Efficient DNA fingerprinting of Clostridium botulinum types A, B, E, and F by amplified fragment length polymorphism analysis.
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Amplified fragment length polymorphism (AFLP) analysis was applied to characterize 33 group I and 37 group II Clostridium botulinum strains. Four restriction enzyme and 30 primer combinations were screened to tailor the AFLP technique for optimal characterization of C. botulinum. The enzyme combination HindIII and HpyCH4IV, with primers having one selective nucleotide apiece (Hind-C and Hpy-A), was selected. AFLP clearly differentiated between C. botulinum groups I and II; group-specific clusters showed <10% similarity between proteolytic and nonproteolytic C. botulinum strains. In addition, group-specific fragments were detected in both groups. All strains studied were typeable by AFLP, and a total of 42 AFLP types were identified. Extensive diversity was observed among strains of C. botulinum type E, whereas group I had lower genetic biodiversity. These results indicate that AFLP is a fast, highly discriminating, and reproducible DNA fingerprinting method with excellent typeability, which, in addition to its suitability for typing at strain level, can be used for C. botulinum group identification. (+info)
Expression of botulinum neurotoxins A and E, and associated non-toxin genes, during the transition phase and stability at high temperature: analysis by quantitative reverse transcription-PCR.
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Production of botulinum neurotoxin A (BoNT/A) and associated non-toxic proteins (ANTPs), which include a non-toxic non-haemagglutinin (NTNH/A) as well as haemagglutinins (HAs), was found previously to be dependent upon an RNA polymerase alternative sigma factor (BotR/A). Expression of the botR/A, bont/A and antp genes, monitored by reverse transcription and real-time PCR analysis, occurred concomitantly at the transition between the exponential and stationary growth phases of Clostridium botulinum A. The botR/A expression level was about 100-fold less than those of the bont/A and antp genes. Therefore, BotR/A is an alternative sigma factor controlling the botulinum A locus genes during the transition phase. The highest toxin concentration was released into the culture supernatant 12 h after maximum expression of the botR/A, bont/A and antp genes, without any apparent bacterial lysis. Toxin levels were then stable over 5 days in cultures at 37 degrees C, whereas a dramatic decrease in lethal activity was observed between 24 and 48 h in cultures at 44 degrees C. High temperature did inhibit transcription, since expression levels of the botR/A, bont/A and antp genes were similar in cultures at 37 and 44 degrees C. However, incubation at 44 degrees C triggered a calcium-dependent protease that degraded BoNT/A and NTNH/A, but not HAs. In C. botulinum E, which contains no gene related to botR, the bont/E and p47 genes were also expressed during the transition phase, and no protease activation at 44 degrees C was evident. (+info)
Comparison of DNA fingerprinting methods for use in investigation of type E botulism outbreaks in the Canadian Arctic.
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Pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) analysis, and automated ribotyping were compared for epidemiological typing of Clostridium botulinum type E using clinical and food isolates associated with four botulism outbreaks occurring in the Canadian Arctic. All type E strains previously untypeable by PFGE, even with the use of a formaldehyde fixation step, could be typed by the addition of 50 microM thiourea to the electrophoresis running buffer. Digestion with SmaI or XhoI followed by PFGE was used to link food and clinical isolates from four different type E botulism outbreaks and differentiate them from among 39 group II strains. Strain differentiation was unsuccessful with the automated ribotyping system, producing a single characteristic EcoRI fingerprint common to all group II strains. RAPD analysis of C. botulinum group II strains was not consistently reproducible with primer OPJ-6 or OPJ-13, apparently discriminating between epidemiologically related strains. A modified PFGE protocol was judged to be the most useful method for typing epidemiologically related C. botulinum type E strains, based on its ability to type all strains reproducibly and with an adequate level of discrimination. (+info)
Outbreak of clinically mild botulism type E illness from home-salted fish in patients presenting with predominantly gastrointestinal symptoms.
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Five persons consumed home-salted fish and then presented with gastrointestinal symptoms to 3 hospitals; 2 of the patients had minimal cranial nerve palsies. Early serum samples obtained from all patients were negative for botulinum toxin. Remnant fish tested positive for botulinum toxin type E. In patients exposed to low doses of botulinum toxin type E, gastrointestinal symptoms may predominate. (+info)
First case of type E wound botulism diagnosed using real-time PCR.
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Wound botulism is a growing problem among injecting drug users. The condition is often difficult to diagnose, with laboratory confirmation in only 50% of the cases. Here we present a real-time PCR-based method for the diagnosis of wound botulism caused by Clostridium botulinum. The assay includes an internal amplification control which is amplified simultaneously with the genes encoding neurotoxin types A, B, and E. This method was used to detect the first case of wound botulism in an injecting drug user in Sweden. In addition, to the best of our knowledge, this is the first reported case of wound botulism caused by C. botulinum type E. (+info)
Development of enrichment semi-nested PCR for Clostridium botulinum types A, B, E, and F and its application to Korean environmental samples.
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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. (+info)
Effects of carbon dioxide on neurotoxin gene expression in nonproteolytic Clostridium botulinum Type E.
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