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

Abstract

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.

An Atypical Outbreak of Food-Borne Botulism Due to Clostridium botulinum Types B and E from Ham

“…3B). Strain 84-10, as well as the other C. botulinum type E strains, seems to have derived from an ancestral strain related to C. botulinum type B by insertion of the bont/E gene cluster and an intact rarA gene into the rarA operon as previously reported (7,12,13). However, the botulinum locus genes of strain 84-10 as well as the flanking genes showed sequence variations with the corresponding genes of Alaska E43 (Fig.…”

“…Preimmune rabbit sera and immunopurified antibodies against rHc proteins of BoNT/A1 and BoNT/B2 are included as controls. geographic localization or source of the strains, at least in northern regions (13). Thus, the genetic variation of the strain 84-10 cannot give useful information on its origin.…”

“…C. botulinum type E strains show a high genetic variation, as tested by pulsedfield gel electrophoresis and randomly amplified polymorphic DNA (RAPD) (14,15). A previous analysis by MLST indicates that C. botulinum type E strains are split into 4 clades, with most strains belonging to the same clade (13). Compared to the previous C. botulinum type E strains analyzed by MLST (13), 14 of 15 housekeeping gene sequences of strain 84-10 show from 1 to more than 20 single nucleotide polymorphisms (SNP) with the most related C. botulinum strains and are considered new alleles (see Table S1).…”

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

“…This heterogeneity led to the introduction of different subtypes for a given serotype, which vary up to 36 % at the amino acid level. Until today, six subtypes of serotype A have been described (A1-A6), seven subtypes of serotype B (B1-B7), eight subtypes of serotype E (E1-E8), and seven subtypes of serotype F (F1-F7), and more are still to be expected (Hill et al 2007;Lúquez et al 2009;Umeda et al 2009;Raphael et al 2010a;Macdonald et al 2011;Kalb et al 2012a). The differences within the subtypes of a given serotype are greatest in serotype A (16 %) and serotype F (36 %) as compared to serotype B (7 %) and serotype E (6 %) at the amino acid level.…”

Restriction Fragment Length Polymorphism Analysis of PCR-Amplified Fragments (PCR-RFLP) and Gel Electrophoresis - Valuable Tool for Genotyping and Genetic Fingerprinting

“…AFLP has been used for a variety of purposes including genomic profiling of bacteria (Macdonald et al 2011), phylogenetic analysis (Arrigo et al, 2011, quantitative trait locus mapping (Heidari et al, 2011), and population genetics (Chybicki et al, 2011). A major disadvantage of AFLP is that it is relatively laborious.…”