Occurrence of Cronobacter spp. in retail foods.

AIMS: To study the occurrence of Cronobacter spp. in foods and to investigate the phenotypic properties of the strains isolated. METHODS AND RESULTS: A total of 53 strains of Cronobacter spp. isolated from 399 food samples were identified using conventional biochemical methods and MALDI-TOF mass spectrometry. Foods of plant origin were the most frequently contaminated samples. No Cronobacter spp. were found in infant milk formula, wheat-based infant food, pasteurized and raw cow milk, mincemeat, chicken, chickpea and potato dumpling powder. The individual species were identified as Cronobacter sakazakii (54·7%), Cronobacter malonaticus (28·4%), Cronobacter dublinensis (7·5%), Cronobacter muytjensii (7·5%) and Cronobacter turicensis (1·9%). Cronobacter sakazakii and C. malonaticus belong to biotype 1, 2, 2a, 3, 4 and 5, 5a, respectively. Cronobacter dublinensis strains were subdivided into biotypes 6 and 12. All strains were resistant to erythromycin and two of them were resistant to both erythromycin and tetracycline. CONCLUSIONS: Cronobacter spp. were isolated from various food samples pre-eminently of plant origin and dried food ingredients. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings will increase and detail our knowledge of the presence and diversity of Cronobacter spp. in foods.

Characterization of rabbit antibodies for immunochemical detection of Yersinia enterocolitica.

Rabbit IgG raised against whole cells of Yersinia enterocolitica O:3, O:9 and against a group of pathogenic Y. enterocolitica strains (serotypes O:3, O:5,27, O:8. and O:9) were prepared. The antibody limiting titers were within the range of 1:9.5 x 10(4)-1:7.5 x 10(5). The immunoblotting analysis of Yersinia lipopolysacchides separated by SDS-PAGE showed that IgG against the single serotype O:3 interacted with high-molar-mass LPS of O:3 whereas other antibodies were bound to low-molar-mass LPS of serotypes O:3, O:5,27, O:9 and strain Y. enterocolitica (CNCTC Y 2/68). IgG against the group of pathogenic serotypes also weakly interacted with low-molar-mass LPS of serotypes O:5, O:6,30, and O:10. The cross-reactivity of the antibodies with Y. pseudotuberculosis Ia and/or Y. rohdei b, d, e, f, i, which was observed by means of dot-blotting procedure using the whole bacterial cells as an antigen, was shown not to be caused by LPS of these bacteria. The prepared antibodies were used in the development of indirect competitive ELISA. At the optimum concentration of the immunoreactants the detection limits were within the range of 3-7 x 10(6) colony-forming units per mL.

Immunochemical determination of gluten in malts and beers.

The gluten content in different varieties of barley and malts, and in different types of beers, was determined by a 'sandwich' enzyme immunoassay (RIDASCREEN Gliadin kit). The gluten levels in barley wheat, rye and spelt malts ranged 18.8-45.0, 44.0-68.0, 41.6 and 21.2 g kg-1, respectively. When various types of beer were compared, the gluten concentration increased as follows: alcohol-free beer (<3.0), lager beers (<3.0-8.7 mg l-1), stouts (9.0-15.2 mg l-1) and wheat beers (10.6-41.2 mg l-1). When 10 Czech lager beers were analysed, using both sandwich and competitive ELISA, the results showed that the latter method provided values several times higher than the former. Gluten balance was carried out during the brewing process, starting from the raw materials and terminating at the final beer. Gluten levels decreased due to precipitation during the mashing process, primary and secondary fermentation and, lastly, as a result of adsorption during beer stabilization. The gluten content in beer is, thus, approximately three orders of magnitude lower than in the raw malt.

Development of an indirect competitive ELISA for detection of Campylobacter jejuni subsp.jejuni O:23 in foods.

An indirect enzyme immunoassay for rapid detection of Campylobacter jejuni subsp. jejuni 0:23 has been developed. Optimum concentrations of immobilized cells, polyclonal chicken IgY, and rabbit anti-IgY antibody-horseradish peroxidase conjugate were 3.1 CFU/nL, 10 microg/mL, and 8 microg/mL, respectively. Under such conditions, the detection limit reached 50 CFU/microL, limit of quantification being 480 CFU/microL. By testing 5 chromogens, viz. 1,2-benzenediamine, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), 3,3',5,5'-tetramethylbenzidine, bi(4,4'-anisidine) and 3-methyl-2-benzothiazolinone hydrazone, in horseradish peroxidase substrate, 1,2-benzenediamine or 3,3',5,5'-tetramethylbenzidine as H-donors in the enzyme substrate provided the highest ELISA sensitivity. The applied polyclonal antibody was specific for homogeneous antigen. The cross-reactions were observed only with one strain of C. sputorum subsp. sputorum (21.5 %) and with G+ bacterium Micrococcus luteus (6.1 %). Preliminary tests have been performed with a limited number of artificially contaminated food samples. No matrix effects on the ELISA sensitivity were observed. The results (by means of ELISA) were comparable with those given by both a standard cultivation method performed according to CSN ISO 10272 and commercially available Singlepath Campylobacter GLISA-Rapid Test.

Enhanced chemiluminescent sandwich enzyme immunoassay for hen egg lysozyme.

A sensitive chemiluminescent sandwich-type enzyme immunoassay for hen egg lysozyme was developed. The assay was performed on polystyrene microtitre plates using immobilized specific polyclonal rabbit antibody against lysozyme, a peroxidase conjugate and the H2O2/luminol-enhanced chemiluminescence detection reagent. The chemiluminescent signal was detected using either a microplate luminometer, or photographic film in a camera luminometer. The detection limit for lysozyme was 0.3 ng/mL, and this was three times lower than that obtained using a colorimetric method with H2O2 and o-phenylendiamine as substrates. Recovery of the assay was 97-112% and the relative standard deviation ranged from 3.6% to 10.3%. The immunoassay overcame interference from the food sample matrix when lysozyme, used as a bacteriostatic agent, was measured.