Thierry Clavel

Survival of Bacillus cereus spores and vegetative cells in acid media simulating human stomach

Aims:  To determine the fate of Bacillus cereus spores or vegetative cells in simulated gastric medium.

Effect of temperature on growth characteristics of Bacillus cereus TZ415

The effect of temperature on the maximal specific growth rate was studied in Bacillus cereus between 5 and 40 degrees C cultivated in courgette broth and rich medium (J broth). B. cereus grown from 5 to 38 degrees C in rich medium. No growth was observed in courgette broth below 10 degrees C. The Arrhenius plot was fitted from experimental data of B. cereus grown in rich medium and at regulated pH, oxygen and temperature. Two domains which are separated by a critical temperature around 13 degrees C can be distinguished with regard to temperature dependence of maximal specific growth rate. Over the cold domain from 5 to 13 degrees C, the temperature characteristic was 2.6 fold higher than over the sub-optimal domain from 13 to 38 degrees C suggesting that the growth temperature regulates several metabolic pathways.

Acid tolerance response is low-pH and late-stationary growth phase inducible in Bacillus cereus TZ415.

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.

The Production of Bacillus cereus Enterotoxins Is Influenced by Carbohydrate and Growth Rate

Enterotoxin production is a key factor in Bacillus cereus food poisoning. Herein, the effect of the growth rate (μ) 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 μ = 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 μ = 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 μ or the carbon source, B. cereus used the mixed acid fermentation to ferment the different carbohydrates. The enterotoxin productions by this strain at μ = 0.2 h−1 are highly influenced by the carbohydrates that do not involve any fermentative metabolism changes.

Influence of Anaerobiosis and Low Temperature on Bacillus cereus Growth, Metabolism, and Membrane Properties

ABSTRACT 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.

Effects of porcine bile on survival of Bacillus cereus vegetative cells and Haemolysin BL enterotoxin production in reconstituted human small intestine media

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).

Fnr mediates carbohydrate-dependent regulation of catabolic and enterotoxin genes in Bacillus cereus F4430/73

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.

Lactate dehydrogenase A promotes communication between carbohydrate catabolism and virulence in Bacillus cereus

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.

Fructose and glucose mediates enterotoxin production and anaerobic metabolism of Bacillus cereus ATCC14579T.

Aims:  To determine the effects of carbohydrates on Bacillus cereus ATCC14579T anaerobic metabolism and enterotoxin production in amino acids rich medium.

Unsaturated fatty acids from food and in the growth medium improve growth of Bacillus cereus under cold and anaerobic conditions.

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.