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.     

Neurotoxin Gene Clusters in Clostridium botulinum Type Ab Strains

There is limited knowledge of the neurotoxin gene diversity among Clostridium botulinum type Ab strains. Only the sequences of the bont/A and bont/B genes in C. botulinum type Ab strain CDC1436 and the sequence of the bont/B gene in C. botulinum type Ab strain CDC588 have been reported. In this study, we sequenced the entire bont/A- and bont/B-associated neurotoxin gene clusters of C. botulinum type Ab strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370 and the bont/A gene of strain CDC588. In addition, we analyzed the organization of the neurotoxin gene clusters in strains CDC588 and CDC1436. The bont/A nucleotide sequence of strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370 differed from those of the known bont/A subtypes A1 to A4 by 2 to 7%, and the predicted amino acid sequence differed by 4% to 14%. The bont/B nucleotide sequence in strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370 showed 99.7% identity to the sequence of subtype B1. The bont/A nucleotide sequence of strain CDC588 was 99.9% identical to that of subtype A1. Although all of the C. botulinum type Ab strains analyzed contained the two sets of neurotoxin clusters, similar to what has been found in other bivalent strains, the intergenic spacing of p21-orfX1 and orfX2-orfX3 varied among these strains. The type Ab strains examined in this study had differences in their toxin gene cluster compositions and bont/A and bont/B nucleotide sequences, suggesting that they may have arisen from separate recombination events.Clostridium botulinum is a gram-positive anaerobic bacterium that produces an extremely potent toxin, the botulinum neurotoxin (BoNT). There are seven serologically distinct types of BoNT (serotypes A through G). Although most strains of C. botulinum express a single toxin serotype, some isolates have been shown to produce more than one, namely, Ab, Af, Ba, and Bf (11). In addition, many strains designated type A by mouse bioassay harbor nucleotide sequences for both type A and B toxins (6). These strains have been designated A(B) to indicate the presence of the bont/B gene without type B-specific toxicity.Based on phylogenetic analysis of the neurotoxin gene sequences, four subtypes have been identified within serotype A and five subtypes within serotype B (12). The neurotoxin gene nucleotide sequences of these subtypes differ by up to 8%, and the predicted amino acid sequences differ by up to 16%. In addition, the genes encoding components of the toxin complexes are arranged in clusters that differ in composition and organization (14) (Fig. ​(Fig.1).1). The toxin gene cluster of subtype A1 (termed ha cluster) includes the gene encoding the nontoxic nonhemagglutinin (ntnh), a regulatory gene (botR), and an operon encoding three hemagglutinins (ha70, ha33, and ha17). The toxin gene clusters containing bont/A2 or bont/A3 (termed orfX cluster) include the ntnh and p21 (analogous to botR) genes and several genes of unknown function (orfX1, orfX2, orfX3, and p47). Type Ba and A(B) strains contain two sets of neurotoxin cluster genes in which ha70, ha33, and ha17 are associated with the bont/B gene, and orfX1, orfX2, orfX3, and p47 are associated with the bont/A gene. In addition, some A1 strains contain a neurotoxin gene cluster that is similar to those in A2 and A3, but the bont/A nucleotide sequence is 99.9% identical to that in other A1 strains. These strains have been designated HA⁻ Orfx⁺ A1 (14). The neurotoxin gene cluster in type B strains includes the ntnh, botR, ha70, ha33, and ha17 genes. Notably, no differences in the neurotoxin gene cluster arrangements among the subtypes within serotype B have been reported.Open in a separate windowFIG. 1.Toxin gene cluster arrangements for BoNT type A-producing strains, including Ab, A(B), and Ba strains.Although several studies have described the organization and the nucleotide sequences of the neurotoxin gene cluster components among type A and B strains [including type Ba and A(B) strains], there is limited information regarding the diversity of the neurotoxin cluster genes among C. botulinum type Ab strains. The nucleotide sequences of the bont/A and bont/B genes in C. botulinum type Ab strain CDC1436 and the sequence of the bont/B gene of C. botulinum type Ab strain CDC588 have been previously reported; strain CDC1436 harbors a bont/A2 gene, and both strains CDC1436 and CDC588 harbor a bont/bvB gene (12, 15). Four additional type Ab strains from Italy have been analyzed by a restriction fragment length polymorphism method to determine the bont/A and bont/B subtypes (7, 9). To the best of our knowledge, the complete nucleotide sequences of the neurotoxin gene clusters in C. botulinum type Ab strains have not been reported. Thus, the objective of this study was to analyze the neurotoxin gene cluster composition in three C. botulinum type Ab strains ({"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370, CDC588, and CDC1436) available in the CDC strain collection. We report differences in bont/A gene sequence among type Ab strains, including the identification of a novel bont/A nucleotide sequence in strain {"type":"entrez-protein","attrs":{"text":"CDC41370","term_id":"524503451","term_text":"CDC41370"}}CDC41370, and describe differences in the organization of the neurotoxin gene clusters among these strains.     

Genetic Diversity among Clostridium botulinum Strains Harboring bont/A2 and bont/A3 Genes

Clostridium botulinum type A strains are known to be genetically diverse and widespread throughout the world. Genetic diversity studies have focused mainly on strains harboring one type A botulinum toxin gene, bont/A1, although all reported bont/A gene variants have been associated with botulism cases. Our study provides insight into the genetic diversity of C. botulinum type A strains, which contain bont/A2 (n = 42) and bont/A3 (n = 4) genes, isolated from diverse samples and geographic origins. Genetic diversity was assessed by using bont nucleotide sequencing, content analysis of the bont gene clusters, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sequences of bont genes obtained in this study showed 99.9 to 100% identity with other bont/A2 or bont/A3 gene sequences available in public databases. The neurotoxin gene clusters of the subtype A2 and A3 strains analyzed in this study were similar in gene content. C. botulinum strains harboring bont/A2 and bont/A3 genes were divided into six and two MLST profiles, respectively. Four groups of strains shared a similarity of at least 95% by PFGE; the largest group included 21 out of 46 strains. The strains analyzed in this study showed relatively limited genetic diversity using either MLST or PFGE.     

Rapid, Quantitative PCR Monitoring of Growth of Clostridium botulinum Type E in Modified-Atmosphere-Packaged Fish

A rapid, quantitative PCR assay (TaqMan assay) which quantifies Clostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of C. botulinum type E and 18 genera of 42 non-C. botulinum type E strains, including strains of C. botulinum types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C. botulinum DNA could be monitored (the quantifiable range was 10² to 10⁸ CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of C. botulinum cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.     

Whole-Genome Single-Nucleotide-Polymorphism Analysis for Discrimination of Clostridium botulinum Group I Strains

Clostridium botulinum is a genetically diverse Gram-positive bacterium producing extremely potent neurotoxins (botulinum neurotoxins A through G [BoNT/A-G]). The complete genome sequences of three strains harboring only the BoNT/A1 nucleotide sequence are publicly available. Although these strains contain a toxin cluster (HA⁺ OrfX⁻) associated with hemagglutinin genes, little is known about the genomes of subtype A1 strains (termed HA⁻ OrfX⁺) that lack hemagglutinin genes in the toxin gene cluster. We sequenced the genomes of three BoNT/A1-producing C. botulinum strains: two strains with the HA⁺ OrfX⁻ cluster (69A and 32A) and one strain with the HA⁻ OrfX⁺ cluster (CDC297). Whole-genome phylogenic single-nucleotide-polymorphism (SNP) analysis of these strains along with other publicly available C. botulinum group I strains revealed five distinct lineages. Strains 69A and 32A clustered with the C. botulinum type A1 Hall group, and strain CDC297 clustered with the C. botulinum type Ba4 strain 657. This study reports the use of whole-genome SNP sequence analysis for discrimination of C. botulinum group I strains and demonstrates the utility of this analysis in quickly differentiating C. botulinum strains harboring identical toxin gene subtypes. This analysis further supports previous work showing that strains CDC297 and 657 likely evolved from a common ancestor and independently acquired separate BoNT/A1 toxin gene clusters at distinct genomic locations.     

Arrangement of the Clostridium baratii F7 Toxin Gene Cluster with Identification of a σ Factor That Recognizes the Botulinum Toxin Gene Cluster Promoters

Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bont gene that is part of a toxin gene cluster that includes several accessory genes. We sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ⁷⁰ family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. This TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.     

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.     

Sequence Diversity of Genes Encoding Botulinum Neurotoxin Type F

Botulism due to type F botulinum neurotoxin (BoNT/F) is rare (<1% of cases), and only a limited number of clostridial strains producing this toxin type have been isolated. As a result, analysis of the diversity of genes encoding BoNT/F has been challenging. In this study, the entire bont/F nucleotide sequences were determined from 33 type F botulinum toxin-producing clostridial strains isolated from environmental sources and botulism outbreak investigations. We examined proteolytic and nonproteolytic Clostridium botulinum type F strains, bivalent strains, including Bf and Af, and Clostridium baratii type F strains. Phylogenetic analysis revealed that the bont/F genes examined formed 7 subtypes (F1 to F7) and that the nucleotide sequence identities of these subtypes differed by up to 25%. The genes from proteolytic (group I) C. botulinum strains formed subtypes F1 through F5, while the genes from nonproteolytic (group II) C. botulinum strains formed subtype F6. Subtype F7 was composed exclusively of bont/F genes from C. baratii strains. The region of the bont/F5 gene encoding the neurotoxin light chain was found to be highly divergent compared to the other subtypes. Although the bont/F5 nucleotide sequences were found to be identical in strains harboring this gene, the gene located directly upstream (ntnh/F) demonstrated sequence variation among representative strains of this subtype. These results demonstrate that extensive nucleotide diversity exists among genes encoding type F neurotoxins from strains with different phylogenetic backgrounds and from various geographical sources.Botulism is a potentially fatal disease caused solely by the action of serologically distinct neurotoxins (BoNT/A, -B, -C, -D, -E, -F, or -G) which prevent acetylcholine release at neuromuscular junctions, resulting in paralysis. Food-borne botulism may result from the ingestion of a preformed toxin that is produced in inadequately preserved food. Under certain conditions, botulinum neurotoxin-producing Clostridium sp. may colonize and produce toxin in wounds (wound botulism) or in the intestine (infant botulism or adult colonization). Globally, human botulism cases are associated with botulinum neurotoxin serotypes A, B, E, and rarely F. The Centers for Disease Control and Prevention (CDC) maintains active surveillance for botulism cases in the United States. Of 1,269 U.S. cases of botulism reported to the CDC between 1981 and 2002, approximately 1% were due to type F toxin (13). An additional 10 cases of type F botulism were reported to the CDC from 2003 to 2007 (http://www.cdc.gov/nationalsurveillance/botulism_surveillance.html).Type F botulism was first described in 1960 following an outbreak occurring in Denmark involving liver paste (30). The organism isolated in this outbreak metabolically resembled proteolytic Clostridium botulinum strains of types A and B. In a subsequent outbreak, type F toxin was found to be produced by a nonproteolytic C. botulinum strain isolated from venison jerky (29). Bivalent toxin-producing strains have been described, including Bf strains isolated from infants in the United States and England (1, 16, 17, 35) and an Af strain isolated from individuals in Argentina with food-borne botulism (11). Bivalent strains may produce higher titers of one toxin type, which are denoted with a capital letter. The only reported organism isolated from infants with botulism due to type F toxin alone (i.e., not associated with additional serotypes as in bivalent strains) is Clostridium baratii (2, 14, 24). In addition, C. baratii type F has been isolated from adults with botulism (28) as well as suspect foods associated with botulism cases (15; CDC, unpublished data).Botulinum neurotoxin genes (bont) are typically found within toxin gene clusters that include other genes encoding components of the toxin complex (ha70, ha17, ha33, ntnh), regulatory proteins (botR), or proteins with unknown functions (p47, orfX1, orfX2, orfX3). Two general toxin gene cluster arrangements have been described, including the orfX cluster (orfX3-orfX2-orfX1-botR-p47-ntnh-bont) and the ha cluster (ha70-ha17-ha33-botR-ntnh-bont) (21, 22). The bont/F genes of type F and type Bf strains examined by Hill et al. (21) were found in an orfX cluster.The amino acid sequence identities of the BoNT serotypes A to G range from approximately 35 to 70% (36). In addition, within nearly all toxin serotypes, various levels of amino acid sequence variation have been observed, resulting in the identification of toxin subtypes (20, 36, 37).Although a limited number of genes encoding type F botulinum neurotoxin have been sequenced, a comparison of sequences available in public databases indicates that significant diversity exists within this serotype. The nucleotide sequence identity of the type F neurotoxin gene from the proteolytic strain Langeland differs from that of the gene in the nonproteolytic strain 202F by 7%. The type F gene from C. baratii strain ATCC 43756 differs from those of Langeland and 202F by 18% and 20%, respectively. Although the bivalent (Bf) strain CDC3281 is phenotypically proteolytic, the toxin gene shows greater similarity to those from nonproteolytic strains (34). In addition to metabolic differences observed between proteolytic and nonproteolytic C. botulinum strains as well as C. baratii, these organisms are phylogenetically distinct based on differences among their 16S rRNA sequences (5, 20).In order to define the degree of genetic diversity among strains encoding botulinum neurotoxin type F, we sequenced the bont/F gene and partially characterized the toxin gene cluster by using a panel of 33 strains with diverse origins. These strains were selected from those available in the CDC culture collection as well as several isolated in Argentina. The only reported Af strains have been isolated in Argentina. Among 68 outbreaks of serotype-confirmed food-borne botulism in Argentina between 1922 and 2007, type F was isolated in two outbreaks, and type Af was isolated in one outbreak. In addition, Lúquez et al. (26) reported isolation of type F and Af strains from Argentine soils.Here, we report that analysis of the bont/F genes from the strains examined in this study revealed a high degree of nucleotide sequence heterogeneity and the identification of seven type F subtypes (F1 to F7). In addition, the nucleotide sequence of one subtype (F5) has not been previously reported and contains evidence of recombination compared to the other subtypes.     

Clostridium botulinum Strain Af84 Contains Three Neurotoxin Gene Clusters: Bont/A2, bont/F4 and bont/F5

Sanger and shotgun sequencing of Clostridium botulinum strain Af84 type Af and its botulinum neurotoxin gene (bont) clusters identified the presence of three bont gene clusters rather than the expected two. The three toxin gene clusters consisted of bont subtypes A2, F4 and F5. The bont/A2 and bont/F4 gene clusters were located within the chromosome (the latter in a novel location), while the bont/F5 toxin gene cluster was located within a large 246 kb plasmid. These findings are the first identification of a C. botulinum strain that contains three botulinum neurotoxin gene clusters.     

Gene Organization and Sequence Determination of the Two Botulinum Neurotoxin Gene Clusters in Clostridium botulinum Type A(B) Strain NCTC 2916

The gene organization and nucleotide sequence of the type A and B BoNT-gene clusters in Clostridium botulinum strain NCTC 2916 were studied. The aim was to clarify the organization of genes within C. botulinum type A strains possessing an unexpressed BoNT/B gene. The BoNT/A-gene cluster includes genes encoding BoNT, NTNH and a part of P-47 (the gene for this protein was reported in strains of C. botulinum types E and F). Clustered with the silent BoNT/B gene were genes encoding NTNH, P-21 and HA-33. Sequencing analysis of the NTNHs revealed the presence of 471 amino acids identical in the type B and A gene clusters. This gene organization contrasts markedly with the purported organization in strain NCTC 2916 described by Henderson et al. (FEMS Microbiol. Lett. 140, 151–158). In type A(B) strain NCTC 2916, the neurotoxin gene is of type BoNT/A1 within a gene cluster that has identical organization to that found in BoNT/A2 type strains; these observations may be significant in establishing the origin of the BoNT-gene cluster. Received: 28 July 1997 / Accepted: 15 October 1997     

Analysis of the neurotoxin complex genes in Clostridium botulinum A1-A4 and B1 strains: BoNT/A3, /Ba4 and /B1 clusters are located within plasmids

Background

Clostridium botulinum and related clostridial species express extremely potent neurotoxins known as botulinum neurotoxins (BoNTs) that cause long-lasting, potentially fatal intoxications in humans and other mammals. The amino acid variation within the BoNT is used to categorize the species into seven immunologically distinct BoNT serotypes (A–G) which are further divided into subtypes. The BoNTs are located within two generally conserved gene arrangements known as botulinum progenitor complexes which encode toxin-associated proteins involved in toxin stability and expression.

Methodology/Principal Findings

Because serotype A and B strains are responsible for the vast majority of human botulism cases worldwide, the location, arrangement and sequences of genes from eight different toxin complexes representing four different BoNT/A subtypes (BoNT/A1-Ba4) and one BoNT/B1 strain were examined. The bivalent Ba4 strain contained both the BoNT/A4 and BoNT/bvB toxin clusters. The arrangements of the BoNT/A3 and BoNT/A4 subtypes differed from the BoNT/A1 strains and were similar to those of BoNT/A2. However, unlike the BoNT/A2 subtype, the toxin complex genes of BoNT/A3 and BoNT/A4 were found within large plasmids and not within the chromosome. In the Ba4 strain, both BoNT toxin clusters (A4 and bivalent B) were located within the same 270 kb plasmid, separated by 97 kb. Complete genomic sequencing of the BoNT/B1 strain also revealed that its toxin complex genes were located within a 149 kb plasmid and the BoNT/A3 complex is within a 267 kb plasmid.

Conclusions/Significance

Despite their size differences and the BoNT genes they contain, the three plasmids containing these toxin cluster genes share significant sequence identity. The presence of partial insertion sequence (IS) elements, evidence of recombination/gene duplication events, and the discovery of the BoNT/A3, BoNT/Ba4 and BoNT/B1 toxin complex genes within plasmids illustrate the different mechanisms by which these genes move among diverse genetic backgrounds of C. botulinum.     

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).     

Identification of Type A, B, E, and F Botulinum Neurotoxin Genes and of Botulinum Neurotoxigenic Clostridia by Denaturing High-Performance Liquid Chromatography

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.     

Comparative Genomic Hybridization Analysis of Two Predominant Nordic Group I (Proteolytic) Clostridium botulinum Type B Clusters

Comparative genomic hybridization analysis of 32 Nordic group I Clostridium botulinum type B strains isolated from various sources revealed two homogeneous clusters, clusters BI and BII. The type B strains differed from reference strain ATCC 3502 by 413 coding sequence (CDS) probes, sharing 88% of all the ATCC 3502 genes represented on the microarray. The two Nordic type B clusters differed from each other by their response to 145 CDS probes related mainly to transport and binding, adaptive mechanisms, fatty acid biosynthesis, the cell membranes, bacteriophages, and transposon-related elements. The most prominent differences between the two clusters were related to resistance to toxic compounds frequently found in the environment, such as arsenic and cadmium, reflecting different adaptive responses in the evolution of the two clusters. Other relatively variable CDS groups were related to surface structures and the gram-positive cell wall, suggesting that the two clusters possess different antigenic properties. All the type B strains carried CDSs putatively related to capsule formation, which may play a role in adaptation to different environmental and clinical niches. Sequencing showed that representative strains of the two type B clusters both carried subtype B2 neurotoxin genes. As many of the type B strains studied have been isolated from foods or associated with botulism, it is expected that the two group I C. botulinum type B clusters present a public health hazard in Nordic countries. Knowing the genetic and physiological markers of these clusters will assist in targeting control measures against these pathogens.Clostridium botulinum produces a potent neurotoxin during its growth. The toxin causes a potentially lethal paralytic disease, botulism, in humans and animals. The classical food-borne botulism follows the consumption of toxin-containing food or drink, while infant and adult intestinal botulism results from in vivo spore germination, outgrowth, and toxin production in the gut. Apart from attenuated intestinal microbial population, other factors affecting the colonization of C. botulinum in the intestinal forms of botulism are not known.Based on their physiology and genetic background, C. botulinum strains are divided into groups I to IV (13). Strains of groups I and II are associated with human disease. Group I strains produce neurotoxin serotypes A, B, and/or F, while the group II strains produce type B, E, or F toxin. Physiologically, groups I and II differ markedly from each other as well as from groups III and IV. Genomic analysis of group I and II C. botulinum strains by 16S rrn sequencing (13), ribotyping (10), and amplified fragment length polymorphism (11, 15, 16) is consistent with the divergent physiologies of the two groups (18).Nordic C. botulinum group I strains show a remarkable homogeneity (15, 20, 21, 23). In a large pulsed-field gel electrophoresis (PFGE) analysis, the majority of group I strains isolated from various sources from Finland, Norway, and Denmark formed type B neurotoxin and clustered into two large groups, with the members of each group sharing identical or nearly identical restriction patterns (20, 23). Many of these strains were recovered from honey for human consumption (23), and one strain was related to an infant botulism case (22). Apart from a recent study showing that strains of the two type B clusters, further referred to as clusters BI and BII, differ in their abilities to grow at extreme temperatures (12), the physiological, epidemiological, and genetic markers of the two clusters are not known. An understanding of such traits will assist in designing control measures against these potential food- and environment-borne pathogens.The availability of group I C. botulinum genome sequences has enabled the construction of whole-genome DNA microarrays and a comprehensive genomic analysis of C. botulinum strains (26, 27). In this paper, we describe a comparative genomic hybridization (CGH) analysis of 32 Nordic group I C. botulinum type B cluster BI or BII strains with a DNA microarray based on the protein-coding sequences (CDS) in the ATCC 3502 genome. Strains within each cluster showed no substantial variation. Furthermore, strains belonging to the two clusters differed by their responses to 145 CDS probes, suggesting differential resistance to toxic compounds and a relatively large antigenic variability. Sequencing of botB in a representative cluster BI strain and a representative cluster BII strain revealed subtype B2 neurotoxin genes in both strains.     

Diversity of Proteolytic Clostridium botulinum Strains, Determined by a Pulsed-Field Gel Electrophoresis Approach

Pulsed-field gel electrophoresis (PFGE) was applied to the study of the similarity of 55 strains of proteolytic Clostridium botulinum (C. botulinum group I) types A, AB, B, and F. Rare-cutting restriction enzymes ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI were tested for their suitability for the cleavage of DNA of five proteolytic C. botulinum strains. Of these enzymes, SacII, followed by SmaI and XhoI, produced the most convenient number of fragments for genetic typing and were selected for analysis of the 55 strains. The proteolytic C. botulinum species was found to be heterogeneous. In the majority of cases, PFGE enabled discrimination between individual strains of proteolytic C. botulinum types A and B. The different toxin types were discriminated at an 86% similarity level with both SacII and SmaI and at an 83% similarity level with XhoI. Despite the high heterogeneity, three clusters at a 95% similarity level consisting of more than three strains of different origin were noted. The strains of types A and B showed higher diversity than the type F organisms which formed a single cluster. According to this survey, PFGE is to be considered a useful tool for molecular epidemiological analysis of proteolytic C. botulinum types A and B. However, epidemiological conclusions based on PFGE data only should be made with discretion, since highly similar PFGE patterns were noticed, especially within the type B strains.     

In Situ Detection of the Clostridium botulinum Type C1 Toxin Gene in Wetland Sediments with a Nested PCR Assay

A nested PCR was developed for detection of the Clostridium botulinum type C₁ toxin gene in sediments collected from wetlands where avian botulism outbreaks had or had not occurred. The C₁ toxin gene was detected in 16 of 18 sites, demonstrating both the ubiquitous distribution of C. botulinum type C in wetland sediments and the sensitivity of the detection assay.     

Plasmid-Borne Type E Neurotoxin Gene Clusters in Clostridium botulinum Strains

A collection of 36 Clostridium botulinum type E strains was examined by pulsed-field gel electrophoresis (PFGE) and Southern hybridization with probes targeted to botE and orfX1 in the neurotoxin gene cluster. Three strains were found to contain neurotoxin subtype E1 gene clusters in large plasmids of about 146 kb in size.     

Conserved structure of genes encoding components of botulinum neurotoxin complex M and the sequence of the gene coding for the nontoxic component in nonproteolyticClostridium botulinum type F

For investigation of the genes of proteins associated in vivo with botulinum neurotoxin (BoNT), polymerase chain reaction (PCR) experiments were carried out with oligonucleotide primers designed to regions of the nontoxic-nonhemagglutinin (NTNH) gene ofClostridium botulinum type C. The primers were used to amplify a DNA fragment from genomic DNA ofC. botulinum types A, B, E, F, G and toxigenic strains ofClostridium barati andClostridium butyricum. The amplified product from all of these strains hybridized with an internal oligonucleotide probe, whereas all nontoxigenic clostridia tested gave no PCR product and showed no reaction with the probe. TheNTNH gene was shown to be located upstream of the gene encoding BoNT, thereby revealing a conserved structure for genes encoding the proteins of the M complex of the progenitor botulinum toxin in these organisms. The sequence of theNTNH gene of nonproteolyticC. botulinum type F was determined by PCR amplification and sequencing of overlapping cloned fragments. NTNH/F showed 71% and 61% identity with NTNH ofC. botulinum type E and type C respectively.     

Inhibition of Clostridium botulinum by Strains of Clostridium perfringens Isolated from Soil

Thirty-one soil samples were examined for the presence of organisms capable of inhibiting growth and toxin production of strains of Clostridium botulinum type A. Such organisms were found in eight samples of soil. Inhibiting strains of C. perfringens were found in five samples, of C. sporogenes in three and of Bacillus cereus in three. Three of the C. perfringens strains produced an inhibitor effective on all 11 strains of C. botulinum type A against which they were tested, seven of eight proteolytic type B strains, one nonproteolytic type B strain, five of nine type E strains and all seven type F strains, whether proteolytic or nonproteolytic. They did not inhibit any of 26 type C strains, 6 type D strains, 4 type E strains, or 24 C. sporogenes strains. In mixed culture, an inhibitor strain of C. perfringens repressed growth and toxin production by a C. botulinum type A strain even though it was outnumbered by the latter about 40 times. It also repressed growth and toxin production of C. botulinum in mixed culture of soils in which this latter organism naturally occurred when cooked meat medium but not when trypticase medium was used.