Quantitative analysis of the effect of specific tea compounds on germination and outgrowth of Bacillus subtilis spores at single cell resolution

Tea is one of the most widely consumed beverages in the world and known for its antimicrobial activity against many microorganisms. Preliminary studies have shown that tea polyphenols can inhibit the growth of a wide range of Gram-positive bacteria. However, the effect of these compounds on germination and outgrowth of bacterial spores is unclear. Spore-forming bacteria are an aggravating problem for the food industry due to spore formation and their subsequent returning to vegetative state during food storage, thus posing spoilage and food safety challenges. Here we analysed the effect of tea compounds: gallic acid, gallocatechin gallate, Teavigo (>90% epigallocatechin gallate), and theaflavin 3,3′-digallate on spore germination and outgrowth and subsequent growth of vegetative cells of Bacillus subtilis. To quantitatively analyse the effect of these compounds, live cell images were tracked from single phase-bright spores up to microcolony formation and analysed with the automated image analysis tool “SporeTracker”. In general, the tested compounds had a significant effect on most stages of germination and outgrowth. However, germination efficiency (ability of spores to become phase-dark) was not affected. Gallic acid most strongly reduced the ability to grow out. Additionally, all compounds, in particular theaflavin 3,3′-digallate, clearly affected the growth of emerging vegetative cells.