ergosterol synthesis.

scholarly journals Fluconazole and Voriconazole Resistance in Candida parapsilosis Is Conferred by Gain-of-Function Mutations inMRR1Transcription Factor Gene

2015 ◽  
Vol 59 (10) ◽  
pp. 6629-6633 ◽  
Author(s):  
Joana Branco ◽  
Ana P. Silva ◽  
Raquel M. Silva ◽  
Ana Silva-Dias ◽  
Cidália Pina-Vaz ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Molecular Mechanisms ◽  
Candida Parapsilosis ◽  
Bloodstream Infections ◽  
Azole Resistance ◽  
Transcription Factor Gene ◽  
Gain Of Function ◽  
Content Type ◽  
Factor Gene ◽  
Resistant Strains

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.

scholarly journals In VivoEfficacy of Glycopeptide-Colistin Combination Therapies in aGalleria mellonellaModel ofAcinetobacter baumanniiInfection

2011 ◽  
Vol 55 (7) ◽  
pp. 3534-3537 ◽  
Author(s):  
M. Hornsey ◽  
D. W. Wareham
Keyword(s):  
Galleria Mellonella ◽  
Survival Rates ◽  
Multidrug Resistant ◽  
New Agents ◽  
Content Type ◽  
Highly Active ◽  
Resistant Strains ◽  
Immunomodulatory Action ◽  
Lethal Doses

ABSTRACTThe treatment ofAcinetobacter baumanniiinfections poses a significant clinical challenge, with isolates resistant to all commonly used agents increasingly being reported. With few new agents in the pipeline, clinicians are increasingly turning to combinations of antimicrobials in the hope that they may act synergistically together. In this study we assessed the activities of two glycopeptide-colistin combinations bothin vitroand using aGalleria mellonellacaterpillar model ofA. baumanniiinfection. In checkerboard assays both vancomycin and teicoplanin were highly active against susceptible and multidrug-resistant strains ofA. baumanniiwhen combined with colistin (fractional inhibitory concentration [FIC] of <0.25). Treatment ofG. mellonellacaterpillars infected with lethal doses ofA. baumanniiresulted in significantly enhanced survival rates when either vancomycin or teicoplanin was given with colistin compared to colistin treatment alone (P< 0.05). This effect was most marked when vancomycin was the glycopeptide administered, although this agent was also highly effective as monotherapy, possibly through an immunomodulatory action on theG. mellonellaresponse toA. baumanniiinfection. This work suggests that glycopeptide-colistin combinations are highly active againstA. baumanniibothin vitroand in a simple animal model of infection. They should be considered further as potential treatments for difficult-to-treatA. baumanniiinfections.

scholarly journals Mutations in TAC1B: a Novel Genetic Determinant of Clinical Fluconazole Resistance in Candida auris

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Jeffrey M. Rybak ◽  
José F. Muñoz ◽  
Katherine S. Barker ◽  
Josie E. Parker ◽  
Brooke D. Esquivel ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Clinical Isolate ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Genetic Determinant ◽  
Candida Auris ◽  
Fluconazole Resistance ◽  
Content Type ◽  
Fluconazole Resistant

ABSTRACT Candida auris has emerged as a multidrug-resistant pathogen of great clinical concern. Approximately 90% of clinical C. auris isolates are resistant to fluconazole, the most commonly prescribed antifungal agent, and yet it remains unknown what mechanisms underpin this fluconazole resistance. To identify novel mechanisms contributing to fluconazole resistance in C. auris, fluconazole-susceptible C. auris clinical isolate AR0387 was passaged in media supplemented with fluconazole to generate derivative strains which had acquired increased fluconazole resistance in vitro. Comparative analyses of comprehensive sterol profiles, [3H]fluconazole uptake, sequencing of C. auris genes homologous to genes known to contribute to fluconazole resistance in other species of Candida, and relative expression levels of C. auris ERG11, CDR1, and MDR1 were performed. All fluconazole-evolved derivative strains were found to have acquired mutations in the zinc-cluster transcription factor-encoding gene TAC1B and to show a corresponding increase in CDR1 expression relative to the parental clinical isolate, AR0387. Mutations in TAC1B were also identified in a set of 304 globally distributed C. auris clinical isolates representing each of the four major clades. Introduction of the most common mutation found among fluconazole-resistant clinical isolates of C. auris into fluconazole-susceptible isolate AR0387 was confirmed to increase fluconazole resistance by 8-fold, and the correction of the same mutation in a fluconazole-resistant isolate, AR0390, decreased fluconazole MIC by 16-fold. Taken together, these data demonstrate that C. auris can rapidly acquire resistance to fluconazole in vitro and that mutations in TAC1B significantly contribute to clinical fluconazole resistance. IMPORTANCE Candida auris is an emerging multidrug-resistant pathogen of global concern, known to be responsible for outbreaks on six continents and to be commonly resistant to antifungals. While the vast majority of clinical C. auris isolates are highly resistant to fluconazole, an essential part of the available antifungal arsenal, very little is known about the mechanisms contributing to resistance. In this work, we show that mutations in the transcription factor TAC1B significantly contribute to clinical fluconazole resistance. These studies demonstrated that mutations in TAC1B can arise rapidly in vitro upon exposure to fluconazole and that a multitude of resistance-associated TAC1B mutations are present among the majority of fluconazole-resistant C. auris isolates from a global collection and appear specific to a subset of lineages or clades. Thus, identification of this novel genetic determinant of resistance significantly adds to the understanding of clinical antifungal resistance in C. auris.

scholarly journals In Vitro Susceptibilities of Candida andCryptococcus neoformans Isolates from Blood Cultures of Neutropenic Patients

1999 ◽  
Vol 43 (6) ◽  
pp. 1463-1464 ◽  
Author(s):  
Daryl J. Hoban ◽  
George G. Zhanel ◽  
James A. Karlowsky
Keyword(s):  
Risk Factors ◽  
Candida Parapsilosis ◽  
Clinical Laboratory ◽  
Blood Cultures ◽  
Weight Basis ◽  
National Committee ◽  
Fluconazole Resistance ◽  
Neutropenic Patients ◽  
Fluconazole Resistant

ABSTRACT Fluconazole-resistant Candida albicans and intrinsically fluconazole-resistant Candida species have been reported as bloodstream isolates. However, an association between the isolation of fluconazole-resistant Candida from the bloodstream and patient risk factors for fungemia has not been established. The purpose of this study was to determine the prevalence of fluconazole resistance in bloodstream isolates ofCandida species and Cryptococcus neoformanscollected from patients with neutropenia, one of the most important risk factors for fungemia. MICs of voriconazole, fluconazole, itraconazole, ketoconazole, amphotericin B, and flucytosine were determined by the National Committee for Clinical Laboratory Standards M27-A method (1997). Voriconazole, on a per-weight basis, was the most active azole tested. Fluconazole resistance (MIC ≥ 64 μg/ml) was not identified in any of the C. albicans(n = 513), Candida parapsilosis(n = 78), Candida tropicalis(n = 62), or C. neoformans(n = 38) isolates tested.

scholarly journals Biological Impact of a Large-Scale Genomic Inversion That Grossly Disrupts the Relative Positions of the Origin and Terminus Loci of theStreptococcus pyogenesChromosome

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
Dragutin J. Savic ◽  
Scott V. Nguyen ◽  
Kimberly McCullor ◽  
W. Michael McShan
Keyword(s):  
Dna Sequences ◽  
Parental Strain ◽  
Large Scale ◽  
Galleria Mellonella ◽  
Acute Infection ◽  
Relative Length ◽  
Published Data ◽  
Rich Medium ◽  
Content Type

ABSTRACTA large-scale genomic inversion encompassing 0.79 Mb of the 1.816-Mb-longStreptococcus pyogenesserotype M49 strain NZ131 chromosome spontaneously occurs in a minor subpopulation of cells, and in this report genetic selection was used to obtain a stable lineage with this chromosomal rearrangement. This inversion, which drastically displaces theorisite relative to the terminus, changes the relative length of the replication arms so that one replichore is approximately 0.41 Mb while the other is about 1.40 Mb in length. Genomic reversion to the original chromosome constellation is not observed in PCR-monitored analyses after 180 generations of growth in rich medium. Compared to the parental strain, the inversion surprisingly demonstrates a nearly identical growth pattern in the first phase of the exponential phase, but differences do occur when resources in the medium become limited. When cultured separately in rich medium during prolonged stationary phase or in an experimental acute infection animal model (Galleria mellonella), the parental strain and the invertant have equivalent survival rates. However, when they are coincubated together, bothin vitroandin vivo, the survival of the invertant declines relative to the level for the parental strain. The accompanying aspect of the study suggests that inversions taking place nearoriCalways happen to secure the linkage oforiCto DNA sequences responsible for chromosome partition. The biological relevance of large-scale inversions is also discussed.IMPORTANCEBased on our previous work, we created to our knowledge the largest asymmetric inversion, covering 43.5% of theS. pyogenesgenome. In spite of a drastic replacement of origin of replication and the unbalanced size of replichores (1.4 Mb versus 0.41 Mb), the invertant, when not challenged with its progenitor, showed impressive vitality for growthin vitroand in pathogenesis assays. The mutant supports the existing idea that slightly deleterious mutations can provide the setting for secondary adaptive changes. Furthermore, comparative analysis of the mutant with previously published data strongly indicates that even large genomic rearrangements survive provided that the integrity of theoriCand the chromosome partition cluster is preserved.

scholarly journals Pharmacodynamics of Itraconazole against Aspergillus fumigatus in anIn VitroModel of the Human Alveolus: Perspectives on the Treatment of Triazole-Resistant Infection and Utility of Airway Administration

2012 ◽  
Vol 56 (8) ◽  
pp. 4146-4153 ◽  
Author(s):  
Zaid Al-Nakeeb ◽  
Ajay Sudan ◽  
Adam R. Jeans ◽  
Lea Gregson ◽  
Joanne Goodwin ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Therapeutic Strategies ◽  
Wild Type ◽  
Content Type ◽  
Exposure Response ◽  
Prevention And Treatment ◽  
Resistant Infection ◽  
Resistant Strains ◽  
Type Strains

ABSTRACTItraconazole is used for the prevention and treatment of infections caused byAspergillus fumigatus. An understanding of the pharmacodynamics of itraconazole against wild-type and triazole-resistant strains provides a basis for innovative therapeutic strategies for treatment of infections. Anin vitromodel of the human alveolus was used to define the pharmacodynamics of itraconazole. Galactomannan was used as a biomarker. The effect of systemic and airway administration of itraconazole was assessed, as was a combination of itraconazole administered to the airway and systemically administered 5FC. Systemically administered itraconazole against the wild type induced a concentration-dependent decline in galactomannan in the alveolar and endothelial compartments. No exposure-response relationships were apparent for the L98H, M220T, or G138C mutant. The administration of itraconazole to the airway resulted in comparable exposure-response relationships to those observed with systemic therapy. This was achieved without detectable concentrations of drug within the endothelial compartment. The airway administration of itraconazole resulted in a definite but submaximal effect in the endothelial compartment against the L98H mutant. The administration of 5FC resulted in a concentration-dependent decline in galactomannan in both the alveolar and endothelial compartments. The combination of airway administration of itraconazole and systemically administered 5FC was additive. Systemic administration of itraconazole is ineffective against Cyp51 mutants. The airway administration of itraconazole is effective for the treatment of wild-type strains and appears to have some activity against the L98H mutants. Combination with other agents, such as 5FC, may enable the attainment of near-maximal antifungal activity.

scholarly journals DAP12 Inhibits Pulmonary Immune Responses to Cryptococcus neoformans

2016 ◽  
Vol 84 (6) ◽  
pp. 1879-1886 ◽  
Author(s):  
Lena J. Heung ◽  
Tobias M. Hohl
Keyword(s):  
Immune Response ◽  
Nk Cells ◽  
Cryptococcus Neoformans ◽  
Molecular Mechanisms ◽  
Content Type ◽  
Effector Cells ◽  
Opportunistic Fungal Pathogen ◽  
Efficient Uptake ◽  
Bacteria And Fungi

Cryptococcus neoformansis an opportunistic fungal pathogen that is inhaled into the lungs and can lead to life-threatening meningoencephalitis in immunocompromised patients. Currently, the molecular mechanisms that regulate the mammalian immune response to respiratory cryptococcal challenge remain poorly defined. DAP12, a signaling adapter for multiple pattern recognition receptors in myeloid and natural killer (NK) cells, has been shown to play both activating and inhibitory roles during lung infections by different bacteria and fungi. In this study, we demonstrate that DAP12 plays an important inhibitory role in the immune response toC. neoformans. Infectious outcomes in DAP12−/−mice, including survival and lung fungal burden, are significantly improved compared to those in C57BL/6 wild-type (WT) mice. We find that eosinophils and macrophages are decreased while NK cells are increased in the lungs of infected DAP12−/−mice. In contrast to WT NK cells, DAP12−/−NK cells are able to repressC. neoformansgrowthin vitro. Additionally, DAP12−/−macrophages are more highly activated than WT macrophages, with increased production of tumor necrosis factor (TNF) and CCL5/RANTES and more efficient uptake and killing ofC. neoformans. These findings suggest that DAP12 acts as a brake on the pulmonary immune response toC. neoformansby promoting pulmonary eosinophilia and by inhibiting the activation and antifungal activities of effector cells, including NK cells and macrophages.

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