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

Abstract

Aims: 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 μg 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 μg ml−1 OIT and to 25–100 μg 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.

Resistance of bacterial biofilms to disinfectants: a review

“…Cells with distinctive metabolic rates were present throughout the threedimensional structure, thus constituting a physiologically heterogeneous population. Alterations to growth and activity rates induced modifications to membrane composition and the expression of defense mechanisms that could lead to an increased resistance of bacteria to biocides (Stewart and Olson 1992;Lisle et al 1998;Saby et al 1999;Taylor et al 2000;Sabev et al 2006). Indeed, it is now widely accepted that the development of a stress response is an important feature of the life cycle of biofilms (Beloin and Ghigo 2005;Coenye 2010).…”

New Antiadhesive Hydrophobic Polysiloxanes

“…A. pullulans cells show high adhesion abilities. This yeast-like fungus forms chlamydoconidia and blastoconidia with high environmental resistance, colonizes different surfaces and extensively produces extracellular polysaccharide pullulan (poly-α-1,6-maltotriose) [57]. Its biofilm and spore structures play an important role in protecting cells against adverse environmental conditions.…”

Comparative transcriptomic analysis ofClostridium perfringensbiofilms and planktonic cells

“…It has been stated that this chemical heterogeneity is the source of the physiological heterogeneity (Stewart & Franklin, 2008) where cells with distinctive metabolic rates are present throughout the biofilm, indicating variety in gene expressions. This induces modifications to membrane composition and in defence mechanism expressions leading to an increase in tolerance of bacteria to environmental stresses (Taylor et al, 2000;Sabev et al, 2006). Also, it has been reported that a small part of the bacterial population within a biofilm may develop into a highly protected state with drastic resistance and referred to as persisters (Harrison et al, 2005;Lewis, 2005).…”