Biodiversity of heteroresistance in food spoilage microorganisms
Publication Date
December 11, 2024
Creator
Violet, Joseph
Abstract
Spoilage of food by microorganisms is a growing problem in food supply chains. Food preservation methods are employed to prevent microbial spoilage of food, however the efficacy of these preservatives is challenged by specialised species which can grow in high concentrations of preservatives, and so spoil preserved foodstuffs. One such species is Zygosaccharomyces bailii and its interspecies hybrids Z. parabailii and Z. pseudobailii, which have extreme resistance to Weak Organic Acids (WOA). Z. bailii and its hybrids also display high cell-cell variation in resistance (heteroresistance), possibly as a bet-hedging strategy to allow a sub-population of resistant cells to resist stress should a sudden onset of stress occur. This thesis aimed to assay heteroresistance to common preservatives across a range of spoilage microorganisms and uncover mechanistic insights as to what determines heteroresistance in Z. bailii and its hybrids.
As existing methodologies of heteroresistance measurement such as dose-response curves were too laborious, the inoculum effect principle was used to develop a new, high-throughput method for assaying heteroresistance. Application of this assay to diverse spoilage yeasts revealed that heteroresistance and especially population-average resistance (IC50) are related to Minimum inhibitory concentration (MIC), whereas such a relationship with heteroresistance was not observed when considering a panel only of Zygosaccharomyces spp. isolates. The sorbic acid MIC was not significantly different between Z. bailii and its hybrids, however Z. parabailii possessed high IC50 and Z. pseudobailii possessed high heteroresistance. Overall, the IC50 appeared to be a strong indicator of the MIC, but heteroresistance was also related to MICs between distantly related species or in contexts where IC50 variation is low (i.e. Z. parabailii isolates).
The inoculum effect methodology was further applied to common food preservative stressors sorbic acid, acetic acid and sodium chloride in common food spoilage yeasts Z. bailii and its hybrids, and P. kudriavsevii, to ask whether heteroresistance varied between stressors and species. There was no difference between a factory or product isolation source for resistance parameters to NaCl, acetic acid and sorbic acid, for the isolates of P. kudriavsevii and Z. bailii (and its hybrids). Heteroresistance values tended to occupy similar ranges between species and stressors, with most values falling between 0.02 and 0.1 (SD, mM). The variation in physiological internal pH (pHi) distribution between Z. bailii isolates was also unrelated to heteroresistance to the three stressors, contrary to previous hypotheses in the field. Across the three stressors Z. bailii had higher IC50 and MIC, however P. kudriavsevii had greater heteroresistance to NaCl and sorbic acid, hinting further at a strategy of using a heteroresistance to compensate for a low IC50.
Finally, to identify genetic elements potentially implicated in sorbic acid heteroresistance, the inoculum effect methodology was used to screen for sorbic acid heteroresistance across 71 isolates of Z. parabailii. Resistance parameters tended to occupy a similar range across the Z. parabailii panel, with the exception of ‘Clade 2’ a distinct branch of the phylogenetic tree with extremely high heteroresistance. Preliminary optimisation of homologous recombination in Z. bailii showed a deletion of both copies of Z. bailii’s MSN4 homologue gave no phenotype to stress responses (including acetic acid) associated with the MSN2/MSN4 system in S. cerevisiae. S. cerevisiae deletant strains for open reading frames (ORFs) implicated by the genome wide association study (GWAS) for sorbic acid heteroresistance in Z. parabailii gave more sorbic acid heteroresistance phenotypes than deletants randomly selected for sorbate sensitivity from previous literature. Validation of GWAS candidates with the S. cerevisiae deletants implicated VPS45, PDX3 and COG8 in sorbic acid heteroresistance in S. cerevisiae, by putative mechanisms of ergosterol regulation in the plasma membrane, vesicle trafficking and varied Golgi-associated mechanisms (altered glycoproteins, cell-wall or vesicle trafficking) for PDX3, VPS45 and COG8 respectively.
Taken together these results suggest heteroresistance is a ubiquitous quality across spoilage yeast fungi, is specific to the stressor, however occupies a similar range of values across stressors and spoilage yeast species. Heteroresistance parameters are unrelated to isolation source, at least in contexts limited to the food supply chain. Our results also indicate heteroresistance, like other phenotypes, can be associated with genetic elements, and this dataset could lay the groundwork for a more mechanistic understanding of heteroresistance in Z. parabailii.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Life Sciences
eprints ID
78793
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