Properties of Bacillus cereus spores in reference materials prepared from artificially contaminated spray dried milk.

A reference material for Bacillus cereus was developed based on spray drying of milk artificially contaminated with B. cereus spores. Various properties of the B. cereus spores in the milk powder were determined. The stability of the materials was good with no detectable decrease in the contamination level during 1 1/2 years storage at -20 degrees C or 4 weeks at 22, 30 or 37 degrees C. The homogeneity of the material was found acceptable for use as a reference material. Heat treatments (10 min at 70 or 80 degrees C) and addition of lysozyme to the enumeration medium did not influence the number of spores counted. The germination of the spores depended on the type of medium in which the milk powder was reconstituted, and on the storage period of the material. The suitability of the material was confirmed in a collaborative study. From the results obtained it was concluded that the material developed meets the general requirements set for reference materials and can therefore be used for, among others, testing laboratory performance.

Formation of antigenic extracellular polysaccharides by selected strains of Mucor spp., Rhizopus spp., Rhizomucor spp., Absidia corymbifera and Syncephalastrum racemosum.

In this study, polyclonal IgG antibodies raised against extracellular polysaccharides (EPS) of Mucor racemosus were characterised as almost specific for moulds belonging to the order of Mucorales. Cross-reactivity in the ELISA could be observed only towards the yeast Pichia membranaefaciens. EPS were isolated from various cultures of M. hiemalis growing on six different carbon sources and two nitrogen sources, with ratios varying from 0.13 to 0.44 relative to the amount of biomass. Other strains including Mucor spp., Rhizopus spp., Rhizomucor spp., Absidia corymbifera and Syncephalastrum racemosum also excreted EPS, with ratios varying from 0.05 to 0.23. In all cases, the excreted EPS had similar antigenic properties as determined by ELISA. No enzymatic degradation of the antigenic parts of the polysaccharides could be observed upon prolonged incubation. Considering that all tested strains formed similar amounts of antigenic EPS there might be scope for the specific detection of biomass of Mucoralean moulds using ELISA techniques for example in food.

Successful approach for detection of low numbers of enterotoxigenic Escherichia coli in minced meat by using the polymerase chain reaction.

The polymerase chain reaction (PCR) was used as a tool for the detection of enterotoxigenic Escherichia coli in minced meat. With two synthetic 29-mer oligonucleotides, a 195-bp fragment from the E. coli heat-labile enterotoxin (LT) gene could be amplified specifically. When 6 CFU was added to the reaction mixture as a template, the PCR yielded sufficient amplified product for visualization on an agarose gel. Prior to PCR amplification, the minced meat samples were subjected to enrichment culturing for E. coli. From these cultures, 10 microliters was used in the PCR assay. All 20 25-g samples that were examined in this assay were negative for E. coli LT. However, when 3 CFU of E. coli LT was added to the 25-g samples of minced meat prior to enrichment culturing, the PCR assay yielded positive results.

Adaptation of Listeria in liquid egg containing sucrose resulting in survival and outgrowth.

Survival of Listeria species was tested in liquid egg products such as albumen, yolk, whole egg and whole egg containing 25% sucrose. At an incubation temperature of 4 degrees C Listeria survived in all products, and even increased slightly in yolk, whole egg and whole egg containing 25% sucrose. However, at 20-22 degrees C, in whole egg containing sucrose, a rapid an dramatic decrease in numbers of Listeria was observed. Following this initial decrease, an increase in the numbers of Listeria was observed. The ability to grow in liquid egg containing sucrose was maintained upon reinoculation into a freshly prepared product. However, this property disappeared after subculturing in brain heart infusion broth. The significance of these findings is discussed.

DNA hybridization and latex agglutination for detection of heat-labile- and shiga-like toxin-producing Escherichia coli in meat.

DNA-hybridization and latex-agglutination tests were used for screening of a group of Escherichia coli isolates for heat-labile enterotoxin (LT)- and shiga-like toxin (SLT1 or VT1) -producing strains, respectively. Strains tested originated from 162 meat samples (poultry, pigs and beef) chosen at random. Additionally LT- and SLT1-producing reference strains were tested. The DNA-hybridization technique allowed screening of large numbers of strains, whereas large scale testing of strains by latex agglutination was laborious. Of 800 E. coli strains tested by DNA hybridization none contained the gene encoding LT. Production of LT as tested by latex agglutination was not found. The gene encoding SLT1 was detected in 10 of the 800 isolates tested. None of these strains, however, showed cytotoxicity on Vero cells. Serotyping was done with sorbitol-negative E. coli strains, first by using the latex-agglutination test for O157 followed by complete serotyping. No E. coli of serogroup O157 were found. Therefore the results obtained also indicate that routine screening of E. coli isolated randomly from food for toxin production is not useful and should be limited to food-borne disease outbreaks with an etiology resembling an E. coli infection.

Survival of pathogenic microorganisms in an egg-nog-like product containing 7% ethanol.

A liquor consisting of whole egg, saccharose (25% w/v) and ethanol (7.0% w/v) was artificially contaminated with Salmonella enteritidis, S. typhimurium, Staphylococcus aureus (three different strains), Bacillus cereus and Listeria monocytogenes. After 3 weeks of incubation at 22 degrees C the numbers of Salmonella, S. aureus and L. monocytogenes decreased more than 3 log10 units. Under such conditions, however, the total number of microorganisms increased 3 log10 units. At 4 degrees C the decrease of pathogenic microorganisms was much slower and a decrease of 3 log10 units was observed only after 7 weeks of incubation. Egg-nog, without ethanol, incubated at 22 degrees C allowed growth of Salmonella and S. aureus, while the numbers of B. cereus spores remained unchanged. Vegetative cells of B. cereus as well as L. monocytogenes decreased in numbers. However, after prolonged incubation the numbers of L. monocytogenes increased significantly.

Method for the detection of Shigella in foods.

A method for the detection of Shigella in foods was evaluated. The method consisted of selective enrichment at 37 degrees C in GN-broth supplemented with novobiocin (10 micrograms/ml) followed by subculture after 6 and 24 h incubation of 0.1 ml volumes to SS-agar with and without streptomycin (7.5 micrograms/ml). Test samples were contaminated by adding shigellae to 1:10 suspensions of foods (minced meat, fresh cut vegetables and cooked peeled shrimp) in the GN-broth with novobiocin. Shigella could be detected at a contamination level of 1 per 25 g in the presence of shrimp (in all 20 samples) and at 25 per 25 g in the presence of nearly all samples of fresh cut vegetables (in 13 out of 18 samples). Using this procedure Shigella could not be detected when the organism was present at a relatively high contamination level of 10(2) per 25 g in the presence of minced meat (with all 10 samples). However, the procedure described is an improvement on methods currently available for the detection of Shigella in foods.

Immunological relationship of extra-cellular polysaccharide antigens produced by different mould species.

Moulds are able to produce extracellular polysaccharide antigens which are heat-stable and almost genus specific. Of 44 different strains of Penicillium 41 (93%) and all 12 strains of Aspergillus tested produced detectable quantities of an immunologically related antigen. Additionally 10 of these 56 strains produced an antigen immunologically related to the antigen produced by the genera Mucor and Fusarium. Immunologically different, but genus-specific antigens were produced by each of the species belonging to the genus Geotrichum, Fusarium, Cladosporium, Mucor and Rhizopus. The antigens produced by Mucor and Rhizopus, however, were immunologically related.