Characterization of six type A strains of<i>Clostridium botulinum</i>that contain type B toxin gene sequences

Kirma, Nameer B.; Ferreira, Joseph; Baumstark, Barbara R.

doi:10.1016/s0378-1097(03)00911-x

Cited by 18 publications

(15 citation statements)

References 15 publications

(27 reference statements)

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“…Sequences were generated from six of the silent BoNT/B genes in these A1(B) strains and compared. All six of the silent BoNT/B sequences were similar to those reported under GenBank accession number AF300467 (26), which generate a truncated protein from a stop codon at amino acid 128. Four of the sequences (A148, A397, A404, and A406) were identical to each other but differed from that reported under accession number AF300467 by two single nucleotide polymorphisms.…”

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confidence: 61%

Genetic Diversity among Botulinum Neurotoxin-Producing Clostridial Strains

et al. 2007

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Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore-forming rod-shaped bacteria that have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and death in humans and other animal species. A collection of 174 C. botulinum strains was examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine the genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT/A to BoNT/G). Analysis of the16S rRNA gene sequences confirmed previous identifications of at least four distinct genomic backgrounds (groups I to IV), each of which has independently acquired one or more BoNT genes through horizontal gene transfer. AFLP analysis provided higher resolution and could be used to further subdivide the four groups into subgroups. Sequencing of the BoNT genes from multiple strains of serotypes A, B, and E confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven toxin genes of the serotypes were compared and showed various degrees of interrelatedness and recombination, as was previously noted for the nontoxic nonhemagglutinin gene, which is linked to the BoNT gene. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for the treatment of botulism.Clostridium botulinum is a taxonomic collection of several distinct species of anaerobic gram-positive spore-forming bacteria that produce the most poisonous substance known, botulinum neurotoxin (BoNT) (1,8). These organisms, along with related neurotoxin-producing species that, for a variety of reasons, were not included under the C. botulinum taxon, pose global health problems that affect both infant and adult humans and can also affect wildlife, waterfowl, and domestic animals. They cause intoxication through ingestion of the neurotoxin in contaminated foods. Toxicoinfections can also occur after contact with bacteria or bacterial spores (6, 17). These pathogens are ubiquitous and can be found in soils and sedi-

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Section: Fig 2 Aflp-based Dendrogram Of 174supporting

confidence: 61%

Genetic Diversity among Botulinum Neurotoxin-Producing Clostridial Strains

et al. 2007

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“…In addition, some Clostridium botulinum type A strains (e.g., NCTC2916) contain a silent type B gene in their chromosome, designated A(B). The silent B toxin genes described to date contain the full length of the active type B gene cluster and one or more mutations that result in an unexpressed type B neurotoxin (3,8).Here we report the first infant botulism case resulting from a rare bont/A5 subtype, which we unexpectedly found to be part of a novel neurotoxin type A/B gene arrangement. Our bont/A5 gene sequence differs by 2 bp from the bont/A5 sequence previously identified in four wound botulism cases (2).…”

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Novel Clostridium botulinum Toxin Gene Arrangement with Subtype A5 and Partial Subtype B3 Botulinum Neurotoxin Genes

2009

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ᰔBotulinum neurotoxin, the most poisonous substance known and a potential biothreat agent (1), causes human and animal botulism worldwide (4,9). This toxin is encoded by the bont gene as part of a cluster that includes nontoxic accessory genes (10). Two main gene clusters are known: the hemagglutinincontaining (haϩ) cluster in toxin serotypes A1, B, C, D, and G and the orfX cluster in toxin serotypes A, E, and F (7, 10). In addition, some Clostridium botulinum type A strains (e.g., NCTC2916) contain a silent type B gene in their chromosome, designated A(B). The silent B toxin genes described to date contain the full length of the active type B gene cluster and one or more mutations that result in an unexpressed type B neurotoxin (3,8).Here we report the first infant botulism case resulting from a rare bont/A5 subtype, which we unexpectedly found to be part of a novel neurotoxin type A/B gene arrangement. Our bont/A5 gene sequence differs by 2 bp from the bont/A5 sequence previously identified in four wound botulism cases (2). The California infant strain, designated IBCA94-0216, was characterized by sequencing the toxin complex genes and their flanking regions (GenBank accession number FJ959094). Sequence analysis confirmed that the bont/A5 is located within a hemagglutinin complex (haϩ) that contains the genes ha70, ha17, ha33, botR, ntnh, and bont/A5 (Fig. 1). This type of toxin cluster is commonly associated with strains bont/A1 and bont/B but is not found in strains bont/A2, bont/A3, and bont/A4 (7, 10). The IBCA94-0216 neurotoxin gene cluster has an unexpected 76-bp deletion between the genes ha33 and botR. This is the first example of such a deletion.Downstream from bont/A5 are two copies of transposases belonging to the is3 family, a structure identical to the downstream flanking sequence of the type A1 Hall neurotoxin gene cluster ( Fig. 1) (10). Interestingly, 2,041 bp downstream from the bont/A5 gene and immediately following the second is3 is a partial bont/B gene that is only 65% of a full-length bont/B (6). The partial bont/B gene lacks the nucleotide sequence coding for the light chain of the toxin and begins at nucleotide 1362 of the 3,876-nucleotide full-length toxin gene. Comparisons of this region (1,362 to 3,876 nucleotides) revealed it is most similar to the rare bont/B3 subtype (5). The entire IBCA94-0216 bont/A/B cluster is flanked by a 5Ј flagellin gene and a 3Ј transposase, as is found in the silent type B gene cluster of strain NCTC2916 (7).Nucleic acid pairwise identities (using the Kimura two-parameter method in MEGA4 software) of the toxin cluster genes show that the IBCA94-0216 A5 neurotoxin and ntnh genes are most similar to the type A1 Hall neurotoxin and ntnh genes, respectively, while the hemagglutinin genes are most similar to the hemagglutinin genes of type B strains. This mosaic gene arrangement and the presence of the partial type B neurotoxin gene lead us to speculate that some form of genetic rearrangement occurred between type A and type B neurotoxin gene clusters in the c...

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“…Based on its phenotypic and genetic traits, group IV C. botulinum has been proposed to be renamed Clostridium argentinense (203). Of the human pathogenic strains, group I cultures produce toxin A, B, or F and group II cultures produce toxin B, E, or F. Dual-toxin-producing strains have also been reported (72,80,121), as have those producing only one type of toxin but carrying a silent gene for another (73,74,121). Other clostridia, namely, C. butyricum and C. baratii, are also known to produce type E and F toxins, respectively.…”

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confidence: 99%

Laboratory Diagnostics of Botulism

2006

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SUMMARY Botulism is a potentially lethal paralytic disease caused by botulinum neurotoxin. Human pathogenic neurotoxins of types A, B, E, and F are produced by a diverse group of anaerobic spore-forming bacteria, including Clostridium botulinum groups I and II, Clostridium butyricum, and Clostridium baratii. The routine laboratory diagnostics of botulism is based on the detection of botulinum neurotoxin in the patient. Detection of toxin-producing clostridia in the patient and/or the vehicle confirms the diagnosis. The neurotoxin detection is based on the mouse lethality assay. Sensitive and rapid in vitro assays have been developed, but they have not yet been appropriately validated on clinical and food matrices. Culture methods for C. botulinum are poorly developed, and efficient isolation and identification tools are lacking. Molecular techniques targeted to the neurotoxin genes are ideal for the detection and identification of C. botulinum, but they do not detect biologically active neurotoxin and should not be used alone. Apart from rapid diagnosis, the laboratory diagnostics of botulism should aim at increasing our understanding of the epidemiology and prevention of the disease. Therefore, the toxin-producing organisms should be routinely isolated from the patient and the vehicle. The physiological group and genetic traits of the isolates should be determined.

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Characterization of six type A strains ofClostridium botulinumthat contain type B toxin gene sequences

Cited by 18 publications

References 15 publications

Genetic Diversity among Botulinum Neurotoxin-Producing Clostridial Strains

Genetic Diversity among Botulinum Neurotoxin-Producing Clostridial Strains

Novel Clostridium botulinum Toxin Gene Arrangement with Subtype A5 and Partial Subtype B3 Botulinum Neurotoxin Genes

Laboratory Diagnostics of Botulism

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