Differentiation between types and strains of Clostridium botulinum by riboprinting

The ability of automated ribotyping to differentiate between major types an d individual strains of Clostridium botulinum was tested using the Qualicon Riboprinter Microbial Characterization System. Pure spores of C. botulinum type A, proteolytic type B, nonproteolytic type B, and type E strains were inoculated onto modified anaerobic egg yolk agar and incubated 24 h at 35 degrees C. Plates were rinsed with buffer (2 mM Tris + 20 mM EDTA) to remov e vegetative cells that were heated for 10 min at 80 degrees C, treated wit h a lysing agent, and ribotyped in the Qualicon Riboprinter utilizing the e nzyme EcoRI. Riboprint patterns were obtained for 30 strains of the four ma jor types of C. botulinum most commonly involved in human foodborne botulis m. Proteolytic strains yielded the best and most consistent results. Fiftee n ribogroups were identified among the 31 strains tested. Interestingly, in two cases, a single ribogroup contained patterns from isolates belonging t o evolutionarily distinct Clostridium lineages. This degree of differentiat ion between strains of C. botulinum may be useful in hazard analysis and id entification, hazard analysis and critical control point monitoring and val idation, environmental monitoring, and in inoculation studies.