Influence of starvation, surface attachment and biofilm growth on the biocide susceptibility of the biodeteriogenic yeast Aureobasidium pullulans.

AIM: To investigate the effect of starvation, surface attachment and growth in a biofilm on the susceptibility of Aureobasidium pullulans to the biocides 2-n-octyl-4-isothiazolin-3-one (OIT) and sodium hypochlorite (NaOCl). METHODS AND RESULTS: Fluorescence loss from a green fluorescent protein (GFP)-transformed strain was used to monitor real-time loss in viability as previously described in situ in 96-well plates. Exponential phase, yeast-like (YL) cells were settled in the bottom of the wells as a low-density monolayer (LDM) and were susceptible to all biocide concentrations (25-100 mug ml(-1)). The exponential phase YL cells were either starved for 48 h in suspension or starved for 48 h as LDMs in the wells. Starvation in both cases led to a small reduction in susceptibility to the biocides. In contrast, 48-h biofilms grown in malt extract broth showed an apparent lack of susceptibility to 25 and 50 mug ml(-1) OIT and to 25-100 mug ml(-1) NaOCl. However, when the OIT concentration was increased to compensate for the higher cell density in the biofilm, the biofilms were found to be equally susceptible to the LDM. CONCLUSIONS: Starvation of A. pullulans YL cells either in suspension or as attached LDM resulted in a decrease in susceptibility to low concentrations of both OIT and NaOCl while the apparent reduced susceptibility of mature biofilms was due to the increase in biofilm cell density rather than true biofilm resistance per se. SIGNIFICANCE AND IMPACT OF THE STUDY: Monitoring fluorescence loss from the GFP-transformed strain of A. pullulans can be used as a fast and reliable method for monitoring cell death in real time as a response to biocide and antimicrobial challenge.

Fungal colonization of soil-buried plasticized polyvinyl chloride (pPVC) and the impact of incorporated biocides.

Plasticized polyvinyl chloride (pPVC) with or without incorporated biocides was buried in grassland and forest soil for up to 10 months. The change with time in viable counts of fungi on the plastic surface was followed, together with the percentage capable of clearing the two plasticizers dioctyl adipate (DOA) and dioctyl phthalate (DOP). With time fungal total viable counts (TVC) on control pPVC increased and the fraction able to clear DOA was considerably higher than the average estimated in both soil types. A total of 92 fungal morphotypes were isolated from grassland soil and 42 from forest soil with the greatest variety of fungal isolates observed on control pPVC. The incorporation of biocides into pPVC affected both fungal TVC and the richness of species isolated. The biocides NCMP [n-(trichloromethylthio)phthalimide], OBPA (10,10'-oxybisphenoxarsine) and OIT (2-n-octyl-4-isothiazolin-3-one) were the most effective in grassland soil, and TCMP [2,3,5,6-tetrachloro-4-(methylsulphonyl)pyridine] and NCMP the most effective in forest soil. In grassland soil, Penicillium janthinellum established as a principal colonizer and was recovered from all pPVC types. DOP clearers were found at much lower levels than DOA clearers, with Doratomyces spp. being the most efficient. At the end of 10 months the physical properties of the pPVC were altered; changes in stiffness were the most significant for heavily colonized grassland-buried pPVC samples, whereas in forest soil, the extensibility of the pPVC was affected more than the stiffness. These results suggest that fungi are important colonizers of pPVC buried in soil and that enrichment of soil fungi capable of clearing DOA occurs during colonization of the plastic surface. The results also demonstrate that incorporated biocides have a marked impact on the richness of species colonizing the pPVC surface.

In situ quantification of biocide efficacy using GFP transformed Aureobasidium pullulans.

AIMS: To develop a real-time in situ method to quantify loss of viability of Aureobasidium pullulans PRAFS8 cells attached to plasticized polyvinyl chloride (pPVC) with incorporated biocides, and to use the method to compare biocide efficacy in situ. METHODS AND RESULTS: A. pullulans PRAFS8, transformed with green fluorescent protein (GFP), was used to quantify the efficacy of a range of biocides incorporated into pPVC. Experimentally, it was found that a density of 1.53 x 10(6) yeast cells per cm(2) of pPVC was optimal as increasing the density of the yeast cells to 6.12 x 10(6) cm(-2) attached to pPVC containing the biocide 2-n-octyl-4-isothiazolin-3-one (OIT) decreased the rate of fluorescence loss. A strong positive correlation between fluorescence and viable yeast cell number was observed and fluorescence was used as a direct indicator of cell viability. The effectiveness of five commercial biocides, commonly incorporated into pPVC at their in-use concentrations, was tested against yeast cells attached to the pPVC surface. The loss of fluorescence and hence viability in situ was quantified using image analysis. The biocides N-(trichloromethylthio) phthalimide (NCMP), 10,10'-oxybisphenoxarsine (OBPA), OIT and 2,3,5,6-tetrachloro-4-(methylsulphonyl) pyridine (TCMP) caused complete loss of fluorescence within 30-50 h. In contrast the biocide dichloro-octyl-isothiazoline caused only 55 +/- 15% fluorescence loss after 50 h. Starvation of the yeast cells in suspension for 24 h prior to attachment reduced their initial sensitivity to OBPA, NCMP, OIT and TCMP by 15-20%, but eventually the fluorescence was also completely lost. CONCLUSIONS: The use of A. pullulans expressing cytosolic GFP enables the in situ quantification of loss of viability when cells are attached to pPVC with incorporated biocides. SIGNIFICANCE AND IMPACT OF THE STUDY: GFP fluorescence was used as a real-time indicator of cell viability and thus can be applied for direct quantification of the effectiveness of a broad range of biocides, incorporated into the polymer mass and used to protect a variety of plastics or other materials from microbial growth.

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

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.