Directed evolution by UV-C treatment of Bacillus cereus spores

Bacterial endospores are exposed to a broad variety of sublethal and lethal stresses in the food production chain. Generally, these stresses will not completely eliminate the existing spore populations, and thus constitute a selection pressure on the spores. One stress that is frequently used in the food production chains to disinfect (food) contact surfaces is UV-C. At a wavelength of 254 nm, UV-C has germicidal properties. The aim of this research is to investigate the impact of UV-C stress on the evolution of endospore recalcitrance and germination in B. cereus. A directed evolution experiment was set up in which B. cereus was repeatedly subjected to a cycle of sporulation, sporicidal UV-C treatment, germination and outgrowth. We show here that three independent lineages of UV-C cycled B. cereus spores reproducibly acquired a 30-fold or higher increase in UV-C resistance at 164 mJ/cm². Surprisingly, the UV-C resistant spores of the clones isolated from each of the lineages also became significantly more sensitive to wet heat as a normally non-lethal heat treatment at 70 °C for 15 min resulted in an average 1.8 log cfu/mL reduction. From time-lapse phase contrast microscopy analysis, UV-C resistant mutant spores also showed a distinctive heterogeneity in refractility and a severe germination defect compared to the wild type. However, UV-C resistance of the corresponding vegetative cells was not altered. In conclusion, this work shows that UV-C resistance of endospores is an adaptive trait that can readily be improved, although at an apparent cost for heat resistance and germination efficiency. As such, these results provide novel insights in the evolvability of, and correlation between, some endospore properties.