Characterization of the Viable but Nonculturable (VBNC) State in Saccharomyces cerevisiae

Abstract

The Viable But Non Culturable (VBNC) state has been thoroughly studied in bacteria. In contrast, it has received much less attention in other microorganisms. However, it has been suggested that various yeast species occurring in wine may enter in VBNC following sulfite stress.In order to provide conclusive evidences for the existence of a VBNC state in yeast, the ability of Saccharomyces cerevisiae to enter into a VBNC state by applying sulfite stress was investigated. Viable populations were monitored by flow cytometry while culturable populations were followed by plating on culture medium. Twenty-four hours after the application of the stress, the comparison between the culturable population and the viable population demonstrated the presence of viable cells that were non culturable. In addition, removal of the stress by increasing the pH of the medium at different time intervals into the VBNC state allowed the VBNC S. cerevisiae cells to “resuscitate”. The similarity between the cell cycle profiles of VBNC cells and cells exiting the VBNC state together with the generation rate of cells exiting VBNC state demonstrated the absence of cellular multiplication during the exit from the VBNC state. This provides evidence of a true VBNC state. To get further insight into the molecular mechanism pertaining to the VBNC state, we studied the involvement of the SSU1 gene, encoding a sulfite pump in S. cerevisiae. The physiological behavior of wild-type S. cerevisiae was compared to those of a recombinant strain overexpressing SSU1 and null Δssu1 mutant. Our results demonstrated that the SSU1 gene is only implicated in the first stages of sulfite resistance but not per se in the VBNC phenotype. Our study clearly demonstrated the existence of an SO2-induced VBNC state in S. cerevisiae and that the stress removal allows the “resuscitation” of VBNC cells during the VBNC state.

Viable But Not Culturable (VBNC) state of Brettanomyces bruxellensis in wine: New insights on molecular basis of VBNC behaviour using a transcriptomic approach

“…According to these authors, the recovery of culturability is due to the presence and sudden growth of a few residual cells with a normal metabolism in a population predominantly non-culturable. Recently, Salma et al (2013) demonstrated that the removal of environmental stresses was sufficient to induce the exit from the VBNC state. Indeed, the recovery of the culturable state was shown as a true 'resuscitation' character and not a simple growth of a few residual cells with a normal metabolism (Salma et al, 2013).…”

“…About the 'resuscitation' character of these strains, they were differently able to exit from the VBNC state. The ability of cells to exit from this state was investigated choosing as model the 0.8 mg/ L concentration of molecular SO 2 , which was reduced to 0 by increasing the pH from 3.5 to 4.0 (Salma et al, 2013;Serpaggi et al, 2012). One day after removal of the stress, all the strains showed a concentration of culturable cells ranging from 10 3 to 10 4 CFU/mL, which was lower than that observed before the entry in the VBNC state.…”

“…Although the entrance in the VBNC state is a serious problem in order to manage microbial stability and safety in fermented food (Millet and Lonvaud-Funel, 2000;Li et al, 2014), few studies have investigated the VBNC behaviour in yeasts (Du Toit et al, 2005;Agnolucci et al, 2010;Serpaggi et al, 2012;Zuehlke and Edwards, 2013;Salma et al, 2013). Referring to the winery environment, several studies have shown that sulphur dioxide, an antimicrobial agent used in food preservation, induces the VBNC state in B. bruxellensis (Du Toit et al, 2005;Agnolucci et al, 2010;Serpaggi et al, 2012;Zuehlke and Edwards, 2013;Agnolucci et al, 2013).…”

State of the art of nonthermal and thermal processing for inactivation of micro-organisms

“…VBNC was mainly observed among Gram‐negative bacteria and has been proposed as a strategy for survival in natural environments (Salma et al . ). More than 60 bacterial species are described as being able to enter into a VBNC state, among them Gram‐positive (e.g.…”

Effects of Exogenous Yeast and Bacteria on the Microbial Population Dynamics and Outcomes of Olive Fermentations

“…Although the significance of this distinction is not yet known, the disparity could be due to the presence of dead but intact C. boidinii in the fermentations or the result of viable but nonculturable cells that were unable to grow on the laboratory culture medium used here. For the latter, other yeast species were also found to enter into nonculturable states (21, 22). Ultimately, refinements to yeast-specific DNA sequencing methods combined with other techniques such as fluorescent in situ hybridization (FISH) will help to resolve this discrepancy and can be used for more thorough measurements of yeast communities than culture-dependent molecular typing methods which are of limited throughput and both costly and time-intensive.…”