Relationship between In Vitro Activities of Amphotericin B and Flucytosine and pH for Clinical Yeast and Mold Isolates

Dorsthorst, D. T. A. Te; Verweij, Paul E.; Meis, Jacques F.; Mouton, Johan W.

doi:10.1128/aac.49.8.3341-3346.2005

Cited by 18 publications

(8 citation statements)

References 14 publications

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“…Similar discrepancies between Etest and the broth microdilution technique have been reported previously, such as in a multicenter study that reported that Etest performed on RPMI misclassified C. neoformans as resistant to flucytosine in 22.5 to 60% of the cases (29). It seems that pH is a critical parameter for flucytosine susceptibility testing with both yeasts and some molds (26). In a recent study, the median MICs of flucytosine determined by Etest against 16 strains of C. neoformans were Ͼ32 g/ml at pH 7.0 and 0.12 g/ml at pH 5.4 (28).…”

Section: Discussionmentioning

confidence: 57%

Results Obtained with Various Antifungal Susceptibility Testing Methods Do Not Predict Early Clinical Outcome in Patients with Cryptococcosis

Dannaoui

Abdul

Arpin

et al. 2006

Antimicrob Agents Chemother

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The in vitro susceptibilities of Cryptococcus neoformans isolates from consecutive human immunodeficiency virus-positive and -negative patients to the antifungal agents fluconazole, amphotericin B, and flucytosine were determined by different techniques, including the CLSI method, Etest, and broth microdilution in yeast nitrogen base (YNB) medium, during a multicenter prospective study in France. The relationship between the in vitro data and the clinical outcome 2 weeks after the initiation of antifungal therapy was assessed. In addition, the correlation between the strain serotype and the in vitro activities of the antifungals was determined, and the susceptibility results obtained with the different techniques were also compared. Thirty-seven patients received a combination of amphotericin B with flucytosine as first-line therapy, 22 were treated with amphotericin B alone, and 15 received fluconazole alone. Whatever the antifungal tested, there was no trend toward higher MICs for strains isolated from patients who failed to respond to a given therapy compared to those from patients who did not with either the CLSI method, Etest, or broth microdilution in YNB medium. The MICs obtained by the CLSI or Etest method were significantly lower for serotype D strains than for serotype A strains for both fluconazole and amphotericin B, while flucytosine MICs were not different according to serotype. These findings suggest that the in vitro antifungal susceptibility of C. neoformans, as determined with the techniques used, is not able to predict the early clinical outcome in patients with cryptococcosis.

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Section: Discussionmentioning

confidence: 57%

Results Obtained with Various Antifungal Susceptibility Testing Methods Do Not Predict Early Clinical Outcome in Patients with Cryptococcosis

Dannaoui

Abdul

Arpin

et al. 2006

Antimicrob Agents Chemother

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“…RPMI contains the pH indicator phenol red; the more yellow that the medium is, the more acidic that the solution is. This factor could be critical for susceptibility testing in this setting, as pH has previously been shown to have a profound effect on MICs (21,48,49). In industry, A. niger is a citric acid producer, so it is not entirely surprising that the organism might produce acid during incubation.…”

Section: Discussionmentioning

confidence: 99%

Cryptic Species and Azole Resistance in the Aspergillus niger Complex

Howard

Harrison

Bowyer

et al. 2011

Antimicrob Agents Chemother

121

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Aspergillus niger is a common clinical isolate. Multiple species comprise the Aspergillus section Nigri and are separable using sequence data. The antifungal susceptibility of these cryptic species is not known. We determined the azole MICs of 50 black aspergilli, 45 from clinical specimens, using modified EUCAST (mEUCAST) and Etest methods. Phylogenetic trees were prepared using the internal transcribed spacer, beta-tubulin, and calmodulin sequences to identify strains to species level and the results were compared with those obtained with cyp51A sequences. We attempted to correlate cyp51A mutations with azole resistance. Etest MICs were significantly different from mEUCAST MICs (P < 0.001), with geometric means of 0.77 and 2.79 mg/liter, respectively. Twenty-six of 50 (52%) isolates were itraconazole resistant by mEUCAST (MICs > 8 mg/liter), with limited cross-resistance to other azoles. Using combined beta-tubulin/calmodulin sequences, the 45 clinical isolates grouped into 5 clades, A. awamori (55.6%), A. tubingensis (17.8%), A. niger (13.3%), A. acidus (6.7%), and an unknown group (6.7%), none of which were morphologically distinguishable. Itraconazole resistance was found in 36% of the isolates in the A. awamori group, 90% of the A. tubingensis group, 33% of the A. niger group, 100% of the A. acidus group, and 67% of the unknown group. These data suggest that cyp51A mutations in section Nigri may not play as important a role in azole resistance as in A. fumigatus, although some mutations (G427S, K97T) warrant further study. Numerous cryptic species are found in clinical isolates of the Aspergillus section Nigri and are best reported as "A. niger complex" by clinical laboratories. Itraconazole resistance was common in this data set, but azole cross-resistance was unusual. The mechanism of resistance remains obscure.

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“…Among other fungal species, A. fumigatus is susceptible to 5FC (19,20) and is therefore anticipated to harbor genes encoding CD and UPRT activities in addition to 5FC uptake. BLASTP-based in silico predictions revealed A. fumigatus FcyA (AFUB_005410) and Uprt (AFUB_053020) as putative orthologs of S. cerevisiae Fcy1p and Fur1p, respectively.…”

Section: Resultsmentioning

confidence: 99%

Multiplex Genetic Engineering Exploiting Pyrimidine Salvage Pathway-Based Endogenous Counterselectable Markers

Birštonas

Dallemulle

López‐Berges

et al. 2020

mBio

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Selectable markers are indispensable for genetic engineering, yet their number and variety are limited. Most selection procedures for prototrophic cells rely on the introduction of antibiotic resistance genes. New minimally invasive tools are needed to facilitate sophisticated genetic manipulations. Here, we characterized three endogenous genes in the human fungal pathogen Aspergillus fumigatus for their potential as markers for targeted genomic insertions of DNAs of interest (DOIs). Since these genes are involved in uptake and metabolization of pyrimidines, resistance to the toxic effects of prodrugs 5-fluorocytosine and 5-fluorouracil can be used to select successfully integrated DOIs. We show that DOI integration, resulting in the inactivation of these genes, caused no adverse effects with respect to nutrient requirements, stress resistance, or virulence. Beside the individual use of markers for site-directed integration of reporter cassettes, including the 17-kb penicillin biosynthetic cluster, we demonstrate their sequential use by inserting three genes encoding fluorescent proteins into a single strain for simultaneous multicolor localization microscopy. In addition to A. fumigatus, we validated the applicability of this novel toolbox in Penicillium chrysogenum and Fusarium oxysporum. Enabling multiple targeted insertions of DOIs without the necessity for exogenous markers, this technology has the potential to significantly advance genetic engineering. IMPORTANCE This work reports the discovery of a novel genetic toolbox comprising multiple, endogenous selectable markers for targeted genomic insertions of DNAs of interest (DOIs). Marker genes encode proteins involved in 5-fluorocytosine uptake and pyrimidine salvage activities mediating 5-fluorocytosine deamination as well as 5-fluorouracil phosphoribosylation. The requirement for their genomic replacement by DOIs to confer 5-fluorocytosine or 5-fluorouracil resistance for transformation selection enforces site-specific integrations. Due to the fact that the described markers are endogenously encoded, there is no necessity for the exogenous introduction of commonly employed markers such as auxotrophy-complementing genes or antibiotic resistance cassettes. Importantly, inactivation of the described marker genes had no adverse effects on nutrient requirements, growth, or virulence of the human pathogen Aspergillus fumigatus. Given the limited number and distinct types of selectable markers available for the genetic manipulation of prototrophic strains such as wild-type strains, we anticipate that the proposed methodology will significantly advance genetic as well as metabolic engineering of fungal species.

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Relationship between In Vitro Activities of Amphotericin B and Flucytosine and pH for Clinical Yeast and Mold Isolates

Cited by 18 publications

References 14 publications

Results Obtained with Various Antifungal Susceptibility Testing Methods Do Not Predict Early Clinical Outcome in Patients with Cryptococcosis

Results Obtained with Various Antifungal Susceptibility Testing Methods Do Not Predict Early Clinical Outcome in Patients with Cryptococcosis

Cryptic Species and Azole Resistance in the Aspergillus niger Complex

Multiplex Genetic Engineering Exploiting Pyrimidine Salvage Pathway-Based Endogenous Counterselectable Markers

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