Strains Of Clostridium Botulinum Type Research Articles

Development of a technique for DNA detection and identification of toxigenic strains of Clostridium botulinum types A, B, E by the Real-Time PCR method

Botulism is dangerous toxic infection caused by a toxin produced by the bacterium Clostridium botulinum. The mortality rate from botulism can reach 70% of all cases of illness in case of untimely initiation of treatment. The pathogenesis of botulism involves the damage to the central nervous system by a toxin produced by C. botulinum. Currently there are seven recognized antigenic types of this toxin. Botulinum toxin is included into the group of biological agents and it is one of the most likely agents to be used in a biological attack. Since botulinum neurotoxin is a complex nucleoprotein complex and the traces of DNA can be detected even in purified toxin preparations, we have elaborated a technique for detecting and identifying DNA of toxigenic strains of Clostridium botulinum types A, B, E, that cause human botulism in most cases. This technique is based on the the detection of residual amounts of this DNA in botulinum toxin using multiplex real-time polymerase chain reaction (PCR) assay with fluorescent hybridization detection. The main obstacle to development of a technique for the detection and identification of DNA of toxigenic strains is the high variability of the genes responsible for the synthesis of botulinum toxin. We have established a region of the gene with the lowest homology in all strains. This requirement is met by a fragment of the bont gene that encodes a light chain of a neurotoxin and is highly conserved in the strains of C. botulinum producing one type of toxin. The paper represents the results of the definition of analytical sensitivity and specific activity of the developed method. The specificity of the determination is 100%, the analytical sensitivity – 1×10 2 mc./ml. The method can be used to analyze food, samples of clinical materials and environmental samples suspected of being contaminated with toxigenic strains of C. botulinum

Draft Genome Sequences of Five Brazilian Clostridium botulinum Group III Type D/C Strains.

ABSTRACTAnimal botulism is mainly associated with Clostridium botulinum group III-producing neurotoxin types C, C/D, D, and D/C. In this report, we present the draft genome sequences of the first five strains of Clostridium botulinum type D/C isolated in Brazil and used for vaccination purposes.

Combined High Pressure and Thermal Processing on Inactivation of Type A and Proteolytic Type B Spores of Clostridium botulinum

The aim of this study was to determine the resistance of multiple strains of Clostridium botulinum type A and proteolytic type B spores exposed to combined high pressure and thermal processing and compare their resistance with Clostridium sporogenes PA3679 and Bacillus amyloliquefaciens TMW-2.479-Fad-82 spores. The resistance of spores suspended in N-(2acetamido)-2-aminoethanesulfonic acid (ACES) buffer (0.05 M, pH 7.0) was determined at a process temperature of 105°C, with high pressures of 600, 700, and 750 MPa by using a laboratory-scale pressure test system. No surviving spores of the proteolytic B strains were detected after processing at 105°C and 700 MPa for 6 min. A . 7-log reduction of B. amyloliquefaciens spores was observed when processed for 4 min at 105°C and 700 MPa. D-values at 105°C and 700 MPa for type A strains ranged from 0.57 to 2.28 min. C. sporogenes PA3679 had a D-value of 1.48 min at 105°C and 700 MPa. Spores of the six type A strains with high D-values along with C. sporogenes PA3679 and B. amyloliquefaciens were further evaluated for their pressure resistance at pressures 600 and 750 MPa at 105°C. As the process pressure increased from 600 to 750 MPa at 105°C, D-values of some C. botulinum strains and C. sporogenes PA3679 spores decreased (i.e., 69-A, 1.91 to 1.33 min and PA3679, 2.35 to 1.29 min). Some C. botulinum type A strains were more resistant than C. sporogenes PA3679 and B. amyloliquefaciens to combined high pressure and heat, based on D-values determined at 105°C. Pulsed-field gel electrophoresis (PFGE) was also performed to establish whether strains with a similar restriction banding pattern also exhibited similar D-values. However, no correlation between the genomic background of a strain and its resistance to high pressure processing was observed, based on PFGE analysis. Spores of proteolytic type B strains of C. botulinum were less resistant to combined high pressure and heat (700 MPa and 105°C) treatment when compared with spores of type A strains.

Comparison of IgG antibody levels to Clostridium botulinum antigens between euthanased and surviving cases of chronic grass sickness

Serum from 12 horses suffering from chronic grass sickness (CGS) were assayed for IgG antibodies against botulinum neurotoxins C and D (BoNT/C and BoNT/D) and to a surface antigen extract of a neurotoxin negative strain of Clostridium botulinum type C. Collectively, the six surviving CGS cases demonstrated significantly higher initial IgG levels ( P = 0.05) against surface antigens than the six that were subsequently euthanased. The surviving animals also demonstrated higher initial IgG levels against the BoNT/C but not reaching significance ( P = 0.06). The two groups demonstrated no difference between IgG levels against BoNT/D. This study supports existing evidence of the involvement of C. botulinum type C in the aetiology of grass sickness.

A rapid chemiluminescent slot blot immunoassay for the detection and quantification of Clostridium botulinum neurotoxin type E

A simple, rapid, cost-effective in vitro slot blot immunoassay was developed for the detection and quantification of botulinum neurotoxin type E (BoNT/E) in cultures. Culture supernatants of 36 strains of clostridia, including 12 strains of Clostridium botulinum type E, 12 strains of other C. botulinum neurotoxin serotypes, and 12 strains of other clostridial species were tested. Samples containing BoNT/E were detected using affinity-purified polyclonal rabbit antisera prepared against BoNT/E with subsequent detection of secondary antibodies using chemiluminescence. All strains of C. botulinum type E tested positive, while all non C. botulinum type E strains tested negative. The sensitivity of the slot blot immunoassay for detection of BoNT/E was approximately four mouse lethal doses (MLD). The intensity of chemiluminescence was directly correlated with the concentration of BoNT/E up to 128 MLD, allowing quantification of BoNT/E between 4 and 128 MLD. The slot blot immunoassay was compared to the mouse bioassay for detection of BoNT/E using cultures derived from fish samples inoculated with C. botulinum type E, and cultures derived from naturally contaminated environmental samples. A total of 120 primary enrichment cultures derived from fish samples, of which 103 were inoculated with C. botulinum type E, and 17 were uninoculated controls, were assayed. Of the 103 primary enrichment cultures derived from inoculated fish samples, all were positive by mouse bioassay, while 94 were also positive by slot blot immunoassay, resulting in a 7.5% false-negative rate. All 17 primary enrichment cultures derived from the uninoculated fish samples were negative by both mouse bioassay and slot blot immunoassay. A total of twenty-six primary enrichment cultures derived from environmental samples were tested by mouse bioassay and slot blot immunoassay. Of 13 primary enrichment cultures positive by mouse bioassay, 12 were also positive by slot blot immunoassay, resulting in a 3.8% false-negative rate. All 13 primary enrichment cultures that tested negative by mouse bioassay also tested negative by slot blot immunoassay. The slot blot immunoassay could be used routinely as a positive screen for BoNT/E in primary enrichment cultures, and could be used as a replacement for the mouse bioassay for pure cultures.

Differentiation of the gene clusters encoding botulinum neurotoxin type A complexes in Clostridium botulinum type A, Ab, and A(B) strains.

We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources. Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis. Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains. By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident. In two C. botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C. botulinum type A(B) and Ab strains. Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.

Selection of primers for specific detection of Clostridium botulinum types B and E neurotoxin genes using PCR method

Improved oligonucleotide primers were designed to flank 370- and 307-bp fragments of the bont genes encoding botulinum neurotoxins types B and E, respectively. Primer specificity was confirmed for reference strains of Clostridium botulinum types B and E for strains representing bacterial species common in food, and for the DNA mixtures of C. botulinum types B and E in the presence of background DNA isolated from cold smoked salmon and ham. The detection limit of template DNAs of C. botulinum types B and E from the DNA mixtures increased from 1 to 0.1 ng by raising annealing temperature from 50°C to 62°C.

Rapid identification of Clostridium botulinum and botulinal toxin in food.

Samples of green beans and mushrooms were inoculated with a toxigenic strain of Clostridium botulinum type A and incubated anaerobically at 37 degrees C. At various time intervals, the seeded food samples were tested for the presence of botulinal toxin and C. 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.

Growth and Production of Toxin of Clostridium botulinum Type E in Rainbow Trout under Various Storage Conditions

Rainbow trout (Oncorhynchus mykiss) were inoculated with 3 to 4 1og10 spores per g of fish of a mixed pool of four strains of Clostridium botulinum type E (Beluga, Minnesota, G21-5, and 070). The trout were vacuum-skin packaged with either oxygen-barrier or oxygen-permeable films. Trout packaged with oxygen-permeable film were stored at 4°C for 21 days, while trout packaged with oxygen-barrier film were stored either at 4°C for 21 days or at 10°C for 15 days. Storage at 10°C was used to simulate commercial temperature abuse. Clostridium botulinum outgrowth was determined by a most probable-number (MPN) method using (tryptone peptone yeast extract glucose trypsin) anaerobic broth. Toxin production was evaluated using a mouse bioassay. Psychrotrophic and anaerobic populations increased with time regardless of packaging type. After 6 days at l0°C, botulinum toxin was detected in the packaged trout; however, the fish was noticeably spoiled before that time. No botulinum toxin was detected in trout packaged with either barrier or permeable films and stored at 4°C for 21 days, although the product was considered spoiled by day 12.

Clostridium botulinum type E outgrowth and toxin production in vacuum-skin packaged shrimp

White and brown shrimp (Penaeus spp.), harvested from the Georgia coast, were inoculated with log 3-4 spores of a mixed pool of four strains of Clostridium botulinum type E (Beluga, Minnesota, G21-5, and 070). Oxygen barrier film was used to vacuum-skin package the shrimp. Packaged shrimp was stored at either 4°C or 10°C. Storage at 10°C was used to simulate temperature abuse. Anaerobes were enumerated by Most Probable Number method using TPYGT broth incubated in anaerobic jars. Clostridium botulinum toxin production was evaluated using the mouse bioassay. After six days at 10°C, botulinum toxin was detected in the packaged shrimp, but the shrimp was noticeably spoiled at this point. No botulinum toxin was detected in the packaged shrimp stored at 4°C for 21 days, although spoilage was noted after 6 days.

Two different types of ADP-ribosyltransferase C3 from Clostridium botulinum type D lysogenized organisms.

We examined production of ADP-ribosyltransferase C3 in 11 strains of Clostridium botulinum type C and D and their nontoxigenic derivatives. Antisera to C3 proteins of type C organisms divided C3 proteins roughly into at least two groups, bearing no relation to their bacterial types. The C3 gene of type D strain South African was isolated from a toxigenic phage library, and the complete sequence of the C3 gene was determined. The C3 protein of type D strain South African had 98% homology to the C3 protein of type C strain 003-9 and 66% homology to that of type D strain 1873. These results indicate that there are two types of C3 protein in type D organisms, as there are in type C organisms.

Cloning of a DNA sequence unique to Clostridium botulinum group I by selective hybridization.

Nucleic acid sequences were isolated from a strain of Clostridium botulinum type A by a selective hybridization method known as deletion enrichment. Nontoxigenic C. sporogenes was used to produce a C. botulinum type A sequence-enriched library. A probe, pCBM44, which showed specific hybridization to a 4.0-kb HindIII fragment present in all of the C. botulinum type A strains tested was isolated, and there was no hybridization to any strains of C. sporogenes. Upon further investigation, pCBM44 was found to hybridize to all of the group I proteolytic C. botulinum strains tested (toxin types A, B, and F) but not to hybridize to groups II, III, and IV (toxin types B, C, D, or E). The probe did not cross-react with nine other Clostridium spp. Such a probe, which differentiates between nontoxigenic C. sporogenes and neurotoxigenic C. botulinum group I strains, should prove extremely useful.

Sequence of the gene coding for the neurotoxin of Clostridium botulinum type A associated with infant botulism: comparison with other clostridial neurotoxins

The neurotoxin gene from a strain of Clostridium botulinum type A causing infant botulism was cloned as a series of overlapping polymerase chain reaction (PCR) fragments generated using primers designed to conserved regions of published botulinal toxin (BoNT) sequences. Translation of the nucleotide sequence derived from cloned PCR fragments demonstrated that the toxin gene encodes a protein of 1,296 amino acid residues. Comparative alignment of the derived infant BoNT/A sequence with those of other published neurotoxins revealed highest sequence relatedness with BoNT/A of classical food-borne botulism. The sequence identity between infant and classical BoNT/A was 94.9% for the light chain (corresponding to 23 amino acid changes) and 87.1% for the heavy chain (corresponding to 109 amino acid changes).

NAD+ binding site of Clostridium botulinum C3 ADP-ribosyltransferase. Identification of peptide in the adenine ring binding domain using 2-azido NAD.

C3 ADP-ribosyltransferase is an exoenzyme produced by certain strains of Clostridium botulinum types C and D, which specifically ADP-ribosylates rho proteins in eukaryotic cells. Using the photoaffinity probe [alpha-32P]nicotinamide-2-azidoadenine dinucleotide, we have identified the adenine ring binding domain of the NAD+ binding site. The specificity of labeling was demonstrated by saturation effects and protection by the natural compound at physiologically relevant concentrations. Saturation of labeling was observed at 50 microM. Protection experiments indicated an 80% protection of labeling by 100 microM NAD+ when protein was photolyzed in the presence of 10 microM probe. Trypsin or Staphylococcus aureus V8 protease digestion of the photolabeled protein, along with boronate affinity chromatography and immobilized metal affinity chromatography, was used to specifically isolate the peptide region photolabeled with the probe. The peptide corresponded to Phe9-Gly19 near the N terminus.

Clostridium botulinum C3 ADP-ribosyltransferase gene. Cloning, sequencing, and expression of a functional protein in Escherichia coli.

C3 ADP-ribosyltransferase is an exoenzyme produced by certain strains of Clostridium botulinum types C and D, which specifically ADP-ribosylates rho and rac proteins in eukaryotic cells. The enzyme was purified from a culture filtrate of C. botulinum type C strain 003-9, and the amino acid sequence from the amino-terminal Ser to Asn192 was determined by Edman degradation. Using a set of degenerate primers based on the sequence, we amplified a part of the gene for this enzyme by polymerase chain reaction. A 2.1-kilobase pair HincII fragment of C. botulinum DNA containing the whole structural gene was then identified by Southern analysis with the polymerase chain reaction product as a probe, and the complete nucleotide structure of the gene together with flanking regions was determined by cloning and DNA sequencing the HincII fragment. The gene encodes a protein of 244 amino acids with a Mr of 27,362 which begins with a putative signal peptide of 40 amino acids. Escherichia coli carrying this gene produced the active enzyme, and about 60% of it was found in the culture medium. Immunoblot analysis with antiserum against the enzyme revealed the presence of two immunoreactive proteins of 27 and 23 kDa in the cytoplasmic/membrane fraction and only the 23-kDa protein in the periplasm and the medium, suggesting that the enzyme expressed is processed in the E. coli, exported into the periplasm and released into the culture medium.

Distinction between Clostridium botulinum type A strains associated with food-borne botulism and those with infant botulism in Japan in intraintestinal toxin production in infant mice and some other properties

Two strains of Clostridium botulinum type A associated with food-borne botulism and six strains associated with infant botulism in Japan were compared in intraintestinal toxin production in infant mice, in vitro toxin and hemagglutinin production, molecular sizes of the toxins, and some other properties. The infant botulism-associated strains, producing M toxin (Mr 300 kDa) but no hemagglutinin, showed significantly lower 50% infective doses in infant mouse intestines. The antigenicities of the toxin differed between the two groups, while the biochemical properties of the cultures did not. Besides infant botulism-associated strains, this set of properties were found only in a strain isolated from honey of South American origin.

Distinction betweenClostridium botulinumtype A strains associated with food-borne botulism and those with infant botulism in Japan in intraintestinal toxin production in infant mice and some other properties

Two strains of Clostridium botulinum type A associated with food-borne botulism and six strains associated with infant botulism in Japan were compared in intraintestinal toxin production in infant mice, in vitro toxin and hemagglutinin production, molecular sizes of the toxins, and some other properties. The infant botulism-associated strains, producing M toxin (Mr 300 kDa) but no hemagglutinin, showed significantly lower 50% infective doses in infant mouse intestines. The antigenicities of the toxin differed between the two groups, while the biochemical properties of the cultures did not. Besides infant botulism-associated strains, this set of properties were found only in a strain isolated from honey of South American origin.

Isolation and Characterization of Converting and Non-Converting Phages, Harbored in the Strains of Clostridium botulinum Types C and D Isolated in Japan.

Isolation and Characterization of Converting and Non-Converting Phages, Harbored in the Strains of Clostridium botulinum Types C and D Isolated in Japan.

Distinct characters of Clostridium botulinum type A strains and their toxin associated with infant botulism in Japan

Four strains of Clostridium botulinum type A having been associated with infant botulism in Japan, and another strain isolated from honey not associated with infant botulism, were found to be hemagglutinin (HA) negative. These strains do not produce L ( M r 500 kDa) nor LL toxin ( M r 900 kDa) but M toxin ( M r 300 kDa) only. No marked difference was found between the HA-positive and HA-negative strains in other biochemical properties, but the HA-negative strains tended to colonize more easily in the intestines of infant mice than did HA-positive strains. The toxin of HA-positive strains and that of HA-negative strains differed in the antigenicity of part of the toxic component and that of the nontoxic component, and in the molecular size of the toxic component.

A simple method of detection of toxigenic Clostridium botulinum type B strains

39 spontaneous nontoxigenic (tox −) mutants were isolated from 20 toxigenic (tox +) strains of Clostridium botulinum types B, A and F. The growth of tox + strains different from that of tox − mutants on a medium supplemented with Tween 80. On VL-Tween agar, tox − mutants formed irregular, glisteing, translucent colonies whereas in tox + colonies the centers were covered by an opaque (‘hoarfrosting’) substance. In Tween-liquid medium, tox + cultures were distinguished from tox − cultures by the formation of a turbid and flocculent growth. Of four different Tween preparations tested (Tweens 20, 40, 60 and 80), only Tween 80 proved to be capable of detecting toxigenic strains. The results indicated a correlation between toxigenicity of C. botulinum and its ability to metabolize Tween 80. The metabolites form the characteristic ‘hoarfrosting’ substance on Tween agar. We recommend the use of Tween 80 for the detection of tox + C. botulinum strains in genetic studies.