The term “toxin subtype” has been applied to strains producing toxins with multiple antigenic components (e.g., Af) and more recently to different clades of highly similar neurotoxin gene sequences encoding specific toxin serotypes (e.g., A1 to A5) (11
). In 2005, Smith et al. (37
) reported that among the 49 complete neurotoxin sequences (A through G) available at that time, the lowest level of amino acid sequence variability within a serotype was 2.6%. In that study, the minimum level of BoNT/F amino acid sequence differences among type F strains (including proteolytic, nonproteolytic, and bivalent C. botulinum
as well as C. baratii
strains) was 10.7%, indicating that all of the sequences were different subtypes. Hill et al. (20
) examined the diversity of neurotoxin genes within serotypes A, B, and E and clustered toxin genes into subtypes by using a phylogenetic approach. The levels of subtype nucleotide sequence variation differ among serotypes. For instance, the nucleotide sequences of subtypes E1 to E5 differ by up to 3%, while subtypes A1 to A5 vary by up to 8% (3
The objective of this study was to define the sequence diversity of type F botulinum neurotoxin genes found in strains isolated from different sources and geographical origins. Although type F strains are infrequently isolated from individuals with botulism and have a low prevalence in the environment, a surprisingly high level of diversity was found among bont/F genes, resulting in the identification of seven subtypes (F1 to F7). These subtypes differed from each other by up to 25% at the nucleotide level, indicating that serotype F strains contain the highest level of nucleotide sequence diversity reported to date. Although a high level of genetic diversity exists among bont/F genes, the phylogenetic diversity of organisms harboring subtypes F1 to F7 is more limited. Strains harboring subtypes F1 to F5 belonged to C. botulinum group I, while subtypes F6 and F7 were found in C. botulinum group II and C. baratii strains, respectively.
Subtypes F1 and F6 contain strains isolated from food associated with human botulism. Strain Langeland was isolated from liver paste, and VPI7943 was isolated from venison jerky (29
). Remarkably, both subtypes F1 and F6 contain several strains isolated from marine environments.
The F2 and F3 subtypes share the highest level of nucleotide sequence identity. Subtype F2 contains nearly identical bont/F
genes from bivalent (Bf) strains (CDC4013 and CDC3281) which were isolated from different infants with botulism (16
). Subtype F3 strains are monovalent and contain bont/F
genes from two different strains (CDC54086 and VPI4257) isolated from soil separated by 400 miles in Buenos Aires province (Argentina). The BoNT/F amino acid sequences of the representative subtype F2 strain (CDC4013) differed from those of the subtype F3 strains (CDC54086 and VPI4257) by 2.9%.
All but one of the subtype F4 strains was isolated from Argentina. Strain CDC49930 was isolated from honey that was examined during an investigation of a laboratory-confirmed (type A) infant botulism case in Ohio. Since the toxin serotypes of the organism isolated from the infant and the isolate from the honey were different, the honey was not the source of infant botulism in this case. Subtype F4 also includes strains isolated from soil, dust, chamomile, and canned anchovies (not associated with a botulism case). The strains isolated from soil (CDC54076, CDC54091, CDC54093) originated in Mendoza province, which included locations with different types of land use (cultivated, uncultivated, etc.).
All of the subtype F5 strains were isolated from Argentina. These strains included those isolated from the stools of different individuals with botulism (CDC54090, CDC54096) as well as from soil. Notably, the strains isolated from soil originated in different locations in Mendoza and Tucuman provinces.
We examined five C. baratii strains isolated from separate botulism cases. Four of the bont/F7 genes which were isolated from C. baratii cases occurring in the United States had identical nucleotide sequences. One isolate from food associated with a botulism outbreak occurring in Thailand harbored a bont/F7 gene that differed from the others by <1%.
Botulinum neurotoxins are composed of a 50-kDa light chain and a 100-kDa heavy chain. The heavy chain possesses a binding domain that enables binding and uptake of the toxin into the neuronal cell. The light chain, which possesses endopeptidase activity, cleaves specific proteins associated with synaptic vesicle docking at the membrane of the host cell. Comparison of the amino acid sequences of the light chain and heavy chain of BoNT/F1 to -F7 revealed that the light chain of BoNT/F5 was highly divergent compared to those of the other subtypes. Although known functional domains of BoNT/F5 are intact, the activity of the light chain in this subtype compared to that of the others remains to be determined. Two lines of evidence indicate that the light chain is functional: (i) the F5 strain CDC54090 was isolated from an individual with botulism, and (ii) F5 strains produced culture supernatants that required neutralization with F antitoxins in the mouse bioassay.
The heavy chain of BoNT/F5 is most similar to that of BoNT/F2 (89.1% identity). As a result, BoNT/F5 appears to be a hybrid composed of a unique light chain and an F2-like heavy chain. Therefore, we sequenced the ntnh/F genes in representative monovalent and bivalent (Af) subtype F5 strains to determine if there was evidence of recombination in this gene, which is located directly upstream of bont/F. While ntnh/F genes found in three subtype F5 strains (CDC54085, CDC54090, and CDC54096) were identical, the gene in strain CDC54079 differs from the others by approximately 4%. This finding is surprising as the bont/F5 genes are 100% identical to each other.
We also sequenced the ntnh/A
gene from Af strains CDC54079 and CDC54096. The ntnh/F
gene in strain CDC54079 was nearly identical to the ntnh/A
gene until nucleotide position 3418. At this position, the ntnh/F
sequence in strain CDC54079 becomes 100% identical with the gene found in strains CDC54085, CDC54090, and CDC54096 through the stop codon. These results indicate that a similar recombination event likely occurred in the 3′ end of the ntnh/F
genes in all of the subtype F5 strains examined. Similarly, the ntnh/F
genes from strains Langeland and Kyoto, respectively, share a high degree of nucleotide identity but have divergent 3′ termini followed by different neurotoxin genes (8
Further work is needed to define the genotypic relationships of strains within and among the F subtypes identified. Nevas et al. (31
) demonstrated that three distinct proteolytic (group I) type F strains had indistinguishable SacII pulsed-field gel electrophoresis (PFGE) patterns, while another study (19
) indicated that two out of three nonproteolytic (group II) strains had indistinguishable SmaI PFGE profiles. It is possible that the level of genotypic diversity among strains harboring different bont/F
The extent of the diversity in bont/F nucleotide sequences determined in this study underscores the importance of examining C. botulinum strains isolated from multiple sources and geographical locations. These findings have particular impact for the design of detection assays for both the bont/F gene and type F botulinum neurotoxin.