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Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis
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Clostridium botulinum group I strain genotyping by 15-locus multilocus variable-number tandem-repeat analysis.

Silvia Fillo1, Francesco Giordani, Fabrizio Anniballi

  • 1Histology and Molecular Biology Section, Army Medical & Veterinary Research Centre, Via Santo Stefano Rotondo 4, 00184 Rome, Italy.

Journal of Clinical Microbiology
|October 21, 2011
PubMed
Summary

This study introduces a new 15-locus MLVA method to genetically characterize Clostridium botulinum strains. The developed MLVA scheme effectively differentiates diverse Clostridium botulinum group I serotypes, crucial for epidemiology and forensic microbiology.

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Area of Science:

  • Microbiology
  • Genetics
  • Epidemiology

Background:

  • Clostridium botulinum produces botulinum neurotoxin (BoNT), causing botulism, a severe neuroparalytic disease.
  • Accurate genetic characterization of C. botulinum is vital for epidemiological surveillance and forensic investigations.
  • Previous methods, like a 10-locus MLVA, were limited in resolving diverse serotypes.

Purpose of the Study:

  • To develop and validate an enhanced multiple-locus variable-number tandem-repeat analysis (MLVA) scheme for high-resolution genetic typing of Clostridium botulinum group I.
  • To assess the genetic diversity across different BoNT serotypes (A, B, F) within C. botulinum group I.
  • To propose a simplified MLVA assay for rapid, first-line screening.

Main Methods:

  • A 15-locus MLVA scheme (MLVA-15) was designed, incorporating five additional loci to a previous 10-locus system.
  • The MLVA-15 scheme was applied to 79 C. botulinum group I strains (serotypes A, B, AB, F) from European human and food samples, plus 5 C. sporogenes strains.
  • In silico analysis of 10 fully sequenced genomes and development of a lab-on-a-chip MLVA-7 assay were also performed.

Main Results:

  • The MLVA-15 scheme successfully identified 86 distinct genotypes among the analyzed C. botulinum group I strains.
  • Genotyping results showed strong concordance with previous amplified fragment length polymorphism (AFLP) and MLVA data.
  • A subset MLVA-7 scheme demonstrated comparable phylogenetic grouping to MLVA-15, suitable for a first-line assay.

Conclusions:

  • The developed MLVA-15 scheme provides a robust tool for fine-resolution genetic characterization of all C. botulinum group I serotypes.
  • This study reveals significant genetic diversity within C. botulinum group I isolates globally.
  • The findings enhance our understanding of C. botulinum population structure, aiding epidemiological and forensic applications.