Analysis of the Botulinum Neurotoxin Type F Gene Clusters in Proteolytic and Nonproteolytic Clostridium botulinum and Clostridium barati

Comparison of genes encoding type F botulinum neurotoxin progenitor complex in strains of proteolytic Clostridium botulinum strain Langeland, nonproteolytic Clostridium botulinum strain 202F, and Clostridium barati strain ATCC 43256 reveals an identical organization of genes encoding a protein of molecular mass of approx. 47 kDa (P-47), nontoxic-nonhemagglutinin (NTNH) and botulinum toxin (BoNT). Although homology between the protein components of the complexes encoded by these different species all producing botulinum neurotoxin type F is considerable (approx. 69–88% identity), exceptionally high homology is observed between the C-termini of the P-47s (approx. 96% identity) and the NTNHs (approx. 94% identity) encoded by Clostridium botulinum type F strain Langeland and Clostridium botulinum type A strain Kyoto. Such a region of extremely high sequence identity is strongly indicative of recombination in these strains synthesizing botulinum neurotoxins of different antigenic types. Received: 13 April 1998 / Accepted: 9 May 1998     

Antigenic Relationships Among the Proteolytic and Nonproteolytic Strains of Clostridium botulinum

Relationships of the somatic antigens among Clostridium botulinum strains have been investigated by tube agglutination and agglutinin absorption tests. Results revealed a relationship by which strains of C. botulinum are grouped by their proteolytic capacity rather than by the type of specific toxin produced. Thus, C. botulinum type E and its nontoxigenic variants, which are nonproteolytic, share common somatic antigens with the nonproteolytic strains of types B and F. Absorption of antiserum of a strain of any one type with antigen of any of the others removes the antibody to all three types. In the same manner, C. botulinum type A shares somatic antigens with the proteolytic strains of types B and F, and absorption of any one antiserum with an antigen of either of the other two types removes the antibody to all three types. Partial cross-agglutination of C. sporogenes, C. tetani, and C. histolyticum with the somatic antisera of the proteolytic group was also observed.     

Molecular Characterization of the Clusters of Genes Encoding the Botulinum Neurotoxin Complex in Clostridium botulinum (Clostridium argentinense) Type G and Nonproteolytic Clostridium botulinum Type B

The cluster of genes encoding components of the progenitor botulinum neurotoxin complex has been mapped and cloned in Clostridium botulinum type G strain ATCC 27322. Determination of the nucleotide sequence of the region has revealed open reading frames encoding nontoxic components of the complex, upstream of the gene encoding BoNT/G (botG). The arrangement of these genes differs from that in strains of other antigenic toxin types. Immediately upstream of botG lies a gene encoding a protein of 1198 amino acids, which shows homology with the nontoxic-nonhemagglutinin (NTNH) component of the progenitor complex. Further upstream there are genes encoding proteins with homology to hemagglutinin components (HA-17, HA-70) and a putative positive regulator of gene expression (P-21). Sequence comparison has shown that BoNT/G has highest homology with BoNT/B. The sequence of the BoNT-cluster of genes in non-proteolytic C. botulinum type B strain Eklund 17B has been extended to include the complete NTNH and HA-17, and partial HA-70 gene sequences. Comparison of NTNH/G with other NTNHs reveals that it shows highest homology with NTNH/B consistent with the genealogical affinity shown between BoNT/G and BoNT/B genes. Received: 28 January 1997 / Accepted: 24 March 1997     

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.     

Proteolytic mutants obtained from Clostridium botulinum type E.

Proteolytic mutants were isolated from toxigenic strains of Clostridium botulinum type E after several transfers. When these cultures were plated on blood agar, almost all of the colonies obtained were proteolytic, and there were fewer toxigenic colonies than nontoxigenic colonies. The proteolytic mutants and nonproteolytic original strains were different in their biological properties.     

Development and Application of a New Method for Specific and Sensitive Enumeration of Spores of Nonproteolytic Clostridium botulinum Types B,E, and F in Foods and Food Materials

The highly potent botulinum neurotoxins are responsible for botulism, a severe neuroparalytic disease. Strains of nonproteolytic Clostridium botulinum form neurotoxins of types B, E, and F and are the main hazard associated with minimally heated refrigerated foods. Recent developments in quantitative microbiological risk assessment (QMRA) and food safety objectives (FSO) have made food safety more quantitative and include, as inputs, probability distributions for the contamination of food materials and foods. A new method that combines a selective enrichment culture with multiplex PCR has been developed and validated to enumerate specifically the spores of nonproteolytic C. botulinum. Key features of this new method include the following: (i) it is specific for nonproteolytic C. botulinum (and does not detect proteolytic C. botulinum), (ii) the detection limit has been determined for each food tested (using carefully structured control samples), and (iii) a low detection limit has been achieved by the use of selective enrichment and large test samples. The method has been used to enumerate spores of nonproteolytic C. botulinum in 637 samples of 19 food materials included in pasta-based minimally heated refrigerated foods and in 7 complete foods. A total of 32 samples (5 egg pastas and 27 scallops) contained spores of nonproteolytic C. botulinum type B or F. The majority of samples contained <100 spores/kg, but one sample of scallops contained 444 spores/kg. Nonproteolytic C. botulinum type E was not detected. Importantly, for QMRA and FSO, the construction of probability distributions will enable the frequency of packs containing particular levels of contamination to be determined.Food-borne botulism is a severe and deadly intoxication caused by the consumption of food containing as little as 30 to 100 ng of preformed botulinum neurotoxin (45). More than 2,500 cases of botulism were reported in Europe in 1999 and 2000, with the majority of cases in the east of the continent (44). Currently, 25 to 50 food-borne botulism cases are diagnosed annually in the United States (27). There are seven distinct botulinum neurotoxins (types A to G) and a number of subtypes (6, 26, 45). In view of the potency of the botulinum neurotoxin and the severity of botulism, four phylogenetically distinct bacteria are grouped together as the Clostridium botulinum species, solely on the basis of their ability to form botulinum neurotoxin. The divergence between these four distinct bacteria is strong enough to merit their classification as distinct species and in some cases is significantly greater than that between bacteria belonging to different genera, e.g., Bacillus subtilis and Staphylococcus aureus (7). Two of these bacteria (proteolytic C. botulinum and nonproteolytic C. botulinum) are responsible for the majority of cases of food-borne botulism. Strains of proteolytic C. botulinum produce neurotoxins of type A, B, or F, form spores of high heat resistance, and have a minimum growth temperature of approximately 12°C (39). Strains of nonproteolytic C. botulinum produce neurotoxins of type B, E, or F, form spores of moderate heat resistance, and are able to grow and form toxin at 3°C (18, 48) and are recognized as the major hazard associated with minimally heated refrigerated foods (4, 37, 43, 44, 48). These new foods meet consumer demand for high-quality, convenient foods that are low in preservatives, and sales are presently increasing by about 10% per annum in many countries (3, 47).Quantitative microbiological risk assessment (QMRA) is now established as an important microbiology food safety tool (42). Process risk models have been used to assess the safety of specific foods with respect to nonproteolytic C. botulinum and the food-borne botulism hazard (e.g., 2, 41). These process risk models benefit from high-quality information, including that on the incidence of spores of nonproteolytic C. botulinum spores in food materials. The implementation of food safety objectives (FSO) also benefits from the availability of high-quality information on the microbial contamination of foods and food materials (24). This information is most effective in the form of probability distributions rather than as average spore concentrations or other statistics.The difficulty with enumerating nonproteolytic C. botulinum in foods is that there is no effective selective culture medium available. Surveys of the extent of contamination of foods and food materials have used a nonselective enrichment followed by either testing for neurotoxin using a mouse test or enzyme-linked immunosorbent assay (ELISA) or testing for the presence of neurotoxin genes using a PCR test (3, 10, 13, 35, 38, 39). This approach, however, is not optimized for nonproteolytic C. botulinum or proteolytic C. botulinum (therefore potentially failing to recover all spores of either organism) and may also not distinguish nonproteolytic C. botulinum from proteolytic C. botulinum. Heating at 80°C for 10 min followed by incubation at 35°C (54) may be reasonably selective for proteolytic C. botulinum, but there is no similar approach for nonproteolytic C. botulinum, although incubation at 28°C (54) may offer an element of selection. It is necessary, therefore, to develop a method to enumerate spores of nonproteolytic C. botulinum in food materials that is robust and optimized, as well as sensitive and specific for this particular pathogen (and does not also detect proteolytic C. botulinum). When enumerating bacteria in foods, it is essential to demonstrate the efficiency of the method by verifying that small concentrations (in the present study, spores of nonproteolytic C. botulinum) can be detected following addition to test samples.This paper describes the development, validation, and application of a new method to enumerate spores of nonproteolytic C. botulinum in foods and in food materials. This method has been designed to generate data for the construction of probability distributions that can be used in QMRA and FSO settings. Most of the effort has been dedicated to the development and evaluation of the enrichment procedure rather than the PCR test, as the PCR test has received much attention from others (e.g., 3, 10, 16, 36, 38). A low-temperature selective-enrichment procedure is described that has been optimized specifically for nonproteolytic C. botulinum over proteolytic C. botulinum and other bacteria. In order to detect low concentrations of spores, large quantities (200 g) of food materials and foods have been tested. Specific detection of neurotoxin genes is achieved by the use of an established multiplex PCR (36), with an internal amplification control now included (25). By the use of a set of control samples inoculated with defined concentrations of spores of nonproteolytic C. botulinum, the detection limit has been estimated for each food material and food tested. The method has been used in an extensive survey of raw materials intended for use in pasta ready meals, as well as the final meals themselves. The implications for risk assessment and risk management of chilled foods are discussed.     

Thermal Inactivation of Nonproteolytic Clostridium botulinum Type E Spores in Model Fish Media and in Vacuum-Packaged Hot-Smoked Fish Products

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93°C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93°C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90°C, respectively. The z values were 10.4°C in trout medium and 10.1°C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10³. An inoculated-pack study revealed that a time-temperature combination of 42 min at 85°C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10⁶ spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8°C. In Finland it is recommended that hot-smoked fish be stored at or below 3°C, further extending product safety. However, heating whitefish for 44 min at 85°C with 10% RH resulted in growth and toxicity in 5 weeks at 8°C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processes rainbow trout were observed.     

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.     

Clostridium botulinum Type F in the Soil of Argentina

One strain of Clostridium botulinum belonging to type F has been isolated from a soil sample taken in Buenos Aires Province, Argentina. Comparative studies carried out with strain Langeland of C. botulinum type F have shown a few differences between the two strains.     

Quantification of Nonproteolytic Clostridium botulinum Spore Loads in Food Materials

We have produced data and developed analysis to build representations for the concentration of spores of nonproteolytic Clostridium botulinum in materials that are used during the manufacture of minimally processed chilled foods in the United Kingdom. Food materials are categorized into homogenous groups which include meat, fish, shellfish, cereals, fresh plant material, dairy liquid, dairy nonliquid, mushroom and fungi, and dried herbs and spices. Models are constructed in a Bayesian framework and represent a combination of information from a literature survey of spore loads from positive-control experiments that establish a detection limit and from dedicated microbiological tests for real food materials. The detection of nonproteolytic C. botulinum employed an optimized protocol that combines selective enrichment culture with multiplex PCR, and the majority of tests on food materials were negative. Posterior beliefs about spore loads center on a concentration range of 1 to 10 spores kg⁻¹. Posterior beliefs for larger spore loads were most significant for dried herbs and spices and were most sensitive to the detailed results from control experiments. Probability distributions for spore loads are represented in a convenient form that can be used for numerical analysis and risk assessments.     

Purification of Clostridium botulinum Type F Progenitor Toxin

Clostridium botulinum type F progenitor toxin was purified to a homogeneous state as judged by gel filtration on Sephadex G-200, ultracentrifugation, and disc electrophoresis. The sedimentation constant, corrected to water at 20 C, of type F progenitor toxin was determined to be 10.3 and the molecular weight to be 235,000 by ultracentrifugation at pH 6.0. The purified toxin contained a toxicity of 1.2 x 10(8) 50% lethal doses/mg of N. In agar gel double diffusion, it formed two precipitin lines at pH 6.0. The progenitor toxin of type F differs from that of type A in that it contains no hemagglutinin and from that of type E in that it is not activable.     

Purification and Properties of Clostridium botulinum Type F Toxin

Clostridium botulinum type F toxin of proteolytic Langeland strain was purified. Toxin in whole cultures was precipitated with (NH₄)₂SO₄. Extract of the precipitate was successively chromatographed on diethylaminoethyl-cellulose at pH 6.0, O-(carboxymethyl) cellulose at pH 4.9, Sephadex G-200 at pH 8.1, quaternary aminoethyl-Sephadex at pH 4.9, and finally diethylaminoethyl-cellulose at pH 8.1. The procedure recovered 14% of the toxin assayed in the starting culture. The toxin was homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, double gel diffusion serology, and isoelectric focusing. Purified toxin had a molecular weight of 150,000 by gel filtration and 155,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specific toxicity was 9.6 × 10⁶ mean lethal doses per absorbancy (278 nm) unit. Sub-units of 105,000 and 56,000 molecular weight are found when purified toxin is treated with a disulfide reducing agent and electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Reciprocal cross neutralizations were demonstrated when purified type F and E toxins were reacted with antitoxins which were obtained with immunizing toxoids prepared with purified toxins.     

Efficient DNA Fingerprinting of Clostridium botulinum Types A, B, E, and F by Amplified Fragment Length Polymorphism Analysis

Amplified fragment length polymorphism (AFLP) analysis was applied to characterize 33 group I and 37 group II Clostridium botulinum strains. Four restriction enzyme and 30 primer combinations were screened to tailor the AFLP technique for optimal characterization of C. botulinum. The enzyme combination HindIII and HpyCH4IV, with primers having one selective nucleotide apiece (Hind-C and Hpy-A), was selected. AFLP clearly differentiated between C. botulinum groups I and II; group-specific clusters showed <10% similarity between proteolytic and nonproteolytic C. botulinum strains. In addition, group-specific fragments were detected in both groups. All strains studied were typeable by AFLP, and a total of 42 AFLP types were identified. Extensive diversity was observed among strains of C. botulinum type E, whereas group I had lower genetic biodiversity. These results indicate that AFLP is a fast, highly discriminating, and reproducible DNA fingerprinting method with excellent typeability, which, in addition to its suitability for typing at strain level, can be used for C. botulinum group identification.     

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 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.     

Clostridium botulinum Type F: Isolation from Venison Jerky

A Clostridium botulinum type F was isolated from the venison jerky responsible for the only type F botulism outbreak reported in the United States. The isolate differed from the prototype Langeland type F strain in being nonproteolytic.