Isolation and characterisation of neurotoxigenic Clostridium butyricum from soil in China

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.

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confidence: 99%

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confidence: 99%

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

Wang

Maegawa

Karasawa

et al. 2000

“…In Italy, the toxigenic C. butyricum type E strains have been associated with infant cases, while the toxigenic C. butyricum type E strains in China have been associated either with food-borne botulism in food products such as fermented soybean paste or with environmental samples (2,20,21,27). Comparison of C. butyricum type E strains by various methods, including randomly amplified polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and bont/E gene sequencing, divided the strains into three clusters: strains associated with (i) infant botulism cases in Italy, (ii) food-borne botulism cases in China, and (iii) soil specimens from the Weishan Lake area in China (22). The results showed the conservation among the C. butyricum type E strains, suggesting a clonal distribution (27).…”

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confidence: 99%

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

MacDonald

Helma

Shou

et al. 2011

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.

“…C. botulinum and neurotoxigenic C. butyricum share the ability to produce botulinum neurotoxin E, while both C. botulinum and neurotoxigenic C. baratii can produce botulinum neurotoxin F; the differentiation of the three species by conventional methods is rather difficult (13,23). Neurotoxigenic C. butyricum type E has been implicated in infectious botulism (2,10) and has recently been shown to be an emergent food-borne pathogen (1,25,26,28); indeed, like C. botulinum, it can grow and produce toxin in food under favorable conditions (1). In Italy, C. butyricum type E appears to be more widespread than C. botulinum type E, based on isolation from several cases of botulism (9).…”

Section: Resultsmentioning

confidence: 99%

“…Neurotoxigenic C. butyricum type E strains JP/LCL155 and JP/KZ1890 were kindly provided by Shinichi Nakamura, Kanazawa University, Japan: they had been isolated from food (JP/LCL155) and soil (JP/KZ1890) samples isolated during investigation of two different outbreaks of food-borne botulism in China (25,26). The remaining C. butyricum type E strains had been isolated in Italy from clinical samples from patients suffering from botulism (1,2,10).…”

Section: Methodsmentioning

confidence: 99%

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

Franciosa

Pourshaban

Luca

et al. 2004

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.