Genotypic Characterization of Clostridium botulinum Strains Producing Type A Neurotoxin Complexes by Random Amplified Polymorphic DNA and PCR-RFLP Analysis

In order to genotypically characterize Clostridium botulinum type A strains, we examined 23 strains from various origins (food-borne botulism, infant botulism, honey, honey-related materials and soil), by random amplified polymorphic DNA (RAPD) analysis and characterized the type A botulinum neurotoxin complex genes by PCR and PCR-RFLP analysis. By RAPD analysis, C.botulinum strains associated with infant botulism in Japan and a strain 804-1H, isolated from Brazilian honey, were clearly differentiated from the other strains examined. PCR and PCR-RFLP analysis were performed for the neurotoxin complex genes (type A neurotoxin (BoNT/A), type B neurotoxin (BoNT/B), nontoxic-nonhemagglutinin (NTNH), hemaggulutinin (HA), and p47 protein). All of 23 strains tested gave positive amplification for BoNT/A and NTNH genes, and the PCR-RFLP analysis differentiated six strains associated with infant botulism in Japan, a strain from Brazilian honey and a strain AF84 (type 2) from other strains (type 1). For HA gene, the type 1 strains gave amplification while the type 2 strains gave no amplification. For BoNT/B gene, the type 1 strains (a strain Renkon and three American infant botulism strains) gave amplification (silent B strains). For p47 gene, the type 2 strains and the silent B strains were positive. The combinations of the PCR of HA, p47, and BoNT/B genes and the PCR-RFLP of BoNT/A, NTNH genes demonstrated three genotypes (A1, A2, and A3) of neurotoxin complex genes. Thus, these PCR-based methods are useful for simple and rapid differentiation of the BoNT-producing C.botulinum strains.