Differentiation of Bacillus cereus isolates from milk and milk products with biochemical, immunological, AP-PCR and PCR-RFLP methods

Physiological features including lecithinase and haemolytic activity, API b iotyping and immunodetection of diarrhoeal enterotoxin were compared with A P-PCR genotyping and PCR-RFLP analysis of hblA and cerAB gene fragments for differentiation of 82 Bacillus cereus isolates from raw milk and milk prod ucts. The amplification of the cerAB gene with selected primers was success ful in 78 out of 82 (95 %) of lecithinase positive strains. An hblA amplifi cation product was obtained in 66 (80.5 %) strains. By using BCET-RPLA immu noassay kit the same result was achieved in 97.5 % of isolates tested. A co mparative analysis of phenotypic expression and PCR amplification of: genes coding for lecithinase and diarrhoeal enterotoxin synthesis in Bacillus ce reus milk isolates reveal a high level of correlation and confirm the usefu lness of rapid molecular detection and/or identification methods for toxino genic Bacillus cereus strains from milk and milk products. Furthermore, res triction analysis of toxin coding gene sequences in Bacillus cereus strains revealed a very high heterogeneity and thus the usefulness of PCR-RFLP typ ing of strains on the basis of these sequences. No correlation was found be tween the clustering of strains on the basis of API biotyping and AP-PCR ge notyping. However, high discrimination indexes were calculated for both typ ing methods, so they could be successfully used for differentiation of Baci llus cereus isolated from milk and milk products. We found PCR-RFLP analysi s of toxin coding gene sequences as a preferable method for detection, iden tification and/or typing and therefore tracing the repositories and distrib ution routes of toxinogenic Bacillus cereus strains at raw milk supply and manufacturing process in a dairy plant.