Isolation of Clostridium botulinum type G from Swiss soil specimens by using sequential steps in an identification scheme

After Clostridium botulinum type G organisms and toxin were identified in necropsy specimens in cases of unexplained death in adults and infants (O. Sonnabend, W. Sonnabend, R. Heinzle, T. Sigrist, R Dirnhofer, and U. Krech, J. Infect. Dis. 143:22-27, 1981), extensive research to detect C. botulinum type G in soil samples from Switzerland was done. A total of 41 specimens from virgin soil and from cultivated land were examined for the presence of C. botulinum type G and other toxin types. Because of the lack of the lipase marker in type G, the detection of C. botulinum type G was based on the demonstration of type G organisms in enrichment cultures by a type G-specific enzyme-linked immunosorbent assay to detect both the type G toxin and antigen; enrichment cultures in which type G toxin or antigen was identified by enzyme-linked immunosorbent assay were then tested by a type G-specific gel immunodiffusion agar procedure. This method not only isolated strains of type G but also strains of Clostridium subterminale, a nontoxigenic variant of C. botulinum type G. As a consequence of the observed cross-reactions caused by strains of C. subterminale within this test system, all isolates of type G had to be definitively confirmed by mouse bioassay. The sequential steps of these methods seem to be very useful for detecting C. botulinum type G organisms. C. botulinum type G strains were isolated in five soil samples from different locations in close association with cultivated land.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of Clostridium botulinum type G toxin by enzyme-linked immunosorbent assay.

Clostridium botulinum type G toxin was detected and quantified readily with the enzyme-linked immunosorbent assay. With the double-sandwich technique and alkaline phosphatase as the enzyme indicator, C. botulinum toxin type G was detected in quantities equaling those required for one mouse intraperitoneal median lethal dose. The time required for the procedure was approximately 6.5 h, but this requirement could have been reduced to 5.5 h or less with the use of precoated plates stored at -70 degrees C. Cross-reactions did not occur with culture extracts of C. sporogenes of C. botulinum types B, C, D, E, and F. Acidic preparations of C. botulinum type A exhibited nonspecific reactivity. Likewise, 50% of the C. subterminale isolates tested were cross-reactive in the assay. These latter isolates express similar metabolic and physiological characteristics with C. botulinum type G.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80 degrees C/5 min + 70 degrees C/2 h, before storage at 15 degrees, 20 degrees or 27 degrees C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5–4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80°C/5 min + 70°C/2 h, before storage at 15°, 20° or 27°C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), aiid 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.     

Examination of Prepared Foods in Plastic Packages for Clostridium botulinum

The incidence of Clostridium botulinum organisms was determined in a variety of plastic-packaged "vulnerable" foods (food requiring little or no heating prior to consumption). A total of 113 foods were examined by use of an enrichment recovery procedure followed by toxin testing in animals. Results of the survey indicate that the incidence of C. botulinum organisms in these vulnerable foods is extremely low. The ability of inoculated food products to support growth and toxigenesis of C. botulinum type E was then tested. The 64 packaged foods were inoculated with type E spores and incubated anaerobically at 30 C for 11 days. A slurry of each food was prepared, smears for fluorescent-antibody testing were made, and animal tests were performed for toxin. If the animal tests were negative, enrichment cultures were prepared from the slurry and incubated at 30 C. On direct examination of the slurries for toxin, only samples of turkey roll and soybean cake supported growth and toxigenesis by C. botulinum type E. However, the enrichment culture method was able to induce growth and toxin production in 60 of the remaining 62 samples.     

Real-time PCR detection of the nontoxic nonhemagglutinin gene as a rapid screening method for bacterial isolates harboring the botulinum neurotoxin (A-G) gene complex

Botulinum neurotoxin (BoNT) producing clostridia contain genes encoding a specific neurotoxin serotype (A-G) and nontoxic associated proteins that form the toxin complex. The nontoxic nonhemagglutinin (NTNH) is a conserved component of the toxin complex in all seven toxin types. A real-time PCR assay that utilizes a locked nucleic acid hydrolysis probe to target the NTNH gene was developed to detect bacterial strains harboring the botulinum neurotoxin gene cluster. The specificity of the assay for Clostridium botulinum types A-G, Clostridium butyricum type E and Clostridium baratii type F was demonstrated using a panel of 73 BoNT producing clostridia representing all seven toxin serotypes. In addition, exclusivity of the assay was demonstrated using non-botulinum toxin producing clostridia (7 strains) and various enteric bacterial strains (n=27). Using purified DNA, the assay had a sensitivity of 4-95 genome equivalents. C. botulinum type A was detected directly in spiked stool samples at 10(2)-10(3) CFU/ml. Stool spiked with 1 CFU/ml was detected when the sample was inoculated into enrichment broth and incubated for 24 h. These results indicate that the NTNH real-time PCR assay can be used to screen enrichment cultures of primary specimens at earlier time points (24 h) than by toxin detection of unknown culture supernatants (up to 5 days).     

Prevalence of Clostridium botulinum type E and coexistence of C. botulinum nonproteolytic type B in the river soil of Japan.

Soil samples from 98 sites in the whole systems of four rivers in Japan were examined for the presence of Clostridium botulinum. Type E organism was prevalently shown throughout the whole river systems including upper part; detection rates of type E toxin in soil culture ranged from 33 to 82%. This type was also detected in soil of adjacent mountainous district. Type B and C toxins were detected at 7% and 9% of the sites examined, respectively. C. botulinum type E and nonproteolytic type B strains were isolated from enrichment cultures of soil samples. These results suggest that the terrestrial origin of type E organism would be considered as one of the reasons for the high incidence of this organism in the sea areas, and prove that C. botulinum nonproteolytic type B exists in the soil of Japan.     

Monoclonal antibody to type F Clostridium botulinum toxin

Hybridomas synthesizing monoclonal antibodies (MAbs) against type F Clostridium botulinum toxin were developed. MAb from one stable hybridoma, hybridoma 223, consisted of kappa light chains and an immunoglobulin G subclass 2a heavy chain. This MAb was used in a double-sandwich enzyme-linked immunosorbent assay to detect type F toxin in foods, culture fluids, and purified toxin preparations. The sensitivity of the double-sandwich enzyme-linked immunosorbent assay was approximately 10 mouse lethal doses of toxin per ml of toxic fluid.     

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.     

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.     

Colony immunoblot assay of botulinal toxin.

Botulinal neurotoxin in and around colonies of Clostridium botulinum types A, B, and E and of toxigenic Clostridium butyricum was detected by an enzyme-linked immunoassay procedure whereby the toxin was transferred from the agar medium to a nitrocellulose support and the immobilized toxin was probed with type-specific antibodies. The method identified the toxin types of the colonies grown from a mixed inoculum of C. botulinum serotypes. The specificity of the antitoxins for type A and B toxins was improved by adsorption of the antitoxins with the antigens of heterologous type cultures.     

Plasmids in Clostridium botulinum and related Clostridium species.

Toxigenic Clostridium botulinum and nontoxigenic C. sporogenes, C. subterminale, and C. botulinum-like organisms from a variety of sources were screened for plasmids. Of the 68 toxigenic C. botulinum isolates, 56% carried one or more plasmids, ranging in mass from 2.1 to 81 megadaltons. Within individual groups (based on the type of neurotoxin produced), many strains showed identical plasmid banding patterns on agarose gels. Of the 15 nontoxigenic strains tested, 40% also carried one or more plasmids ranging from 1.7 to 25.0 megadaltons, with both unique and common banding patterns represented. A total of 67 plasmids from both toxigenic and nontoxigenic strains were detected. At this time, no phenotypic functions have been assessed for these plasmids, and they must therefore be considered cryptic. A variety of lysing and extraction techniques were necessary to detect plasmids in the different C. botulinum groups.     

Transfer of neurotoxigenicity from Clostridium butyricum to a nontoxigenic Clostridium botulinum type E-like strain.

Two Clostridium butyricum strains from infant botulism cases produce a toxic molecule very similar to C. botulinum type E neurotoxin. Chromosomal, plasmid, and bacteriophage DNAs of toxigenic and nontoxigenic strains of C. butyricum and C. botulinum type E were probed with (i) a synthesized 30-mer oligonucleotide encoding part of the L chain of type E botulinum toxin and (ii) the DNA of phages lysogenizing these cultures. The toxin gene probe hybridized to the chromosomal DNA of toxigenic strains but not to their plasmid DNA. All toxigenic and most nontoxigenic strains tested were lysogenized by a prophage on the chromosome. Prophages of toxigenic strains, irrespective of species, had related or identical DNAs which differed from the DNAs of prophages in nontoxigenic strains. The prophage of toxigenic strains was adjacent or close to the toxin gene on the chromosome. Phage DNAs purified from toxigenic strains did not hybridize with the toxin gene probe but could act as the template of the polymerase chain reaction to amplify the toxin gene. The toxin gene was not transferred between C. botulinum and C. butyricum (either direction) when different pairs of a possible gene donor and a recipient strain were grown as mixed cultures. Nontoxigenic C. butyricum or C. botulinum type E-like strains did not become toxigenic when grown in broth containing the phage induced from a toxigenic strain of the other species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Real-time PCR detection of the nontoxic nonhemagglutinin gene as a rapid screening method for bacterial isolates harboring the botulinum neurotoxin (A–G) gene complex

Botulinum neurotoxin (BoNT) producing clostridia contain genes encoding a specific neurotoxin serotype (A–G) and nontoxic associated proteins that form the toxin complex. The nontoxic nonhemagglutinin (NTNH) is a conserved component of the toxin complex in all seven toxin types. A real-time PCR assay that utilizes a locked nucleic acid hydrolysis probe to target the NTNH gene was developed to detect bacterial strains harboring the botulinum neurotoxin gene cluster. The specificity of the assay for Clostridium botulinum types A–G, Clostridium butyricum type E and Clostridium baratii type F was demonstrated using a panel of 73 BoNT producing clostridia representing all seven toxin serotypes. In addition, exclusivity of the assay was demonstrated using non-botulinum toxin producing clostridia (7 strains) and various enteric bacterial strains (n = 27). Using purified DNA, the assay had a sensitivity of 4–95 genome equivalents. C. botulinum type A was detected directly in spiked stool samples at 10²–10³ CFU/ml. Stool spiked with 1 CFU/ml was detected when the sample was inoculated into enrichment broth and incubated for 24 h. These results indicate that the NTNH real-time PCR assay can be used to screen enrichment cultures of primary specimens at earlier time points (24 h) than by toxin detection of unknown culture supernatants (up to 5 days).     

Production of toxin by Clostridium botulinum type A strains cured by plasmids

Twelve strains of Clostridium botulinum type A and seven strains of Clostridium sporogenes were screened for plasmids by agarose gel electrophoresis of cleared lysates of cells from 5 ml of mid-log-phase culture. Nine type A strains had one or more plasmids of 4.3, 6.8, or 36 megadaltons (MDa); several strains showed a large plasmid of 61 MDa, but it was not consistently recovered. Four C. sporogenes strains had one or more plasmids of 4.3, 5.6 or 36 MDa. Isolates obtained from cultures of plasmid-containing C. botulinum type A strains grown in ionic detergent broth and from spontaneously arising variants were screened both for toxin production and for plasmid content. Toxigenicity of C. botulinum could not be correlated with the presence of any one plasmid.     

Production of toxin by Clostridium botulinum type A strains cured by plasmids.

Twelve strains of Clostridium botulinum type A and seven strains of Clostridium sporogenes were screened for plasmids by agarose gel electrophoresis of cleared lysates of cells from 5 ml of mid-log-phase culture. Nine type A strains had one or more plasmids of 4.3, 6.8, or 36 megadaltons (MDa); several strains showed a large plasmid of 61 MDa, but it was not consistently recovered. Four C. sporogenes strains had one or more plasmids of 4.3, 5.6 or 36 MDa. Isolates obtained from cultures of plasmid-containing C. botulinum type A strains grown in ionic detergent broth and from spontaneously arising variants were screened both for toxin production and for plasmid content. Toxigenicity of C. botulinum could not be correlated with the presence of any one plasmid.     

Detection of the genes encoding botulinum neurotoxin types A to E by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used as the basis for the development of highly sensitive and specific diagnostic tests for organisms harboring botulinum neurotoxin type A through E genes. Synthetic DNA primers were selected from nucleic acid sequence data for Clostridium botulinum neurotoxins. Individual components of the PCR for each serotype (serotypes A through E) were adjusted for optimal amplification of the target fragment. Each PCR assay was tested with organisms expressing each of the botulinum neurotoxin types (types A through G), Clostridium tetani, genetically related nontoxigenic organisms, and unrelated strains. Each assay was specific for the intended target. The PCR reliably identified multiple strains having the same neurotoxin type. The sensitivity of the test was determined with different concentrations of genomic DNA from strains producing each toxin type. As little as 10 fg of DNA (approximately three clostridial cells) was detected. C. botulinum neurotoxin types A, B, and E, which are most commonly associated with human botulism, could be amplified from crude DNA extracts, from vegetative cells, and from spore preparations. This suggests that there is great potential for the PCR in the identification and detection of botulinum neurotoxin-producing strains.     

Clostridium botulinum in the soil of Paraguay

Seventeen soil samples of Paraguay were examined for the presence of Clostridium botulinum. Botulinum type A, C1 and F toxins were detected in soil cultures. Type E toxin was not detected in any of soil cultures including those from river and lake shores.