Mechanistic insights into the adaptive evolvability of spore heat resistance in Bacillus cereus sensu lato.

Wet heat treatment is a commonly applied method in the food and medical industries for the inactivation of microorganisms, and bacterial spores in particular. While many studies have delved into the mechanisms underlying wet heat killing and spore resistance, little attention has so far been dedicated to the capacity of spore-forming bacteria to tune their resistance through adaptive evolution. Nevertheless, a recent study from our group revealed that a psychrotrophic strain of the Bacillus cereus sensu lato group (i.e. Bacillus weihenstephanensis LMG 18989) could readily and reproducibly evolve to acquire enhanced spore wet heat resistance without compromising its vegetative cell growth ability at low temperatures. In the current study, we demonstrate that another B. cereus strain (i.e. the mesophilic B. cereus sensu stricto ATCC 14579) can acquire significantly increased spore wet heat resistance as well, and we subjected both the previously and currently obtained mutants to whole genome sequencing. This revealed that five out of six mutants were affected in genes encoding regulators of the spore coat and exosporium pathway (i.e. spoIVFB, sigK and gerE), with three of them being affected in gerE. A synthetically constructed ATCC 14579 ΔgerE mutant likewise yielded spores with increased wet heat resistance, and incurred a compromised spore coat and exosporium. Further investigation revealed significantly increased spore DPA levels and core dehydration as the likely causes for the observed enhanced spore wet heat resistance. Interestingly, deletion of gerE in Bacillus subtilis 168 did not impose increased spore wet heat resistance, underscoring potentially different adaptive evolutionary paths in B. cereus and B. subtilis.

UV-C and wet heat resistance of Bacillus thuringiensis biopesticide endospores compared to foodborne Bacillus cereus endospores.

Bacillus endospores (spores) are generally resistant to environmental and food processing-related stress including thermal and non-thermal processing in the food industry, such as pasteurization, and UV-C inactivation. Bacillus thuringiensis insecticidal crystals and spores as the active substances in commercial biopesticides can also be introduced to vegetable foods and their food processing environment due to pre-harvest treatment of edible crops. The resistance of B. thuringiensis biopesticide spores in comparison to the genetically closely related foodborne B. cereus against heat and UV-C treatment is investigated in this study. The results show that B. thuringiensis biopesticide spores with the commercial granulated product formulation are better protected and as such more resistant to both wet heat (D values at 90 °C: 50.1-79.5 min) and UV-C treatment (D values at 0.6 mW/cm2: 7.5-8.9 min) than the pure spore suspension. The enhanced UV-C resistance properties of B. thuringiensis-formulated spores also indicate that the B. thuringiensis spores in powder or granule formulation applied in the field might not be effectively inactivated by solar radiation (UV-A and UV-B) in a short period. Furthermore, the spores of one emetic B. cereus toxin-producing strain (LFMFP 254; a Belgian outbreak strain) were found more resistant to the wet heat at 90 °C (D90-value = 71.2 min) than other tested pure spore suspensions, although the spores of B. cereus 254 did not show different behavior against UV-C treatment. This result suggests that UV-C treatment can be applied as an effective inactivation method against B. cereus 254 spores.

Detection of Enterotoxigenic Psychrotrophic Presumptive Bacillus cereus and Cereulide Producers in Food Products and Ingredients.

In the last decade, foodborne outbreaks and individual cases caused by bacterial toxins showed an increasing trend. The major contributors are enterotoxins and cereulide produced by Bacillus cereus, which can cause a diarrheal and emetic form of the disease, respectively. These diseases usually induce relatively mild symptoms; however, fatal cases have been reported. With the aim to detected potential toxin producers that are able to grow at refrigerator temperatures and subsequently produce cereulide, we screened the prevalence of enterotoxin and cereulide toxin gene carriers and the psychrotrophic capacity of presumptive B. cereus obtained from 250 food products (cereal products, including rice and seeds/pulses, dairy-based products, dried vegetables, mixed food, herbs, and spices). Of tested food products, 226/250 (90.4%) contained presumptive B. cereus, which communities were further tested for the presence of nheA, hblA, cytK-1, and ces genes. Food products were mainly contaminated with the nheA B. cereus carriers (77.9%), followed by hblA (64.8%), ces (23.2%), and cytK-1 (4.4%). Toxigenic B. cereus communities were further subjected to refrigerated (4 and 7 °C) and mild abuse temperatures (10 °C). Overall, 77% (94/121), 86% (104/121), and 100% (121/121) were able to grow at 4, 7, and 10 °C, respectively. Enterotoxin and cereulide potential producers were detected in 81% of psychrotrophic presumptive B. cereus. Toxin encoding genes nheA, hblA, and ces gene were found in 77.2, 55, and 11.7% of tested samples, respectively. None of the psychrotrophic presumptive B. cereus were carriers of the cytotoxin K-1 encoding gene (cytK-1). Nearly half of emetic psychrotrophic B. cereus were able to produce cereulide in optimal conditions. At 4 °C none of the examined psychrotrophs produced cereulide. The results of this research highlight the high prevalence of B. cereus and the omnipresence of toxin gene harboring presumptive B. cereus that can grow at refrigerator temperatures, with a focus on cereulide producers.

Bacillus weihenstephanensis can readily evolve for increased endospore heat resistance without compromising its thermotype.

Proper elimination of bacterial endospores in foods and food processing environment is challenging because of their extreme resistance to various stresses. Often, sporicidal treatments prove insufficient to eradicate the contaminating endospore population as a whole, and might therefore serve as a selection pressure for enhanced endospore resistance. In the sporeforming Bacillus cereus group, Bacillus weihenstephanensis is an important food spoilage organism and potential cereulide producing pathogen, due to its psychrotolerant growth ability at 7 °C. Although the endospores of B. weihenstephanensis are generally less heat resistant compared to their mesophilic or thermotolerant relatives, our data now show that non-emetic B. weihenstephanensis strain LMG 18989T can readily and reproducibly evolve to acquire much enhanced endospore heat resistance. In fact, one of the B. weihenstephanensis mutants from directed evolution by wet heat in this study yielded endospores displaying a > 4-fold increase in D-value at 91 °C compared to the parental strain. Moreover, these mutant endospores retained their superior heat resistance even when sporulation was performed at 10 °C. Interestingly, increased endospore heat resistance did not negatively affect the vegetative growth capacities of the evolved mutants at lower (7 °C) and upper (37 °C) growth temperature boundaries, indicating that the correlation between cardinal growth temperatures and endospore heat resistance which is observed among bacterial sporeformers is not necessarily causal.

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.