Short-Chain and Unsaturated Fatty Acids Increase Sequentially From the Lag Phase During Cold Growth of Bacillus cereus.

Fatty acids of two mesophilic and one psychrotrophic strains of the foodborne pathogen Bacillus cereus were analyzed by gas chromatography coupled to mass spectrometry during growth at cold (10 and 12°C) vs. optimal (30°C) temperatures and during the whole growth process (6-7 sampling times) from lag to stationary phase. In all these strains, a sequential change of fatty acids during cold growth was observed. Fatty acids were modified as soon as the end of lag, with an increase of the short-chain fatty acids (less than 15 carbons), particularly i13. These short-chain fatty acids then reached a maximum at the beginning of growth and eventually decreased to their initial level, suggesting their importance as a rapid cold adaptation mechanism for B. cereus. In a second step, an increase in Δ5,10 di-saturated fatty acids and in monounsaturated fatty acids in Δ5 position, at the expense of unsaturation in Δ10, started during exponential phase and continued until the end of stationary phase, suggesting a role in growth consolidation and survival at cold temperatures. Among these unsaturated fatty acids, those produced by unsaturation of n16 increased in the three strains, whereas other unsaturated fatty acids increased in some strains only. This study highlights the importance of kinetic analysis of fatty acids during cold adaptation.

Impact of temperature and oxygen on the fate of Bacillus weihenstephanensis in a food-based medium.

The capacity of the Bacillus weihenstephanensis KBAB4 strain, a psychrotolerant species of the B. cereus sensu lato group, to multiply in carrot broth at 8 °C and 30 °C, in presence or absence of oxygen was determined. In aerobic carrot broth tyndallized in presence of oxygen, at both temperatures, the population of vegetative cells of B. weihenstephanensis inoculated at a level of 103 or 106 CFU/ml dropped immediately. After 16 h at 30 °C, B. weihenstephanensis reached around 103 CFU/ml, indicating that some vegetative cells had survived and multiplied, with lipid inclusions accumulated in cells, indicating possible stressing conditions. At 8 °C, no multiplication of B. weihenstephanensis was observed during 3 days to at least 12 days, depending of carrot broth batches. In anaerobic carrot broth tyndallized without oxygen, the vegetative cells of B. weihenstephanensis were not killed upon inoculation and multiplied in the broth at both 30 °C and 8 °C. Comparison with results from previous studies shows that B. weihenstephanensis behaves differently in carrot broth and in laboratory media at 8 °C with regards to presence or absence of oxygen.

Cereulide production by Bacillus weihenstephanensis strains during growth at different pH values and temperatures.

Besides Bacillus cereus, some strains of the psychrotolerant, potentially foodborne pathogen Bacillus weihenstephanensis can produce the emetic toxine (cereulide). This toxin is a heat- and acid-stable cyclic dodecadepsipeptide that causes food intoxication with vomiting. However, some severe clinical cases with lethal outcomes have been described. If cereulide can be produced during refrigerated storage, it will not be inactivated by reheating food, representing an important risk of food intoxication for consumers. In this paper, we determined the capacity of the B. weihenstephanensis strains BtB2-4 and MC67 to grow and produce cereulide on agar media at temperatures from 8 °C to 25 °C and at a pH from 5.4 to 7.0. At 8 °C, strain BtB2-4 produced quantifiable amounts of cereulide, whereas the limit of detection was reached for strain MC67. For BtB2-4, cereulide production increased 5-fold between 8 °C and 10-15 °C and by more than 100-fold between 15 °C and 25 °C. At temperatures of 10 °C and higher, cereulide concentrations were within the range of those reported by previous works in foods implicated in emetic poisoning. At 25 °C, decreasing the pH to 5.4 reduced cereulide production by strain BtB2-4 by at least 20-fold.

Heat-resistance of psychrotolerant Bacillus cereus vegetative cells.

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 log10 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 log10 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 log10 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.

Combined effect of anaerobiosis, low pH and cold temperatures on the growth capacities of psychrotrophic Bacillus cereus.

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.

The Water Cycle, a Potential Source of the Bacterial Pathogen Bacillus cereus.

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.

Spores of Bacillus cereus strain KBAB4 produced at 10 °C and 30 °C display variations in their properties.

Spores of the psychrotrophic Bacillus cereus KBAB4 strain were produced at 10 °C and 30 °C in fermentors. Spores produced at 30 °C were more resistant to wet heat at 85 °C, 1% glutaraldehyde, 5% hydrogen peroxide, 1M NaOH and pulsed light at fluences between 0.5 and 1.75 Jcm(-2) and to a lesser extent to monochromatic UV-C at 254 nm. No difference in resistance to 0.25 mM formaldehyde, 1M nitrous acid and 0.025 gl(-1) calcium hypochlorite was observed. Spores produced at 10 °C germinated more efficiently with 10 mM and 100 mM l-alanine than spores produced at 30 °C, while no difference in germination was observed with inosine. Dipicolinic acid (DPA) content in the spore was significantly higher for spores prepared at 30 °C. Composition of certain fatty acids varied significantly between spores produced at 10 °C and 30 °C.

Identification of Bacillus cereus genes specifically expressed during growth at low temperatures.

The mechanisms involved in the ability of Bacillus cereus to multiply at low temperatures were investigated. It was assumed that many genes involved in cold acclimation would be upregulated at low temperatures. Recombinase-based in vivo expression technology (IVET) was adapted to the detection of the transient activation of B. cereus promoters during growth at 10 degrees C. Four independent screenings of a promoter library from type strain ATCC 14579 were performed, and 17 clones were isolated. They corresponded to 17 promoter regions that displayed reproducibly elevated expression at 10 degrees C relative to expression at 30 degrees C. This analysis revealed several genes that may be important for B. cereus to grow successfully under the restrictive conditions of cold habitats. Among them, a locus corresponding to open reading frames BC5402 to BC5398, harboring a lipase-encoding gene and a putative transcriptional regulator, was identified three times. While a mutation in the putative regulator-encoding gene did not cause any particular phenotype, a mutant deficient in the lipase-encoding gene showed reduced growth abilities at low temperatures compared with the parental strain. The mutant did not change its fatty acid profiles in the same way as the wild type when grown at 12 degrees C instead of 37 degrees C. This study demonstrates the feasibility of a promoter trap strategy for identifying cold-induced genes. It outlines a first picture of the different processes involved in B. cereus cold acclimation.

The YvfTU two-component system is involved in plcR expression in Bacillus cereus.

BACKGROUND: Most extracellular virulence factors produced by Bacillus cereus are regulated by the pleiotropic transcriptional activator PlcR. Among strains belonging to the B. cereus group, the plcR gene is always located in the vicinity of genes encoding the YvfTU two-component system. The putative role of YvfTU in the expression of the PlcR regulon was therefore investigated. RESULTS: Expression of the plcR gene was monitored using a transcriptional fusion with a lacZ reporter gene in a yvfTU mutant and in its B. cereus ATCC 14579 parental strain. Two hours after the onset of the stationary phase, a stage at which the PlcR regulon is highly expressed, the plcR expression in the yvfTU mutant was only 50% of that of its parental strain. In addition to the reduced plcR expression in the yvfTU mutant, a few members of the PlcR regulon showed a differential expression, as revealed by transcriptomic and proteomic analyses. The virulence of the yvfTU mutant in a Galleria mellonella insect model was slightly lower than that of the parental strain. CONCLUSION: The YvfTU two-component system is not required for the expression of most of the virulence factors belonging to the PlcR regulon. However, YvfTU is involved in expression of plcR, a major regulator of virulence in B. cereus.

Growth and germination of proteolytic Clostridium botulinum in vegetable-based media.

The growth of proteolytic Clostridium botulinum from spore inocula and changes in spore counts in mushroom, broccoli, and potato purées were monitored. Four strains of proteolytic C. botulinum types A and B were inoculated separately at approximately 10(4) spores per ml in nutrient broth and vegetable purées incubated at 15, 20, and 30 degrees C for up to 52 days. The times for the cell populations to increase 1,000-fold (T1,000) in the tested vegetables (1 to 5 days at 30 degrees C, 3 to 16 days at 20 degrees C, 7 to > 52 days at 15 degrees C) were similar to those for meat or fish. Only temperature significantly influenced growth rate. In contrast, the lag phase depended on the strains and media tested, in addition to temperature. Lag times and T1,000S for proteolytic C. botulinum were longer for potato and broccoli purées than for mushroom purée. These differences were not related to different pHs or redox potentials. The germination level, evaluated as the decrease in the spore count, was low. The addition of a germinant mixture (L-cysteine, L-alanine, and sodium lactate) to some strains inoculated in vegetable purées resulted in an increase in germination, suggesting a lack of germination-triggering agents in the vegetable purées.

Detection by PCR-enzyme-linked immunosorbent assay of Clostridium botulinum in fish and environmental samples from a coastal area in northern France.

The prevalence of Clostridium botulinum types A, B, E, and F was determined in 214 fresh fish and environmental samples collected in Northern France. A newly developed PCR-enzyme-linked immunosorbent assay (ELISA) used in this survey detected more than 80% of samples inoculated with fewer than 10 C. botulinum spores per 25 g and 100% of samples inoculated with more than 30 C. botulinum spores per 25 g. The percent agreement between PCR-ELISA and mouse bioassay was 88.9%, and PCR-ELISA detected more positive samples than the mouse bioassay did. The prevalence of C. botulinum in seawater fish and sediment was 16.6 and 4%, respectively, corresponding to 3.5 to 7 and 1 to 2 C. botulinum most-probable-number counts, respectively, and is in the low range of C. botulinum contamination reported elsewhere. The toxin type identification of the 31 naturally contaminated samples was 71% type B, 22.5% type A, and 9.6% type E. Type F was not detected. The high prevalence of C. botulinum type B in fish samples is relatively unusual compared with the high prevalence of C. botulinum type E reported in many worldwide and northern European surveys. However, fish processing and fish preparation in France have not been identified as a significant hazard for human type B botulism.

Antimicrobial activity of foodborne Paenibacillus and Bacillus spp. against Clostridium botulinum.

The saprophytic Paenibacillus and Bacillus spp. found in cooked chilled foods may have an effect on the growth of Clostridium botulinum, a major microbiological hazard, especially for pasteurized vacuum-packaged products. Culture supernatants of 200 strains of Paenibacillus and Bacillus strains isolated from commercial cooked chilled foods containing vegetables were screened for activity against C. botulinum type A, proteolytic type B, and type E strains in a well diffusion assay. Nineteen strains were positive against C. botulinum. Among those, seven Paenibacillus polymyxa strains showed the highest antibotulinal activity and the largest antimicrobial spectrum against C. botulinum strains. The antibotulinal activity was evaluated throughout the growth of a representative strain of the positive P. polymyxa strains. The antimicrobial activity was detected in the culture supernatant from late-log/early stationary phase of the bacteria, which occurred after 7 to 10 days of incubation at 10 degrees C and after 2 to 3 days at 20 degrees C in nutrient broth and in vegetable purées under aerobic or anaerobic conditions. In co-cultures with the positive strain of P. polymyxa in nutrient broth and vegetable purées, a C. botulinum type E strain was inhibited whenever P. polymyxa reached stationary phase and produced its antimicrobial activity before C. botulinum began its exponential growth phase. The antimicrobial activity of P. polymyxa against C. botulinum was attributed to the production of antimicrobial peptides resistant to high temperature and acidity. Other gram-positive and -negative bacteria (Escherichia coli, Streptococcus mutans, Leuconostoc mesenteroides, and Bacillus subtilis) were also sensitive to these antimicrobial peptides.