Background: This study aimed to evaluate the safety of raw vegetable products present on the German market regarding toxin-producing Bacillus cereus sensu lato (s.l.) group bacteria. Results: A total of 147 B. cereus s.l. group strains isolated from cucumbers, carrots, herbs, salad leaves and ready-to-eat mixed salad leaves were analyzed. Their toxinogenic potential was assessed by multiplex PCR targeting the hemolysin BL (hbl) component D (hblD), non-hemolytic enterotoxin (nhe) component A (nheA), cytotoxin K-2 (cytK-2) and the cereulide (ces) toxin genes. In addition, a serological test was used to detect Hbl and Nhe toxins. On the basis of PCR and serological results, none of the strains were positive for the cereulide protein/genes, while 91.2, 83.0 and 37.4% were positive for the Hbl, Nhe and CytK toxins or their genes, respectively. Numerous strains produced multiple toxins. Generally, strains showed resistance against the β-lactam antibiotics such as penicillin G and cefotaxim (100%), as well as amoxicillin/clavulanic acid combination and ampicillin (99.3%). Most strains were susceptible to ciprofloxacin (99.3%), chloramphenicol (98.6%), amikacin (98.0%), imipenem (93.9%), erythromycin (91.8%), gentamicin (88.4%), tetracycline (76.2%) and trimethoprim/sulfamethoxazole combination (52.4%). The genomes of eight selected strains were sequenced. The toxin gene profiles detected by PCR and serological test mostly agreed with those from whole-genome sequence data. Conclusions: Our study showed that B. cereus s.l. strains encoding toxin genes occur in products sold on the German market and that these may pose a health risk to the consumer if present at elevated levels. Furthermore, a small percentage of these strains harbor antibiotic resistance genes. The presence of these bacteria in fresh produce should, therefore, be monitored to guarantee their safety.

In this study, nine Gram-negative, motile and rod-shaped bacteria were isolated during a Germany-wide investigation of raw milk microbiota. The strains could be differentiated from their closest relatives by phenotypic and chemotaxonomic characterization and average nucleotide identity (ANIb) values calculated from draft genome assemblies. Strains MBT-1^T, MBT-8, MBT-9, MBT-10, MBT-11 and MBT-12 were related to the Pseudomonas chlororaphis subgroup. Isolates MBT-2^T, MBT-13 and MBT-14 were closely related to Pseudomonas rhizosphaerae DSM 16299^T with an ANIb of 88.2 % and a genome-to-genome distance result of 36.0 %. The G+C content of the DNA of strains MBT-1^T and MBT-2^T was 60.84 and 62.48 mol%, respectively. The major fatty acids were C_16 : 1 ω7c (summed feature 3), C_16 : 0 and C_18 : 1 ω7c (summed feature 8). The strains were catalase-positive, while production of urease, β-galactosidase and indole were negative. Growth occurred at 4–30 °C and at pH values of pH 6.0–8.0. Based on these results, we conclude that the strains belong to two novel species, for which the names Pseudomonas kielensis sp. nov. and Pseudomonas baltica sp. nov. are proposed. The type strains are MBT-1^T (=DSM 111668^T= LMG 31954^T) and MBT-2^T (=DSM 111761^T=LMG 31955^T).

Here we present the description of a novel Pseudomonas species, designated Pseudomonas rustica sp. nov., which was isolated from raw milk samples obtained from Germany. Results of initial 16S rRNA gene sequence analysis assigned the strain into the genus Pseudomonas and showed Pseudomonas helmanticensis , Pseudomonas neuropathica and Pseudomonas atagonensis to be its closest relatives. Further studies including sequence analysis of the rpoB gene, multi-gene phylogenetic tree reconstruction, whole-genome sequence comparisons, cellular fatty acid analysis and chemotaxonomic characterization showed a clear separation from the known Pseudomonas species. Isolate MBT-4^T was closely related to Pseudomonas helmanticensis , 'Pseudomonas crudilactis' and Pseudomonas neuropathica with average nucleotide identities based on blast values of 88.8, 88.8 and 88.6%, respectively. Therefore, the strain can be classified into the Pseudomonas koreensis subgroup of the Pseudomonas fluorescens group. The G+C content of strain MBT-4^T was 58.9 mol%. The strain was catalase- and oxidase-positive, while the β-galactosidase reaction was negative. Growth occurred between 4 and 30 °C and at pH values from pH 6.0 to 8.0. In conclusion, strain MBT-4^T belongs to a novel species, for which the name Pseudomonas rustica sp. nov. is proposed. The type strain is MBT-4^T (=DSM 112348^T=LMG 32241^T) and strain MBT-17 is also a representative of this species.

The genome of the intimin ( eae )-harboring Escherichia albertii strain MBT-EA1, isolated from lettuce in Germany, was sequenced. Sequence analysis showed the assembled draft genome size to be 4,560,948 bp, containing a predicted total of 4,414 protein-encoding genes, 11 rRNAs, and 82 tRNAs. Furthermore, three plasmid sequences were found.

The genetic heterogeneity of Heyndrickxia sporothermodurans (formerly Bacillussporothermodurans) was evaluated using whole genome sequencing. The genomes of 29 previously identified Heyndrickxiasporothermodurans and two Heyndrickxia vini strains isolated from ultra-high-temperature (UHT)-treated milk were sequenced by short-read (Illumina) sequencing. After sequence analysis, the two H. vini strains could be reclassified as H. sporothermodurans. In addition, the genomes of the H.sporothermodurans type strain (DSM 10599^T) and the closest phylogenetic neighbors Heyndrickxiaoleronia (DSM 9356^T) and Heyndrickxia vini (JCM 19841^T) were also sequenced using both long (MinION) and short-read (Illumina) sequencing. By hybrid sequence assembly, the genome of the H. sporothermodurans type strain was enlarged by 15% relative to the short-read assembly. This noticeable increase was probably due to numerous mobile elements in the genome that are presumptively related to spore heat tolerance. Phylogenetic studies based on 16S rDNA gene sequence, core genome, single-nucleotide polymorphisms and ANI/dDDH, showed that H. vini is highly related to H. sporothermodurans. When examining the genome sequences of all H.sporothermodurans strains from this study, together with 4 H. sporothermodurans genomes available in the GenBank database, the majority of the 36 strains examined occurred in a clonal lineage with less than 100 SNPs. These data substantiate previous reports on the existence and spread of a genetically highly homogenous and heat resistant spore clone, i.e., the HRS-clone.

Pasteurized whey concentrate is used as a base for the production of ingredients for various food products. Whey concentrate (30% dry matter) was used to assess the thermal inactivation of Salmonella (S.) enterica serovar Senftenberg 775W (DSM 10062) and Escherichia (E.) coli AW1.7 (DSM 108612) strains in a pilot-scale pasteurizer mimicking industrial heat processing. These strains, chosen for their exceptional heat resistance, represent the most challenging scenario for pasteurization within the context of S. enterica and E. coli. Heat resistance was tested at temperatures of 56, 60, 64, 68, and 72 °C at an average holding time of 17.5 s. These exceptionally heat-resistant strains showed a relatively low reduction in numbers of between 0 and 4.2 log₁₀ CFU/mL at lower inactivation temperatures of ≤68 °C. A reduction of at least 5 log₁₀ CFU/mL, as required for adequate heat processing, was achieved for both species after heating at 72 °C for 17.5 s. This study shows that whey concentrate should not lead to contamination of food ingredients and can be considered safe after pasteurization at 72 °C for at least 17.5 s with respect to the pathogens tested.