Morphological and genetic characterization of group I Clostridium botulinum type B strain 111 and the transcriptional regulator spoIIID gene knockout mutant in sporulation.

Clostridium botulinum is a heat-resistant spore-forming bacterium that causes the serious paralytic illness botulism. Heat-resistant spores may cause food sanitation hazards and sporulation plays a central role in the survival of C. botulinum. We observed morphological changes and investigated the role of the transcriptional regulator SpoIIID in the sporulation of C. botulinum type B strain 111 in order to elucidate the molecular mechanism in C. botulinum. C. botulinum type B formed heat-resistant spores through successive morphological changes corresponding to those of Bacillus subtilis, a spore-forming model organism. An analysis of the spoIIID gene knockout mutant revealed that the transcriptional regulator SpoIIID contributed to heat-resistant spore formation by C. botulinum type B and activated the transcription of the sigK gene later during sporulation. Transcription of the spoIIID gene, which differed from that in B. subtilis and Clostridium difficile, was observed in the sigE gene knockout mutant of C. botulinum type B. An analysis of the sigF gene knockout mutant showed that the sporulation-specific sigma factor SigF was essential for transcription of the spoIIID gene in C. botulinum type B. These results suggest that the regulation of sporulation in C. botulinum is not similar to that in B. subtilis and other clostridia.

Complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in Clostridium botulinum type B strain 111 isolated from an infant patient in Japan.

Botulinum neurotoxins (BoNTs) are highly potent toxins that are produced by Clostridium botulinum. We determined the complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in C. botulinum type B strain 111 in order to obtain an insight into the toxigenicity and evolution of the bont gene in C. botulinum. Group I C. botulinum type B strain 111 was isolated from the first case of infant botulism in Japan in 1995. In previous studies, botulinum neurotoxin subtype B2 (BoNT/B2) produced by strain 111 exhibited different antigenic properties from those of authentic BoNT/B1 produced by strain Okra. We have recently shown that the isolates of strain 111 that lost toxigenicity were cured of the plasmid containing the bont/B2 gene. In the present study, the plasmid (named pCB111) was circular 265,575 bp double-stranded DNA and contained 332 predicted open reading frames (ORFs). 85 gene products of these ORFs could be functionally assigned on the basis of sequence homology to known proteins. The bont/B2 complex genes were located on pCB111 and some gene products may be involved in the conjugative plasmid transfer and horizontal transfer of bont genes. pCB111 was similar to previously identified plasmids containing bont/B1, /B5, or/A3 complex genes in other group I C. botulinum strains. It was suggested that these plasmids had been derived from a common ancestor and had played important roles for the bont gene transfer between C. botulinum.

Distinguishing highly-related outbreak-associated Clostridium botulinum type A(B) strains.

BACKGROUND: In the United States, most Clostridium botulinum type A strains isolated during laboratory investigations of human botulism demonstrate the presence of an expressed type A botulinum neurotoxin (BoNT/A) gene and an unexpressed BoNT/B gene. These strains are designated type A(B). The most common pulsed-field gel electrophoresis (PFGE) pattern in the C. botulinum PulseNet database is composed of A(B) strains. The purpose of this study was to evaluate the ability of genome sequencing and multi-loci variable number of tandem repeat analysis (MLVA) to differentiate such strains. RESULTS: The genome sequences of type A(B) strains evaluated in this study are closely related and cluster together compared to other available C. botulinum Group I genomes. In silico multilocus sequence typing (MLST) analysis (7-loci) was unable to differentiate any of the type A(B) strains isolated from seven different outbreak investigations evaluated in this study. A 15-locus MLVA scheme demonstrated an improved ability to differentiate these strains, however, repeat unit variation among the strains was restricted to only two loci. Reference-free single nucleotide polymorphism (SNP) analysis demonstrated the ability to differentiate strains from all of the outbreaks examined and a non-outbreak associated strain. CONCLUSIONS: This study confirms that type A(B) strains that share the same PFGE pattern also share closely-related genome sequences. The lack of a complete type A(B) strain representative genome sequence hinders the ability to assemble genomes by reference mapping and analysis of SNPs at pre-identified sites. However, compared to other methods evaluated in this study, a reference-free SNP analysis demonstrated optimal subtyping utility for type A(B) strains using de novo assembled genome sequences.

Multi-locus variable number tandem repeat analysis for Clostridium botulinum type B isolates in Japan: comparison with other isolates and genotyping methods.

Clostridium botulinum produces botulinum neurotoxin (BoNT) and causes botulism in humans and animals. Recently, 15-loci multi-locus variable number tandem repeat analysis (MLVA) for C. botulinum was developed for high-resolution and inter-lab comparative genotyping. This study examines the relation between MLVA and other genotyping methods such as pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), BoNT/B subtyping and bont/b gene location to evaluate MLVA as a method applicable to the genetic markers for C. botulinum type B. Japanese isolates were genotyped using MLVA and were compared with strains from other sources reported previously. Results show that the discriminatory power of MLVA was comparable to that of PFGE and higher than that of MLST. The topology of the minimum spanning tree (MST) constructed using MLVA data was very consistent with the phylogenetic classifications of PFGE and MLST. The MST topology also represented genetic diversity between the strains possessing bont/b gene on chromosomes and plasmids. Some Japanese isolates including those associated with infant botulism were inferred to be related to isolates of Europe origin from MLVA genotyping results. The MLVA scheme used for this study is apparently useful not only for high-resolution molecular typing, but also for phylogenetic characterization of C. botulinum type B.

Towards an international standard for detection and typing botulinum neurotoxin-producing Clostridia types A, B, E and F in food, feed and environmental samples: a European ring trial study to evaluate a real-time PCR assay.

A real-time PCR method for detection and typing of BoNT-producing Clostridia types A, B, E, and F was developed on the framework of the European Research Project "Biotracer". A primary evaluation was carried out using 104 strains and 17 clinical and food samples linked to botulism cases. Results showed 100% relative accuracy, 100% relative sensitivity, 100% relative specificity, and 100% selectivity (inclusivity on 73 strains and exclusivity on 31 strains) of the real-time PCR against the reference cultural method combined with the standard mouse bioassay. Furthermore, a ring trial study performed at four different European laboratories in Italy, France, the Netherlands, and Sweden was carried out using 47 strains, and 30 clinical and food samples linked to botulism cases. Results showed a concordance of 95.7% among the four laboratories. The reproducibility generated a relative standard deviation in the range of 2.18% to 13.61%. Considering the high level of agreement achieved between the laboratories, this real-time PCR is a suitable method for rapid detection and typing of BoNT-producing Clostridia in clinical, food and environmental samples and thus support the use of it as an international standard method.

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.

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.