Maria A. Andersson

Identification and partial characterization of the nonribosomal peptide synthetase gene responsible for cereulide production in emetic Bacillus cereus.

ABSTRACT Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Due to its chemical structure, (d-O-Leu-d-Ala-l-O-Val-l-Val)3, cereulide might be synthesized nonribosomally. Therefore, degenerate PCR primers targeted to conserved sequence motifs of known nonribosomal peptide synthetase (NRPS) genes were used to amplify gene fragments from a cereulide-producing B. cereus strain. Sequence analysis of one of the amplicons revealed a DNA fragment whose putative gene product showed significant homology to valine activation NRPS modules. The sequences of the flanking regions of this DNA fragment revealed a complete module that is predicted to activate valine, as well as a putative carboxyl-terminal thioesterase domain of the NRPS gene. Disruption of the peptide synthetase gene by insertion of a kanamycin cassette through homologous recombination produced cereulide-deficient mutants. The valine-activating module was highly conserved when sequences from nine emetic B. cereus strains isolated from diverse geographical locations were compared. Primers were designed based on the NRPS sequence, and the resulting PCR assay, targeting the ces gene, was tested by using a panel of 143 B. cereus group strains and 40 strains of other bacterial species showing PCR bands specific for only the cereulide-producing B. cereus strains.

Quantitative Analysis of Cereulide, the Emetic Toxin of Bacillus cereus, Produced under Various Conditions

ABSTRACT This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 μg ml−1. The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 μg of cereulide g−1 (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 μg of cereulide ml−1 in liquid medium at room temperature (21 ± 1°C) in 1 to 3 days, during the stationary growth phase when cell density was 2 × 108 to 6 × 108 CFU ml−1. Cereulide production at temperatures at and below 8°C or at 40°C was minimal.

In vitro assay for human toxicity of cereulide, the emetic mitochondrial toxin produced by food poisoning Bacillus cereus

The in vitro boar spermatozoon test was compared with the LC ion trap MS analysis for measuring the cereulide content of a pasta dish, implemented in serious emetic food poisoning caused by Bacillus cereus. Both assays showed that the poisonous food contained approximately 1.6 microg of cereulide g(-1) implying the toxic dose in human as < or =8 microg kg(-1) body weight. The threshold concentration of cereulide provoking visible mitochondrial damage in boar sperm exposed in vitro was 2 ng of cereulide ml(-1) of extended boar sperm. The same threshold value was found for cereulide extracted from the food and from the cultured bacteria. This shows that other constituents of the food did not enhance or mask the effects of cereulide. Exposure of four human cell lines (HeLa, Caco-2, Calu-3 and Paju) to cereulide showed that the threshold concentration for the loss of mitochondrial membrane potential in human cells was similar to that observed in boar sperm. Human cells and boar sperm were equally sensitive to cereulide. The results show that boar spermatozoan assay is useful for detecting cereulide concentrations toxic to humans. Spermatozoa in commercially available extended fresh boar and cryopreserved bull semen were compared, boar sperms were 100 times more sensitive to cereulide than bull sperms.

Evaluation of Methods for Recognising Strains of the Bacillus cereus Group with Food Poisoning Potential Among Industrial and Environmental Contaminants

Toxin production, biochemical properties and ribotypes of Bacillus cereus group (B. cereus, B. thuringiensis, B. mycoides) strains originating from industrial and environmental sources (n = 64), from food poisoning incidents (n = 22) and from reference sources (n = 7) were analysed. Forty ribotypes were found among the 93 strains. Eleven strains from food poisoning incidents produced emetic (mitochondrio) toxin, as determined by the boar spermatozoa toxicity test. These strains possessed closely similar ribotypes which were rare among strains of other origins. Sperm toxin producing (cereulide positive) strains did not hydrolyse starch and did not produce haemolysin BL, as determined by the reverse passive latex agglutination test. Sixteen different ribotypes were found among B. cereus strains from board machines (n = 16) and from packaging board (n = 16), indicating many different sources of B. cereus contamination in board mills. Strains originating from packaging board had predominantly different ribotypes from those of dairy and dairy product originating strains. Nine (53%) out of 17 strains from a single dairy process shared the same ribotype whereas strains from milk and milk products from different dairies had different ribotypes indicating that B. cereus group populations were dairy specific. Twenty-two percent of strains isolated from the paperboard industry on non-selective medium were lecithinase negative, including enterotoxin producing strains. This stresses the importance of other detection methods not based on a positive lecithinase reaction.

Toxic-Metabolite-Producing Bacteria and Fungus in an Indoor Environment

ABSTRACT Toxic-metabolite-emitting microbes were isolated from the indoor environment of a building where the occupant was suffering serious building-related ill-health symptoms. Toxic substances soluble in methanol and inhibitory to spermatozoa at <10 μg (dry weight) ml−1 were found from six bacterial isolates and one fungus. The substances from isolates of Bacillus simplexand from isolates belonging to the actinobacterial generaStreptomyces and Nocardiopsis were mitochondriotoxic. These substances dissipated the mitochondrial membrane potential (Δψ) of boar spermatozoa. The substances from the Streptomyces isolates also swelled the mitochondria. The substances from isolates of Trichoderma harzianum Rifai and Bacillus pumilus damaged the cell membrane barrier function of sperm cells.

A new method for in vitro detection of microbially produced mitochondrial toxins

Sperm motility inhibition assay, earlier shown valuable for the detection of food poisoning non-protein toxins of Bacillus species was developed into an assay useful for specific detection of mitochondria damaging toxins. This was done by assessing the dissipation of the mitochondrial inner membrane transmembrane potential, Deltapsim under conditions where the plasma membrane permeability barrier remained intact. The Deltapsim was estimated as the intensity of orange JC-1 fluorescence in the mitochondrial sheath of the exposed spermatozoa. The plasma membrane integrity of the same cells was assessed by observing the exclusion of propidium iodide from the cytoplasm. Three types of mitochondrial toxic responses to microbially made bioactive substances were recognised. Mitochondrial toxicity by gramicidin (A, B, C, D), nigericin, salinomycin, narasin, monensin, calcimycin and antimycin A was characterised by gradual fading of the JC-1 fluorescence in the mitochondria. Dissipation of the Deltapsim by cereulide, valinomycin and enniatin (A, A1, B, B1) was visible as spotwise quenching of the mitochondrial JC-1 fluorescence. In addition these substances caused hyperpolarisation of the plasma membrane. Oligomycin (A, B, C), ionomycin and staurosporine inhibited the spermatozoan motility, but Deltapsim was fully preserved. Surfactin and lichenysin A caused mitochondrial damage at concentrations where the plasma membrane was also damaged.

Williamsia muralis gen. nov., sp. nov., isolated from the indoor environment of a children's day care centre.

The taxonomic status of an actinomycete (MA 140/96T) isolated from indoor building materials of a childrens day care centre was studied using the polyphasic approach. The cell morphology was atypical for an actinomycete, electron microscopy revealed a hairy surface, highly unusual for Gram-positive bacteria. The organisms grew at 10-37 degrees C, no growth was visible at 5 degrees C and 45 degrees C in 5 d. The cell wall contained the diamino acid meso-diaminopimelic acid and the sugars arabinose, galactose, mannose and ribose. The phospholipids phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol and diphosphatidylglycerol were detected. The only menaquinone found was MK-9(H2). The fatty acid pattern was composed of palmitic acid (23.6%) palmitoleic acid (16.5%) and another hexadecenoic acid 16:1cis11 (1.4%), oleic acid (29.9%), stearic acid (2.9%) and the 10-methyl-branched tuberculostearic acid (23.3%). A gas-chromatographic analysis of the mycolic acid revealed a carbon-chain length of C50-C56. The G + C was 64.8 mol%. The results of 16S rDNA sequence comparisons revealed that strain MA 140/96T represents a new lineage in the suborder Corynebacterineae of the order Actinomycetales. Therefore, it was concluded that strain MA 140/96T should be assigned to a new genus and species, for which the name Williamsia muralis gen. nov., sp. nov. is proposed. The type strain of the species is MA 140/96T (= DSM 44343T).

The Fusarium mycotoxins enniatins and beauvericin cause mitochondrial dysfunction by affecting the mitochondrial volume regulation, oxidative phosphorylation and ion homeostasis.

The mechanisms of cell toxicity of mycotoxins of the enniatin family produced by Fusarium sp. enniatin B, a mixture of enniatin homologues (3% A, 20% A(1), 19% B, 54% B1) and beauvericin, were investigated. In isolated rat liver mitochondria, exposure to submicromolar concentrations of the enniatin mycotoxins depleted the mitochondrial transmembrane potential, uncoupled oxidative phosphorylation, induced mitochondrial swelling and decreased calcium retention capacity of the mitochondria. The mitochondrial effects were strongly connected with the potassium (K(+)) ionophoric activity of the enniatins. The observed enniatins induced K(+) uptake by mitochondria. This shows that the enniatins acted as ionophores highly selective for potassium ions. The effects were observed in potassium containing media whereas less or no effect remained to be observed when K(+) was partially or totally replaced by isomolar concentrations of Na(+). The rank order of enniatin induced mitochondrial impairment was beauvericin>enniatin mixture>enniatin B. Exposure to the enniatins depleted the mitochondrial membrane potential also in intact human neural (Paju), murine insulinoma (Min-6) cells as well as boar spermatozoa. Exposure to enniatin B in media with physiological (4mM) or low (<1mM) but not in high (60mM) external concentration of K(+) induced hyperpolarization of the spermatozoal plasma membrane indicating enniatin that catalysed efflux of the cytosolic K(+) ions. These results indicate that the cellular toxicity targets of the enniatin mycotoxins are the mitochondrion and the homeostasis of potassium ions.

Frigoribacterium faeni gen. nov., sp. nov., a novel psychrophilic genus of the family Microbacteriaceae.

The taxonomic position of five actinobacterial strains isolated from dust, an animal shed, the air inside a museum and soil was investigated using a polyphasic approach. The growth characteristics were unusual for actinomycetes. Optimal growth was at temperatures ranging from 2 to 10 degrees C. After small-step adaptation (5 degrees C steps) to higher temperatures, the strains were also able to grow at 20 degrees C. Cell wall analyses revealed that the organisms showed a hitherto undescribed, new group B-type peptidoglycan [type B2beta according to Schleifer & Kandler (1972), but with lysine instead of ornithine]. All strains contained menaquinone MK-9. Mycolic acids were not detected. Diphosphatidylglycerol, phosphatidylglycerol and an unknown glycolipid were detected in the polar lipid extracts. The main fatty acids were 12-methyl-tetradecanoic acid (15:0 anteiso), 12-methyl-tetradecenoic acid (15:1 anteiso), 14-methyl-pentadecanoic acid (16:0 iso) and 14-methyl-hexadecanoic acid (17:0 iso), as well as an unusual compound identified as 1,1-dimethoxy-anteiso-pentadecane (15:0 anteiso-DMA). The G+C content of DNA was approximately 71 mol%. The results of 16S rRNA gene sequence comparisons revealed that the strains represent a new lineage in the suborder Micrococcineae and the family Microbacteriaceae of the order Actinomycetales. On the basis of these results the new genus Frigoribacterium gen. nov. is proposed, harbouring the new species Frigoribacterium faeni sp. nov. (type strain = 801T = DSM 10309T).

Potential of Bacillus cereus for Producing an Emetic Toxin, Cereulide, in Bakery Products: Quantitative Analysis by Chemical and Biological Methods

A method for the direct quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, in bakery products was developed. The analysis was based on robotized extraction followed by quantitation of cereulide by liquid chromatography-mass spectrometry and an assay of toxicity by the boar sperm motility inhibition test. The bioassay and the chemical assay gave comparable results, demonstrating that the extracted cereulide was in a biologically active form. Cereulide was formed when cereulide-producing B. cereus strains were present at > or = 10(6) CFU/g in products with water activity values of > 0.953 and pHs of > 5.6. Rice-containing pastries accumulated high contents of cereulide (0.3 to 5.5 microg/g [wet weight]) when stored at nonrefrigeration temperatures (21 to 23 degrees C). Cereulide was not formed in products stored at refrigeration temperatures (4 to 8 degrees C). Cereulide is not inactivated by heating during food processing. Therefore, direct analysis of this toxin in food is preferable to cultivating methods for assessing the risk of food poisoning by emetic B. cereus.