Jacques Mahillon

Fatal Family Outbreak of Bacillus cereus-Associated Food Poisoning

ABSTRACT Bacillus cereus is a well-known cause of food-borne illness, but infection with this organism is not commonly reported because of its usually mild symptoms. A fatal case due to liver failure after the consumption of pasta salad is described and demonstrates the possible severity of the emetic syndrome.

Sudden death of a young adult associated with Bacillus cereus food poisoning.

ABSTRACT A lethal intoxication case, which occurred in Brussels, Belgium, is described. A 20-year-old man died following the ingestion of pasta contaminated with Bacillus cereus. Emetic strains of B. cereus were isolated, and high levels of cereulide (14.8 μg/g) were found in the spaghetti meal.

Distribution, Diversity, and Potential Mobility of Extrachromosomal Elements Related to the Bacillus anthracis pXO1 and pXO2 Virulence Plasmids

ABSTRACT The presence of a pXO1- and/or pXO2-like plasmid(s) in clinical isolates of Bacillus cereus sensu stricto and in strains of the biopesticide Bacillus thuringiensis has been reported recently, and the pXO2-like plasmid pBT9727 and another pXO2-like plasmid, pAW63, were found to be conjugative. In this study, a total of 1,000 B. cereus group isolates were analyzed for the presence of pXO1- and pXO2-like replicons and for the presence of pXO2-related conjugative modules. pXO1- and pXO2-like replicons were present in ca. 6.6% and 7.7% of random environmental samples, respectively, and ca. 1.54% of the strains were positive for pXO2-like transfer module genes. Only the strains harboring a pXO2-like replicon also contained the corresponding transfer genes. For the strains which contained a pXO1- and/or pXO2-like replicon(s), a large plasmid(s) whose size was similar to that of pXO1-like and/or pXO2-like plasmids was also observed, but none of these isolates were found to carry the Bacillus anthracis toxin or capsule virulence genes. Furthermore, 17 of 22 pXO2-like plasmids containing the transfer modules were able to self-transfer and to mobilize small plasmids. No pXO1- or pXO2-like plasmid lacking the cognate transfer modules has been found to have transfer potential. In the strains possessing the putative pXO2-like conjugative apparatus, variations in the presence of the group II introns B.th.I.1 and B.th.I.2 were observed, suggesting that there is important flexibility in the conjugation modules and their regulation. There was no consistent correlation between a pXO2-like repA dendrogram and the presence of the tra region or between a virB4 dendrogram and transfer ability. Discrepancies between pXO2-like repA and virB4 dendrograms were also observed, indicating that the evolution of pXO2 is an active process.

Development and validation of qualitative SYBR®Green Real-Time PCR for detection and discrimination of Listeria spp. and Listeria monocytogenes

A combination of four qualitative SYBR®Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) programme. The paper also proposes a new validation procedure to specifically validate qPCR assays applied to food microbiology according to two guidelines: the ISO 22118 norm and the “Definition of minimum performance requirements for analytical methods of GMO testing”. The developed assays target the iap, prs and hlyA genes that belong to or neighbour the virulence cluster of Listeria spp. The selected primers were designed to amplify short fragments (60 to 103xa0bp) in order to obtain optimal PCR efficiency (between 97 and 107xa0% efficiency). The limit of detection of the SYBR®Green qPCR assays is two to five copies of target genes per qPCR reaction. These assays are highly accurate (98.08 and 100xa0% accuracy for the Listeria spp. and L. monocytogenes assays, respectively).

Evaluation of viability-qPCR detection system on viable and dead Salmonella serovar Enteritidis

The propidium monoazide (PMA) coupled with PCR (viability PCR) is used in foodborne pathogen detection in order to detect only viable bacteria. Originally presented to fully remove the signal of dead bacteria, the limits of the viability PCR rapidly came out in the literature. In this study, the use of PMA in a viability-qPCR (v-qPCR) was assessed on viable and dead cells of Salmonella enterica subsp. enterica serovar Enteritidis. The PMA treatment protocol was modified (dark incubation duration, concentration of PMA) to evaluate if a complete negative signal of dead Salmonella was possible. However, none of these modifications was found to improve the removal of the remaining qPCR signal observed in the presence of dead bacteria. The present research also underlines that PMA may unexpectedly decrease the qPCR signal observed on living S. Enteritidis at low concentration. Finally, the use of S. Enteritidis cells killed by processes altering or not the cell-wall/membrane gives us a clue to answering the question about the non-total extinction of the signal of dead cells sample in the v-qPCR assay. Indeed, the data strongly indicate that the remaining qPCR signal observed in non-culturable cells does not only depend on the cell-wall/membrane integrity of the bacteria. According to these results, the authors suggest that for a rapid and reliable foodborne bacteria detection system, an enrichment followed by a qPCR analysis should be preferred to a v-qPCR.

Whole-Genome Sequences of 94 Environmental Isolates of Bacillus cereus Sensu Lato

ABSTRACT Bacillus cereus sensu lato is a species complex that includes the anthrax pathogen Bacillus anthracis and other bacterial species of medical, industrial, and ecological importance. Their phenotypes of interest are typically linked to large plasmids that are closely related to the anthrax plasmids pXO1 and pXO2. Here, we present the draft genome sequences of 94 isolates of B. cereus sensu lato, which were chosen for their plasmid content and environmental origins.

Particular Biochemical Profiles for Enterohemorrhagic Escherichia coli O157:H7 Isolates on the ID 32E System

ABSTRACT The ability of the ID 32E system to identify and discriminate 74Escherichia coli O157 isolates among 106 E. coli non-O157 isolates was evaluated. The results showed atypical biochemical reactions but accurate identification at the species level and no unique biochemical profile numbers for E. coli O157, although these numbers were distinct from those of other serotypes.

Differential transfer dynamics of pAW63 plasmid among members of the Bacillus cereus group in food microcosms.

Aim:u2002 To assess the dynamics of plasmid transfer between Bacillus thuringiensis and B. cereus in various food microcosms using the B. thuringiensis pAW63 and Staphylococcus aureus pUB110 plasmids as models.

Characterization of Bacilli Isolated from the Confined Environments of the Antarctic Concordia Station and the International Space Station

Bacillus and related genera comprise opportunist and pathogen species that can threaten the health of a crew in confined stations required for long-term missions. In this study, 43 Bacilli from confined environments, that is, the Antarctic Concordia station and the International Space Station, were characterized in terms of virulence and plasmid exchange potentials. No specific virulence feature, such as the production of toxins or unusual antibiotic resistance, was detected. Most of the strains exhibited small or large plasmids, or both, some of which were related to the replicons of the Bacillus anthracis pXO1 and pXO2 virulence elements. One conjugative element, the capacity to mobilize and retromobilize small plasmids, was detected in a Bacillus cereus sensu lato isolate. Six out of 25 tested strains acquired foreign DNA by conjugation. Extremophilic bacteria were identified and exhibited the ability to grow at high pH and salt concentrations or at low temperatures. Finally, the clonal dispersion of an opportunist isolate was demonstrated in the Concordia station. Taken together, these results suggest that the virulence potential of the Bacillus isolates in confined environments tends to be low but genetic transfers could contribute to its capacity to spread.

Fast and discriminative CoSYPS detection system of viable Salmonella spp. and Listeria spp. in carcass swab samples

In this study, the complete CoSYPS Path Food workflow including all steps, namely swab sample enrichment, SYBR®Green qPCR detection of Salmonella spp. and Listeria spp., isolation and confirmation of the detected strain, was validated on beef carcass swabs. To perform the validation, the results of the complete workflow were compared, according to the ISO 16140:2003, with the ISO reference methods for detection, isolation and confirmation of Listeria monocytogenes and Salmonella spp. The results showed that the relative level of detection and the limit of detection of the complete workflow and ISO reference methods are in a range from 2 to 16 CFU/swab for both bacteria. The relative specificity, sensitivity and accuracy identified during this validation were all 100% since the results obtained with the complete CoSYPS Path Food workflow and the ISO reference methods were identical (Cohens kappa index=1.00). In addition the complete CoSYPS Path Food workflow is able to provide detection results (negative or presumptive positive) in half the time needed as for the ISO reference methods. These results demonstrate that the performance of the complete CoSYPS Path Food workflow is not only comparable to the ISO reference methods but also provides a faster response for the verification of beef carcasses before commercial distribution.