Risultati finali
The first purpose of this result is to permit a rapid detection of Bacillus cereus srains carrying enterotoxin genes (i.e. potentially able to cause diarrhoea). Three toxins coded by one gene (cytK) and by two operons of three genes each (hbl and nhe) have been showed to be able to cause diarrhoeal poisoning. New primers and a multiplex PCR method to amplify simultaneously three genes (cytK gene and one gene of the two operons hbl and nhe) have been developed.
The second purpose of this result is to permit a reliable PCR detection of Bacillus cereus strains carrying enterotoxin genes. Previously published primers for PCR amplification of nhe and hbl operon did not detect the operons in all Bacillus cereus strains, leading to false negative results. The sequence of enterotoxin genes from diverse Bacillus cereus strains was obtained to design more universal primers, able to detect the previously false negative strains. To increase reliabilityn, these primers were also designed to amplify in one-reaction long fragments covering the 3 genes of the hbl and nhe operons (the three genes are necessary for enterotoxic activity), whereas previously published primers detected only one gene for one operon.
As new analytical tools monoclonal antibodies (mab) against the components of the NHE (and CytK)enterotoxins were developed which enable the specific and sensitive detection of these exoproteins in cell-free culture supernatants of Bacillus cereus isolates.
These mabs were successfully employed in a broad range of immunochemical techniques:
(a) by immunoaffinity chromatography highly pure toxin preparations could be obtained
(b) for the simple differentiation of HBL and/or NHE producing B. cereus isolates a colony immunoblot assay was established
(c) different EIA methods enabled the reliable detection of diarrhoeal B. cereus strains and their discrimination in high and low producers.
Particularly the developed sandwich enzyme immunoassays for the L2 component of HBL and the B component of NHE, respectively, represent a considerable improvement in sensitivity and specificity compared to the currently used assays. They permit a quantitative measure of the amount of NHE and HBL produced by a strain, thereby providing an estimate of the virulence of this strain with regard to diarrhoeal poisoning.
Molecular assay for rapid and conclusive identification of emetic toxin (cereulide) producing Bacillus cereus.
A gene fragment of the non-ribosomal peptide synthetase that is responsible for emetic toxin production in Bacillus cereus was identified and used for detection and identification of emetic toxin producing B. cereus. The resulting assay is based on polymerase chain reaction (PCR) techniques and its specificity has been proven with a test panel of 200 bacterial isolates. Bacillus cereus group isolates as well as non-B. Cereus group isolates, with special emphasis on food pathogens, were included in this survey.
The developed molecular assay allows a rapid detection of emetic B. cereus strains within a day. The simplicity of this assay renders it applicable for most routine labs and it is expected that this will improve food safety and diagnosis of the emetic type of illness caused by B. cereus.
A hundred strains of Bacillus cereus have been selected from food borne outbreaks (emetic and diarrhoeic), from various foods, from various environments, and from various European countries. Care was taken to maximise the diversity of this set of 100 strains. These strains have been characterised for minimum and maximum growth temperatures, for growth kinetics at 37°C and various pH, for heat resistance of spores, for spore adhesion on epithelial cell cultures (some strains) for emetic toxin genes and emetic toxin production, for enterotoxins genes and enterotoxin production, for enterotoxic activity measured on cell cultures. These 100 strains have been integrated in a large taxonomical investigation on 400 strains of B. cereus and related species to specify their taxonomical position.
This database provides an overview of the diversity of B. cereus for features important in virulence and behaviour during food processing and storage. The strain collection provides a set of strains already characterized and available for the scientific and professional community for future work on B. cereus.
The novel in vitro sperm micro assay distinguishes mitochondrial toxin producing Bacillus cereus from other B. cereus. It is based on the easily recorded response, motility inhibition, exhibited by boar spermatozoa exposed to mitochondrial toxins. Cereulide, the emetic toxin of B. cereus, is a mitochondrial toxin and is detected by this assay. The assay is nonlaborious, robust, not sensitive to age or storage of the culture plates and can be executed with equipment present in most laboratories. The assay can be executed in 15 min with a loop of biomass from culture plate. The full details and validation are described in Andersson et al 2004.
The test distinguishes mitochondrial toxins from other toxins when supplemented with functional staining (JC-1, calcein AM, PI). Full details are described in Hoornstra et al 2003.The presence of cereulide in the bacterial extract can be confirmed by chemical analysis. Bacterial extracts are analysed by liquid chromatography-ion trap mass spectrometry (LC-ion trap MS) using the mass ions m/z 1175 (Na+ adduct), m/z 1192, (K+ adduct), m/z 1154 (H+ adduct) and m/z 1171 (NH4+ adduct). The detection limit was 10 pg of cereulide per injection.
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