Green fluorescent protein as a novel indicator of antimicrobial susceptibility in Aureobasidium pullulans

Presently there is no method available that allows noninvasive and real-tim e 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. Aurcobasidium pullulans wa s transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Van den Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, B ioTechniques 23:686-690, 1997). The transformed strain Apl gfp showed brigh t fluorescence that was amenable to quantification using fluorescence spect rophotometry. Fluorescence levels in Ap I grp 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 fluo rescence was investigated by adding a range of concentrations of each of th e biocides sodium hypochlorite and 2-n-octylisothiozolin-3-one (OIT) to sus pensions of Apt gfp blastospores (pH 5 buffer). These biocides each caused a rapid (< 25-min) loss of fluorescence of greater than 90%. when used at c oncentrations of 150 mug of available chlorine ml(-1) and 500 mug ml(-1), r espectively. 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 associat ed with a lower reduction in viability. When A. pullulans was attached to t he surface of plasticized poly(vinylchloride) containing 500 ppm of OIT, fl uorescence decreased more slowly than in cell suspensions, with > 95% loss of fluorescence after 27 h. This technique should have broad applications i n testing the susceptibility, of A. pullulans and other fungal species to a ntimicrobial compounds.