Véronique Broussolle

Enterotoxigenic Profiles of Food-Poisoning and Food-Borne Bacillus cereus Strains

ABSTRACT The enterotoxigenic profiles of 51 B. cereus food-related strains were compared to those of 37 B. cereus food-poisoning strains. cytK and association of hbl-nhe-cytK enterotoxin genes were more frequent among diarrheal strains (73 and 63%) than among food-borne strains (37 and 33%). Unlike diarrheal strains, food-borne strains showed frequent nhe and hbl gene polymorphisms and were often low toxin producers.

Detection by PCR-Enzyme-Linked Immunosorbent Assay of Clostridium botulinum in Fish and Environmental Samples from a Coastal Area in Northern France

ABSTRACT 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.

Variability in spore germination response by strains of proteolytic Clostridium botulinum types A, B and F

Aims: The objective of the study was to evaluate the variability of germination response of 10 strains of proteolytic Clostridium botulinum.

Prevalence of Clostridium botulinum in food raw materials used in REPFEDs manufactured in France

Food raw materials used in refrigerated processed foods of extended durability (REPFEDs) manufactured in France were surveyed for Clostridium botulinum types A, B and E using PCR-Enzyme-linked Immunosorbent assay (PCR-ELISA) and mouse bioassay for detection respectively of cells and toxins in enrichment broth. Portions of 25 to 50 g of food were analysed. A total of 8 out of the 102 samples of fish and shellfish, 12 out of the 143 samples of meat and poultry, 1 out of the 62 samples of aroma, sauce and gravy, 4 out of the 25 samples of thickening agents, 3 out of the 26 samples of dehydrated dairy ingredients, and none of the 65 samples of spices, herbs and dehydrated mushroom were positive for C. botulinum in PCR-ELISA, i.e., 6.6% of all the samples tested. The 28 positive samples comprised 10 type A, 10 type B, 4 with both types A and B, and 4 undetermined by PCR typing. No sample positive for type E was detected. Of the 28 samples positive in PCR-ELISA, 15 were also positive in the mouse bioassay. The MPN count was between 1 and 3 C. botulinum/kg of food, which is similar to or in the lower range of values reported in the literature.

Differential involvement of the five RNA helicases in adaptation of Bacillus cereus ATCC 14579 to low growth temperatures.

ABSTRACT Bacillus cereus ATCC 14579 possesses five RNA helicase-encoding genes overexpressed under cold growth conditions. Out of the five corresponding mutants, only the ΔcshA, ΔcshB, and ΔcshC strains were cold sensitive. Growth of the ΔcshA strain was also reduced at 30°C but not at 37°C. The cold phenotype was restored with the cshA gene for the ΔcshA strain and partially for the ΔcshB strain but not for the ΔcshC strain, suggesting different functions at low temperature.

Screening for clostridium botulinum type A, B, and E in cooked chilled foods containing vegetables and raw material using polymerase chain reaction and molecular probes.

A molecular method was used for the detection of Clostridium botulinum spores of type A, B, and E in commercial cooked and pasteurized vegetable purées and in the raw materials (vegetables and other ingredients). The method allowed the detection of less than 8 spores/g of product for C. botulinum type A, less than 1 spore/g for proteolytic type B, less than 21 spores/g for nonproteolytic type B, and less than 0.1 spore/g for type E. Thirty-seven samples of raw vegetables and ingredients were tested for the presence of C. botulinum type A, B, and E; 88 and 90 samples of vegetable purées were tested, respectively, for the presence of C. botulinum type A and B and for the presence of C. botulinum type E. All samples were negative, suggesting that the prevalence of C. botulinum in these vegetable purées and the raw ingredients is probably low.

The adaptive response of bacterial food-borne pathogens in the environment, host and food: Implications for food safety

Bacteria are constantly faced to stress situations in their ecological niches, the food and the host gastrointestinal tract. The capacity to detect and respond to surrounding changes is crucial for bacterial pathogens to survive or grow in changing environments. To this purpose, cells have evolved various sophisticated networks designed to protect against stressors or repair damage caused by them. Challenges can occur during production of foods when subjected to processing, and after food ingestion when confronted with host defensive barriers. Some pathogenic bacteria have shown the capacity to develop stable resistance against extreme conditions within a defined genomic context and a limited number of generations. On the other hand, bacteria can also respond to adverse conditions in a transient manner, through the so-called stress tolerance responses. Bacterial stress tolerance responses include both structural and physiological modifications in the cell and are mediated by complex genetic regulatory machinery. Major aspects in the adaptive response are the sensing mechanisms, the characterization of cell defensive systems, such as the operation of regulatory proteins (e.g. RpoS), the induction of homeostatic and repair systems, the synthesis of shock response proteins, and the modifications of cell membranes, particularly in their fatty acid composition and physical properties. This article reviews certain strategies used by food-borne bacteria to respond to particular stresses (acid, cold stress, extreme pressure) in a permanent or transient manner and discusses the implications that such adaptive responses pose for food safety.

Molecular cloning, expression, and characterization of a new endoglucanase gene fromFibrobacter succinogenes S85

A DNA fragment coding for a carboxymethylcellulase (CMCase) ofFibrobacter succinogenes S85 was isolated from a pUC18 gene library inEscherichia coli JM109. The CMCase gene was present as a single copy in theF. succinogenes S85 genome and was found in all the otherF. succinogenes strains tested. The gene was expressed from an endogenous promoter inE. coli and was not subject to glucose repression. Most of the CMCase activity was located in the membrane ofE. coli. Zymogram analysis and35S labeling of the proteins encoded by the CMCase gene-containing plasmid indicated that the enzyme has a molecular mass of 58,000. The optimal pH and temperature of activity on CMC were respectively 6.4 and 30°C. The enzyme was active on CMC, barley β-glucan, and lichenan but would not hydrolyze laminarin and exhibited no exoglucanase-type activity, suggesting that it is an endo-(1,4)-β-d-glucanase.

The YvfTU Two-component System is involved in plcR expression in Bacillus cereus

BackgroundMost 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.ResultsExpression 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.ConclusionThe 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.

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.