Cereulide production capacities and genetic properties of 31 emetic Bacillus cereus group strains.

The highly potent toxin cereulide is a frequent cause of foodborne intoxications. This extremely resistant toxin is produced by Bacillus cereus group strains carrying the plasmid encoded cesHPTABCD gene cluster. It is known that the capacities to produce cereulide vary greatly between different strains but the genetic background of these variations is not clear. In this study, cereulide production capacities were associated with genetic characteristics. For this, cereulide levels in cultures of 31 strains were determined after incubation in tryptic soy broth for 24 h at 24 °C, 30 °C and 37 °C. Whole genome sequencing based data were used for an in-depth characterization of gene sequences related to cereulide production. The taxonomy, population structure and phylogenetic relationships of the strains were evaluated based on average nucleotide identity, multi-locus sequence typing (MLST), core genome MLST and single nucleotide polymorphism analyses. Despite a limited strain number, the approach of a genome wide association study (GWAS) was tested to link genetic variation with cereulide quantities. Our study confirms strain-dependent differences in cereulide production. For most strains, these differences were not explainable by sequence variations in the cesHPTABCD gene cluster or the regulatory genes abrB, spo0A, codY and pagRBc. Likewise, the population structure and phylogeny of the tested strains did not comprehensively reflect the cereulide production capacities. GWAS yielded first hints for associated proteins, while their possible effect on cereulide synthesis remains to be further investigated.

Occurrence of selected bacterial pathogens in insect-based food products and in-depth characterisation of detected Bacillus cereus group isolates.

Insects are increasingly used as alternative protein sources and ingredients of foodstuffs produced in industrial scale. Previous studies on the microbial status of insect-based foods revealed that classical foodborne pathogens such as Salmonella spp., Campylobacter spp., Listeria monocytogenes or pathogenic Escherichia coli are rarely detected, whereas particularly spore-forming bacteria with pathogenic potential such as species of the Bacillus cereus group or Clostridium species may pose a food safety risk. However, detailed descriptions of the encountered pathogenic bacteria in insect foods are scarce. We investigated a variety of 73 food products with insect or other arthropod ingredients on the occurrence of potential bacterial pathogens. These included B. cereus (sensu lato (s.l.)), Clostridium perfringens and Clostridioides difficile as representatives of spore-formers and Salmonella spp. and Shiga toxin producing and enteropathogenic E. coli (STEC/EPEC) as representatives of non-spore-forming Enterobacteriaceae. Most of the investigated food products complied with food safety standards regarding the presence of pathogens considered. However, one cricket product contained two Salmonella enterica subspecies enterica serovars (S. Wandsworth and S. Stanley). B. cereus (s.l.) was found in 42 samples (58 %), of which six contained B. cereus (s.l.) at levels higher than 103 cfu/g. The highest B. cereus (s.l.) counts of 3.8 × 105 cfu/g were found in a product with boiled and dried scorpions. Clostridium perfringens was detected in twelve samples (16 %), whereas Clostridioides difficile and STEC/EPEC were not detected in any of the samples. Remarkably, five samples contained the B. cereus (s.l.) species B. cytotoxicus. Moreover, strikingly high numbers of B. cereus (s.l.) isolates carried the capsule syntheses genes capBCADE, which were presumably located on the B. cereus pBFI_2 plasmid. Whole genome sequencing-based phylogenetic analysis suggested a high relatedness for only very few of the B. cytotoxicus and cap-positive isolates, respectively.

Establishment and Validation of a Two-Step LAMP Assay for Detection of Bacillus cereus-Group Isolates in Food and Their Possibility of Non-haemolytic Enterotoxin Production.

The closely related members of the Bacillus cereus-group can mainly only be differentiated by whole genome sequencing. Among them, there are potentially toxin-producing bacteria. When consumed with food, these can cause vomiting or diarrhea and abdominal cramps. To date, although no EU-wide threshold exists, a bacterial count of 105 CFU/g can be regarded as critical. Specific and rapid detection of the bacteria is difficult due to their close relationship, and no loop-mediated isothermal amplification (LAMP) assay has been developed so far to detect potentially toxin-producing members of the B. cereus-group. Aim of this study was to develop a LAMP method to detect critical cell counts specifically and rapidly of potentially non-haemolytic enterotoxin (NHE)-producing cells of this group. A two-step LAMP assay was developed. First, the target sequence groEL was used to determine the representatives of the B. cereus-group. Second, since bacteria in which nheB is present are basically capable of producing enterotoxins, this gene was chosen for detection. The specificity of the developed assay was 100% for B. cereus-group isolates and 93.7% for the detection of nheB. The analytical sensitivity was 0.1 pg DNA/µl. Using simplified DNA extraction by boiling, cell-based sensitivity was determined. Targeting groEL and nheB, 11.35-27.05 CFU/reaction and 11.35-270.5 CFU/reaction were detectable, respectively. Artificially contaminated samples were investigated to prove the application in foods. Direct detection of the critical value of B. cereus-group cells was possible in 83.3% of samples and detecting the toxin-gene 50% thereof. After a 6-h incubation period, the detection rate increased to 100 and 91.7%, respectively. Additionally, 100 natively contaminated food samples were tested, also quantitatively and culturally. Samples with relevant contamination levels were reliably detected using groEL-LAMP. After a 6-h incubation period, isolates bearing the toxin gene nheB could also be reliably detected. In addition, colony material was boiled and used as a LAMP template for simple detection. Specificity for the B. cereus-group was 100 and 93.22% detecting nheB. The study demonstrated that screening of food samples with the groEL/nheB-LAMP assay can be performed within 1 day, making it possible to detect critical levels of potentially NHE-toxin-producing cells of the B. cereus-group.

The Pore-Forming Hemolysin BL Enterotoxin from Bacillus cereus: Subunit Interactions in Cell-Free Systems.

The tripartite enterotoxin Hemolysin BL (Hbl) has been widely characterized as a hemolytic and cytotoxic virulence factor involved in foodborne diarrheal illness caused by Bacillus cereus. Previous studies have described the formation of the Hbl complex and aimed to identify the toxin's mode of action. In this study, we analyzed the assembly of Hbl out of its three individual subunits L1, L2 and B in a soluble as well as a putative membrane bound composition using a Chinese hamster ovary (CHO) cell-free system. Subunits were either coexpressed or synthesized individually in separate cell-free reactions and mixed together afterwards. Hemolytic activity of cell-free synthesized subunits was demonstrated on 5% sheep blood agar and identified both synthesis procedures, coexpression as well as individual synthesis of each subunit, as functional for the synthesis of an active Hbl complex. Hbl's ability to perforate cell membranes was evaluated using a propidium iodide uptake assay. These data suggested that coexpressed Hbl subunits augmented cytotoxic activity with increasing concentrations. Further, a pre-pore-complex of L1-L2 showed cytotoxic effects suggesting the possibility of an interaction between the cell membrane and the pre-pore-complex. Overall, this study shows that cell-free protein synthesis is a fast and efficient way to study the assembly of multiple protein subunits in soluble as well as vesicular fractions.

Temperature-Dependent Growth Characteristics of Bacillus thuringiensis in a Ratatouille Food Model.

ABSTRACT: In contrast to Bacillus cereus, the role of Bacillus thuringiensis in foodborne illness has been controversially discussed. As B. thuringiensis-based biopesticides containing a mixture of crystal toxins and viable spores are widely used, a current European Food Safety Authority opinion underlines the need for additional data to enable risk assessment. However, it is currently poorly understood if B. thuringiensis is able to multiply in food, which is crucial to sound risk assessment. Therefore, the aim of this study was to investigate growth of selected B. thuringiensis strains from food and insecticides in a ratatouille food model. To this end, the growth parameters of three B. thuringiensis strains were determined: insecticide strain ABTS-351 (CH_119, B. thuringiensis serovar kurstaki), insecticide strain ABTS-1857 (CH_121, B. thuringiensis serovar aizawai), and CH_48 (wild-type B. thuringiensis isolated from rosemary), producing extremely high levels of enterotoxins. After an initial drop in colony counts, we observed a statistically significant growth for the tested B. thuringiensis strains between 6 and 24 h at 22, 30, and 37°C, conditions mimicking prolonged holding times. We were also able to show that the enterotoxin overproducer CH_48 can grow up to 108 CFU/g in the ratatouille matrix within 24 h at 37°C. The two midlevel enterotoxin formers ABTS-351 (CH_119) and ABTS-1857 (CH_121) isolated from biopesticides exhibited growth between 6 and 24 h, with one of the strains growing to 107 CFU/g. To our knowledge, this is the first study providing evidence of B. thuringiensis growth in a food model with intact competitive flora. Our findings suggest strain-specific variation and stress the complexity of assessing the risk related to B. thuringiensis in food, indicating that some strains can represent a risk to consumer health when vegetable-based foods are stored under conditions of prolonged temperature abuse.

Mammalian cell-free protein expression promotes the functional characterization of the tripartite non-hemolytic enterotoxin from Bacillus cereus.

Bacillus cereus is increasingly recognized as an opportunistic pathogen causing local and systemic infections. The causative strains typically produce three pore-forming enterotoxins. This study focusses on the tripartite non-hemolytic enterotoxin (Nhe). Until today, studies have tried to elucidate the structure, complex formation and cell binding mechanisms of the tripartite Nhe toxin. Here, we demonstrate the synthesis of the functional tripartite Nhe toxin using eukaryotic cell-free systems. Single subunits, combinations of two Nhe subunits as well as the complete tripartite toxin were tested. Functional activity was determined by hemolytic activity on sheep blood agar plates, planar lipid bilayer measurements as well as cell viability assessment using the MTT assay. Our results demonstrate that cell-free protein synthesis based on translationally active eukaryotic lysates is a platform technology for the fast and efficient synthesis of functionally active, multicomponent toxins.

Indications of biopesticidal Bacillus thuringiensis strains in bell pepper and tomato.

Members of the Bacillus cereus group are common contaminants of vegetables. One potential source of contamination is the application of B. thuringiensis based biopesticides. Although evidence of the presence of biopesticidal strains on food products is scarce, this information is essential for assessing potential risks associated with the application of these biopesticides. In order to contribute to knowledge about the presence of biopesticidal B. thuringiensis strains in foodstuffs, we investigated the occurrence of B. thuringiensis on tomatoes and bell pepper. We analyzed 99 samples of fresh bell pepper for B. cereus group members, while 426 samples of tomatoes were tested by the competent food control laboratories of the federal states in Germany. The isolates recovered from these samples were further characterized in terms of their capability to produce parasporal crystals as well as enterotoxins. A possible correlation between the B. thuringiensis isolates and biopesticidal strains was investigated by multilocus sequence typing (MLST) and whole genome Single Nucleotide Polymorphism (wgSNP) analyses. The prevalence of B. cereus group members was 41% for bell pepper and 28% for tomato samples. Isolates recovered from these samples were dominated by B. thuringiensis (93% and 99%, respectively). All B. thuringiensis isolates carried the enterotoxin genes nheA, hblD and cytK-2. In a subset of 83 B. thuringiensis isolates analyzed by MLST, 99% of the isolates matched the sequence types (ST) 8 and 15, which are also shared by the biopesticidal strains B. thuringiensis kurstaki ABTS-351 and B. thuringiensis aizawai ABTS-1857. Of the 82 isolates assigned to ST 8 or ST 15, a selection of 42 isolates was further characterized by wgSNP analysis. Of these, seven isolates differed from strain ABTS-351 by ≤4 core SNPs and 18 isolates differed from strain ABTS-1857 by ≤2 core SNPs, indicating a relationship of these isolates with the respective biopesticidal strain. These isolates originated from samples with maximum colony counts of 5.3 × 103 cfu/g for bell pepper and 1.0 × 105 cfu/g for tomatoes.

Decontamination of high-risk animal and zoonotic pathogens.

Preparedness for the decontamination of affected environments, premises, facilities, and products is one prerequisite for an immediate response to an animal disease outbreak. Various information sources provide recommendations on how to proceed in an outbreak situation to eliminate biological contaminants and to stop the spread of the disease. In order to facilitate the identification of the right decontamination strategy, we present an overview of relevant references for a collection of pathogenic agents. The choice of pathogens is based on a survey of lists containing highly pathogenic agents and/or biological agents considered to be potential vehicles for deliberate contamination of food, feed, or farm animals. European legislation and guidelines from national and international institutions were screened to find decontamination protocols for each of the agents. Identified recommendations were evaluated with regard to their area of application, which could be facilities and equipment, wastes, food, and other animal products. The requirements of a disinfectant for large-scale incidents were gathered, and important characteristics (eg, inactivating spectrum, temperature range, toxicity to environment) of the main recommended disinfectants were summarized to assist in the choice of a suitable and efficient approach in a crisis situation induced by a specific high-risk animal or zoonotic pathogen. The literature search revealed numerous relevant recommendations but also legal gaps for certain diseases, such as Q fever or brucellosis, and legal difficulties for the use of recommended disinfectants. A lack of information about effective disinfectants was identified for some agents.

Control of the deliberate spread of foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is one of the most feared of transboundary animal diseases. Accidental or deliberate release of the causative agent can have both direct and indirect effects that result in massive economic losses and disruption. The direct effects of an FMD outbreak include immediate losses to agricultural production and disruption of local economies, while the indirect effects are mainly related to disease control measures such as restriction of market access at local and global levels and the high costs of disease control. To improve the capacity of the European Union (EU) to counter animal bioterrorism threats, AniBioThreat was launched with a special focus on threats to living animals, feed, and food of animal origin. As part of this project, several zoonotic or animal pathogenic agents are considered from different perspectives. FMD virus was selected as one agent to be scrutinized because it is highly contagious and an outbreak can have a severe economic impact. Ways to fight a deliberate outbreak can be demonstrated through the example of FMD. In this article, the virology and epidemiology of FMD virus are discussed with special attention to the related law enforcement aspects.

Development of a comparative risk ranking system for agents posing a bioterrorism threat to human or animal populations.

Various systems for prioritizing biological agents with respect to their applicability as biological weapons are available, ranging from qualitative to (semi)quantitative approaches. This research aimed at generating a generic risk ranking system applicable to human and animal pathogenic agents based on scientific information. Criteria were evaluated and clustered to create a criteria list. Considering availability of data, a number of 28 criteria separated by content were identified that can be classified in 11 thematic areas or categories. Relevant categories contributing to probability were historical aspects, accessibility, production efforts, and possible paths for dispersion. Categories associated with impact are dealing with containment measures, availability of diagnostics, preventive and treatment measures in human and animal populations, impact on society, human and veterinary public health, and economic and ecological consequences. To allow data-based scoring, each criterion was described by at least 1 measure that allows the assignment of values. These values constitute quantities, ranges, or facts that are as explicit and precise as possible. The consideration of minimum and maximum values that can occur due to natural variations and that are often described in the literature led to the development of minimum and maximum criteria and consequently category scores. Missing or incomplete data, and uncertainty resulting therefrom, were integrated into the scheme via a cautious (but not overcautious) approach. The visualization technique that was used allows the description and illustration of uncertainty on the level of probability and impact. The developed risk ranking system was evaluated by assessing the risk originating from the bioterrorism threat of the animal pathogen bluetongue virus, the human pathogen Enterohemorrhagic Escherichia coli O157:H7, the zoonotic Bacillus anthracis, and Botulinum neurotoxin.