Genetic Homogeneity of Clostridium botulinum Type A1 Strains with Unique Toxin Gene Clusters (original) (raw)
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Genetic Homogeneity of Clostridium botulinum Type A1 Strains with Unique Toxin Gene Clusters
Applied and Environmental Microbiology, 2008
A group of five clonally related Clostridium botulinum type A strains isolated from different sources over a period of nearly 40 years harbored several conserved genetic properties. These strains contained a variant bont/A1 with five nucleotide polymorphisms compared to the gene in C. botulinum strain ATCC 3502. The strains also had a common toxin gene cluster composition (ha؊/orfX؉) similar to that associated with bont/A in type A strains containing an unexpressed bont/B [termed A(B) strains]. However, bont/B was not identified in the strains examined. Comparative genomic hybridization demonstrated identical genomic content among the strains relative to C. botulinum strain ATCC 3502. In addition, microarray data demonstrated the absence of several genes flanking the toxin gene cluster among the ha؊/orfX؉ A1 strains, suggesting the presence of genomic rearrangements with respect to this region compared to the C. botulinum ATCC 3502 strain. All five strains were shown to have identical flaA variable region nucleotide sequences. The pulsed-field gel electrophoresis patterns of the strains were indistinguishable when digested with SmaI, and a shift in the size of at least one band was observed in a single strain when digested with XhoI. These results demonstrate surprising genomic homogeneity among a cluster of unique C. botulinum type A strains of diverse origin. Clostridium botulinum is a gram-positive anaerobic bacterium that is identified by the production of botulinum neurotoxin (BoNT). BoNTs are extremely potent neurotoxins that induce flaccid paralysis and are potentially fatal because they can cause respiratory failure. There are seven serological types of BoNT (A to G) that are defined by the ability of serotypespecific equine antitoxins to neutralize the BoNT and prevent signs of botulism in the mouse bioassay. The species C. botulinum is genetically diverse, containing four phylogenetic groups that have been identified based on 16S rRNA nucleotide sequence comparison (2). These groupings also coincide with specific metabolic differences. Group I strains are proteolytic and produce BoNT types A, B, and F. This group also includes Clostridium sporogenes, which is a nontoxic species that metabolically resembles C. botulinum. Group II contains strains that are nonproteolytic and produce BoNT types B, E, and F. Group III strains produce BoNT types C and D and typically cause botulism in animals. Finally, group IV strains produce the rare toxin type G and are generally referred to as C. argentinense. Rarely, strains of Clostridium butyricum and Clostridium baratii produce BoNT types E and F, respectively (5, 12). The nucleotide sequences of the genes encoding BoNT/A-G differ by as much as 50%. Recently, Hill et al. (6) reported differences in the nucleotide sequence of the bont gene among type A, B, and E strains. Based on phylogenetic analysis of the bont/A sequence, four subtypes were identified (A1 to A4).
Genetic Diversity among Clostridium botulinum Strains Harboring bont/A2 and bont/A3 Genes
Applied and Environmental Microbiology, 2012
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.
Applied and environmental microbiology, 2004
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 ge...
Applied and Environmental Microbiology, 2008
Ten variable-number tandem-repeat (VNTR) regions identified within the complete genomic sequence of Clostridium botulinum strain ATCC 3502 were used to characterize 59 C. botulinum strains of the botulism neurotoxin A1 (BoNT/A1) to BoNT/A4 (BoNT/A1-A4) subtypes to determine their ability to discriminate among the serotype A strains. Two strains representing each of the C. botulinum serotypes B to G, including five bivalent strains, and two strains of the closely related species Clostridium sporogenes were also tested. Amplified fragment length polymorphism analyses revealed the genetic diversity among the serotypes and the high degree of similarity among many of the BoNT/A1 strains. The 10 VNTR markers amplified fragments within all of the serotype A strains but were less successful with strains of other serotypes. The composite multiple-locus VNTR analysis of the 59 BoNT/A1-A4 strains and 3 bivalent B strains identified 38 different genotypes. Thirty genotypes were identified among the 53 BoNT/A1 and BoNT/A1(B) strains, demonstrating discrimination below the subtype level. Contaminating DNA within crude toxin preparations of three BoNT/A subtypes (BoNT/A1 to BoNT/A3) also supported amplification of all of the VNTR regions. These markers provide clinical and forensics laboratories with a rapid, highly discriminatory tool to distinguish among C. botulinum BoNT/A1 strains for investigations of botulism outbreaks.
Applied and environmental microbiology, 2014
Genetic characterization was performed for 10 group I Clostridium botulinum strains isolated from botulism cases in Japan between 2006 and 2011. Of these, 1 was type A, 2 were type B, and 7 were type A(B) {carrying a silent bont/B [bont/(B)] gene} serotype strains, based on botulinum neurotoxin (BoNT) production. The type A strain harbored the subtype A1 BoNT gene (bont/A1), which is associated with the ha gene cluster. The type B strains carried bont/B5 or bont/B6 subtype genes. The type A(B) strains carried bont/A1 identical to that of type A(B) strain NCTC2916. However, bont/(B) genes in these strains showed single-nucleotide polymorphisms (SNPs) among strains. SNPs at 2 nucleotide positions of bont/(B) enabled classification of the type A(B) strains into 3 groups. Pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat analysis (MLVA) also provided consistent separation results. In addition, the type A(B) strains were separated into 2 lineages ba...
Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 2014
The whole genomes for six botulinum neurotoxin-producing clostridial strains were sequenced to provide references for under-represented toxin types, bivalent strains or unusual toxin complexes associated with a bont gene. The strains include three Clostridium botulinum Group I strains (CDC 297, CDC 1436, and Prevot 594), a Group II C. botulinum strain (Eklund 202F), a Group IV Clostridium argentinense strain (CDC 2741), and a Group V Clostridium baratii strain (Sullivan). Comparisons of the Group I genomic sequences revealed close relationships and conservation of toxin gene locations with previously published Group I C. botulinum genomes. The bont/F6 gene of strain Eklund 202F was determined to be a chimeric toxin gene composed of bont/F1 and bont/F2. The serotype G strain CDC 2741 remained unfinished in 20 contigs with the bont/G located within a 1.15Mb contig, indicating a possible chromosomal location for this toxin gene. Within the genome of C. baratii Sullivan strain, direct r...
Pulsotype Diversity of Clostridium botulinum Strains Containing Serotypes A and/or B Genes
Foodborne pathogens and disease, 2017
Clostridium botulinum strains are prevalent in the environment and produce a potent neurotoxin that causes botulism, a rare but serious paralytic disease. In 2010, a national PulseNet database was established to curate C. botulinum pulsotypes and facilitate epidemiological investigations, particularly for serotypes A and B strains frequently associated with botulism cases in the United States. Between 2010 and 2014 we performed pulsed-field gel electrophoresis (PFGE) using a PulseNet protocol, uploaded the resulting PFGE patterns into a national database, and analyzed data according to PulseNet criteria (UPGMA clustering, Dice coefficient, 1.5% position tolerance, and 1.5% optimization). A retrospective data analysis was undertaken on 349 entries comprised of type A and B strains isolated from foodborne and infant cases to determine epidemiological relevance, resolution of the method, and the diversity of the database. Most studies to date on the pulsotype diversity of C. botulinum ...
Journal of Clinical Microbiology, 2011
Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse.