Clostridium botulinum Group I Strain Genotyping by 15-Locus Multilocus Variable-Number Tandem-Repeat Analysis

Abstract

Clostridium botulinum is a taxonomic designation that encompasses a broad variety of spore-forming, Gram-positive bacteria producing the botulinum neurotoxin (BoNT). C. botulinum is the etiologic agent of botulism, a rare but severe neuroparalytic disease. Fine-resolution genetic characterization of C. botulinum isolates of any BoNT type is relevant for both epidemiological studies and forensic microbiology. A 10-locus multiple-locus variable-number tandem-repeat analysis (MLVA) was previously applied to isolates of C. botulinum type A. The present study includes five additional loci designed to better address proteolytic B and F serotypes. We investigated 79 C. botulinum group I strains isolated from human and food samples in several European countries, including types A (28), B (36), AB (4), and F (11) strains, and 5 nontoxic Clostridium sporogenes. Additional data were deduced from in silico analysis of 10 available fully sequenced genomes. This 15-locus MLVA (MLVA-15) scheme identified 86 distinct genotypes that clustered consistently with the results of amplified fragment length polymorphism (AFLP) and MLVA genotyping in previous reports. An MLVA-7 scheme, a subset of the MLVA-15, performed on a lab-on-a-chip device using a nonfluorescent subset of primers, is also proposed as a first-line assay. The phylogenetic grouping obtained with the MLVA-7 does not differ significantly from that generated by the MLVA-15. To our knowledge, this report is the first to analyze genetic variability among all of the C. botulinum group I serotypes by MLVA. Our data provide new insights into the genetic variability of group I C. botulinum isolates worldwide and demonstrate that this group is genetically highly diverse.

Multilocus variable-number of tandem repeat analysis (MLVA) for Clostridium tyrobutyricum strains isolated from cheese production environment

“…MLVA is thus a DNA typing technique that is simple, rapid, and highly reproducible. Although earlier studies using MLVA have extensively examined pathogens such as Staphylococcus aureus (Sabat et al, 2003), Listeria monocytogenes (Murphy et al, 2007;Miya et al, 2008), and C. botulinum (Fillo et al, 2011), reports on food spoilage organisms other than Geobacillus species (Seale et al, 2012) and Bacillus licheniformis (Dhakal et al, 2013), which are isolated from milk powders, are scarce.…”

Overview of molecular typing methods for outbreak detection and epidemiological surveillance

“…In 2005, Francois and colleagues [30] reported on the use of automated microfluidic electrophoresis with the Agilent 2100 bioanalyzer 'lab-on-achip' for the VNTR typing of S. aureus isolates. Since then, there have been a growing number of studies that have shown the clear advantage of microfluidic chips over the standard agarose gels for the MLVA/MLVF typing in terms of electrophoretic separation resolution, reproducibility, rapidity and automated data analysis [31,32].…”

Use of multilocus variable-number tandem repeat analysis (MLVA) in eight European countries, 2012

“…The analysis was extended to B and F toxin serotype strains, in addition to A serotype strains: five newly characterised MLVA loci were added to the previous 10-MLVA scheme and new groups were described. To date, MLVA data have been obtained for about 300 international C. botulinum strains, whereas profiles from 79 strains across Europe have been published [40].…”