Diversity of Proteolytic Clostridium botulinum Strains, Determined by a Pulsed-Field Gel Electrophoresis Approach

Abstract

Pulsed-field gel electrophoresis (PFGE) was applied to the study of the similarity of 55 strains of proteolytic Clostridium botulinum (C. botulinum group I) types A, AB, B, and F. Rare-cutting restriction enzymes ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI were tested for their suitability for the cleavage of DNA of five proteolytic C. botulinum strains. Of these enzymes, SacII, followed by SmaI and XhoI, produced the most convenient number of fragments for genetic typing and were selected for analysis of the 55 strains. The proteolytic C. botulinum species was found to be heterogeneous. In the majority of cases, PFGE enabled discrimination between individual strains of proteolytic C. botulinum types A and B. The different toxin types were discriminated at an 86% similarity level with both SacII and SmaI and at an 83% similarity level with XhoI. Despite the high heterogeneity, three clusters at a 95% similarity level consisting of more than three strains of different origin were noted. The strains of types A and B showed higher diversity than the type F organisms which formed a single cluster. According to this survey, PFGE is to be considered a useful tool for molecular epidemiological analysis of proteolytic C. botulinum types A and B. However, epidemiological conclusions based on PFGE data only should be made with discretion, since highly similar PFGE patterns were noticed, especially within the type B strains.The most commonly reported forms of botulism are foodborne botulism, wound botulism, and infant botulism. Foodborne botulism is an intoxication caused by consumption of preformed botulinum neurotoxin, while wound and infant botulism are both infections in which neurotoxin is formed in vivo (13). Proteolytic Clostridium botulinum (C. botulinum group I) is one of six phylogenetically and physiologically distinct clostridia that forms botulinum neurotoxin. It is responsible for a majority of the cases of infant botulism and wound botulism as well as many cases of classical food-borne botulism (1,2,4,16,17). Nonproteolytic C. botulinum (C. botulinum group II) is responsible for most other cases of food-borne botulism (13). The confirmation of food-borne botulism outbreaks has been based on the detection of botulinum toxin or C. botulinum organisms of the same toxin type from both the patient and the suspected food item. In order to be able to confirm the epidemiological relatedness of the strains isolated from patients and from food, a method for genotyping proteolytic C. botulinum isolates is needed. Such a method would also be of utility when investigating cases of wound or infant botulism.Since the first paper on pulsed-field gel electrophoresis (PFGE) typing of C. botulinum type A strains Hall A and 62 A was published (10), no reports on the genetic diversity of strains of proteolytic of C. botulinum as determined by PFGE have been published. With nonproteolytic C. botulinum, a high genetic diversity determined by PFGE and randomly amplified polymorphic DNA (RAPD) analysis has been report...

Historical and current perspectives on Clostridium botulinum diversity

“…Pulsed-field gel electrophoresis (PFGE) has also provided a tool that can be used in multiple laboratories to characterize strains by comparison of DNA fragment sizes from enzyme digested genomic DNAs [63,97,136,130,116,88,8]. Standardization of this technique (for example, using the PulseNet protocol available at http://www.cdc.gov/pulsenet/PDF/cbotulinum-protocol-508c.pdf) has enabled comparisons of multiple strains representing different Groups of C. botulinum bacteria from various laboratories throughout the world, and this continues to be a primary technique for typing C. botulinum strains.…”

Clostridial neurotoxins

“…For example, among proteolytic C. botulinum strains, PFGE analysis differentiates the toxinotypes A, B, and F at a 83-86% similarity level, and enable discrimination of most of individual strains. A greater diversity was observed between type A strains than between type B strains [41]. These studies also indicate that each C. botulinum group is heterogeneous at the genome level.…”

“…Amplified fragment length polymorphism (AFLP) and pulsed-field gel electrophoresis (PFGE) analysis also confirm the classification of proteolytic types A, B, and F strains in group I and the nonproteolytic types B, E, and F strains in group II, but can differentiate individual strains into each group [18,[41][42][43][44][45]. These methods have been used in epidemiological studies and are useful tools to investigate relatedness between strains isolated from patients and food.…”

The Cold-Induced Two-Component System CBO0366/CBO0365 Regulates Metabolic Pathways with Novel Roles in Group I Clostridium botulinum ATCC 3502 Cold Tolerance

“…In our previous studies, an association between arsenic resistance and robustness toward low temperature was observed in C. botulinum (47,48). The Nordic (group I) C. botulinum type B strains form two distinct clusters BI and BII (49,50), which differ in their genomic content and, in particular, in their arsenical resistance operon structure (47). The lack of arsenic resistance-encoding genes in the cluster BI strains was consistent with significantly decreased robustness to the presence of sodium arsenite (47) or low temperature (48).…”