scholarly journals Candida tropicalis Antifungal Cross-Resistance Is Related to Different Azole Target (Erg11p) Modifications

2013 ◽  
Vol 57 (10) ◽  
pp. 4769-4781 ◽  
Author(s):  
A. Forastiero ◽  
A. C. Mesa-Arango ◽  
A. Alastruey-Izquierdo ◽  
L. Alcazar-Fuoli ◽  
L. Bernal-Martinez ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Amphotericin B ◽  
Candida Tropicalis ◽  
Antifungal Drug ◽  
Cross Resistance ◽  
Clinical Samples ◽  
Content Type ◽  
Antifungal Drug Resistance

ABSTRACTCandida tropicalisranks between third and fourth amongCandidaspecies most commonly isolated from clinical specimens. Invasive candidiasis and candidemia are treated with amphotericin B or echinocandins as first-line therapy, with extended-spectrum triazoles as acceptable alternatives.Candida tropicalisis usually susceptible to all antifungal agents, although several azole drug-resistant clinical isolates are being reported. However,C. tropicalisresistant to amphotericin B is uncommon, and only a few strains have reliably demonstrated a high level of resistance to this agent. The resistance mechanisms operating inC. tropicalisstrains isolated from clinical samples showing resistance to azole drugs alone or with amphotericin B cross-resistance were elucidated. Antifungal drug resistance was related to mutations of the azole target (Erg11p) with or without alterations of the ergosterol biosynthesis pathway. The antifungal drug resistance shownin vitrocorrelated very well with the results obtainedin vivousing the model hostGalleria mellonella. Using this panel of strains, theG. mellonellamodel system was validated as a simple, nonmammalian minihost model that can be used to studyin vitro-in vivocorrelation of antifungals inC. tropicalis. The development inC. tropicalisof antifungal drug resistance with different mechanisms during antifungal treatment has potential clinical impact and deserves specific prospective studies.

scholarly journals Environmental Isolates of Azole-Resistant Aspergillus fumigatus in Germany

2015 ◽  
Vol 59 (7) ◽  
pp. 4356-4359 ◽  
Author(s):  
Oliver Bader ◽  
Jana Tünnermann ◽  
Anna Dudakova ◽  
Marut Tangwattanachuleeporn ◽  
Michael Weig ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Aspergillus Fumigatus ◽  
Antifungal Drug ◽  
Clinical Samples ◽  
Environmental Isolates ◽  
Northern Germany ◽  
Content Type ◽  
Antifungal Drug Resistance ◽  
The World

ABSTRACTAzole antifungal drug resistance inAspergillus fumigatusis an emerging problem in several parts of the world. Here we investigated the distribution of such strains in soils from Germany. At a general positivity rate of 12%, most prevalently, we found strains with the TR34/L98H and TR46/Y121F/T289A alleles, dispersed along a corridor across northern Germany. Comparison of the distributions of resistance alleles and genotypes between environment and clinical samples suggests the presence of local clinical clusters.

scholarly journals A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Qusai Al Abdallah ◽  
Wenbo Ge ◽  
Jarrod R. Fortwendel
Keyword(s):  
Drug Resistance ◽  
Gene Deletion ◽  
Gene Replacement ◽  
Antifungal Drug ◽  
Wild Type ◽  
Gene Manipulation ◽  
Content Type ◽  
Antifungal Drug Resistance ◽  
Efficient Gene

ABSTRACT Tackling the multifactorial nature of virulence and antifungal drug resistance in A. fumigatus requires the mechanistic interrogation of a multitude of genes, sometimes across multiple genetic backgrounds. Classical fungal gene replacement systems can be laborious and time-consuming and, in wild-type isolates, are impeded by low rates of homologous recombination. Our simple and universal CRISPR-Cas9 system for gene manipulation generates efficient gene targeting across different genetic backgrounds of A. fumigatus. We anticipate that our system will simplify genome editing in A. fumigatus, allowing for the generation of single- and multigene knockout libraries. In addition, our system will facilitate the delineation of virulence factors and antifungal drug resistance genes in different genetic backgrounds of A. fumigatus. CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is a novel genome-editing system that has been successfully established in Aspergillus fumigatus. However, the current state of the technology relies heavily on DNA-based expression cassettes for delivering Cas9 and the guide RNA (gRNA) to the cell. Therefore, the power of the technology is limited to strains that are engineered to express Cas9 and gRNA. To overcome such limitations, we developed a simple and universal CRISPR-Cas9 system for gene deletion that works across different genetic backgrounds of A. fumigatus. The system employs in vitro assembly of dual Cas9 ribonucleoproteins (RNPs) for targeted gene deletion. Additionally, our CRISPR-Cas9 system utilizes 35 to 50 bp of flanking regions for mediating homologous recombination at Cas9 double-strand breaks (DSBs). As a proof of concept, we first tested our system in the ΔakuB (ΔakuB ku80 ) laboratory strain and generated high rates (97%) of gene deletion using 2 µg of the repair template flanked by homology regions as short as 35 bp. Next, we inspected the portability of our system across other genetic backgrounds of A. fumigatus, namely, the wild-type strain Af293 and a clinical isolate, A. fumigatus DI15-102. In the Af293 strain, 2 µg of the repair template flanked by 35 and 50 bp of homology resulted in highly efficient gene deletion (46% and 74%, respectively) in comparison to classical gene replacement systems. Similar deletion efficiencies were also obtained in the clinical isolate DI15-102. Taken together, our data show that in vitro-assembled Cas9 RNPs coupled with microhomology repair templates are an efficient and universal system for gene manipulation in A. fumigatus. IMPORTANCE Tackling the multifactorial nature of virulence and antifungal drug resistance in A. fumigatus requires the mechanistic interrogation of a multitude of genes, sometimes across multiple genetic backgrounds. Classical fungal gene replacement systems can be laborious and time-consuming and, in wild-type isolates, are impeded by low rates of homologous recombination. Our simple and universal CRISPR-Cas9 system for gene manipulation generates efficient gene targeting across different genetic backgrounds of A. fumigatus. We anticipate that our system will simplify genome editing in A. fumigatus, allowing for the generation of single- and multigene knockout libraries. In addition, our system will facilitate the delineation of virulence factors and antifungal drug resistance genes in different genetic backgrounds of A. fumigatus.

scholarly journals Fungal biofilm architecture produces hypoxic microenvironments that drive antifungal resistance

2020 ◽  
Vol 117 (36) ◽  
pp. 22473-22483 ◽  
Author(s):  
Caitlin H. Kowalski ◽  
Kaesi A. Morelli ◽  
Daniel Schultz ◽  
Carey D. Nadell ◽  
Robert A. Cramer
Keyword(s):  
Drug Resistance ◽  
Fungal Infections ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Drug Resistant ◽  
Antifungal Drug Resistance ◽  
Oxygen Gradients ◽  
Biofilm Architecture

Human fungal infections may fail to respond to contemporary antifungal therapies in vivo despite in vitro fungal isolate drug susceptibility. Such a discrepancy between in vitro antimicrobial susceptibility and in vivo treatment outcomes is partially explained by microbes adopting a drug-resistant biofilm mode of growth during infection. The filamentous fungal pathogenAspergillus fumigatusforms biofilms in vivo, and during biofilm growth it has reduced susceptibility to all three classes of contemporary antifungal drugs. Specific features of filamentous fungal biofilms that drive antifungal drug resistance remain largely unknown. In this study, we applied a fluorescence microscopy approach coupled with transcriptional bioreporters to define spatial and temporal oxygen gradients and single-cell metabolic activity withinA. fumigatusbiofilms. Oxygen gradients inevitably arise duringA. fumigatusbiofilm maturation and are both critical for, and the result of,A. fumigatuslate-stage biofilm architecture. We observe that these self-induced hypoxic microenvironments not only contribute to filamentous fungal biofilm maturation but also drive resistance to antifungal treatment. Decreasing oxygen levels toward the base ofA. fumigatusbiofilms increases antifungal drug resistance. Our results define a previously unknown mechanistic link between filamentous fungal biofilm physiology and contemporary antifungal drug resistance. Moreover, we demonstrate that drug resistance mediated by dynamic oxygen gradients, found in many bacterial biofilms, also extends to the fungal kingdom. The conservation of hypoxic drug-resistant niches in bacterial and fungal biofilms is thus a promising target for improving antimicrobial therapy efficacy.

scholarly journals Environmental Triazole Induces Cross-Resistance to Clinical Drugs and Affects Morphophysiology and Virulence of Cryptococcus gattii and C. neoformans

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Rafael Wesley Bastos ◽  
Hellem Cristina Silva Carneiro ◽  
Lorena Vívien Neves Oliveira ◽  
Karen Maia Rocha ◽  
Gustavo José Cota Freitas ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Efflux Pump ◽  
Study Data ◽  
Cryptococcus Gattii ◽  
Cross Resistance ◽  
Human Pathogens ◽  
Content Type ◽  
Clinical Drugs

ABSTRACTCryptococcus gattiiandCryptococcus neoformansare environmental fungi that cause cryptococcosis, which is usually treated with amphotericin B and fluconazole. However, therapeutic failure is increasing because of the emergence of resistant strains. Because these species are constantly isolated from vegetal materials and the usage of agrochemicals is growing, we postulate that pesticides could be responsible for the altered susceptibility of these fungi to clinical drugs. Therefore, we evaluated the influence of the pesticide tebuconazole on the susceptibility to clinical drugs, morphophysiology, and virulence ofC. gattiiandC. neoformansstrains. The results showed that tebuconazole exposure causedin vitrocross-resistance (CR) between the agrochemical and clinical azoles (fluconazole, itraconazole, and ravuconazole) but not with amphotericin B. In some strains, CR was observed even after the exposure ceased. Further, tebuconazole exposure changed the morphology, including formation of pseudohyphae inC. neoformansH99, and the surface charge of the cells. Although the virulence of both species previously exposed to tebuconazole was decreased in mice, the tebuconazole-exposed colonies recovered from the lungs were more resistant to azole drugs than the nonexposed cells. Thisin vivoCR was confirmed when fluconazole was not able to reduce the fungal burden in the lungs of mice. The tolerance to azoles could be due to increased expression of theERG11gene in both species and of efflux pump genes (AFR1andMDR1) inC. neoformans. Our study data support the idea that agrochemical usage can significantly affect human pathogens present in the environment by affecting their resistance to clinical drugs.

scholarly journals Surface-Engineered Dendrimeric Nanoconjugates for Macrophage-Targeted Delivery of Amphotericin B: Formulation Development andIn VitroandIn VivoEvaluation

2015 ◽  
Vol 59 (5) ◽  
pp. 2479-2487 ◽  
Author(s):  
Keerti Jain ◽  
Ashwni Kumar Verma ◽  
Prabhat Ranjan Mishra ◽  
Narendra Kumar Jain
Keyword(s):  
Drug Release ◽  
Amphotericin B ◽  
Human Erythrocytes ◽  
Antileishmanial Activity ◽  
Cell Uptake ◽  
Antiparasitic Activity ◽  
Content Type ◽  
Drug Localization

ABSTRACTThe present study aimed to develop an optimized dendrimeric delivery system for amphotericin B (AmB). Fifth-generation (5.0G) poly(propylene imine) (PPI) dendrimers were synthesized, conjugated with mannose, and characterized by use of various analytical techniques, including Fourier transform infrared spectroscopy (FTIR),1H nuclear magnetic resonance (1H-NMR) spectroscopic analysis, and atomic force microscopy (AFM). Mannose-conjugated 5.0G PPI (MPPI) dendrimers were loaded with AmB and evaluated for drug loading efficiency,in vitrodrug release profile, stability, hemolytic toxicity to human erythrocytes, cytotoxicity to and cell uptake by J774A.1 macrophage cells, antiparasitic activity against intracellularLeishmania donovaniamastigotes,in vivopharmacokinetic and biodistribution profiles, drug localization index, toxicity, and antileishmanial activity. AFM showed the nanometric size of the MPPI dendrimers, with a nearly globular architecture. The conjugate showed a good entrapment efficiency for AmB, along with pH-sensitive drug release. Highly significant reductions in toxicity toward human erythrocytes and macrophage cells, without compromising the antiparasitic activity of AmB, were observed. The dendrimeric formulation of AmB showed a significant enhancement of the parasiticidal activity of AmB toward intramacrophagicL. donovaniamastigotes. In thein vitrocell uptake studies, the formulation showed selectivity toward macrophages, with significant intracellular uptake. Further pharmacokinetic and organ distribution studies elucidated the controlled delivery behavior of the formulation. The drug localization index was found to increase significantly in macrophage-rich organs.In vivostudies showed a biocompatible behavior of MPPIA, with negligible toxicity even at higher doses, and promising antileishmanial activity. From the results, we concluded that surface-engineered dendrimers may serve as optimized delivery vehicles for AmB with enhanced activity and low or negligible toxicity.

scholarly journals In VitroandIn VivoActivity of Solithromycin (CEM-101) against Plasmodium Species

2011 ◽  
Vol 56 (2) ◽  
pp. 703-707 ◽  
Author(s):  
Sergio Wittlin ◽  
Eric Ekland ◽  
J Carl Craft ◽  
Julie Lotharius ◽  
Ian Bathurst ◽  
...  
Keyword(s):  
Drug Exposure ◽  
Antimalarial Activity ◽  
Plasmodium Species ◽  
Parasite Clearance ◽  
Response To Treatment ◽  
Cross Resistance ◽  
Asexual Blood Stage ◽  
Content Type

ABSTRACTWith the emergence ofPlasmodium falciparuminfections exhibiting increased parasite clearance times in response to treatment with artemisinin-based combination therapies, the need for new therapeutic agents is urgent. Solithromycin, a potent new fluoroketolide currently in development, has been shown to be an effective, broad-spectrum antimicrobial agent. Malarial parasites possess an unusual organelle, termed the apicoplast, which carries a cryptic genome of prokaryotic origin that encodes its own translation and transcription machinery. Given the similarity of apicoplast and bacterial ribosomes, we have examined solithromycin for antimalarial activity. Other antibiotics known to target the apicoplast, such as the macrolide azithromycin, demonstrate a delayed-death effect, whereby treated asexual blood-stage parasites die in the second generation of drug exposure. Solithromycin demonstrated potentin vitroactivity against the NF54 strain ofP. falciparum, as well as against two multidrug-resistant strains, Dd2 and 7G8. The dramatic increase in potency observed after two generations of exposure suggests that it targets the apicoplast. Solithromycin also retained potency against azithromycin-resistant parasites derived from Dd2 and 7G8, although these lines did demonstrate a degree of cross-resistance. In anin vivomodel ofP. bergheiinfection in mice, solithromycin demonstrated a 100% cure rate when administered as a dosage regimen of four doses of 100 mg/kg of body weight, the same dose required for artesunate or chloroquine to achieve 100% cure rates in this rodent malaria model. These promisingin vitroandin vivodata support further investigations into the development of solithromycin as an antimalarial agent.

scholarly journals Dysfunction of Prohibitin 2 Results in Reduced Susceptibility to Multiple Antifungal Drugs via Activation of the Oxidative Stress-Responsive Transcription Factor Pap1 in Fission Yeast

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Qiannan Liu ◽  
Fan Yao ◽  
Guanglie Jiang ◽  
Min Xu ◽  
Si Chen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Drug Resistance ◽  
Transcription Factor ◽  
Fission Yeast ◽  
Mitochondrial Protein ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Gene Encoding ◽  
Content Type ◽  
Antifungal Drug Resistance

ABSTRACT The fight against resistance to antifungal drugs requires a better understanding of the underlying cellular mechanisms. In order to gain insight into the mechanisms leading to antifungal drug resistance, we performed a genetic screen on a model organism, Schizosaccharomyces pombe, to identify genes whose overexpression caused resistance to antifungal drugs, including clotrimazole and terbinafine. We identified the phb2+ gene, encoding a highly conserved mitochondrial protein, prohibitin (Phb2), as a novel determinant of reduced susceptibility to multiple antifungal drugs. Unexpectedly, deletion of the phb2+ gene also exhibited antifungal drug resistance. Overexpression of the phb2+ gene failed to cause drug resistance when the pap1+ gene, encoding an oxidative stress-responsive transcription factor, was deleted. Furthermore, pap1+ mRNA expression was significantly increased when the phb2+ gene was overexpressed or deleted. Importantly, either overexpression or deletion of the phb2+ gene stimulated the synthesis of NO and reactive oxygen species (ROS), as measured by the cell-permeant fluorescent NO probe DAF-FM DA (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate) and the ROS probe DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate), respectively. Taken together, these results suggest that Phb2 dysfunction results in reduced susceptibility to multiple antifungal drugs by increasing NO and ROS synthesis due to dysfunctional mitochondria, thereby activating the transcription factor Pap1 in fission yeast.

scholarly journals Pre-metastatic niche triggers SDF-1/CXCR4 axis and promotes organ colonisation by hepatocellular circulating tumour cells via downregulation of Prrx1

2019 ◽  
Vol 38 (1) ◽  
Author(s):  
Yujun Tang ◽  
Yishi Lu ◽  
Yuan Chen ◽  
Lei Luo ◽  
Lei Cai ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Line ◽  
Lentiviral Vector ◽  
Tumour Cells ◽  
Clinical Samples ◽  
Circulating Tumour Cells ◽  
Metastatic Niche ◽  
Metastatic Sites

Abstract Background Circulating tumour cells (CTCs), especially mesenchymal CTCs, are important determinants of metastasis, which leads to most recurrence and mortality in hepatocellular carcinoma (HCC). However, little is known about the underlying mechanisms of CTC colonisation in pre-metastatic niches. Methods Detection and classification of CTCs in patients were performed using the CanPatrol™ system. A lentiviral vector expressing Prrx1-targeting shRNA was constructed to generate a stable HCC cell line with low expression of Prrx1. The effect of Prrx1 knockdown on stemness, migration, and drug resistance of the cell line was assessed, including involvement of SDF-1/CXCR4 signalling. Promising clinical applications of an inhibitor of STAT3 tyrosine phosphorylation, C188–9, and specific blockade with CXCR4 antibody were explored. Results The number of mesenchymal CTCs in blood was closely associated with tumour recurrence or metastasis. Pre-metastatic niche-derived SDF-1 could downregulate Prrx1, which induced the stemness, drug resistance, and increased expression of CXCR4 in HCC cells through the STAT3 pathway in vitro. In vivo, mice bearing tumours of Prrx1 low-expressing cells had significantly shorter survival. In xenograft tumours and clinical samples, loss of Prrx1 was negatively correlated with increased expression of CXCR4 in lung metastatic sites compared with that in the primary foci. Conclusions These findings demonstrate that decreased expression of Prrx1 stimulates SDF-1/CXCR4 signalling and contributes to organ colonisation with blood CTCs in HCC. STAT3 inhibition and specific blockade of CXCR4 have clinical potential as therapeutics for eliminating organ metastasis in advanced HCC.

scholarly journals New Triazole NT-a9 Has Potent Antifungal Efficacy against Cryptococcus neoformans In Vitro and In Vivo

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Ren-Yi Lu ◽  
Ting-Jun-Hong Ni ◽  
Jing Wu ◽  
Lan Yan ◽  
Quan-Zhen Lv ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Pathogenic Fungi ◽  
Control Group ◽  
Survival Times ◽  
Content Type ◽  
Fungal Burden ◽  
Wide Range

ABSTRACT In the past decades, the incidence of cryptococcosis has increased dramatically, which poses a new threat to human health. However, only a few drugs are available for the treatment of cryptococcosis. Here, we described a leading compound, NT-a9, an analogue of isavuconazole, that showed strong antifungal activities in vitro and in vivo. NT-a9 showed a wide range of activities against several pathogenic fungi in vitro, including Cryptococcus neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, and Candida parapsilosis, with MICs ranging from 0.002 to 1 μg/ml. In particular, NT-a9 exhibited excellent efficacy against C. neoformans, with a MIC as low as 0.002 μg/ml. NT-a9 treatment resulted in changes in the sterol contents in C. neoformans, similarly to fluconazole. In addition, NT-a9 possessed relatively low cytotoxicity and a high selectivity index. The in vivo efficacy of NT-a9 was assessed using a murine disseminated-cryptococcosis model. Mice were infected intravenously with 1.8 × 106 CFU of C. neoformans strain H99. In the survival study, NT-a9 significantly prolonged the survival times of mice compared with the survival times of the control group or the isavuconazole-, fluconazole-, or amphotericin B-treated groups. Of note, 4 and 8 mg/kg of body weight of NT-a9 rescued all the mice, with a survival rate of 100%. In the fungal-burden study, NT-a9 also significantly reduced the fungal burdens in brains and lungs, while fluconazole and amphotericin B only reduced the fungal burden in lungs. Taken together, these data suggested that NT-a9 is a promising antifungal candidate for the treatment of cryptococcosis infection.

scholarly journals In Vitro Evaluation of Radiolabeled Amphotericin B for Molecular Imaging of Mold Infections

2020 ◽  
Vol 64 (7) ◽  
Author(s):  
Lukas Page ◽  
Andrew J. Ullmann ◽  
Fabian Schadt ◽  
Sebastian Wurster ◽  
Samuel Samnick
Keyword(s):  
Molecular Imaging ◽  
Amphotericin B ◽  
Invasive Pulmonary Aspergillosis ◽  
Nuclear Imaging ◽  
Molecular Probes ◽  
Content Type ◽  
Diagnostic Potential ◽  
Gallium 68

ABSTRACT Invasive pulmonary aspergillosis and mucormycosis are life-threatening complications in immunocompromised patients. A rapid diagnosis followed by early antifungal treatment is essential for patient survival. Given the limited spectrum of biomarkers for invasive mold infections, recent studies have proposed the use of radiolabeled siderophores or antibodies as molecular probes to increase the specificity of radiological findings by nuclear imaging modalities. While holding enormous diagnostic potential, most of the currently available molecular probes are tailored to the detection of Aspergillus species, and their cost-intensive and sophisticated implementation restricts their accessibility at less specialized centers. In order to develop cost-efficient and broadly applicable tracers for pulmonary mold infections, this study established streamlined and high-yielding protocols to radiolabel amphotericin B (AMB) with the gamma emitter technetium-99m (99mTc-AMB) and the positron emitter gallium-68 (68Ga-AMB). The radiochemical purity of the resulting tracers consistently exceeded 99%, and both probes displayed excellent stability in human serum (>98% after 60 to 240 min at 37°C). The uptake kinetics by representative mold pathogens were assessed in an in vitro Transwell assay using infected endothelial cell layers. Both tracers accumulated intensively and specifically in Transwell inserts infected with Aspergillus fumigatus, Rhizopus arrhizus, and other clinically relevant mold pathogens compared with their accumulation in uninfected inserts and inserts infected with bacterial controls. Inoculum-dependent enrichment was confirmed by gamma counting and autoradiographic imaging. Taken together, this pilot in vitro study proposes 99mTc-AMB and 68Ga-AMB to be facile, stable, and specific probes, meriting further preclinical in vivo evaluation of radiolabeled amphotericin B for molecular imaging in invasive mycoses.

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