Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridium species by PCR: evidence of unexpressed type B toxin genes in type A toxigenic organisms

Abstract

We studied the eflectiveness of the PCR in detecting the type A, B, and E botulism neurotoxin genes in 209 strains of Clostridium botulinum and 29 strains of other Clostridium spp. All 79 strains that produced type A toxin, 77 strains that produced type B toxin, and 51 organisms that produced type E toxin (46 C. botulinum and 5 C. butyricum) were PCR positive in reactions with primers targeting sequences specific for their respective toxin genes. The PCR for type A toxin was positive for one type B toxin-producing strain that produced a small amount of type A toxin in addition to a large amount of type B toxin. Surprisingly, the type B toxin gene was detected in addition to the type A toxin gene in 43 type A toxin-producing strains, only 1 of which could be shown by bioassay to produce biologically active type B toxin in culture. The type B gene was also detected in two strains of C. subterminale, which were determined to be nontoxigenic by bioassay. While the PCR was sensitive and specific in detecting the neurotoxin genes, the discovery of unexpressed toxin genes indicates that PCR results may not be adequate for establishing type B neurotoxigenicity.

The detection of a deletion in the type B neurotoxin gene of Clostridium botulinum A(B) strains by a two‐step PCR

“…Strain CL135, isolated from a fatal case of food-borne botulism in Italy, also corresponded to type Ab (Franciosa et al 1997). The remaining 42 listed strains, although previously thought to be type A strains, were previously shown by PCR to carry genes for both the BoNT/A and BoNT/B (Franciosa et al 1994). The unexpressed BoNT/B gene of strain 667 had been sequenced in a subsequent study (Hutson et al 1996).…”

“…Step inally thought to be a conventional type A strain, was recently shown to produce both BoNT/A and BoNT/B (type Ab) (Franciosa et al 1994). Strain CL135, isolated from a fatal case of food-borne botulism in Italy, also corresponded to type Ab (Franciosa et al 1997).…”

“…The first-step PCR was performed with primers B1a and B2d, using reagents and amplification conditions described elsewhere (Franciosa et al 1994). A 5 ml aliquot of the centrifuged and washed Cl.…”

“…In a recent study, 42 of 79 strains of Cl. botulinum type A isolated in the United States were shown to carry in their genomes two neurotoxin genes, encoding BoNT/A and BoNT/B, even when they apparently did not express the latter (Franciosa et al 1994). Analysis of the unexpressed type B toxin gene sequence of one of the 42 Cl.…”

“…In the present study, a two-step PCR was used to screen all 42 previously isolated strains of Cl. botulinum type A(B) (Franciosa et al 1994) for the presence of the six nucleotides in the BoNT/B gene sequence. The purpose was to investigate the frequency of occurrence of this deletion and its relationship to the failure of the B toxin encoding gene to express toxin.…”