Significant substratum damage can occur when plasticized PVC (pPVC) is colonized by microorganisms. We investigated microbial colonization of pPVC in an in situ, longitudinal study. Pieces of pPVC containing the plasticizers dioctyl phthalate and dioctyl adipate (DOA) were exposed to the atmosphere for up to 2 years. Fungal and bacterial populations were quantified, and colonizing fungi were identified by rRNA gene sequencing and morphological characteristics. Aureobasidium pullulans was the principal colonizing fungus, establishing itself on the pPVC between 25 and 40 weeks of exposure. A group of yeasts and yeast-like fungi, including Rhodotorula aurantiaca and Kluyveromyces spp., established themselves on the pPVC much later (after 80 weeks of exposure). Numerically, these organisms dominated A. pullulans after 95 weeks, with a mean viable count ؎ standard error of 1,000 ؎ 200 yeast CFU cm ؊2 , compared to 390 ؎ 50 A. pullulans CFU cm ؊2 . No bacterial colonization was observed. We also used in vitro tests to characterize the deteriogenic properties of fungi isolated from the pPVC. All strains of A. pullulans tested could grow with the intact pPVC formulation as the sole source of carbon, degrade the plasticizer DOA, produce extracellular esterase, and cause weight loss of the substratum during growth in vitro. In contrast, several yeast isolates could not grow on pPVC or degrade DOA. These results suggest that microbial succession may occur during the colonization of pPVC and that A. pullulans is critical to the establishment of a microbial community on pPVC.Plasticized PVC (pPVC) is highly susceptible to microbial attack in many different environmental situations. The problem was first identified in U.S. government reports of the deterioration of military equipment (8, 44), and subsequent reports described defacement and deterioration of commercial pPVC products (20,50). Biodeterioration of pPVC is now known to occur in a wide range of industrial, commercial, and structural applications (18,19,22).The susceptibility of pPVC results from the presence of plasticizers, commonly organic acid esters such as dioctyl phthalate (DOP) and dioctyl adipate (DOA), added to modify physical or mechanical properties of the polymer. Both bacteria (6,7,14) and fungi (5, 36) can degrade ester-based plasticizers. Loss of plasticizers from pPVC due to microbial degradation results in brittleness, shrinkage, and ultimately failure of the pPVC in its intended application.Microbial deterioration of pPVC has been studied extensively in vitro. Many studies have examined the resistance of pPVC formulations incorporating biocides to colonization by test organisms (38,39,46). Other research has determined biodegradability by measuring changes in the physical properties of pPVC, such as changes in tensile strength (49), mass (9), or electrical properties (42) during biodegradation. Several international standard test methods for microbiological susceptibility of plastics have been established (1, 2, 26).Colonization processes occurring...

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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 g of available chlorine ml ؊1 and 500 g 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.Since the cloning of wild-type green fluorescent protein (GFP) from the jellyfish Aequoria victoria (25), expression of GFP has been demonstrated in numerous organisms, including plants (31), animals (5), bacteria (3), yeasts (7), and filamentous fungi (13, 37). Most applications of GFP have been as a passive label of gene expression and protein localization (for a review, see reference 36). However, GFP and selected mutants are now increasingly used as active sensors of physiological events within cells. In this role, GFP fluorescence is influenced posttranslationally by its chemical environment. For example, the pH sensitivity of GFP has recently been exploited to measure intracellular (16,18,27) and organellar (20) pH, and GFP-based systems have been developed to monitor intracellular calcium (21), microviscosity (34), and protease activity (15).One application of GFP that has not been explored with fungi is its use as an indicator of antimicrobial susceptibility. GFP has several properties that are desirable for this purpose, including simplicity and versatility for in vitro use (9). GF...

Initial adhesion of fungi to plasticized polyvinyl chloride (pPVC) may determine subsequent colonization and biodeterioration processes. The deteriogenic fungus Aureobasidium pullulans was used to investigate the physicochemical nature of adhesion to both unplasticized PVC (uPVC) and pPVC containing the plasticizers dioctyl phthalate (DOP) and dioctyl adipate (DOA). A quantitative adhesion assay using image analysis identified fundamental differences in the mechanism of adhesion of A. pullulans blastospores to these substrata. Adhesion to pPVC was greater than that to uPVC by a maximum of 280% after a 4-h incubation with 108 blastospores ml−1. That plasticizers enhance adhesion to PVC was confirmed by incorporating a dispersion of both DOA and DOP into the blastospore suspension. Adhesion to uPVC was increased by up to 308% in the presence of the dispersed plasticizers. Hydrophobic interactions were found to dominate adhesion to uPVC because (i) a strong positive correlation was observed between substratum hydrophobicity (measured by using a dynamic contact angle analyzer) and adhesion to a range of unplasticized polymers including uPVC, and (ii) neither the pH nor the electrolyte concentration of the suspension buffer, both of which influence electrostatic interactions, affected adhesion to uPVC. In contrast, adhesion to pPVC is principally controlled by electrostatic interactions. Enhanced adhesion to pPVC occurred despite a relative reduction of 13° in the water contact angle of pPVC compared to that of uPVC. Furthermore, adhesion to pPVC was strongly dependent on both the pH and electrolyte concentration of the suspension medium, reaching maximum levels at pH 8 and with an electrolyte concentration of 10 mM NaCl. Plasticization with DOP and DOA therefore increases adhesion ofA. pullulans blastospores to pPVC through an interaction mediated by electrostatic forces.

Many antimicrobial compounds exhibit bacterial cell membrane activity as either potassium ion leakage and/or leakage of material that absorbs at 260 nm from the cell. In this experiment a potassium ion selective electrode and spectophotometric observation of 260-nm leakage were used in order to examine cell membrane effects in a selection of common biocides upon both Escherichia coli NCIMB 10000 and Pseudomonas aeruginosa NCIMB 10548. The observation of potassium ion leakage for pyrithione biocides yielded results which were initially difficult to interpret, but are thought to suggest a species-dependent combination of potassium ion leakage from affected membranes and chelation of those leaked ions in the bathing suspension. Such a result is not, however, supported by the 260-nm material leakage results, which indicate very similar levels of membrane active effects for both species of bacteria.