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.

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.

Spread of a distinct Stx2-encoding phage prototype among Escherichia coli O104:H4 strains from outbreaks in Germany, Norway, and Georgia.

Shiga toxin 2 (Stx2)-producing Escherichia coli (STEC) O104:H4 caused one of the world's largest outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in Germany in 2011. These strains have evolved from enteroaggregative E. coli (EAEC) by the acquisition of the Stx2 genes and have been designated enteroaggregative hemorrhagic E. coli. Nucleotide sequencing has shown that the Stx2 gene is carried by prophages integrated into the chromosome of STEC O104:H4. We studied the properties of Stx2-encoding bacteriophages which are responsible for the emergence of this new type of E. coli pathogen. For this, we analyzed Stx bacteriophages from STEC O104:H4 strains from Germany (in 2001 and 2011), Norway (2006), and the Republic of Georgia (2009). Viable Stx2-encoding bacteriophages could be isolated from all STEC strains except for the Norwegian strain. The Stx2 phages formed lysogens on E. coli K-12 by integration into the wrbA locus, resulting in Stx2 production. The nucleotide sequence of the Stx2 phage P13374 of a German STEC O104:H4 outbreak was determined. From the bioinformatic analyses of the prophage sequence of 60,894 bp, 79 open reading frames were inferred. Interestingly, the Stx2 phages from the German 2001 and 2011 outbreak strains were found to be identical and closely related to the Stx2 phages from the Georgian 2009 isolates. Major proteins of the virion particles were analyzed by mass spectrometry. Stx2 production in STEC O104:H4 strains was inducible by mitomycin C and was compared to Stx2 production of E. coli K-12 lysogens.