Relation between radiation resistance and salt sensitivity of spores of five strains of Clostridium botulinum types A, B, and E.

The NaCl tolerance of different strains of Clostridium botulinum varies over a wide range, and the patterns of NaCl inhibition differ distinctly and characteristically from strain to strain. The more radiation-resistant strains, such as 33A, 62A, and 7272A, are more resistant to NaCl, whereas the more radiation-sensitive strains, such as 51B and 1304E, are more sensitive to NaCl. This rule appears to hold irrespective of whether the spores were unirradiated controls or whether they were radiation damaged prior to exposure to NaCl in the recovery media. The data seem to indicate that radiation doses in the shoulder portion of the radiation survival curves did not noticeably sensitive the spores to NaCl, whereas radiation doses in the exponential-decline portion of the survival curve invariably produced a distinct sensitization. Thus, strains 33A and 62A were not sensitized to NaCl by 0.3 to 0.4 Mrad, i.e., in the shoulder portion of the survival curve. Radiation-sensitive strain 51B, which shows no distinct shoulder in its survival curve, was sensitized to NaCl by 0.1 Mrad, the lowest radiation dose employed in this study. These observations seem to suggest a possible relationship between deoxyribonucleic acid repair capacity and salt tolerance.     

Recombination and insertion events involving the botulinum neurotoxin complex genes in Clostridium botulinum types A, B, E and F and Clostridium butyricum type E strains

Background  

Clostridium botulinum is a taxonomic designation for at least four diverse species that are defined by the expression of one (monovalent) or two (bivalent) of seven different C. botulinum neurotoxins (BoNTs, A-G). The four species have been classified as C. botulinum Groups I-IV. The presence of bont genes in strains representing the different Groups is probably the result of horizontal transfer of the toxin operons between the species.     

Variability in spore germination response by strains of proteolytic Clostridium botulinum types A,B and F

AIMS: The objective of the study was to evaluate the variability of germination response of 10 strains of proteolytic Clostridium botulinum. METHODS AND RESULTS: An automated turbidometric method was used to follow the fall in optical density. Spores of proteolytic Cl. botulinum germinated in response to l-alanine alone, with rate and extent of germination increased by addition of l-lactate or bicarbonate ions. Other hydrophobic amino acids also triggered germination of spores of proteolytic Cl. botulinum but not AGFK and inosine, germinants for Bacillus subtilis or B. cereus. CONCLUSIONS: Unlike spores of nonproteolytic Cl. botulinum, all proteolytic Cl. botulinum germinate in hydrophobic l-amino acids without l-lactate. However, a great variability of response to germinant is evidenced between the species. SIGNIFICANCE AND IMPACT OF THE STUDY: The selection of a model strain to study germination of Cl. botulinum spores should consider the variability in sensitivity to germinants shown in this work. In particular, the sequenced strain ATCC 3502 may not be the most appropriate model for germination studies.     

Chemical manipulation of the heat resistance of Clostridium botulinum spores.

The chemical forms of Clostridium botulinum 62A and 213B were prepared, and their heat resistances were determined in several heating media, including some low-acid foods. The heat resistance of C. botulinum spores can be manipulated up and down by changing chemical forms between the resistant calcium form and the sensitive hydrogen form. The resistant chemical form of type B spores has about three times the classical PO4 resistance at 235 F (112.8 C). As measured in peas and asparagus, both types of C. botulinum spores came directly from the culture at only a small fraction of the potential heat resistance shown by the same spores when chemically converted to the resistant form. The resistant spore form of both types (62A and 213B), when present in a low-acid food, can be sensitized to heating at the normal pH of the food.     

Comparative dose-survival curves of representative Clostridium botulinum type F spores with type A and B spores.

Radiation survival data of proteolytic (Walls 8G-F) and non-proteolytic (Eklund 83F) type F spores of Clostridium botulinum were compared with dose-response data of radiation-resistant type A (33A) and B (40B) spores. Strain Eklund 83F was as resistant as strain 33A, whereas strain Walls 8G-F was the most sensitive of the four strains tested. The methods suggested for computing both an initial shoulder and a D value for the dose-survival curves yielded results comparable to the graphic techniques used to obtain these two parameters.     

Comparative dose-survival curves of representative Clostridium botulinum type F spores with type A and B spores.

Radiation survival data of proteolytic (Walls 8G-F) and non-proteolytic (Eklund 83F) type F spores of Clostridium botulinum were compared with dose-response data of radiation-resistant type A (33A) and B (40B) spores. Strain Eklund 83F was as resistant as strain 33A, whereas strain Walls 8G-F was the most sensitive of the four strains tested. The methods suggested for computing both an initial shoulder and a D value for the dose-survival curves yielded results comparable to the graphic techniques used to obtain these two parameters.     

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.     

Fluorescent Studies in the Genus Clostridium. II. A rapid method for differentiating Clostridium botulinum types A, B and F, types C and D, and type E

S ummary : Fluorescent labelled specific antisera have been used to distinguish between types A, B and F, types C and D, and type E of Clostridium botulinum .     

Detection of Clostridium botulinum types A, B, E and F in foods by PCR and DNA probe

A PCR procedure was developed for the detection of Clostridium botulinum in foods. PCR products were detected in agarose gels and by Southern hybridization. The sensitivity of PCR was tested in broth cultures and in canned asparagus, dry cured ham and honey. The sensitivity of the method in broth was high (2·1–8·1 cfu ml⁻¹) for types A and B, but rather low (10⁴ cfu ml⁻¹) for types E and F. However, after enrichment at 37°C for 18 h, it was possible to detect Cl. botulinum types A, B, E and F in food samples at initial levels of about 1 cfu 10 g⁻¹ of food. This PCR detection protocol provides a sensitive and relatively rapid technique for the routine detection of Cl. botulinum in foods.     

Influence of transition metals added during sporulation on heat resistance of Clostridium botulinum 113B spores.

Sporulation of Clostridium botulinum 113B in a complex medium supplemented with certain transition metals (Fe, Mn, Cu, or Zn) at 0.01 to 1.0 mM gave spores that were increased two to sevenfold in their contents of the added metals. The contents of calcium, magnesium, and other metals in the purified spores were relatively unchanged. Inclusion of sodium citrate (3 g/liter) in the medium enhanced metal accumulation and gave consistency in the transition metal contents of independent spore crops. In citrate-supplemented media, C. botulinum formed spores with very high contents of Zn (approximately 1% of the dry weight). Spores containing an increased content of Fe (0.1 to 0.2%) were more susceptible to thermal killing than were native spores or spores containing increased Zn or Mn. The spores formed with added Fe or Cu also appeared less able to repair heat-induced injuries than the spores with added Mn or Zn. Fe-increased spores appeared to germinate and outgrow at a higher frequency than did native and Mn-increased spores. This study shows that C. botulinum spores can be sensitized to increased thermal destruction by incorporation of Fe in the spores.     

Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum

Aims:  To develop real-time PCR assays for tracking and tracing clostridia responsible for human botulism.
Methods and Results:  Real-time PCR assays based on the detection of the genes ntnh encoding the nontoxin-nonhaemagglutinin (NTNH) proteins or the most homologous regions of the botulinum neurotoxin ( bont ) genes have been developed together with four real-time PCR assays, each being specific of the genes bont/A , bont/B , bont/E , bont/F and enables a toxin type-specific identification. The specificity of the assays was demonstrated using a panel of botulinum toxin producing clostridia (29 strains), nonbotulinum toxin producing clostridia (21 strains) and various other bacterial strains. The toxin type-specific assays had a sensitivity of 100 fg–1000 fg of total DNA in the PCR tube (25–250 genome equivalents) which correspond to 10³ to 10⁴ cells ml⁻¹. After a 48 h enrichment in anaerobic conditions, these PCR assays allowed the detection of Clostridium botulinum type A in a naturally contaminated sample of 'foie gras' suspected in a C. botulinum outbreak.
Conclusion:  These PCR tests are specific and reliable for detection of heterogeneous BoNT producing clostridia responsible for human botulism.
Significance and Impact of the Study:  Adoption of these PCR assays is a step forward a reliable and rapid detection of these clostridia in food samples.     

Germinability and heat resistance of spores of Clostridium difficile strains

Out of 111 Clostridium difficile strains, 108 produced spores in numbers of more than 10(5)/ml and the remaining three did not produce any spores in brain heart infusion medium. The germination frequency in the medium without lysozyme varied widely from strain to strain, ranging from less than 10(-8) to 10(0), and in 77 of the 108 strains the germination frequency was 10(-5) or less. The spores, when treated with sodium thioglycollate and then inoculated into the medium containing lysozyme, germinated in all of the 108 strains at a frequency of 10(-0.5) or more. The spores of two strains germinated at a frequency of more than 10(-0.5) in all methods. Spores of C. difficile strains were fairly highly heat-resistant; D100C values ranged from 2.5 to 33.5 min.     

Development of improved defined media for Clostridium botulinum serotypes A, B, and E.

The minimal nutritional growth requirements were determined for strains Okra B and Iwanai E, which are representatives of groups I and II, respectively, of Clostridium botulinum. These type B and E strains differed considerably in their nutrient requirements. The organic growth factors required in high concentrations by the Okra B strain (group I) were arginine and phenylalanine. Low concentrations (less than or equal to 0.1 g/liter) of eight amino acids (methionine, leucine, valine, isoleucine, glycine, histidine, tryptophan, and tyrosine) and of five vitamins (pyridoxamine, p-aminobenzoic acid, biotin, nicotinic acid, and thiamine) were also essential for biosynthesis. The 10 required amino acids could be replaced by intact protein of known composition by virtue of the bacterium's ability to synthesize proteases. Glucose or other carbohydrates were not essential for Okra B, although they did stimulate growth. Quantitatively, the most essential nutrients for Okra B were arginine and phenylalanine. In contrast, the nonproteolytic strain, Iwanai E (group II), did not require either arginine or phenylalanine. It required glucose or another carbohydrate energy source for growth and did not utilize arginine or intact protein as a substitute source of energy. Iwanai E utilized ammonia as a nitrogen source, although growth was stimulated significantly by organic nitrogenous nutrients, especially glutamate and asparagine. Iwanai E also required biosynthesis levels of seven amino acids (histidine, isoleucine, leucine, tryptophan, tyrosine, valine, and serine), adenine, and six vitamins (biotin, thiamine, pyridoxamine, folic acid, choline, and nicotinamide). Calcium pantothenate also stimulated growth. On the basis of the nutritional requirements, chemically defined minimal media have been constructed for C. botulinum serotypes A, B, E, and F (proteolytic).(ABSTRACT TRUNCATED AT 250 WORDS)