In the 96-well microtitre plates, it was necessary to wait until colonies were 0.5–1 mm in size

to ensure accurate counting. In the absence of preservatives, this required 2–3 days incubation. At higher concentrations of preservatives, the incubation PD-0332991 clinical trial time required increased up to 12–14 days. It was noted that when the resistant sub-populations were re-inoculated into media containing weak-acids, the slow rate of growth remained unchanged, even though all cells (from that resistant population) then grew. This occurred in sorbic acid, benzoic acid and acetic acid and can be regarded as an indication that preservatives were not being degraded by resistant sub-populations, since this would result in faster growth following removal of preservative. Resistant sub-populations were grown over 2 weeks in 6 mM sorbic acid, 8 mM benzoic acid, and 350 mM acetic acid. These populations were then cross-inoculated into all combinations of other preservatives, at a full range of concentrations. Surprisingly, all resistant sub-populations were resistant to all

three Talazoparib preservatives tested (Fig. 4). All cell populations grown in 6 mM sorbic acid were fully resistant to sorbic acid, benzoic acid and acetic acid. Similarly, 100% population resistance was obtained in all nine preservative combinations, i.e. cells grown in 8 mM benzoic acid, 6 mM sorbic acid or in 350 mM acetic acid and then inoculated into any weak acid. These data indicate either a common mechanism of action by all three preservatives against Z. bailii, or a common resistance mechanism in Z. bailii affecting all weak acid preservatives. The data presented have shown that Z. bailii is resistant to a variety of weak acids of different structures but not lipophilic alcohols. Furthermore, that resistance is due to heterogeneity within the yeast population, and the resistance to any single acid confers resistance to other (possibly all) weak acids. The simplest hypothesis explaining

these data is that there is a mechanism lowering uptake of weak acids in the resistant sub-population, which is non-functional in the bulk population. This would result in a lower cytoplasmic accumulation of all acids and minimise toxic effects, irrespective of any mechanism of action. This hypothesis was tested using PDK4 uptake of 14C-acetic acid, using a low concentration that would not significantly disturb the cytoplasmic pH ( Fig. 5). Uptake of acetic acid in populations grown with or without sorbic acid was rapid, reaching a plateau in ~ 3–10 min. This represents the maximum cellular accumulation, a dynamic equilibrium of diffusion into and out from the cell. The initial uptake rate ( Fig. 5) reflected the final equilibrium level, but it is the equilibrium level that determines the accumulated concentration of weak-acid. The maximum uptake level in the normal bulk populations of S. cerevisiae was marginally higher than the bulk population of Z.