BackgroundThis 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.ResultsA 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.ConclusionsOur 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.

Forty-seven Acinetobacter spp. isolates from milk powder obtained from a powdered milk producer in Germany were investigated for their antibiotic resistance susceptibilities, in order to assess whether strains from food harbor multiple antibiotic resistances and whether the food route is important for dissemination of resistance genes. The strains were identified by 16S rRNA and rpoB gene sequencing, as well as by whole genome sequencing of selected isolates and their in silico DNA-DNA hybridization (DDH). Furthermore, they were genotyped by rep-PCR together with reference strains of pan-European groups I, II, and III strains of Acinetobacter baumannii. Of the 47 strains, 42 were identified as A. baumannii, 4 as Acinetobacter Pittii, and 1 as Acinetobacter calcoaceticus based on 16S rRNA gene sequencing. In silico DDH with the genome sequence data of selected strains and rpoB gene sequencing data suggested that the five non-A. baumannii strains all belonged to A. pittii, suggesting that the rpoB gene is more reliable than the 16S rRNA gene for species level identification in this genus. Rep-PCR genotyping of the A. baumannii strains showed that these could be grouped into four groups, and that some strains clustered together with reference strains of pan-European clinical group II and III strains. All strains in this study were intrinsically resistant toward chloramphenicol and oxacillin, but susceptible toward tetracycline, tobramycin, erythromycin, and ciprofloxacin. For cefotaxime, 43 strains (91.5%) were intermediate and 3 strains (6.4%) resistant, while 3 (6.4%) and 21 (44.7%) strains exhibited resistance to cefepime and streptomycin, respectively. Forty-six (97.9%) strains were susceptible to amikacin and ampicillin-sulbactam. Therefore, the strains in this study were generally not resistant to the clinically relevant antibiotics, especially tobramycin, ciprofloxacin, cefepime, and meropenem, suggesting that the food route probably poses only a low risk for multidrug resistant Acinetobacter strains or resistance genes.

Two hundred fresh produce samples (cucumber, carrots, herbs, leaf lettuce, and ready-to-eat mixed salad leaves) were obtained from retail in northern Germany in 2015. These were investigated for microbial contamination and the presence of foodborne pathogens, including Listeria monocytogenes, Salmonella serovars, presumptive Bacillus (B.) cereus, and Shiga toxin-producing Escherichia coli using culture-dependent (enrichment, plating on selective media) and -independent (real-time polymerase chain reaction [PCR]) techniques. Overall, our results showed that the fresh produce samples generally showed high mean aerobic mesophilic bacterial counts of between 7 and 8 log cfu/g. However, there was also a considerable variation in total aerobic bacterial counts between different product samples. Although real-time PCR signals for pathogenic E. coli were detected in 14.0% of total samples analyzed, only one (0.5%) Shiga toxin-producing E. coli isolate of serotype O26:H11 was recovered from mixed salad leaves and contained stx1, stx2, and eae genes. Two L. monocytogenes isolates (1% of total samples) were recovered from packaged mixed salad leaves and belonged to PCR serogroups IIb and IVb, respectively. One Salmonella isolate (0.5%) was recovered after selective enrichment also from mixed salad leaves and it was identified as Salmonella Szentes serotype 16:k:1,2. Overall the incidence of foodborne pathogens on the northern German retail market in 2015 was very low.

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-1T, MBT-8, MBT-9, MBT-10, MBT-11 and MBT-12 were related to the Pseudomonas chlororaphis subgroup. Isolates MBT-2T, MBT-13 and MBT-14 were closely related to Pseudomonas rhizosphaerae DSM 16299T 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-1T and MBT-2T was 60.84 and 62.48 mol%, respectively. The major fatty acids were C16 : 1 ω7c (summed feature 3), C16 : 0 and C18 : 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-1T (=DSM 111668 T= LMG 31954T) and MBT-2T (=DSM 111761 T=LMG 31955T).

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