Inhibition of Clostridium botulinum by Strains of Clostridium perfringens Isolated from Soil

Thirty-one soil samples were examined for the presence of organisms capable of inhibiting growth and toxin production of strains of Clostridium botulinum type A. Such organisms were found in eight samples of soil. Inhibiting strains of C. perfringens were found in five samples, of C. sporogenes in three and of Bacillus cereus in three. Three of the C. perfringens strains produced an inhibitor effective on all 11 strains of C. botulinum type A against which they were tested, seven of eight proteolytic type B strains, one nonproteolytic type B strain, five of nine type E strains and all seven type F strains, whether proteolytic or nonproteolytic. They did not inhibit any of 26 type C strains, 6 type D strains, 4 type E strains, or 24 C. sporogenes strains. In mixed culture, an inhibitor strain of C. perfringens repressed growth and toxin production by a C. botulinum type A strain even though it was outnumbered by the latter about 40 times. It also repressed growth and toxin production of C. botulinum in mixed culture of soils in which this latter organism naturally occurred when cooked meat medium but not when trypticase medium was used.     

Clostridium botulinum type C toxin

Summary The purification and crystallization of type C botulinum toxin along with its physical characteristics are described. The shape of Clostridium botulinum type C toxin molecule is globular like a pressed ball with a 7.4 nm diameter and a 4.3 urn thickness. The molecular volume is approximately 185 nl and the molecular weight is 141 000. The toxin molecule is composed of two parts, which are separable under appropriate conditions. These parts have some differences in the electrophoretic properties, amino acid distribution, immunological, and functional characteristics. The toxin molecule can be reconstituted by association of S-S bond between the two chains. The expression of the toxicity requires that the fragments of the polypeptide chain carrying the necessary information be functionally organized for the proper development of the specific tertiary structure for active conformation.     

Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin

The culture medium of certain strains of Clostridium botulinum type C contains two separable ADP-ribosyltransferases. Besides the ADP-ribosylation of actin due to botulinum C2 I toxin, a second microbial enzyme causes the mono-ADP-ribosylation of a eukaryotic protein with a molecular mass of about 20 kDa found in platelets, neuroblastoma X glioma hybrid cells, S49 lymphoma cells, chick embryo fibroblasts and sperm. The eukaryotic substrate is inactivated by heating and trypsin treatment. In contrast, the novel ADP-ribosyltransferase, which can be separated by DEAE-Sephadex chromatography, is largely resistant in the short term to trypsin digestion.     

Inhibition of type A and type B (proteolytic) Clostridium botulinum by sorbic acid

The effect of sorbic acid in the pH range 4.9 to 7.0 on the probability P of growth of a single vegetative bacterium of proteolytic strains of Clostridium botulinum has been determined by comparison of the most probable number count of the bacteria in media at pH 4.9 to 7.0 containing a series of concentrations of potassium sorbate and in a nutrient medium at pH 6.8 to 7.0. The media were maintained under strictly anaerobic conditions at a redox potential equivalent to lower than -350 mV at pH 7. In medium adjusted to the required pH with HCl, P for strain ZK3 (type A) at pH 5.1 or 5.5 after 2 days at 30 degrees C was similar to that at pH 6.8 to 7.0 but was slightly lower at pH 4.9. Potassium sorbate inhibited growth, the inhibition being a function of the concentration of undissociated sorbic acid. A calculated undissociated sorbic acid concentration of 156 mg/liter delayed growth of strain ZK3 (type A) but did not result in a significant decrease in P after an incubation time of 14 days. Higher concentrations of undissociated sorbic acid caused longer delays before maximum most probable number counts developed, and a calculated undissociated sorbic acid concentration of 282 mg/liter decreased log P for strain ZK3 after an incubation time of 14 days by a factor of 5.5 to 7.5. Four additional type A strains and five type B strains were inhibited to an extent comparable to inhibition of strain ZK3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of type A and type B (proteolytic) Clostridium botulinum by sorbic acid.

The effect of sorbic acid in the pH range 4.9 to 7.0 on the probability P of growth of a single vegetative bacterium of proteolytic strains of Clostridium botulinum has been determined by comparison of the most probable number count of the bacteria in media at pH 4.9 to 7.0 containing a series of concentrations of potassium sorbate and in a nutrient medium at pH 6.8 to 7.0. The media were maintained under strictly anaerobic conditions at a redox potential equivalent to lower than -350 mV at pH 7. In medium adjusted to the required pH with HCl, P for strain ZK3 (type A) at pH 5.1 or 5.5 after 2 days at 30 degrees C was similar to that at pH 6.8 to 7.0 but was slightly lower at pH 4.9. Potassium sorbate inhibited growth, the inhibition being a function of the concentration of undissociated sorbic acid. A calculated undissociated sorbic acid concentration of 156 mg/liter delayed growth of strain ZK3 (type A) but did not result in a significant decrease in P after an incubation time of 14 days. Higher concentrations of undissociated sorbic acid caused longer delays before maximum most probable number counts developed, and a calculated undissociated sorbic acid concentration of 282 mg/liter decreased log P for strain ZK3 after an incubation time of 14 days by a factor of 5.5 to 7.5. Four additional type A strains and five type B strains were inhibited to an extent comparable to inhibition of strain ZK3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Observations on toxin and hemagglutinin produced by Clostridium botulinum type C.

In the culture fluid of a hemagglutinin-positive strain of Clostridium botulinum type C, two toxins of different molecular size, hemagglutinin positive and negative, were separated by sucrose density gradient centrifugation.     

Observations on toxin and hemagglutinin produced by Clostridium botulinum type C.

In the culture fluid of a hemagglutinin-positive strain of Clostridium botulinum type C, two toxins of different molecular size, hemagglutinin positive and negative, were separated by sucrose density gradient centrifugation.     

Characteristics of spore formation in Clostridium botulinum type C]

Electron microscope study of C1. botulinum, tyep C, showed that microbial cells were surrounded with a five-layer wall. Structures characteristic of sporulating cells and phage particles whose intracellular development led to reduction and lysis of the cytoplasm were revelaed in the area of the cytoplasm. Mature spores were encountered rarely. Formation of prespore, cortex was observed, but the elements of the spore membrane were chaotically dispersed in the whole cytoplasm. Such disturbances could be connected with the presence of phage in the culture.     

Molecular composition of progenitor toxin produced by Clostridium botulinum type C strain 6813

The molecular composition of the purified progenitor toxin produced by a Clostridium botulinum type C strain 6813 (C-6813) was analyzed. The strain produced two types of progenitor toxins (M and L). Purified L toxin is formed by conjugation of the M toxin (composed of a neurotoxin and a non-toxic nonhemagglutinin) with additional hemagglutinin (HA) components. The dual cleavage sites at loop region of the dichain structure neurotoxin were identified between Arg444-Ser445 and Lys449-Thr450 by the analyses of C-terminal of the light chain and N-terminal of the heavy chain. Analysis of partial amino acid sequences of fragments generated by limited proteolysis of the neurotoxin has shown to that the neurotoxin protein produced by C-6813 was a hybrid molecule composed of type C and D neurotoxins as previously reported. HA components consist of a mixture of several subcomponents with molecular weights of 70-, 55-, 33-, 26~21- and 17-kDa. The N-terminal amino acid sequences of 70-, 55-, and 26~21-kDa proteins indicated that the 70-kDa protein was intact HA-70 gene product, and other 55- and 26~21-kDa proteins were derived from the 70-kDa protein by modification with proteolysis after translation of HA-70 gene. Furthermore, several amino acid differences were exhibited in the amino acid sequence as compared with the deduced sequence from the nucleotide sequence of the HA-70 gene which was common among type C (strains C-St and C-468) and D progenitor toxins (strains D-CB16 and D-1873).     

Clostridium botulinum type C in healthy swine in Japan.

Healthy cattle and swine bred in a district of Japan were examined for the presence of Clostridium botulinum in their liver. Liver specimens were cultivated in chopped meat-glucose medium and the cultures were examined for botulinum toxin. In cattle, none of the cultures of 100 liver specimens yielded the toxin. In swine, however, C1 or C2 toxin was demonstrated in 8 of 100 liver specimens from 36 farms. One of the five farms where the carrier-state swine were present was surveyed for about 2 years to determine whether the carrier-state was transient or resident. C. botulinum type C was found in swine livers and feces, and environmental specimens at extremely high rates during the surveillances, with 76% of specimens yielding botulinum toxin following the culture. These data suggest that it is not uncommon for healthy swine to carry C. botulinum type C in the liver and that there is a close relationship between C. botulinum carrier-state in swine and the presence of this organism in their raising environments. In 20 cattle and 20 swine suffering from parturient paresis of unknown etiology no evidence for involvement of C. botulinum type C was obtained.     

Comparison of antigenicity of toxins produced by Clostridium botulinum type C and D strains

C1 neurotoxin of Clostridium botulinum strains C-Stockholm (C-ST), C beta-Yoichi, C-468, CD6F, and C-CB19 and type D toxin of strains D-1873 and D-CB16 were purified by gel filtration, ion exchange, and affinity chromatographies. The purified toxins had di-chain structure made of heavy and light chains. The toxins of C beta-Yoichi, C-468, CD6F, and C-CB19 reacted with anti-C-ST heavy chain and anti-C-ST light chain in immunodiffusion tests and enzyme-linked immunosorbent assay, whereas D-CB16 toxin reacted with anti-D-1873 heavy chain and anti-D-1873 light chain. However, C-6813 toxin reacted with anti-D-1873 heavy chain and anti-C-ST light chain but not with anti-C-ST heavy chain or anti-D-1873 light chain immunoglobulin G. These results indicate common antigens in the heavy chains of C-6813 and D-1873 toxins and in the light chains of C-6813 and C-ST toxins. Further, they provide evidence for heterogeneity within type C1 toxin subunits.