ABSTRACT
A nontoxigenic strain isolated from a fatal human case of bacterial sepsis was identified as a Clostridium strain from Clostridium botulinum group III, based on the phenotypic characters and 16S rRNA gene sequence, and was found to be related to the mosaic C. botulinum D/C strain according to a multilocus sequence analysis of 5 housekeeping genes.
Subject(s)
Blood/microbiology , Clostridium botulinum type C/isolation & purification , Clostridium botulinum type D/isolation & purification , Sepsis/diagnosis , Sepsis/microbiology , Aged, 80 and over , Bacterial Proteins/genetics , Clostridium botulinum type C/classification , Clostridium botulinum type C/genetics , Clostridium botulinum type D/classification , Clostridium botulinum type D/genetics , Cluster Analysis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Fatal Outcome , Female , Humans , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNAABSTRACT
Two real-time PCR arrays based on the GeneDisc(®) cycler platform (Pall-GeneDisc Technologies) were evaluated in a multicenter collaborative trial for their capacity to specifically detect and discriminate Clostridium botulinum types C, D and their mosaic variants C-D and D-C that are associated with avian and mammalian botulism. The GeneDisc(®) arrays developed as part of the DG Home funded European project 'AnibioThreat' were highly sensitive and specific when tested on pure isolates and naturally contaminated samples (mostly clinical specimen from avian origin). Results of the multicenter collaborative trial involving eight laboratories in five European Countries (two laboratories in France, Italy and The Netherlands, one laboratory in Denmark and Sweden), using DNA extracts issued from 33 pure isolates and 48 naturally contaminated samples associated with animal botulism cases, demonstrated the robustness of these tests. Results showed a concordance among the eight laboratories of 99.4%-100% for both arrays. The reproducibility of the tests was high with a relative standard deviation ranging from 1.1% to 7.1%. Considering the high level of agreement achieved between the laboratories these PCR arrays constitute robust and suitable tools for rapid detection of C. botulinum types C, D and mosaic types C-D and D-C. These are the first tests for C. botulinum C and D that have been evaluated in a European multicenter collaborative trial.
Subject(s)
Botulism/diagnosis , Botulism/microbiology , Clostridium botulinum type C/classification , Clostridium botulinum type C/genetics , Clostridium botulinum type D/classification , Clostridium botulinum type D/genetics , Real-Time Polymerase Chain Reaction/methods , Animals , Clostridium botulinum type C/isolation & purification , Clostridium botulinum type D/isolation & purification , Europe , Humans , Reproducibility of ResultsABSTRACT
Type C botulinum neurotoxin (BoNT/C)-producing Clostridium botulinum causes animal botulism worldwide and has become a serious problem in poultry flocks and waterfowl in Sweden. The objectives of the present study were to isolate, characterize and subtype C. botulinum type C avian isolates in order to increase the knowledge of the genetic diversity. Isolates from 13 birds were identified by 16S rRNA sequencing and BoNT/C gene detection by real-time polymerase chain reaction (PCR). Conventional PCR was used to distinguish a chimeric BoNTC/D gene, often associated with avian botulism, from the BoNT/C gene. The isolates analysed all contained the gene coding for a chimeric toxin type C/D. Two fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA analysis (RAPD), were optimized and used to investigate the epidemiological relatedness among the strains. The isolates were divided into three different pulsotypes based upon their restriction profiles for SmaI and SalI. The RAPD system proved to be as discriminative as PFGE. This study reveals a small genetic diversity among Swedish type C strains, with a high similarity between strains from broilers and herring gulls.
