Differentiation of Bacillus cereus Isolates from Milk and Milk Products with Biochemical, Immunological, AP-PCR and PCR-RFLP Methods

Karmen Godič Torkar¹* and Sonja Smole Možina<sup>2

1</sup>Institute of Dairying, Zootechnical Dept., Biotechnical Faculty, University of Ljubljana, Groblje 3, SI-1230 Domžale, Slovenia
²Dept. of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia

Article history:
Received February 11, 2000
Accepted April 25, 2000

Key words:
Bacillus cereus, milk, genotyping, biotyping, enterotoxigenity

Summary:
Physiological features including lecithinase and haemolytic activity, API biotyping and immunodetection of diarrhoeal enterotoxin were compared with AP-PCR genotyping and PCR-RFLP analysis of hblA and cerAB gene fragments for differentiation of 82 Bacillus cereus isolates from raw milk and milk products. The amplification of the cerAB gene with selected primers was successful in 78 out of 82 (95 %) of lecithinase positive strains. An hblA amplification product was obtained in 66 (80.5 %) strains. By using BCET-RPLA immunoassay kit the same result was achieved in 97.5 % of isolates tested. A comparative analysis of phenotypic expression and PCR amplification of genes coding for lecithinase and diarrhoeal enterotoxin synthesis in Bacillus cereus milk isolates reveal a high level of correlation and confirm the usefulness of rapid molecular detection and/or identification methods for toxinogenic Bacillus cereus strains from milk and milk products. Furthermore, restriction analysis of toxin coding gene sequences in Bacillus cereus strains revealed a very high heterogeneity and thus the usefulness of PCR-RFLP typing of strains on the basis of these sequences. No correlation was found between the clustering of strains on the basis of API biotyping and AP-PCR genotyping. However, high discrimination indexes were calculated for both typing methods, so they could be successfully used for differentiation of Bacillus cereus isolated from milk and milk products. We found PCR-RFLP analysis of toxin coding gene sequences as a preferable method for detection, identification and/or typing and therefore tracing the repositories and distribution routes of toxinogenic Bacillus cereus strains at raw milk supply and manufacturing process in a dairy plant.

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