Open AccessAnalysis 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
Author(s) -
Thomas MacDonald,
Charles H. Helma,
Yulin Shou,
Yolanda E. Valdez,
Lawrence O. Ticknor,
Brian Foley,
Stephen W. Davis,
George E. Hannett,
Cassandra Kelly-Cirino,
Jason R. Barash,
Stephen S. Ar,
Miia Lindström,
Hannu Korkeala,
Leonard A. Smith,
Theresa Smith,
Karen K. Hill
Publication year - 2011
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.05155-11
Subject(s) - multilocus sequence typing , biology , genetics , clostridium botulinum , amplified fragment length polymorphism , botulism , sequence analysis , gene , microbiology and biotechnology , genotype , genetic diversity , toxin , population , demography , sociology
A total of 41Clostridium 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 recombinaserarA , involved withbont /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 thebont /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, orbont gene sequencing were further examined using three VNTR regions. Both intact and splitrarA 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) theC. botulinum serotype E strains result from the targeted insertion of thebont /E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) withinbont /E result in toxin variants or subtypes within strains.