Heterogeneity in the cellular protein profiles ofClostridium botulinum types A and B

Summary Profiles of cellular proteins from 11 type A and 9 type B strains ofClostridium botulinum were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cellular protein profiles exhibited considerable heterogeneity among strains of the same biotype, as well as among strains of different biotypes. This study also demonstrated the reliability and usefulness of this rapid and inexpensive procedure to determine the variation which may occur amongC. botulinum and related species.References to brand or firm names do not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.     

Effects of irradiation on growth and toxigenicity of Clostridium botulinum types A and B inoculated onto chicken skins

This study was conducted to examine the effects of 0.3-Mrad irradiation on growth and toxigenicity of Clostridium botulinum types A and B on chicken skins. Irradiation followed by aerobic or anaerobic incubation at 30 degrees C extended the shelf life of skin samples and delayed growth and toxin production by C. botulinum. During 2 weeks of incubation at 10 degrees C, the irradiated and nonirradiated C. botulinum spores failed to grow or produce toxin.     

Effects of irradiation on growth and toxigenicity of Clostridium botulinum types A and B inoculated onto chicken skins.

This study was conducted to examine the effects of 0.3-Mrad irradiation on growth and toxigenicity of Clostridium botulinum types A and B on chicken skins. Irradiation followed by aerobic or anaerobic incubation at 30 degrees C extended the shelf life of skin samples and delayed growth and toxin production by C. botulinum. During 2 weeks of incubation at 10 degrees C, the irradiated and nonirradiated C. botulinum spores failed to grow or produce toxin.     

Factors affecting initiation of growth ofClostridium botulinum

Summary When solutions containing glucose and phosphate were autoclaved, substances formed which altered the growth response ofClostridium botulinum 62A. Some of these substances were stimulatory and decreased the time required for the appearance of turbidity in liquid cultures. Other substances were inhibitory and delayed the appearance of growth. The inhibitory substances were removed from solution by adsorption on activated charcoal. The concentration of inhibitory substances increased with time of storage of media and autoclaved phosphate-glucose supplement. Deceased September 23, 1959.     

The stability of toxigenicity in Clostridium botulinum types C and D.

Several type C and D strains of Clostridium botulinum, which had been converted to the toxgenic state by phages, were serially transferred through cooked meat medium with and without specific anti-phage serum. Most of the converted strains lost their toxigenicity even during transfer without antiserum, and the non-toxigenic variants that appeared were resistant to lysis and conversion by the original phage. However, in some combinations of phage and host bacteria toxigenicity was stable after ten transfers, though it showed a transient decrease, and the non-toxigenic variants that arose remained sensitive to lysis and conversion. When converted strains were transferred in medium containing anti-phage serum, toxigenicity was lost more rapidly than in the absence of serum and the non-toxigenic variants that appeared remained sensitive to lysis and conversion by the parent phage. Filtrates of the supernatants of culture fluids of strains transferred without anti-phase serum converted non-toxigenic strains to toxigenicity at varying rates.     

Hydrogen gas accelerates thermal inactivation ofClostridium botulinum 113B spores

Summary Heat inactivation ofClostridium botulinum spores was accelerated in atmospheres containing hydrogen gas. Hydrogen gas also moderately increased the thermal destruction ofBacillus spores. Hydrogen gas may react with components inC. botulinum spores such as transition metals producing hydrogen ions or hydrides, which destroy essential spore components. Thermal processing in modified atmospheres may have applications in food processing and in sterilization of medical supplies.     

A simple procedure for identification ofClostridium botulinum colonies

For direct identification of toxigenic colonies ofClostridium botulinum type E, suspected colonies are uniformly suspended in a phosphate buffer containing 0.5% (w/v) gelatin and 0.05% (w/v) Tween 20. After centrifuging, the supernatant is tested for botulinal toxin by an enzyme-linked immunosorbent assay (ELISA). The assay is specific for this type as it did not react with culture filtrates of otherClostridium species, including non-toxigenic E-like organisms.     

Stability of toxigenicity in proteolytic Clostridium botulinum type B upon serial passage

Proteolytic Clostridium botulinum type B strains were investigated for stability of toxigenicity and bont/b gene upon serial passage. Strains with bont/b gene located on their plasmids showed loss or decrease of toxigenicity during serial passage. Some strains lost the bont/b gene-encoding plasmid. The stability of the plasmids varied between strains.     

Behaviour of proteolytic Clostridium botulinum types A and B near the lower temperature limits of growth

Summary The behaviour of spores of Clostridium botulinum type A and proteolytic C. botulinum type B has been studied in cooked meat medium at 10°C, 12°C, 15°C, and 20°C, using mixed cultures (9 groups of in total 41 strains) and pure cultures (41 strains).At 10°C a decrease of 1–1.5 log cycles for type B and of 2–4 log cycles for type A Clostridia was observed. Neither growth nor toxin formation could be demonstrated.At 12°C spores of some strains developed and formed toxin with 3–4 weeks, whereas other strains did not develop within 7 weeks.At 15°C growth and toxin formation could be observed within 1 week, whereas at 20°C toxin was formed mostly within 2 or 3 days. Incubation at 10°C prior to incubation at 20°C seemed to have some effect on the lag time.     

Rapid identification ofClostridium botulinum and botulinal toxin in food

Samples of green beans and mushrooms were inoculated with a toxigenic strain ofClostridium botulinum type A and incubated anaerobically at 37 °C. At various time intervals, the seeded food samples were tested for the presence of botulinal toxin andC. botulinum by an agar plating method and an enzyme-linked immunosorbent assay.C. botulinum type A that appeared as lipase-positive colonies on selective agar plates, and its elaborated toxin, were identified in all seeded food samples within 1 to 2 d. This procedure can be adapted for rapid screening of suspected food samples. This study was presented in part at the96th General Meeting of the American Society for Microbiology, New Orleans, Louisiana, May 19–23, 1996 (abstract no. P71). Part of the requirements for the MSc degree received by A. Rodriguez.     

Inhibition of Clostridium botulinum types A and B hemagglutinins by sugars.

Chromatographically isolated hemagglutinins of Clostridium botulinum types A and B are serologically related but not identical. Of the sugars (5, 6, 12, 18 carbons, some derivatives, L and D forms) tested, only D-galactose and some of tis derivatives were inhibitors of these hemagglutinins. O-Nitrophenyl-beta-D-galactopyranoside and isopropyl-beta-D-thiogalactoside were the most potent inhibitors. The two hemagglutinins were bound tightly by p-aminophenyl-beta-D-thiogalactopyranoside coupled to CH-Sepharose 4B. The ligands to which these hemagglutinins bind were determined as the sugars which inhibited the hemagglutinating activity.     

Reductive methylation of lysine residues of botulinum neurotoxin types A and B

Summary Reductive methylation of botulinum neurotoxin (NT) serotypes A and B at various ratios of protein to reagent modified up to 75° 10 of the lysine residues. Amino acid analysis of the modified proteins (HCl hydrolysed) confirmed selective modifications of lysine. The derivative N,N-dimethyl lysine was more abundant than monomethyl lysine; trimethyl lysine was not detected. Distribution of modified lysine residues among the heavy and light chains (M_r 100000 and 50000, respectively) of the dichain type A NT (M_r 150000) was approximately proportional to the lysine contents of the two subunit chains of the NT. Toxicity (mouse lethality) and serological reactivity (polyclonal antibody) of serotype A NT were not (or insignificantly) damaged following methylation of up to 72 lysine residues. Modification of 3 additional residues caused precipitous loss in toxicity. Toxicity of serotype B NT, unlike type A, appeared more sensitive to lysine modification. The large number of lysine residues that can be methylated without damaging toxicity of type A NT can be exploited to a) radiolabel the dichain protein exclusively in one chain keeping the other chain unlabelled, b) restrict the number of tryptic cleavage sites of the NT, and c) tag the protein with various markers or reactive ligands.     

Occurrence ofClostridium botulinum in the soil of the vicinity of Rome

After the isolation of two strains ofClostridium butyricum that produced type E botulinal toxin from the feces of two babies in Rome affected by infant botulism (P. Aureli, L. Fenicia, M. Gianfranceschi, L. McCroskey, and C. Hatheway, J Infect Dis 154:207–211, 1986), extensive research was carried out to detect botulinal toxin-producing clostridia in soil samples from the vicinity of Rome. A total of 520 specimens from cultivated and pasture lands, obtained from 52 collection stations, were examined.Enrichment cultures were tested for botulinal toxin, and the isolated neurotoxigenic micro-organisms were characterized both enzymatically and gas chromatographically.Clostridium botulinum type A and B proteolytic, the former being the most common, were found in 7 (1.3%) out of 520 samples from 5 (9.6%) of the 52 locations in close association with pasture lands. Possible relationships between the presence of botulinal organisms and the properties of the soil in which they were found have also been investigated.     

Partial amino acid sequences of botulinum neurotoxins types B and E

Clostridium botulinum type E neurotoxin, a single-chain protein of Mr 147,000, was purified and subjected to amino acid sequencing. The same was done for single-chain botulinum type B neurotoxin (Mr 152,000), and for the heavy and light chains (Mr 104,000 and 51,000 respectively) derived from type B by limited trypsin digestion. Twelve to eighteen residues were identified and the following conclusions were drawn: The light chain of the nicked (dichain) type B is derived from the N-terminal one-third of the single-chain (unnicked) parent neurotoxin; sequence homologies are present between single-chain types B and E and the light chain of the nicked type A [J. J. Schmidt, V. Sathyamoorthy, and B. R. DasGupta (1984) Biochem. Biophys. Res. Commun. 119, 900-904]; the N-terminal regions of the heavy chains of types A and B have some structural similarity; and activation of type B neurotoxin cannot involve removal of amino acids or peptides from the N terminus.     

Rapid and simple detection of Clostridium botulinum types A and B by loop-mediated isothermal amplification

Aims:  To develop a convenient and rapid detection method for toxigenic Clostridium botulinum types A and B using a loop-mediated isothermal amplification (LAMP) method.
Methods and results:  The LAMP primer sets for the type A or B botulinum neurotoxin gene, BoNT / A or BoNT / B , were designed. To determine the specificity of the LAMP assay, a total of 14 C. botulinum strains and 17 other Clostridium strains were tested. The assays for the BoNT/A or BoNT/B gene detected only type A or B C. botulinum strains, respectively, but not other types of C. botulinum or strains of other Clostridium species. Using purified chromosomal DNA, the sensitivity of LAMP for the BoNT/A or BoNT/B gene was 1 pg or 10 pg of DNA per assay, respectively. The assay times needed to detect 1 ng of DNA were only 23 and 22 min for types A and B, respectively. In food samples, the detection limit per reaction was one cell for type A and 10 cells for type B.
Conclusions:  The LAMP is a sensitive, specific and rapid detection method for C. botulinum types A and B.
Significance and Impact of the Study:  The LAMP assay would be useful for detection of C. botulinum in environmental samples.