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The gut microbiota influences human health and the development of chronic diseases. However, our understanding of potentially protective or harmful microbe-host interactions at the molecular level is still in its infancy. To gain further insights into the hidden gut metabolome and its impact, we identified a cryptic non-ribosomal peptide BGC in the genome of Bacillus cereus DSM 28590 from the mouse intestine ( www.dsmz.de/miBC ), which was predicted to encode a thiazol(in)e substructure. Cloning and heterologous expression of this BGC revealed that it produces bacillamide D. In-depth functional evaluation showed potent cytotoxicity and inhibition of cell migration using the human cell lines HCT116 and HEK293, which was validated using primary mouse organoids. This work establishes the bacillamides as selective cytotoxins from a bacterial gut isolate that affect mammalian cells. Our targeted structure-function-predictive approach is demonstrated to be a streamlined method to discover deleterious gut microbial metabolites with potential effects on human health., (© 2024. The Author(s).)

Bacillus cereus is a ubiquitous endospore-forming bacterium, which mainly affects humans as a food-borne pathogen. Bacillus cereus can contaminate groundwater used to irrigate food crops. Here, we examined the ability of the emetic strain B. cereus F4810/72 to survive abiotic conditions encountered in groundwater. Our results showed that vegetative B. cereus cells rapidly evolved in a mixed population composed of endospores and asporogenic variants bearing spo0A mutations. One asporogenic variant, VAR-F48, was isolated and characterized. VAR-F48 can survive in sterilized groundwater over a long period in a vegetative form and has a competitive advantage compared to its parental strain. Proteomics analysis allowed us to quantify changes to cellular and exoproteins after 24 and 72 h incubation in groundwater, for VAR-F48 compared to its parental strain. The results revealed a significant re-routing of the metabolism in the absence of Spo0A. We concluded that VAR-F48 maximizes its energy use to deal with oligotrophy, and the emergence of spo0A-mutated variants may contribute to the persistence of emetic B. cereus in natural oligotrophic environments., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Spores of psychrotolerant strains of the foodborne pathogen Bacillus cereus can multiply during storage of cooked or pasteurized, refrigerated foods and can represent a risk if these cells are not eliminated during reheating of food product before consumption. We determined the heat-resistance of psychrotolerant B. cereus vegetative cells at different heating temperatures in laboratory medium and compared it with that of thermotolerant B. cereus vegetative cells. The z values, based on times for a 3 log 10 reduction, of the vegetative cells of the three psychrotolerant phylogenetic groups of B. cereus varied between 3.02 °C and 4.84 °C. The temperature at which a 3 log 10 reduction was achieved in 10 min varied between 47.6 °C and 49.2 °C for psychrotolerant vegetative cells and it was around 54.8 °C for thermotolerant vegetative cells. Moreover, 0.4 min at 60 °C would be sufficient for a 6 log 10  CFU/ml reduction of the most heat resistant psychrotolerant B. cereus vegetative cells. These data clearly showed that psychrotolerant B. cereus vegetative cells can be rapidly eliminated by a mild heat treatment such as food reheating., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Psychrotrophic strains of the foodborne pathogen Bacillus cereus can multiply during the refrigerated storage of food products. The aim of this study was to determine the impact of anaerobiosis on the growth of two psychrotrophic B. cereus strains exposed to acidic pH at a cold temperature in a laboratory medium. At 10 °C, growth occurred at pH values equal to or higher than 5.7 during anaerobiosis, whereas aerobic growth was observed from pH 5.4. Growth rates during aerobiosis were similar at pH 5.4 and pH 7. No growth was observed for the two tested strains at 8 °C without oxygen regardless of the pH; however, both strains grew at this temperature from pH 5.4 in the presence of oxygen. These pH growth limits in aerobiosis are consistent with those reported for different strains and different foods or media, but no other studies have described anaerobic growth at acidic pH values. The maximal B. cereus concentration was approximately 6.0 log10 CFU/ml for cultures in the absence of oxygen and approximately 8.0 log10 CFU/ml for cultures in the presence of oxygen. In conclusion, we found that the combination of anaerobiosis, pH < 5.7 at 10 °C, or anaerobiosis and temperatures ≤8 °C prevent psychrotrophic B. cereus growth., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The behaviour of the sporulating soil-dwelling Bacillus cereus sensu lato (B. cereus sl) which includes foodborne pathogenic strains has been extensively studied in relation to its various animal hosts. The aim of this environmental study was to investigate the water compartments (rain and soil water, as well as groundwater) closely linked to the primary B. cereus sl reservoir, for which available data are limited. B. cereus sl was present, primarily as spores, in all of the tested compartments of an agricultural site, including water from rain to groundwater through soil. During rain events, leachates collected after transfer through the soil eventually reached the groundwater and were loaded with B. cereus sl. In groundwater samples, newly introduced spores of a B. cereus model strain were able to germinate, and vegetative cells arising from this event were detected for up to 50 days. This first B. cereus sl investigation in the various types of interrelated environments suggests that the consideration of the aquatic compartment linked to soil and to climatic events should provide a better understanding of B. cereus sl ecology and thus be relevant for a more accurate risk assessment of food poisoning caused by B. cereus sl pathogenic strains.

In a chemically defined medium and in Luria broth, cold strongly reduced maximal population density of Bacillus cereus ATCC 14579 in anaerobiosis and caused formation of filaments. In cooked spinach, maximal population density of B. cereus in anaerobiosis was the same at cold and optimal temperatures, with normal cell divisions. The lipid containing fraction of spinach, but not the hydrophilic fraction, restored growth of B. cereus under cold and anaerobiosis when added to the chemically defined medium. This fraction was rich in unsaturated, low melting point fatty acids. Addition of phosphatidylcholine containing unsaturated, low melting point, fatty acids similarly improved B. cereus anaerobic growth at cold temperature. Addition of hydrogenated phosphatidylcholine containing saturated, high melting point, fatty acids did not modify growth. Fatty acids from phospholipids, from spinach and from hydrogenated phosphatidylcholine, although normally very rare in B. cereus, were inserted in the bacterium membrane. Addition of phospholipids rich in unsaturated fatty acids to cold and anaerobic cultures, increased fluidity of B. cereus membrane lipids, to the same level as those from B. cereus normally cold adapted, i.e. grown aerobically at 15 °C. B. cereus is therefore able to use external fatty acids from foods or from the growth medium to adapt its membrane to cold temperature under anaerobiosis, and to recover the maximal population density achieved at optimal temperature., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The impact of fermentative metabolism at low temperature on cell division of Bacillus cereus was studied. Fermentation at 37 °C had no influence on the division of bacteria. Aerobic cultures at 15 °C produced larger cells than at 37 °C, but cell division was normal. In fermentative cultures at 15 °C, no increase in CFU ml(-1) was observed. However, A(600) increased, due to formation of long filaments. Transmission electronic microscopy and light microscopy with fluorescent staining showed several nucleic acid entities separated by a hydrophobic membrane, indicating that each filament contained several individual cells attached by peptidoglycan. When left in air at room temperature, one filament gave several daughter cells, this means that one CFU formed by one filament may represent a greater contamination potential than one CFU formed by a single cell. Division was observed in cultures at 15 °C with anaerobic respiration in the presence of nitrates. Possible filamentous growth must thus be taken into account to avoid underestimating B. cereus growth in vacuum or modified atmosphere packaged foods stored at low temperature., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The impact of simultaneous anaerobiosis and low temperature on growth parameters, metabolism, and membrane properties of Bacillus cereus ATCC 14579 was studied. No growth was observed under anaerobiosis at 12°C. In bioreactors, growth rates and biomass production were drastically reduced by simultaneous anaerobiosis and low temperature (15°C). The two conditions had a synergistic effect on biomass reduction. In anaerobic cultures, fermentative metabolism was modified by low temperature, with a marked reduction in ethanol production leading to a lower ability to produce NAD(+). Anaerobiosis reduced unsaturated fatty acids at both low optimal temperatures. In addition, simultaneous anaerobiosis and low temperatures markedly reduced levels of branched-chain fatty acids compared to all other conditions (accounting for 33% of total fatty acids against more 71% for low-temperature aerobiosis, optimal-temperature aerobiosis, and optimal-temperature anaerobiosis). This corresponded to high-melting-temperature lipids and to low-fluidity membranes, as indicated by differential scanning calorimetry, 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy, and infrared spectroscopy. This is in contrast to requirements for cold adaptation. A link between modification in the synthesis of metabolites of fermentative metabolism and the reduction of branched-chain fatty acids at low temperature under anaerobiosis, through a modification of the oxidizing capacity, is assumed. This link may partly explain the impact of low temperature and anaerobiosis on membrane properties and growth performance.

The diarrheal potential of a Bacillus cereus strain is essentially dictated by the amount of secreted nonhemolytic enterotoxin (Nhe). Expression of genes encoding Nhe is regulated by several factors, including the metabolic state of the cells. To identify metabolic sensors that could promote communication between central metabolism and nhe expression, we compared four strains of the B. cereus group in terms of metabolic and nhe expression capacities. We performed growth performance measurements, metabolite analysis, and mRNA measurements of strains F4430/73, F4810/72, F837/76, and PA cultured under anoxic and fully oxic conditions. The results showed that expression levels of nhe and ldhA, which encodes lactate dehydrogenase A (LdhA), were correlated in both aerobically and anaerobically grown cells. We examined the role of LdhA in the F4430/73 strain by constructing an ldhA mutant. The ldhA mutation was more deleterious to anaerobically grown cells than to aerobically grown cells, causing growth limitation and strong deregulation of key fermentative genes. More importantly, the ldhA mutation downregulated enterotoxin gene expression under both anaerobiosis and aerobiosis, with a more pronounced effect under anaerobiosis. Therefore, LdhA was found to exert a major control on both fermentative growth and enterotoxin expression, and it is concluded that there is a direct link between fermentative metabolism and virulence in B. cereus. The data presented also provide evidence that LdhA-dependent regulation of enterotoxin gene expression is oxygen independent. This study is the first report to describe a role of a fermentative enzyme in virulence in B. cereus.

We had previously demonstrated that Fnr is required for fermentative growth and oxic production of hemolysin BL (Hbl) and non-hemolytic enterotoxin (Nhe) in the food-borne pathogen Bacillus cereus F4430/73. In the present work, the regulatory impact of Fnr on microaerobic growth and enterotoxin production in response to carbohydrates was examined using glucose, fructose, sucrose or a glucose-fructose mixture as carbon and energy sources. Growth parameters, byproduct spectra and transcription levels of catabolic and enterotoxin genes were analyzed in a strain lacking Fnr in comparison to the parental F4430/73 strain. The results showed that B. cereus prefers glucose to other carbohydrates for microaerobic growth, and that lacking of Fnr less strongly affected the respiro-fermentative catabolism of glucose than fructose and, to a lesser extent than sucrose. In addition, lacking of Fnr strongly decreased expression of hbl and nhe genes, leading to the absence of Hbl and low production of Nhe independently of the carbohydrate used as carbon source. We conclude that Fnr is an important element for carbon source regulation in B. cereus F4430/73 and that the regulation of enterotoxin gene expression in response to carbohydrates may represent one aspect of overall catabolite control mediated by Fnr., (Copyright 2009 Elsevier Masson SAS. All rights reserved.)

Aims: To determine the effects of carbohydrates on Bacillus cereus ATCC14579(T) anaerobic metabolism and enterotoxin production in amino acids rich medium., Methods and Results: Bacillus cereus anaerobic growth on different carbohydrates (glucose, fructose, sucrose or glucose-fructose mixture) was examined in synthetic mMOD medium under continuous cultures (mu = 0.2 h(-1)). Fermentation end-products, flux partitioning at each key branch points of the mixed acid pathway and consumption or production of amino acids were determined. On both fructose and sucrose, ATP production was favoured via acetate production from acetyl-CoA. In addition, amino acids present in the growth medium showed significant variations with high consumption of serine and net production of glutamate and alanine on some or all sugars. Enterotoxins Hbl and Nhe production was high during growth on fructose (or mixtures involving a fructose moiety)., Conclusions: Fructose was identified as a key sugar influencing anaerobic metabolism and toxin production of B. cereus., Significance and Impact of the Study: The physiological differences associated with the fermentation of the various carbohydrates clearly modify toxinogenesis indicating that the risk of foodborne pathogens is to some extent dependent upon the prevailing nutritional environment.

Aims: To determine the effects of porcine bile (PB) on Bacillus cereus vegetative cells and Haemolysin BL (HBL) enterotoxin production in reconstituted small intestine media (IM)., Methods and Results: The effects of PB on the growth of B. cereus vegetative cells in reconstituted IM at PB concentrations ranging between 0 and 3.0 g l(-1) were examined. Four gastric media (GM) named GM-J broth (JB), GM-chicken, GM-milk and GM-pea were prepared by mixing equal volumes of a gastric electrolyte solution containing pepsin with JB, chicken, semi-skimmed milk and pea soup, respectively. Bacillus cereus was inoculated at approx. 2 x 10(4) CFU ml(-1) into each GM at pH 5.0 for 30 min at 37 degrees C, then mixed to the same volume of double-strength JB (IM) and PB to give concentrations of between 0 and 3.0 g of PB per litre at pH 6.5 and incubated at 37 degrees C. The diarrhoeal B. cereus strain F4430/73 grew in IM-JB, IM-chicken and IM-milk at PB concentrations of up to 0.6, 1.5 and 1.2 g l(-1), respectively. Growth was observed in IM-pea at all concentrations tested. The highest PB concentrations allowing a 3 log B. cereus increase in IM-JB, IM-chicken, IM-milk and IM-pea after a 7-10 h incubation period were 0.3, 0.9, 0.9 and 3.0 g l(-1), respectively. The effect of PB on B. cereus cells was strongest in IM-JB, followed by IM-chicken, IM-milk and IM-pea. Haemolysin BL enterotoxin was detectable in IM-chicken, IM-whole milk, IM-semi-skimmed milk and IM-pea up to PB concentrations of only 0.6, 0.6, 0.3 and 0.9 g l(-1), respectively. The diarrhoeal B. cereus strain F4433/73 behaved similarly to B. cereus strain F4430/73, whereas the food strain TZ415 was markedly more susceptible to bile., Conclusions: The tolerance of B. cereus cells to PB strongly depends on the type of food contained in the IM. Bile tolerance is also subject to strain variation., Significance and Impact of the Study: The probability that B. cereus cells will grow in the small intestine, produce toxins and cause diarrhoea is likely to depend on the food they are ingested with, on the bile tolerance of the B. cereus strain, and on bile concentration.

Enterotoxin production is a key factor in Bacillus cereus food poisoning. Herein, the effect of the growth rate (mu) on B. cereus toxin production when grown on sucrose was studied and the Hemolytic BL enterotoxin (HBL) and nonhemolytic enterotoxin (Nhe) production by B. cereus was compared according to carbohydrate at mu = 0.2 h(-1). The anaerobic growth was carried out on continuous cultures in synthetic medium supplemented with glucose, fructose, sucrose, or an equimolar mixture of glucose and fructose. Concerning the HBL and Nhe enterotoxin production: (1) the highest enterotoxin production has occurred at mu = 0.2 h(-1) when growing on sucrose; (2) HBL production was repressed when glucose was consumed and the presence of fructose (alone or in mixture) cancelled glucose catabolite repression; (3) the consumption of sucrose increased Nhe production, which was not affected by the catabolite repression. Furthermore, analysis of the fermentative metabolism showed that whatever the mu or the carbon source, B. cereus used the mixed acid fermentation to ferment the different carbohydrates. The enterotoxin productions by this strain at mu = 0.2 h(-1) are highly influenced by the carbohydrates that do not involve any fermentative metabolism changes.

Aims: To determine the fate of Bacillus cereus spores or vegetative cells in simulated gastric medium., Methods and Results: The effects of acidity on the survival of B. cereus in a medium simulating human stomach content was followed on spores at pH 1.0-5.2, and on vegetative cells at pH 2.5-5.7. Gastric media (GM) were prepared by mixing equal volumes of a gastric electrolyte solution with J broth (JB), half-skim milk, pea soup and chicken. At pH 1.0 and 1.4, the number of spores slightly decreased in GM-JB and GM-pea soup and remained stable in GM-milk and GM-chicken. A rapid marked decrease (always higher than 2.0 log CFU ml(-1) in 2 h) in vegetative cell counts was observed at pH below 4.2, 4.0, 3.6 and 3.5 in GM-chicken, GM-JB, GM-milk and GM-pea soup, respectively. Between pH 5.0 and 5.3, B. cereus growth was observed in GM-JB (1.2 log CFU ml(-1) increase after 4 h) and in GM-pea soup (1.8 log CFU ml(-1) increase after 4 h)., Conclusions: Bacillus cereus spores are very much more resistant to gastric acidity than vegetative cells. This resistance strongly depends on the type of food present in the GM., Significance and Impact of the Study: Our results suggest that the probability that viable B. cereus cells enter the small intestine, where they can cause diarrhoea, strongly depends on the form of the ingested cells (spores or vegetative cells), on what food they are ingested with, and on the level of stomach acidity.

The acid tolerance of foodborne pathogen Bacillus cereus TZ415 was examined. B. cereus was more tolerant to an acid challenge at pH 4.0 when cells were grown at low pH in regulated batch cultures of rich J Broth (JB) medium. The pH-inducible acid tolerance response (ATR) was maximal at pH 5.0, a sublethal growth condition inducing a remarkable cell elongation. During growth at regulated pH 7.0 and 6.0, B. cereus TZ415 became more acid sensitive from lag to stationary growth phase and the acid tolerance of cells reached its maximum level in late-stationary growth phase. The ATR induced at pH 5.5 and 5.0 was not affected by growth phase. Cellular protein profiles were analysed as a function of growth phase and medium pH. The Hemolysin BL (HBL) enterotoxin was only detected when cells were grown at pH 7.0.

Aims: To determine the effects of carbohydrates on Bacillus cereus ATCC14579T anaerobic metabolism and enterotoxin production in amino acids rich medium. Methods and Results: Bacillus cereus anaerobic growth on different carbohydrates (glucose, fructose, sucrose or glucose–fructose mixture) was examined in synthetic mMOD medium under continuous cultures (μ = 0·2 h−1). Fermentation end-products, flux partitioning at each key branch points of the mixed acid pathway and consumption or production of amino acids were determined. On both fructose and sucrose, ATP production was favoured via acetate production from acetyl-CoA. In addition, amino acids present in the growth medium showed significant variations with high consumption of serine and net production of glutamate and alanine on some or all sugars. Enterotoxins Hbl and Nhe production was high during growth on fructose (or mixtures involving a fructose moiety). Conclusions: Fructose was identified as a key sugar influencing anaerobic metabolism and toxin production of B. cereus. Significance and Impact of the Study: The physiological differences associated with the fermentation of the various carbohydrates clearly modify toxinogenesis indicating that the risk of foodborne pathogens is to some extent dependent upon the prevailing nutritional environment. [ABSTRACT FROM AUTHOR]

Aims: To determine the effects of carbohydrates on Bacillus cereus ATCC14579T anaerobic metabolism and enterotoxin production in amino acids rich medium. Methods and Results: Bacillus cereus anaerobic growth on different carbohydrates (glucose, fructose, sucrose or glucose–fructose mixture) was examined in synthetic mMOD medium under continuous cultures (μ = 0·2 h−1). Fermentation end-products, flux partitioning at each key branch points of the mixed acid pathway and consumption or production of amino acids were determined. On both fructose and sucrose, ATP production was favoured via acetate production from acetyl-CoA. In addition, amino acids present in the growth medium showed significant variations with high consumption of serine and net production of glutamate and alanine on some or all sugars. Enterotoxins Hbl and Nhe production was high during growth on fructose (or mixtures involving a fructose moiety). Conclusions: Fructose was identified as a key sugar influencing anaerobic metabolism and toxin production of B. cereus. Significance and Impact of the Study: The physiological differences associated with the fermentation of the various carbohydrates clearly modify toxinogenesis indicating that the risk of foodborne pathogens is to some extent dependent upon the prevailing nutritional environment. [ABSTRACT FROM AUTHOR]