Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.     

Plasmid localization of a type E botulinal neurotoxin gene homologue in toxigenic Clostridium butyricum strains, and absence of this gene in non-toxigenic C. butyricum strains

It has been shown recently that two Clostridium butyricum strains (ATCC 43181 and ATCC 43755) contain a botulinal neurotoxin type E (BoNT/E) gene closely related to that of C. botulinum type E. In this study, we show that this gene is located on a large plasmid in the two toxigenic C. butyricum strains and is absent in 18 non-toxigenic C. butyricum and C. beijerinckii strains. Interestingly, the 230 bp upstream and the 1260 bp downstream of the neurotoxin coding sequence are not present in either the non-toxigenic C. butyricum or C. beijerinckii strains. Our data suggest a BoNT/E gene transfer from C. botulinum E to originally non-toxigenic C. butyricum strains.     

Phenotype and genotype of Clostridium sp. strains producing botulism neurotoxin

Neurotoxins produced by strains of Clostridium sp. are belonging to the most toxic biological substances. In the study phenotypes and genotypes of C. botulinum strains in animal studies in vivo and on the DNA level were evaluated, respectively. Additionally, the presence of genes encoding BoNT toxins of A, B, and E types among strains of Clostridium sp. were identified. In case of C. botulinum DNA was isolated from vegetative bacterial cells and from spores. Two different genes encoding two different neurotoxins harboured by three strains of Ae biotype/ae genotype, and by two strains of B biotype/be genotype were detected. Additionally, above E type C. botulinum strains, the presence of gene encoding E type neurotoxin, was found in genome of two C. baratii, two C. butyricum, and C. bifidobacterium, and C. oedematicum strains. C. bifidobacterium and C. oedematicum strains positive for presence of gene encoding E type neurotoxin, were found negative for E neurotoxin production in vivo in TN test. The study indicates that genes encoding BoNT/E neurotoxins are very common among Clostridium species. Phenotype and genotype analysis indicated co-presence of B phenotype together with be genotype and A phenotype together with ae genotype among C. botulinum strains.     

Genetic Analysis of Type E Botulinum Toxin-Producing Clostridium butyricum Strains

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area.     

Analysis of neurotoxin cluster genes in Clostridium botulinum strains producing botulinum neurotoxin serotype A subtypes

Neurotoxin cluster gene sequences and arrangements were elucidated for strains of Clostridium botulinum encoding botulinum neurotoxin (BoNT) subtypes A3, A4, and a unique A1-producing strain (HA(-) Orfx(+) A1). These sequences were compared to the known neurotoxin cluster sequences of C. botulinum strains that produce BoNT/A1 and BoNT/A2 and possess either a hemagglutinin (HA) or an Orfx cluster, respectively. The A3 and HA(-) Orfx(+) A1 strains demonstrated a neurotoxin cluster arrangement similar to that found in A2. The A4 strain analyzed possessed two sets of neurotoxin clusters that were similar to what has been found in the A(B) strains: an HA cluster associated with the BoNT/B gene and an Orfx cluster associated with the BoNT/A4 gene. The nucleotide and amino acid sequences of the neurotoxin cluster-specific genes were determined for each neurotoxin cluster and compared among strains. Additionally, the ntnh gene of each strain was compared on both the nucleotide and amino acid levels. The degree of similarity of the sequences of the ntnh genes and corresponding amino acid sequences correlated with the neurotoxin cluster type to which the ntnh gene was assigned.     

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.     

Cloning of a DNA fragment encoding the 5'-terminus of the botulinum type E toxin gene from Clostridium butyricum strain BL6340

Chromosomal DNA was extracted from toxigenic Clostridium butyricum strain BL6340 isolated from a case of infant botulism. After digestion by EcoRI, a DNA fragment of about 1 kbp was cloned into Escherichia coli using lambda gt11, and was subcloned into pUC118. The E. coli cells transformed with this cloned fragment produced a 33 kDa protein which reacted with monoclonal antibodies recognizing the light chain (Lc) component of botulinum type E toxin. The nucleotide sequence of the cloned fragment was determined. The sequence was similar to that from botulinum type E toxin gene fragments previously determined by our laboratory (strains Mashike, Otaru and Iwanai). Several highly homologous sequences among the botulinum type A, C, E, butyricum and tetanus toxin genes were found in both translated and untranslated regions. These results suggest that the toxin gene of C. butyricum may have evolved by transfer from C. botulinum.     

Cloning and whole nucleotide sequence of the gene for the light chain component of botulinum type E toxin from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike.

Chromosomal DNAs were extracted from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike. The 6.0 Kbp fragment coding for the entire light chain (L) component and the N-terminus of heavy chain (H) component of botulinum type E toxin was obtained from each extracted DNAs after digestion with HindIII. The entire nucleotide sequences for the light chain components of these cloned genes were determined, and the derived amino acid sequences were compared to each other, and with those of botulinum type A, C1, D, and tetanus toxins reported previously. The cleavage site of L and H components of type E toxin was presumed to be Arg-422. In a total of 422 amino acid residues of L component, 17 residues were different between butyricum and type E toxins, and all these differences were found within 200 residues of N-terminus of L component. On the contrary, five regions showing highly homologous sequences were found in L components among these six toxins, and one more region between botulinum type E and tetanus toxins.     

SYBR green real-time PCR method to detect Clostridium botulinum type A

Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 x 10(1) copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%).     

Phylogeny and taxonomy of the food-borne pathogen Clostridium botulinum and its neurotoxins

Until recently, all clostridia producing neurotoxins able to cause paralysis symptomatic of botulism were deemed to be Clostridium botulinum. Defining Cl. botulinum on the basis of this single phenotypic trait has resulted in the species encompassing metabolically very diverse organisms, and four distinct phenotypic groups are recognized within this taxon (designated groups I-IV). Nucleic acid hybridization and 16S ribosomal RNA sequencing studies have revealed the presence of four phylogenetically distinct lineages within the species, which correlate with these phenotypic divisions. In addition to marked phenotypic and genotypic heterogeneity between groups, the taxonomy of the species is further complicated by the existence of strains which are closely related, if not genetically identifiable, to members of each Cl. botulinum group, but are non-toxigenic. Furthermore, strains of species other than Cl. botulinum (viz. Cl. baratii, Cl. butyricum) have been found which express botulinum neurotoxin (BoNT). Great advances have been made in recent years in elucidating the nucleotide sequences of genes encoding the various BoNT antigenic types (A through to G). Genealogical trees derived from BoNTs show marked discordance with those depicting 'natural' relationships inferred from 16S rRNA and phenotypic clusters, and strong evidence exists for BoNT gene transfer between some groups of Cl. botulinum (e.g. groups I and II), and with non-botulinum species. Botulinum neurotoxin is produced by Cl. botulinum as a non-covalently bound progenitor toxin complex of two or more protein components. Information on the evolutionary histories of the various non-toxic progenitor proteins is currently limited, although there is evidence of gene recombination. In particular, chimera-like or mosaic non-toxic-non-haemagglutinins (NTNH) genes in group I Cl. botulinum have been described, and it is now apparent that the phylogeny of the NTNHs is not going to 'mirror' that of botulinal neurotoxins, although their genes are physically contiguous. In this article, the current state of knowledge of the phylogenetics of the species Cl. botulinum and its neurotoxins is reviewed, and a view is presented that a nomenclature based rigidly on BoNT production is no longer tenable.     

The complete amino acid sequence of the Clostridium botulinum type-E neurotoxin, derived by nucleotide-sequence analysis of the encoding gene.

The entire structural gene of the Clostridium botulinum NCTC 11219 type-E neurotoxin (BoNT/E) has been cloned as five overlapping DNA fragments, generated by polymerase chain reaction (PCR). Analysis of triplicate clones of each fragment, derived from three independent PCR, has allowed the derivation of the entire nucleotide sequence of the BoNT/E gene. Translation of the sequence has shown BoNT/E to consist of 1252 amino acids and, as such, represents the smallest BoNT characterised to date. The light chain of the toxin exhibits the highest level of sequence similarity to tetanus toxin (TeTx, 40%). The light chains of BoNT/A and BoNT/D share 33% similarity with BoNT/E, while BoNT/C exhibits 32% similarity. In contrast, the TeTx heavy chain exhibits the lowest degree of similarity (35%) with BoNT/E, with the BoNT heavy chains sharing 46%, 36% and 37%, for neurotoxin types A, C and D, respectively. Comparisons with partial amino acid sequences of the light chain of BoNT/E from C. botulinum strain Beluga and that from the strains Mashike, Iwanai and Otaru, indicate single amino acid differences in each case. Alignment of all characterised neurotoxin sequences (BoNT/A, BoNT/C, BoNT/D, BoNT/E and TeTx) shows them to be composed of highly conserved amino acid domains interspersed with amino acid tracts exhibiting little overall similarity. The most divergent region corresponds to the extreme COOH-terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.     

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 serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing

A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.     

神经毒素原性酪酸梭菌与E型肉毒梭菌毒素的精制及特性比较

对首次自Ｅ型肉毒中毒食品中分离到的一株神经毒素原性酪酸梭菌（ＬＣＬ１５５）所产生的神经毒素,同Ｅ型肉毒梭菌（Ｅ１５３）所产生的神经毒素进行了精制及特性比较,发现（１）两菌神经毒素的分子量,Ｎａｔｉｖｅ－ＰＡＧＥ测试均为３２０ｋＤａ;ＳＤＳ－ＰＡＧＥ测试则均为１４７ｋＤａ,非毒性非血凝素部分均为１２８ｋＤａ;用胰蛋白酶激活神经毒素后发现两菌神经毒素均由分子量为１０３ｋＤａ的Ｈ链和４８ｋＤａ的Ｌ链组成。（２）两菌神经毒素柱层析图像基本一致,但在菌体毒素提取效果及精制效果诸方面,分离的酪酸梭菌却都较差。（３）胰蛋白酶激活试验表明：两菌神经毒素达到最大毒力所需激活时间不等。在相同温度下,分离的酪酸梭菌毒素只需５ｍｉｎ,而Ｅ型肉毒梭菌毒素却需３０ｍｉｎ,提示两菌神经毒素激活动力学上存在差异。（４）琼脂双扩散试验结果表明两菌神经毒素的抗原性是一致的,没有发现沉淀线呈交叉或部分交叉现象。     

Sequencing the botulinum neurotoxin gene and related genes in Clostridium botulinum type E strains reveals orfx3 and a novel type E neurotoxin subtype

Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (≤3%) in the nucleotide sequences of the gene cluster components were observed among the three strains and the published BoNT/E-producing clostridia. The orfx3, orfx2, orfx1, and p47 gene sequences of the three type E strains shared homologies of 81%, 67 to 76%, 78 to 79%, and 79 to 85%, respectively, with published sequences for type A1 and A2 C. botulinum. Analysis of bont/E from the 14 type E strains and 19 previously published BoNT/E-producing clostridia revealed six neurotoxin subtypes, with a new distinct subtype consisting of three Finnish isolates alone. The amino acid sequence of the subtype E6 neurotoxin differed 3 to 6% from the other subtypes, suggesting that these subtype E6 neurotoxins may possess specific antigenic or functional properties.     

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)     

Two Novel Toxin Variants Revealed by Whole-Genome Sequencing of 175 Clostridium botulinum Type E Strains

We sequenced 175 Clostridium botulinum type E strains isolated from food, clinical, and environmental sources from northern Canada and analyzed their botulinum neurotoxin (bont) coding sequences (CDSs). In addition to bont/E1 and bont/E3 variant types, neurotoxin sequence analysis identified two novel BoNT type E variants termed E10 and E11. Strains producing type E10 were found along the eastern coastlines of Hudson Bay and the shores of Ungava Bay, while strains producing type E11 were only found in the Koksoak River region of Nunavik. Strains producing BoNT/E3 were widespread throughout northern Canada, with the exception of the coast of eastern Hudson Bay.     

The complete genome sequence of Clostridium botulinum F str. 230613, insertion sites, and recombination of BoNT gene clusters

The genomic DNA of Clostridium botulinum F str. 230613 includes a chromosome (3?993?083?bp, 3502 coding sequences (CDs)) and a plasmid (17?531?bp, 25 CDs). The arrangement of the botulinum neurotoxin serotype F (BoNT/F) gene cluster, a 15-kb (or longer) fragment including the bont gene and other relevant genes, and its different insertion sites in C. botulinum A2 and C. botulinum F were formulated. Mobile elements and virulence factors were analysed. We also found a cell adhesion and pectin lyase domain-containing protein, which may function in attaching to the host and as a pectin lyase. The nine BoNT gene clusters of group I C. botulinum strains were located at three sites in the chromosome of C. botulinum F str. 230613. This study showed the inserting inclination of BoNT/A1 tend to have gene clusters inserted at site 3, BoNT/F at site 2, and BoNT/A2 at site 1. Additionally, we found the recombination event between the BoNT gene clusters of sites 2 and 3, a mechanism that contributed to the diversity of the BoNT gene cluster arrangement.     

Genetic diversity among Botulinum Neurotoxin-producing clostridial strains

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.