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)     

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.     

Differentiation of Clostridium botulinum Types A, B, and E by Pyrolysis-Gas-Liquid Chromatography

Vegetative cells and spores of 10 strains of Clostridium botulinum representing types A, B, and E were grown in Trypticase-peptone-sucrose-yeast extract (TPSY) medium. Five type E strains were also grown in Multipeptone-sucrose-Nutramino acids (MSN) medium. Lyophilized samples were subjected to pyrolysis-gas-liquid chromatography (PGLC) analysis, and the resulting pyrograms were examined for variations in elution patterns between spores and vegetative cells of types A, B, and E grown in the TPSY medium and spores and vegetative cells of type E grown in the TPSY medium and spores and vegetative cells of type E grown in TPSY and MSN media. Growth and toxin production of all 10 strains of C. botulinum were investigated by using a modified dialysis sac culture technique. The dialysate supernatant fluid (DSF) obtained after centrifugation of the 5-day-old cultures from the dialysate was also subjected to PGLC analysis. Control samples consisting of (i) noninoculated DSF, (ii) noninoculated DSF plus partially purified toxin, and (iii) 1.0 mg of partially purified toxin were also analyzed by PGLC. Differences between pyrograms of cultures were suitable for positive identification at the type level but not at the strain level. Pyrograms permitting differentiation were also obtained between spores and vegetative cells as well as between the same cultures grown in different media. The dialysis sac technique was useful in detecting growth but not toxin production of C. botulinum.     

Analysis of Clostridium botulinum Toxigenic Types A, B, and E for Fatty and Carbohydrate Content

Lyophilized, 48-hr log-phase vegetative cells were extracted with chloroform-methanol (2:1, v/v) and ethanol-ether (3:1, v/v) and then saponified with methanolic KOH. Gas-liquid chromatography of the methyl esters of extractable fatty acids revealed distinctive "pattern profiles" of Clostridium botulinum toxigenic types "A," "B," and "E." C. perfringens type "A" and Escherichia coli strain "B" were also studied in a similar manner and were found to give pattern profiles which were distinct even from those obtained for the C. botulinum microorganisms. Amino sugar content of the five microorganisms was determined by using a Beckman amino acid analyzer. The molar ratio of glucosamine to that of galactosamine was found to be of further assistance in distinguishing the individual microorganisms.     

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.     

Development of enrichment semi-nested PCR for Clostridium botulinum types A, B, E, and F and its application to Korean environmental samples

An enrichment semi-nested PCR procedure was developed for detection of Clostridium botulinum types A, B, E, and F. It was applied to sediment samples to examine the prevalence of C. botulinum in the Korean environment. The first pair of primers for the semi-nested PCR was designed using a region shared by the types A, B, E, and F neurotoxin gene sequences, and the second round employed four nested primers complementary to the BoNT/A, /B, /E, and /F encoding genes for simultaneous detection of the four serotypes. Positive results were obtained from the PCR analysis of five of 44 sediments (11%) collected from Yeong-am Lake in Korea; all were identified as deriving from type B neurotoxin (bontb) genes. Two of the C. botulinum type B organisms were isolated, and their bontb genes sequenced. The deduced amino acid sequences of BoNT/B showed 99.5 and 99.8% identity with the amino acid sequence of accession no. AB084152. Our data suggest that semi-nested PCR is a useful tool for detecting C. botulinum in sediments, and renders it practicable to conduct environmental surveys.     

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 .     

Growth of Clostridium tertium and Clostridium septicum in chemically defined media.

Defined media for the growth of Clostridium tertium and Clostridium septicum are described. The requirements for growth of these two species are compared with each other and with those of Clostridium perfringens.     

Unique substrate recognition by botulinum neurotoxins serotypes A and E

Botulinum neurotoxins (BoNTs) are zinc proteases that cleave SNARE proteins to elicit flaccid paralysis by inhibiting the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. There are seven serotypes of BoNT, termed A-G. BoNT serotype A and serotype E cleave SNAP25 at residues 197-198 and 180-181, respectively. Unlike other zinc proteases, the BoNTs recognize extended regions of SNAP25 for cleavage. The basis for this extended substrate recognition and specificity is unclear. Saturation mutagenesis and deletion mapping identified residues 156-202 of SNAP25 as the optimal cleavage domain for BoNT/A, whereas the optimal cleavage domain for BoNT/E was shorter, comprising residues 167-186 of SNAP25. Two sub-sites were resolved within each optimal cleavage domain, which included a recognition or active site (AS) domain that contained the site of cleavage and a binding (B) domain, which contributed to substrate affinity. Within the AS domains, the P1', P3, and P5 sites of SNAP25 contributed to scissile bond cleavage by LC/A, whereas the P1' and P2 sites of SNAP25 contributed to scissile bond cleavage by LC/E. These studies provide insight into the development of strategies for small molecule inhibitors of the BoNTs.     

Multiplex PCR assay for detection and identification of Clostridium botulinum types A, B, E, and F in food and fecal material.

Botulism is diagnosed by detecting botulinum neurotoxin and Clostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 10(2) cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10(-2) spore/g for types A, B, and F to 10(-1) spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.     

Polymerase chain reaction for detection of Clostridium botulinum types A, B and E in food, soil and infant faeces

The application of the polymerase chain reaction (PCR) for detection of Clostridium botulinum types A, B and E in foods, environmental and clinical samples was evaluated and compared to the mouse bioassay. Samples inoculated with 10, 100 and 1000 spores of Cl. botulinum types A and B included pasteurized milk, UHT milk, infant formula, infant faeces, meat juice, canned tuna, mushrooms, blood sausage and soil. Clostridium botulinum type E spores were inoculated into fish eggs, canned tuna, picked herring, raw fish and soil at similar levels. Spores were added to 2.5 g of each sample with the exception of soil which was inoculated in 10 g samples. The presence of Cl. botulinum in sample enrichments was determined by both PCR and the bioassay. An overall correlation of 95.6% was observed between PCR results and the mouse bioassay. Of the total of 114 samples tested there was disparity between the mouse bioassay and the PCR in three samples of soil inoculated with 100 type A or E spores and 10 type B spores per 10 g, respectively, and two samples of infant faeces inoculated with 10 type A or B spores per 2.5 g. All of these samples gave negative animal results and positive PCR results.     

Simplified purification method for Clostridium botulinum type E toxin.

Clostridium botulinum type E toxin was purified in three chromatography steps. Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer. When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid. When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form. The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex. At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein. These values are significantly higher than those previously reported.     

New equine antitoxins to botulinum neurotoxins serotypes A and B

Hyperimmune monovalent antitoxins to botulinum neurotoxin serotypes A and B have been produced by immunizing horses with newly developed formalin toxoids. After primary immunization, horses developed acceptable prophylactic antibody titers (1-5 IU/mL). Three horses received additional toxoid booster injections to induce hyperimmune antibody titers with antitoxin-A and antitoxin-B titers reaching peaks of approximately 2000 IU/mL and 150-625 IU/mL, respectively. Titers were quantified throughout the process by antigen-capture ELISA and by in-vivo neutralization. ELISA titers and neutralization titers correlated (R2 ～0.62-0.92), however, unique correlations between in-vitro and in-vivo titers were observed for each horse. Monovalent antitoxin pools were made by combining plasma that had been collected twice via plasmaphoresis several months after primary immunization. Neutralizing units were established for each pool relative to the current US and WHO reference standards. Titers were determined at the L(+)/10 and L(+)/40 toxin dose for Toxin types A and B, respectively, and U.S. and international units were assigned to each monovalent antitoxin. Avidity of the new Anti-A pool was equivalent to the WHO Anti-A reference at the L(+), L(+)/10 and L(+)/30 dose. Each monovalent plasma pool failed to cross-neutralize other botulinum neurotoxin serotypes indicating a high degree of specificity of each antitoxin for the toxin serotype used during immunization.