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

Abstract

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 (< 25-min) loss of fluorescence of greater than 90% when used at concentrations of 150 microg of available chlorine ml(-1) and 500 microg 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.

FIG. 1

Influence of the biocides OIT and sodium hypochlorite on GFP fluorescence in Ap1 gfp blastospores. (a) OIT at concentrations of 0 (○), 50 (♦), 100 (▪), 350 (▴), and 500 (●) μg ml⁻¹. (b) Sodium hypochlorite at concentrations of 0 (○), 25 (⧫), 50 (▪), 75(▴), and 125 (●) μg of available chlorine ml⁻¹. Each point represents the mean of three replicate measurements ± standard error of the mean. (Errors were within the heights of the symbols.)

FIG. 2

Influence of the biocides OIT and hypochlorite on loss of GFP fluorescence and cell viability in Ap1 gfp blastospores. (a) Influence of 100 μg of OIT ml⁻¹. (b) Influence of 75 μg of hypochlorite ml⁻¹. (c) Correlation between GFP fluorescence and numbers of viable cells during treatment with OIT (○) and sodium hypochlorite (▪). Each point represents the mean of three replicate measurements ± standard error of the mean. (Errors not shown were within the heights of the symbols.)

FIG. 3

Influence of external pH on percent loss of GFP fluorescence and viability of Ap1 gfp cells after treatment with 100 μg of OIT ml⁻¹ for 1 h. Each point represents the mean of three replicate measurements ± standard error of the mean. (Errors not shown were within the heights of the symbols.)

FIG. 4

Influence of a range of biocides on GFP fluorescence in Ap1 gfp blastospores. Blastospores were exposed to the concentrations of biocide normally incorporated within pPVC: NCMP, 500 μg ml⁻¹ (▪); OBPA, 50 μg ml⁻¹ (▴); TCMP, 50 μg ml⁻¹ (●); BBIT, 750 μg ml⁻¹ (○); OIT, 500 μg ml⁻¹ (✳); no biocide, 2% DMSO (◊). Each point represents the mean of three replicate measurements ± standard error of the mean. (Errors were within the heights of the symbols.)

FIG. 5

Effect of incorporation of 500 ppm of OIT into pPVC on surface-attached AP1 gfp spores. (a) Percentages of fluorescent cells on pPVC (●) and pPVC with 500 ppm of OIT incorporated (▪). Each point represents the mean of five replicate measurements ± standard error of the mean. (b) Fluorescent images taken after 0 h (top left), 2 h (top right), 6 h (bottom left), and 27 h (bottom right).

FIG. 5

Effect of incorporation of 500 ppm of OIT into pPVC on surface-attached AP1 gfp spores. (a) Percentages of fluorescent cells on pPVC (●) and pPVC with 500 ppm of OIT incorporated (▪). Each point represents the mean of five replicate measurements ± standard error of the mean. (b) Fluorescent images taken after 0 h (top left), 2 h (top right), 6 h (bottom left), and 27 h (bottom right).