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.
Summary
The bacterium Streptomyces davaonensis synthesizes the antibiotic roseoflavin in the stationary phase of growth. The starting point for roseoflavin biosynthesis is riboflavin (vitamin B2) and four enzymes (RibCF, RosB, RosA and RosC) are necessary to convert a vitamin (riboflavin) into a potent, broad‐spectrum antibiotic (roseoflavin). In S. davaonensis, seven enzymatic functions are required to synthesize the roseoflavin precursor riboflavin from the central building blocks GTP and ribulose 5‐phosphate. When compared with other bacterial and in particular Streptomyces genomes the S. davaonensis genome contains an unusual high number (21) of putative riboflavin biosynthetic genes (rib genes), including a rib gene encoding an additional riboflavin synthase originating from an Archaeon. We show by complementation analyses and enzyme assays that 17 out of these 21 putative rib genes indeed encode for riboflavin biosynthetic enzymes. Biochemical analyses of selected enzymes support this finding. Transcriptome analyses show that all of the rib genes are expressed either in the exponential or in the stationary phase of growth and thus do not represent silent genes. We conclude that the Rib enzymes produced in the stationary phase represent a physiological adaptation to support roseoflavin biosynthesis.
is a red riboflavin analog with broad antimicrobial activity against Gram-positive bacteria (Otani et al., 1974). Under laboratory conditions RoF is produced in the stationary phase of growth by two different Gram-positive bacterial species, S. davaonensis (formerly S. davawensis (Landwehr et al., 2018)) and Streptomyces cinnabarinus (Jankowitsch et al., 2012). Other RoF producers are not known. RoF is taken up by target cells by riboflavin importers. Thus, RoF only acts against bacteria having riboflavin importers, which appear to be present mostly in Gram-positive bacteria (Grill
Binary toxins are produced by several pathogenic bacteria. Examples are the C2 toxin from Clostridium botulinum, the iota toxin from Clostridium perfringens, and the CDT from Clostridium difficile. All these binary toxins have ADP-ribosyltransferases (ADPRT) as their enzymatically active component that modify monomeric actin in their target cells. The binary C2 toxin was intensively described as a tool for intracellular delivery of allogenic ADPRTs. Here, we firstly describe the binary toxin CDT from C. difficile as an effective tool for heterologous intracellular delivery. Even 60 kDa glucosyltransferase domains of large clostridial glucosyltransferases can be delivered into cells. The glucosyltransferase domains of five tested large clostridial glucosyltransferases were successfully introduced into cells as chimeric fusions to the CDTa adapter domain (CDTaN). Cell uptake was demonstrated by the analysis of cell morphology, cytoskeleton staining, and intracellular substrate glucosylation. The fusion toxins were functional only when the adapter domain of CDTa was N-terminally located, according to its native orientation. Thus, like other binary toxins, the CDTaN/b system can be used for standardized delivery systems not only for bacterial ADPRTs but also for a variety of bacterial glucosyltransferase domains.
Streptomyces davaonensis and Streptomyces cinnabarinus produce the red riboflavin (RF) analog roseoflavin (8-demethyl-8-dimethylamin oriboflavin; RoF) (Figure 1) from RF and currently are the only organisms known to synthesize and export this antibiotic (Jankowitsch et al., 2012). All bacteria able to take up RoF are likely to be sensitive to this antimetabolite as all cells depend on the RF-derived cofactors riboflavin-5′-phosphate(RP)(alsocalledflavinmononucleotide,FMN) andflavinadeninedinucleotide(FAD) (Pedrollietal.,2013).Grampositive bacteria appear to represent the main targets for RoF as RF transporters, which also import the structurally very similar RoF,
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