scholarly journals Rare modification in the ergosterol biosynthesis pathway leads to amphotericin B resistance in Candida auris clinical isolates

2021 ◽  
Author(s):  
Milena Kordalewska ◽  
Kevin D. Guerrero ◽  
Timothy D. Mikulski ◽  
Tony N. Elias ◽  
Rocio Garcia-Rubio ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Ergosterol Biosynthesis ◽  
Biosynthesis Pathway ◽  
Wild Type ◽  
Candida Auris ◽  
Key Genes

We determined amphotericin B (AmB) susceptibility and sequenced key genes of the ergosterol biosynthesis pathway implicated in AmB resistance (ERG2, ERG3, ERG6, ERG11) of 321 clinical isolates of Candida auris. In antifungal susceptibility testing, 19 (5.9%) isolates were categorized as AmB-resistant (MIC ≥2 mg/l). Only one AmB-resistant isolate presented a unique non-wild-type ERG6 genotype that was confirmed to confer amphotericin B resistance (MIC >32 mg/l) when introduced into a susceptible strain (MIC = 0.5 mg/l).

scholarly journals Mutation in the Squalene Epoxidase Gene ofTrichophyton interdigitaleandTrichophyton rubrumAssociated with Allylamine Resistance

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Shivaprakash M. Rudramurthy ◽  
Shamanth A. Shankarnarayan ◽  
Sunil Dogra ◽  
Dipika Shaw ◽  
Khurram Mushtaq ◽  
...  
Keyword(s):  
Antifungal Agents ◽  
Susceptibility Testing ◽  
Trichophyton Rubrum ◽  
Antifungal Susceptibility ◽  
Ergosterol Biosynthesis ◽  
Squalene Epoxidase ◽  
Biosynthesis Pathway ◽  
Wild Type ◽  
Content Type ◽  
Recent Attention

ABSTRACTDermatophytosis, the commonest superficial fungal infection, has gained recent attention due to its change of epidemiology and treatment failures. Despite the availability of several agents effective against dermatophytes, the incidences of chronic infection, reinfection, and treatment failures are on the rise.Trichophyton rubrumandTrichophyton interdigitaleare the two species most frequently identified among clinical isolates in India. Consecutive patients (n= 195) with suspected dermatophytosis during the second half of 2014 were included in this study. Patients were categorized into relapse and new cases according to standard definitions. Antifungal susceptibility testing of the isolatedTrichophytonspecies (n= 127) was carried out with 12 antifungal agents: fluconazole, voriconazole, itraconazole, ketoconazole, sertaconazole, clotrimazole, terbinafine, naftifine, amorolfine, ciclopirox olamine, griseofulvin, and luliconazole. The squalene epoxidase gene was evaluated for mutation (if any) in 15T. interdigitaleand 5T. rubrumisolates exhibiting high MICs for terbinafine. A T1189C mutation was observed in fourT. interdigitaleand twoT. rubrumisolates. This transition leads to the change of phenylalanine to leucine in the 397th position of the squalene epoxidase enzyme. In homology modeling the mutant residue was smaller than the wild type and positioned in the dominant site of squalene epoxidase during drug interaction, which may lead to a failure to block the ergosterol biosynthesis pathway by the antifungal drug.

In vitro activity of olorofim against clinical isolates of Scedosporium species and Lomentospora prolificans using EUCAST and CLSI methodologies

2020 ◽  
Vol 75 (12) ◽  
pp. 3582-3585
Author(s):  
Olga Rivero-Menendez ◽  
Manuel Cuenca-Estrella ◽  
Ana Alastruey-Izquierdo
Keyword(s):  
Amphotericin B ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Vitro Activity ◽  
Clinical Isolates ◽  
Scedosporium Apiospermum ◽  
In Vitro Activity ◽  
Scedosporium Dehoogii ◽  
Scedosporium Species

Abstract Objectives To evaluate the in vitro activity of olorofim, a new broad-spectrum antifungal with a novel mechanism of action, against a collection of 123 Spanish clinical isolates belonging to five Scedosporium species and Lomentospora prolificans. Methods The activity of olorofim against Scedosporium apiospermum (n = 30), Scedosporium boydii (n = 30), Scedosporium ellipsoideum (n = 10), Scedosporium aurantiacum (n = 20), Scedosporium dehoogii (n = 3) and Lomentospora prolificans (n = 30) was compared with that of amphotericin B, voriconazole, isavuconazole and micafungin by performing EUCAST and CLSI reference methods for antifungal susceptibility testing. Results Amphotericin B and isavuconazole showed MICs ≥2 mg/L for all the species evaluated and voriconazole was moderately active (GM, MIC50 and MIC90 values ≤2 mg/L) against all of them except L. prolificans. Micafungin was effective against S. apiospermum complex strains, but exhibited elevated MECs for S. dehoogii and S. aurantiacum. Olorofim showed low MICs for all the Scedosporium strains tested (GM values were lower than 0.130 and 0.339 by the EUCAST method and the CLSI method, respectively, for all of the species), including those belonging to the MDR species L. prolificans, for which GM values were 0.115 and 0.225 mg/L by the EUCAST method and the CLSI method, respectively, while the GMs for the rest of the antifungals evaluated were higher than 3.732 mg/L using both methodologies. Conclusions Olorofim displayed promising in vitro activity against the Scedosporium and L. prolificans strains tested, some of which have reduced susceptibility to the antifungals that are currently in use.

scholarly journals Mutations in the fks1 Gene in Candida albicans, C. tropicalis, and C. krusei Correlate with Elevated Caspofungin MICs Uncovered in AM3 Medium Using the Method of the European Committee on Antibiotic Susceptibility Testing

2008 ◽  
Vol 52 (9) ◽  
pp. 3092-3098 ◽  
Author(s):  
Marie Desnos-Ollivier ◽  
Stéphane Bretagne ◽  
Dorothée Raoux ◽  
Damien Hoinard ◽  
Françoise Dromer ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Antibiotic Susceptibility ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Clinical Isolates ◽  
Antibiotic Susceptibility Testing ◽  
Wild Type ◽  
European Committee ◽  
Rpmi Medium

ABSTRACT Mutations in two specific regions of the Fks1 subunit of 1,3-β-d-glucan synthase are known to confer decreased caspofungin susceptibility on Candida spp. Clinical isolates of Candida spp. (404 Candida albicans, 62 C. tropicalis, and 21 C. krusei isolates) sent to the French National Reference Center were prospectively screened for susceptibility to caspofungin in vitro by the broth microdilution reference method of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST). Twenty-eight isolates (25 C. albicans, 2 C. tropicalis, and 1 C. krusei isolate) for which the caspofungin MIC was above the MIC that inhibited 90% of the isolates of the corresponding species (MIC90) were subjected to molecular analysis in order to identify mutations in the fks1 gene. Substitutions in the deduced protein sequence of Fks1 were found for 8 isolates, and 20 isolates had the wild-type sequence. Among the six C. albicans isolates harboring mutations, six patterns were observed involving amino acid changes at positions 641, 645, 649, and 1358. For C. tropicalis, one isolate showed an L644W mutation, and for one C. krusei isolate, two mutations, L658W and L701M, were found. Two media, RPMI medium and AM3, were tested for their abilities to distinguish between isolates with wild-type Fks1 and those with mutant Fks1. In RPMI medium, caspofungin MICs ranged from 0.25 to 2 μg/ml for wild-type isolates and from 1 to 8 μg/ml for mutant isolates. A sharper difference was observed in AM3: all wild-type isolates were inhibited by 0.25 μg/ml of caspofungin, while caspofungin MICs for all mutant isolates were ≥0.5 μg/ml. These data demonstrate that clinical isolates of C. albicans, C. tropicalis, and C. krusei with decreased susceptibility to caspofungin in vitro have diverse mutations in the fks1 gene and that AM3 is potentially a better medium than RPMI for distinguishing between mutant and wild-type isolates using the AFST-EUCAST method.

scholarly journals ERG6 and ERG2 Are Major Targets Conferring Reduced Susceptibility to Amphotericin B in Clinical Candida glabrata Isolates in Kuwait

2018 ◽  
Vol 63 (2) ◽  
pp. e01900-18 ◽  
Author(s):  
Suhail Ahmad ◽  
Leena Joseph ◽  
Josie E. Parker ◽  
Mohammad Asadzadeh ◽  
Steven L. Kelly ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Candida Glabrata ◽  
Molecular Mechanisms ◽  
Total Sterol ◽  
Resistant Isolate ◽  
Ergosterol Biosynthesis ◽  
Sterol Content ◽  
Wild Type ◽  
Content Type ◽  
Synonymous Mutations

ABSTRACT Candida glabrata is intrinsically less susceptible to azoles, and resistance to echinocandins and reduced susceptibility (RS) to amphotericin B (AMB) have also been detected. The molecular mechanisms of RS to AMB were investigated in C. glabrata strains in Kuwait by sequence analyses of genes involved in ergosterol biosynthesis. A total of 1,646 C. glabrata isolates were tested by Etest, and results for 12 selected isolates were confirmed by reference broth microdilution. PCR sequencing of three genes (ERG2, ERG6, and ERG11) was performed for all isolates with RS to AMB (RS-AMB isolates) and 5 selected wild-type C. glabrata isolates by using gene-specific primers. The total cell sterol content was analyzed by gas chromatography-mass spectrometry. The phylogenetic relationship among the isolates was investigated by multilocus sequence typing. Wild-type isolates contained only synonymous mutations in ERG2, ERG6, or ERG11, and the total sterol content was similar to that of the reference strains. A nonsynonymous ERG6 mutation (AGA48AAA, R48K) was found in both RS-AMB and wild-type isolates. Four RS-AMB isolates contained novel nonsense mutations at Trp286, Tyr192, and Leu341, and 2 isolates contained a nonsynonymous mutation in ERG6 (V126F or C198F); and the sterol content of these isolates was consistent with ERG6 deficiency. Two other RS-AMB isolates contained a novel nonsynonymous ERG2 mutation (G119S or G122S), and their sterol content was consistent with ERG2 deficiency. Of 8 RS-AMB isolates, 1 fluconazole-resistant isolate also contained nonsynonymous Y141H plus L381M mutations, while 7 isolates contained only synonymous mutations in ERG11. All isolates with ERG6, ERG2, and ERG11 mutations were genotypically distinct strains. Our data show that ERG6 and ERG2 are major targets conferring RS-AMB in clinical C. glabrata isolates.

scholarly journals Large-Scale Evaluation of In Vitro Amphotericin B, Triazole, and Echinocandin Activity against Coccidioides Species from U.S. Institutions

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
George R. Thompson ◽  
Bridget M. Barker ◽  
Nathan P. Wiederhold
Keyword(s):  
Amphotericin B ◽  
Patient Outcomes ◽  
Large Scale ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Clinical Isolates ◽  
Content Type ◽  
Scale Evaluation ◽  
Large Scale Testing

ABSTRACT Large-scale testing of Coccidioides isolates has not been performed, and the frequency of clinical isolates with elevated amphotericin B or triazole MICs has not been evaluated. Coccidioides isolates (n = 581) underwent antifungal susceptibility testing. Elevated MIC values were observed for fluconazole (≥16 μg/ml, 37.3% of isolates; ≥32 μg/ml, 7.9% of isolates), itraconazole (≥2 μg/ml, 1.0% of isolates), posaconazole (≥1 μg/ml, 1.0% of isolates), and voriconazole (≥2 μg/ml, 1.2% of isolates). However, mold-active triazoles exhibited low MICs for the majority of isolates tested. Additional correlation with patient outcomes to determine the relevance of elevated MICs in Coccidioides isolates is needed.

scholarly journals In Vitro Activities of Posaconazole, Itraconazole, Voriconazole, Amphotericin B, and Fluconazole against 37 Clinical Isolates of Zygomycetes

2002 ◽  
Vol 46 (5) ◽  
pp. 1581-1582 ◽  
Author(s):  
Qiu N. Sun ◽  
Annette W. Fothergill ◽  
Dora I. McCarthy ◽  
Michael G. Rinaldi ◽  
John R. Graybill
Keyword(s):  
Amphotericin B ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Clinical Development ◽  
Antifungal Susceptibility Testing ◽  
Clinical Isolates ◽  
Significant Potential ◽  
In Vitro Antifungal Susceptibility

ABSTRACT In vitro antifungal susceptibility testing results of a new antifungal triazole, posaconazole (POS), were compared to results with amphotericin B (AMB), itraconazole (ITC), voriconazole (VRC), and fluconazole (FLC) against clinical agents of zygomycosis. The MICs of POS at which 50% and 90% of the isolates were inhibited were 0.25 and 4 μg/ml, respectively. POS was significantly more active than VRC and FLC and slightly more active than ITC. The results suggest that POS has significant potential for clinical development against the zygomycetes.

Use of spectrophotometric reading for in vitro antifungal susceptibility testing ofAspergillusspp.

1999 ◽  
Vol 45 (10) ◽  
pp. 871-874 ◽  
Author(s):  
Eric Dannaoui ◽  
Florence Persat ◽  
Marie-France Monier ◽  
Elisabeth Borel ◽  
Marie-Antoinette Piens ◽  
...  
Keyword(s):  
Amphotericin B ◽  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Antifungal Susceptibility ◽  
Reference Method ◽  
Antifungal Susceptibility Testing ◽  
Aspergillus Species ◽  
National Committee ◽  
Broth Microdilution Method

A comparative study of visual and spectrophotometric MIC endpoint determinations for antifungal susceptibility testing of Aspergillus species was performed. A broth microdilution method adapted from the National Committee for Clinical Laboratory Standards (NCCLS) was used for susceptibility testing of 180 clinical isolates of Aspergillus species against amphotericin B and itraconazole. MICs were determined visually and spectrophotometrically at 490 nm after 24, 48, and 72h of incubation, and MIC pairs were compared. The agreement between the two methods was 99% for amphotericin B and ranged from 95 to 98% for itraconazole. It is concluded that spectrophotometric MIC endpoint determination is a valuable alternative to the visual reference method for susceptibility testing of Aspergillus species.Key words: antifungal, susceptibility testing, Aspergillus, spectrophotometric reading.

scholarly journals Flow Cytometry Antifungal Susceptibility Testing of Pathogenic Yeasts other than Candida albicans and Comparison with the NCCLS Broth Microdilution Test

2000 ◽  
Vol 44 (10) ◽  
pp. 2752-2758 ◽  
Author(s):  
Rama Ramani ◽  
Vishnu Chaturvedi
Keyword(s):  
Flow Cytometry ◽  
Candida Albicans ◽  
Amphotericin B ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Antifungal Susceptibility Testing ◽  
Broth Microdilution ◽  
Nitrogen Base ◽  
Microdilution Method ◽  
Broth Microdilution Method

ABSTRACT Candida species other than Candida albicansfrequently cause nosocomial infections in immunocompromised patients. Some of these pathogens have either variable susceptibility patterns or intrinsic resistance against common azoles. The availability of a rapid and reproducible susceptibility-testing method is likely to help in the selection of an appropriate regimen for therapy. A flow cytometry (FC) method was used in the present study for susceptibility testing ofCandida glabrata, Candida guilliermondii,Candida krusei, Candida lusitaniae,Candida parapsilosis, Candida tropicalis, andCryptococcus neoformans based on accumulation of the DNA binding dye propidium iodide (PI). The results were compared with MIC results obtained for amphotericin B and fluconazole using the NCCLS broth microdilution method (M27-A). For FC, the yeast inoculum was prepared spectrophotometrically, the drugs were diluted in either RPMI 1640 or yeast nitrogen base containing 1% dextrose, and yeast samples and drug dilutions were incubated with amphotericin B and fluconazole, respectively, for 4 to 6 h. Sodium deoxycholate and PI were added at the end of incubation, and fluorescence was measured with a FACScan flow cytometer (Becton Dickinson). The lowest drug concentration that showed a 50% increase in mean channel fluorescence compared to that of the growth control was designated the MIC. All tests were repeated once. The MICs obtained by FC for all yeast isolates except C. lusitaniae were in very good agreement (within 1 dilution) of the results of the NCCLS broth microdilution method. Paired ttest values were not statistically significant (P = 0.377 for amphotericin B; P = 0.383 for fluconazole). Exceptionally, C. lusitaniae isolates showed higher MICs (2 dilutions or more) than in the corresponding NCCLS broth microdilution method for amphotericin B. Overall, FC antifungal susceptibility testing provided rapid, reproducible results that were statistically comparable to those obtained with the NCCLS method.

scholarly journals Characterization of the Proliferating Cell Nuclear Antigen of Leishmania donovani Clinical Isolates and Its Association with Antimony Resistance

2014 ◽  
Vol 58 (6) ◽  
pp. 2997-3007 ◽  
Author(s):  
Rati Tandon ◽  
Sharat Chandra ◽  
Rajendra Kumar Baharia ◽  
Sanchita Das ◽  
Pragya Misra ◽  
...  
Keyword(s):  
Clinical Isolate ◽  
Proliferating Cell Nuclear Antigen ◽  
Leishmania Donovani ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Nuclear Antigen ◽  
Wild Type ◽  
Content Type ◽  
Sensitive Isolate ◽  
Proliferating Cell

ABSTRACTPreviously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate ofLeishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance inL. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by ≥3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ∼5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate.L. donovaniPCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate ofL. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (SbV) and trivalent antimonial (SbIII) drugs was assessedin vitroagainst promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of SbV(from 41.2 ± 0.6 μg/ml to 66.5 ± 3.9 μg/ml) and SbIII(from 24.0 ± 0.3 μg/ml to 43.4 ± 1.8 μg/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon SbIIItreatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance inL. donovaniclinical isolates, which warrants detailed investigations regarding its mechanism.

scholarly journals Epidermophyton floccosum: nucleotide sequence analysis and antifungal susceptibility testing of 40 clinical isolates

2019 ◽  
Vol 68 (11) ◽  
pp. 1655-1663 ◽  
Author(s):  
Saham Ansari ◽  
Bahram Ahmadi ◽  
Maryam Norouzi ◽  
Zohreh Ansari ◽  
Mohammad Hosein Afsarian ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Nucleotide Sequence ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Antifungal Susceptibility Testing ◽  
Clinical Isolates ◽  
Nucleotide Sequence Analysis ◽  
Epidermophyton Floccosum
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