Directed evolution by UV-C treatment of Bacillus cereus spores

Abstract

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 minutes 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 were 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.

Usefulness of a new DUV-LED device for the control of infection by Escherichia coli, Staphylococcus aureus, mycobacteria and spore-forming bacteria

“…After irradiation for 2 s, a ≥90% reduction in the infectious titers of both E. coli and S. aureus was observed; this short irradiation time is expected to be very useful considering the time it takes to clean floors. Even for bacterial species that have been reported to be resistant to DUV ( 17 , 18 ), a ≥90% reduction was observed after 10 s of irradiation for M. gordonae , and after 30 s of irradiation for spore-type B. subtilis . Furthermore, irradiation at intervals showed the same microbial-inactivating effect as that from continuous irradiation, indicating that a sufficient inactivating effect can be obtained by divided irradiation.…”

The Bacillus cereus Food Infection as Multifactorial Process

“…Next to heat treatment, further non-thermal technologies for the elimination/reduction of B. cereus spores in foods were established, with the aim of significantly reducing or inactiving spores without affecting the integrity and quality of the food, alone or in combination with mild heat treatment. These are pulsed light treatment [158], electron beam irradiation [159,160], continuous ohmic heating [161][162][163], dielectric barrier discharge plasma [164], acidic electrolyzed oxidizing (EO) water and slightly acidic EO water [140,165] coupled with ultrasonication [166,167], UV treatment [168,169], microwave-combined cold plasma treatment [170] and combined treatment with germinant compounds and superheated steam [171].…”

Colorimetric aptasensor for detection of Bacillus cytotoxicus spores in milk and ready-to-use food

“…The final solutions containing spores were washed to eliminate the remaining medium and debris of bacterial cells using a protocol described by Ref. [ 19 ] with some modifications. Briefly, the spores were collected by centrifugation (5 min at 5000 rpm) and washed one time with sterile water, and a further three times with 50% ethanol aqueous solution, followed by an overnight incubation in 4 °C water.…”