Green Fluorescent Protein as a Novel Indicator of Antimicrobial Susceptibility in Aureobasidium pullulans
| Autor: | Sarah R. Barratt, Geoffrey D. Robson, I. M. Eastwood, H.A. Sabev, Marianne Nixon, Malcolm Greenhalgh, Jeremy S. Webb, Pauline S. Handley |
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| Jazyk: | angličtina |
| Rok vydání: | 2001 |
| Předmět: |
Green Fluorescent Proteins
Mycology Microbial Sensitivity Tests Applied Microbiology and Biotechnology Microbiology Green fluorescent protein chemistry.chemical_compound Transformation Genetic Ascomycota Blastospore Viability assay Ecology biology Fungal genetics Spores Fungal biology.organism_classification Fluorescence Fungicides Industrial Aureobasidium pullulans Transformation (genetics) Luminescent Proteins Spectrometry Fluorescence chemistry Sodium hypochlorite Biophysics Indicators and Reagents Food Science Biotechnology |
| Popis: | Presently there is no method available that allows noninvasive and real-time monitoring of fungal susceptibility to antimicrobial compounds. The green fluorescent protein (GFP) of the jellyfish Aequoria victoria was tested as a potential reporter molecule for this purpose. Aureobasidium pullulans was transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Vanden Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, BioTechniques 23:686–690, 1997). The transformed strain Ap1 gfp showed bright fluorescence that was amenable to quantification using fluorescence spectrophotometry. Fluorescence levels in Ap1 gfp blastospore suspensions were directly proportional to the number of viable cells determined by CFU plate counts ( r 2 > 0.99). The relationship between cell viability and GFP fluorescence was investigated by adding a range of concentrations of each of the biocides sodium hypochlorite and 2- n -octylisothiozolin-3-one (OIT) to suspensions of Ap1 gfp blastospores (pH 5 buffer). These biocides each caused a rapid (−1 and 500 μg ml −1 , respectively. Further, loss of GFP fluorescence from A. pullulans cells was highly correlated with a decrease in the number of viable cells ( r 2 > 0.92). Losses of GFP fluorescence and cell viability were highly dependent on external pH; maximum losses of fluorescence and viability occurred at pH 4, while reduction of GFP fluorescence was absent at pH 8.0 and was associated with a lower reduction in viability. When A. pullulans was attached to the surface of plasticized poly(vinylchloride) containing 500 ppm of OIT, fluorescence decreased more slowly than in cell suspensions, with >95% loss of fluorescence after 27 h. This technique should have broad applications in testing the susceptibility of A. pullulans and other fungal species to antimicrobial compounds. |
| Databáze: | OpenAIRE |
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