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Appl Environ Microbiol. Dec 2011; 77(24): 8625–8634.
PMCID: PMC3233090

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[down-pointing small open triangle]

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.

INTRODUCTION

Botulinum neurotoxins (BoNTs) consist of seven different serotypes (A to G) found within the genetically diverse bacterial species Clostridium botulinum. Of the seven serotypes, four BoNTs (A, B, E, and F) primarily cause human disease. Serotype A and B strains have been identified worldwide, with the exception of Antarctica, and cause food-borne, infant, and wound botulism. Serotype F strains share the same global distribution as serotypes A and B, but the incidence of botulism from BoNT/F is relatively rare by comparison. In contrast, C. botulinum type E strains have a limited geographical distribution and occur primarily in northern countries such as Canada, Finland, Japan, Norway, Sweden, Russia, and Alaska in the United States. Serotype E was first described in the 1930s in several reports: (i) in 1936 from sturgeon from the Sea of Azov, (ii) in 1937 in botulism cases due to salted seal meat near the Caspian Sea, and (iii) in 1938 in smoked salmon from Labrador (7, 8, 16). These initial cases associated type E botulism with marine mammals and fish in regions north of the 40° parallel. Subsequently, strains have been isolated from freshwater or saltwater sediments and from cases of food-borne, wound, and infant botulism (1, 5, 9, 18).

Unlike other C. botulinum serotypes, the BoNT/E-producing strains are universally nonproteolytic. The bont/E gene is expressed as a single polypeptide chain that requires cleavage with trypsin for complete activation. Type E strains ferment many carbohydrates and, like other nonproteolytic “group II” C. botulinum strains, grow and produce toxin at temperatures as low as 3°C (6). This attribute together with anaerobic growth makes the strains particularly hazardous for refrigerated vacuum-packaged foods.

The bont/E gene has also been discovered within Clostridium butyricum type E strains in Italy and China. In Italy, the toxigenic C. butyricum type E strains have been associated with infant cases, while the toxigenic C. butyricum type E strains in China have been associated either with food-borne botulism in food products such as fermented soybean paste or with environmental samples (2, 20, 21, 27). Comparison of C. butyricum type E strains by various methods, including randomly amplified polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and bont/E gene sequencing, divided the strains into three clusters: strains associated with (i) infant botulism cases in Italy, (ii) food-borne botulism cases in China, and (iii) soil specimens from the Weishan Lake area in China (22). The results showed the conservation among the C. butyricum type E strains, suggesting a clonal distribution (27).

Comparison of the bont/E gene nucleotide or derived amino acid sequences shows that the gene/protein is quite conserved despite its presence in the two species C. botulinum and C. butyricum. Among the six BoNT/E subtypes designated E1 to E6, amino acid differences range from 1.0% to 5.3% (4, 10).

Several molecular methods have been used to examine C. botulinum serotype E strains. These methods include PFGE of strains from the Baltic Sea and the arctic region of Canada, amplified fragment length polymorphism (AFLP) analysis, and RAPD analysis (9, 10, 12, 15, 17). The molecular characterizations illustrate the diversity of serotype E bacterial strains in comparison to other C. botulinum groups. In addition, the flagellin gene has been used as a marker for differentiating group I and group II strains (25).

Recently, whole-genome sequencing of two C. botulinum serotype E strains and one C. butyricum type E strain led to the discovery of a transposon-associated recombinase, rarA, near the bont/E gene cluster. The three sequenced genomes show the presence of an intact rarA gene and a rarA homolog split approximately in half, with the insertion of DNA that contains the bont/E gene (11). To determine whether this targeted insertion event has occurred in other C. botulinum serotype E strains, collaborators have shared their strains from different geographic regions and sources to enable their molecular characterization. A total of 41 C. botulinum serotype E strains were examined by multilocus sequence typing (MLST), AFLP, and variable-number tandem-repeat (VNTR) analysis and bont/E gene sequencing. The strains originate from food-borne botulism cases, an infant botulism case, and environmental samples isolated within Canada, Denmark, Finland, France, Greenland, Japan, and the United States. The results indicate that the C. botulinum serotype E strains appear genetically conserved and that recombination events within the bont/E gene have resulted in the genetic variation now described as toxin subtypes.

MATERIALS AND METHODS

Strains.

Type E DNA preparations were provided from the Lindström and Korkeala collection at the University of Helsinki, Finland (n = 8); the Infant Botulism Treatment and Prevention Program within the California Department of Public Health, Richmond, CA (n = 9); the Smith and Smith Collection at the Integrated Toxicology Division, United States Army Medical Institute of Infectious Diseases (USAMRIID), Fort Detrick, MD (n = 22); and the New York State Department of Health Wadsworth Center Strain Collection in Albany, NY (n = 2). The ability of each strain to produce botulinum neurotoxin was previously confirmed by the mouse bioassay or by enzyme-linked immunosorbent assay (ELISA). Table 1 lists the available information for each strain, representing environmental sources and food-borne and infant botulism cases from different geographic regions.

Table 1.
C. botulinum serotype E strains analyzed by different methods

MLST analysis.

Fifteen genes were selected for MLST analysis in the serotype E strains. Seven genes were included to complement a previous MLST study by Jacobson et al. (13), who performed MLST analysis within serotype A strains; eight other genes were selected because of their utility in distinguishing strains of other species within the MLST database at www.mlst.net.

The same primer sequences used to amplify the seven MLST genes in serotype A strains could not be used because of the sequence diversity between serotype A (group I) and serotype E (group II) strains (13). The primer sequences for both PCR amplification and amplicon sequencing in the serotype E strains were designed using the two completed genomes of C. botulinum serotype E strains (Beluga or E542, ACSC01000001-6; and Alaska E43 or E185, CP001078). The MLST genes and primer sequences are listed in Table 2.

Table 2.
MLST genes and primer sequences (5′-3′)a

Primers were designed using Oligo 6.8 (Molecular Biological Insights, Inc., Cascade, CO). PCR amplification was performed in separate reactions of a final volume of 20 μl, containing 1× PCR buffer, 2 mM MgCl2, 200 nM deoxynucleoside triphosphates (dNTPs), 0.025 U AmpliTaq Gold (Applied Biosystems Inc., Foster City, CA), 225 nM forward and reverse primers, and 1 ng of template DNA. Thermocycling conditions included an initial melt at 95°C for 10 min followed by 45 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final extension step of 72°C for 5 min. A 3-μl aliquot of the PCR mixture was loaded onto a 2% agarose gel with electrophoresis conditions of 80 V for 90 min followed by ethidium bromide staining and visualization using an Alpha Innotech UV transilluminator. PCR products were purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA) and then sequenced on an ABI 3730 automated fluorescent sequencer. Alignments were performed using Sequencher 4.9 (Gene Codes Corp., Ann Arbor, MI).

AFLP analysis.

AFLP reactions and analysis were performed as previously described (7). Briefly, DNA preparations were digested with two restriction endonucleases, EcoRI and MseI, and the resulting fragments were ligated to linkers. Dye-labeled primers homologous to the linkers were used to selectively amplify the fragments by PCR. The resulting products were mixed with DNA size standards and loaded onto an ABI 3130 genetic analyzer. Forty DNA fragments present in triplicate runs were used as a “fingerprint” to represent a strain. The AFLP-based dendrogram (see Fig. 2) was generated from the comparison of strain fingerprints/fragment sizes by using the Jaccard similarity index.

Fig. 2.
AFLP-based dendrogram of 41 C. botulinum serotype E strains and 2 C. butyricum type E strains. The dendrogram shows the clustering of 41 C. botulinum serotype E strains and their distant relationship to the two C. butyricum type E strains. The dendrogram ...

bont/E and rarA gene sequencing.

The full-length bont/E gene was amplified by PCR using six sets of previously published primer pairs that span bont/E (10). The PCR amplification and sequencing of the rarA gene used three sets of primers designed to detect the split rarA (5′ or 3′) or the intact rarA. An additional set of primers was designed to amplify the split rarA or intact rarA and their flanking region to ensure that the split rarA and intact rarA were in unique locations. The primers and their location within the sequenced genome C. botulinum Alaska E43 are provided in Table 3. Primers were designed using Oligo 6.8 (Molecular Biological Insights, Inc., Cascade, CO), and the conditions for PCR amplification and sequencing of the PCR products are as described for the MLST gene analysis. Each of the 41 strains was tested with the six primer sets, and the amplicons were sequenced to confirm the presence of the intact or partial gene in 11 strains: E134, E178, E183, E216, E546, IBCA07-0062, IBCA97-0192, K15, K35, K37, and K44.

Table 3.
rarA primer sequencesa

VNTR analysis.

The genomic sequences of two C. botulinum serotype E strains (Beluga, E42, ACSC01000001-6; and Alaska E43, E185, CP001078) were analyzed using the software Tandem Repeats Finder to identify VNTR regions (3). Three VNTR regions that discriminated among the strains were identified. The primers in Table 4 were designed using Oligo 6.8 and were synthesized (Invitrogen Corp., Carlsbad, CA) with a fluorescent 6-carboxyfluorescein (6-FAM) label on the forward primer. PCR amplification of the VNTR regions and fragment sizing on an ABI 3130 genetic analyzer were performed as previously described (19).

Table 4.
VNTR primer sequences and characteristicsa

Representatives of each fragment size were sequenced to verify the fragment length and tandem-repeat unit. Fragments amplified in PCR experiments were purified using a QIAquick PCR purification kit and then sequenced on an ABI 3730 automated fluorescent sequencer. Sequence alignments were performed using Sequencher 4.9 software.

The diversity index for each of the three VNTR loci was calculated for the strains, and the values are reported in Table 4 (28). The diversity index (D) for VNTR “l” was calculated as follows: Dl = 1 − uPlu2, where Plu2 is the frequency of the uth allele at the lth VNTR.

Nucleotide sequence accession numbers.

The MLST gene sequences have the following GenBank accession numbers (in parentheses): 16S rRNA genes (JN617053 to JN617093), 23S rRNA genes (JN617094 to JN617134), atpD (JN616520 to JN616560), guaA (JN616561 to JN616601), gyrB (JN616602 to JN616642), ilvD (JN616643 to JN616683), lepA (JN616684 to JN761624), mutL (JN616725 to JN616765), oppB (JN616766 to JN616806), pta (JN616807 to JN616847), pyc (JN616848 to JN616888), recA (JN616889 to JN616929), rpoB (JN616930 to JN616970), trpB (JN616971 to JN617011), and tuf (JN617012 to JN617052). The MLST dendrogram was generated by sequence alignments using Sequencher 4.9 (Gene Codes Corp., Ann Arbor, MI) and unweighted-pair group method using average linkages (UPGMA) analysis within PAUP 4.0 software (Sinauer Associates, Inc., Sunderland, MA). Two GenBank accession numbers (JN695729 and JN695730) represent the newly identified variants of bont/E7 and bont/E8.

RESULTS

Forty-one C. botulinum serotype E strains from different geographic regions, environmental sources, and food-borne or infant botulism cases were analyzed using different molecular methods. The various methods detect changes in (i) nucleotides within conserved genes (MLST) or within the bont gene, (ii) fragment sizes generated from restriction enzyme digestion (AFLP), or (iii) the number of repeated units at different sites within the genome (VNTR). The methods capture different rates of mutation, such as the slowly changing single nucleotide polymorphisms (SNPs) in conserved genes (MLST), the moderately changing SNPs within coding and noncoding regions in AFLP, and the more rapidly changing repeat units within VNTR regions (14).

Table 1 lists the 41 strains, their BoNT/E subtype designation, and other available information. The dates of isolation, or publication of the information, span the 75 years from 1932 to 2007. The strains were compared by MLST using 15 genes, AFLP analysis, VNTR regions, and full-length bont/E gene sequencing. In addition, to determine whether the serotype E strains may have resulted from a transposase insertion event, the rarA genes and their flanking regions were amplified by PCR in all strains and verified by sequencing in 11 strains. The results from analyzing the strains using the different methods are described below.

MLST.

Fifteen genes were selected from the whole-genome sequences of two serotype E C. botulinum strains (Beluga, E542, ACSC01000001-6; and Alaska E43, E185, CP001078) for MLST. A list of the genes and the primer sequences for PCR amplification is provided in Table 2. The genes included the 16S rRNA gene, which is fairly conserved but sometimes used for bacterial species identification. The number of SNPs from the other 14 MLST genes is listed in Table 2 and shows that the other genes offered greater discrimination among the strains.

The composite MLST dendrogram in Fig. 1 is based upon 188 SNPs within the 15 genes and illustrates that the 41 strains representing different geographic regions and spanning a 75-year period are conserved. Four clades can be distinguished based on branch length (nucleotide differences) in Fig. 1. Most of the strains (78%) cluster into one large clade. Geographic information is available for 30 of the 41 strains, and seven countries are represented: Canada (n = 4), Denmark (n = 1), Finland (n = 6), France (n = 1), Greenland (n = 1), Japan (n = 2), and the United States (n = 15). The dendrogram illustrates that strains from different geographic regions do not cluster by their location of origin but are widely distributed throughout the tree. The sources of the strains also vary and include fish (n = 15), environmental soils (n = 3), and a single isolate each from a bird and a patient with infant botulism (18). None of the strains clustered in the MLST dendrogram based upon their source, i.e., no clustering of strains is unique to a fish species, to soils, or to the infant botulism case. Interestingly, the environmental sources include strains isolated from lake sediment in northern Japan, from soil from the Olympia National Forest in Washington, and from sea mud from Agluitsok Bay in Greenland at a depth of 300 to 400 m. The strain from a bird was isolated from stomach contents of a bald eagle. Strains from these various sources (infant, fish, bird, and environment) and geographic locations are distributed throughout the MLST dendrogram.

Fig. 1.
MLST dendrogram with VNTR profiles of 41 C. botulinum serotype E strains. A composite MLST dendrogram based upon 188 SNPs was generated from the sequences of 15 genes in 41 serotype E strains. The UPGMA dendrogram shows four clades with 32 strains (78%) ...

Twenty of the serotype E strains in the dendrogram have recorded dates of isolation, thereby indicating that the 20 strains span 75 years, 1932 to 2007 (Table 1). The 1932 strain (E675) is from a food-borne botulism outbreak associated with smoked salmon in Nova Scotia (8). This strain is located in the largest clade within the MLST dendrogram and includes the most recent strain (located at the top of the dendrogram) isolated from an infant botulism case in Illinois in 2007, IBCA07-0062 (also isolated from a different stool specimen as CDC52256 [18]). The distribution of the strains in the dendrogram indicates there is no clustering of the strains by year.

Because strain DNA was received from different collections, it is possible that some strains might have originated from the same source, such as a researcher sharing a strain with colleagues. For example, two strains from 1966 appear to represent the same strain in two separate collections. E540 within the USAMRIID collection is labeled “FDA066” and “066B” within the California Department of Public Health collection; both strains share similar sample names, and they may be the same strain that was associated with Great Lakes smoked whitefish chubs in 1966. Their identical MLST and VNTR profiles support their similar provenances. Similar findings were observed by Nevas et al. (24), where the same strain from different sources shared identical PFGE patterns.

In addition, three “Beluga” strains in the most distant clade are from the USAMRIID collection; two (E538 and E542) were provided to USAMRIID by Virginia Polytechnic Institute, and the third, E213, is of unknown origin. The three strains are identical by MLST and VNTR analysis and therefore could represent samples from the same source. The fourth strain within the clade was isolated in 1996 from vacuum-packaged smoked whitefish in Canada. The presence of the whitefish strain in the clade indicates that the cluster is not unique to the Beluga strains.

The location of E542 Beluga in the clade at the base of the MLST dendrogram is noteworthy because it is one of two C. botulinum serotype E strains with a completed genomic sequence. The neighboring clade contains four strains, Kalamazoo, Minnesota, E183, and Alaska E43 (E185), another serotype E strain with a completed genome sequence. It is interesting that the two sequenced strains, Beluga (E542) and Alaska E43 (E185), are distant from the clade at the top of the dendrogram, which contains 78% of the strains.

One clade contains a single strain, K35, isolated in Finland from a freshwater whitefish, Coregonus albula. This strain is unique; none of the other strains in this study were found to be similar by MLST or AFLP analysis. K35 also contains the bont/E6 subtype (4).

In summary, most of the strains cluster in a large clade at the top of the MLST dendrogram. These strains are from different sources (fish, infant, and soils) and geographic locations (Canada, Denmark, Finland, France, Greenland, Japan and the United States) and contain the earliest (1932) and most recent (2007) strains. The composite dendrogram and the presence of only one SNP within the 16S rRNA gene illustrate the genetic conservation within the serotype E strains examined.

VNTR analysis.

Three variable-number tandem-repeat (VNTR) regions were identified using two serotype E whole-genome sequences (Table 4). PCR amplification and sequencing of the three VNTR regions in the strains verified the presence of the tandem-repeat units and the PCR fragment sizes (Fig. 1). VNTR data for the 41 strains in Fig. 1 illustrate how these regions differ between strains.

VNTR regions provide another method to compare strains by using a region that may mutate faster than SNPs within conserved MLST genes (14). Some of the strains that appear identical by MLST may also share the same VNTR profiles, such as E343 and E545, 066B and E540, and E182 and E184. The similar VNTR profiles among these strains suggest that they may have originated from the same source and may have been shared between colleagues or institutions. Strains that cannot be distinguished by MLST may also have different VNTR profiles. For example, E185 Minnesota and Kalamazoo strains differ in VNTR 24 and 25 and E547 and E548 differ in VNTR 21. The VNTR markers are relatively easy and rapid to use and provide additional information to compare strains.

AFLP analysis.

The 41 strains were examined by AFLP analysis to determine the genetic diversity of the bacteria and to compare the AFLP clustering of the strains to the clustering within the MLST dendrogram. The AFLP dendrogram in Fig. 2 shows the relatively conserved clustering of the 41 C. botulinum serotype E strains (within approximately 0.2 genetic distance units), in contrast to the distant two C. butyricum type E strains.

The overall clustering of the C. botulinum serotype E strains indicates that the relationships in the MLST dendrogram are shared in the AFLP-based dendrogram. The three Beluga strains cluster with the K37 whitefish strain, while K35 is unique within its own branch, distant from the other strains. Similar clusterings of strains in both dendrograms include (E539, E675, K8), (IBCA07-0062, E343, E545), (K549, K126, K3), (Hazen-Salmon, E216), (IBCA97-0192, Detroit, Canada), (E544, E546), (E182, E84), (Minnesota, Kalamazoo), and (E547, E548); also, according to both methods, K35 is unique, as is the clade containing E213, E538, E542, and K37. However, the relationship between K15 and K185, in contrast to what was seen for the other strains, differs between the AFLP and MLST dendrograms. The difference might reflect rearrangements in the genomes, such as inversion, insertion, or the presence of phage or plasmids that have the restriction enzyme sites used in the AFLP method. In general, the comparison shows that AFLP and MLST provide results that are consistent with each other when applied to this set of strains with relatively conserved bacteria.

BoNT/E comparisons.

Full-length bont gene sequences were generated for each of the 41 strains. Figure 3 shows a bont/E dendrogram that includes reference GenBank accession numbers and C. butyricum bont/E4 and bont/E5 sequences for comparison. Thirty-four of the 41 strains (83%) were either bont/E1 (n = 18) or bont/E3 (n = 16) subtypes. No additional strains containing the bont/E2 or E6 subtype were identified among the 41 strains. However, three strains (IBCA97-0192, Canada, Detroit) had a unique bont/E variant designated bont/E7. In addition, strain E134, from a round goby, contained a novel variant, bont/E8. Table 5 shows the amino acid and nucleic acid identities of the subtypes compared in the light chain, translocation domain, receptor-binding domain, or holotoxin. The comparison shows the greatest amino acid variation (5.3 to 5.9%) when comparing the C. butyricum BoNT/E5 to newly identified BoNT/E6, E7, and E8. Historically, the original five subtypes, BoNT/E1 to E5, were identified in the work of Hill et al. (10), with E6 identified in the 2007 work of Chen et al. (4). The newest, BoNT/E7 and E8, form distinct groups that differ from the other E subtypes by 2.2 to 5.9% at the amino acid level. The variants or subtypes were not found to be unique to a geographic region or source, such as species of fish or soils within the environment. Instead the subtypes occupy a variety of geographic locations, sources, and types of botulism.

Fig. 3.
Dendrogram of bont/E gene sequences. The full-length coding region of 70 bont/E gene sequences from the 41 strains in this study and GenBank accessions were aligned. Eight clusters are shown, with the bont/E1- and bont/E3-subtype clusters containing the ...
Table 5.
Nucleotide and amino acid identities among BoNT/E subtypes by domain

Figure 4 shows an alignment of the nucleotide sequences of the full-length bont/E genes representing the subtypes compared to bont/E1. The comparison indicates that the differences within the bont/E gene are found within specific regions. For example, the bont/E1 subtype differs from the bont/E3 subtype in the light-chain region (5.2% amino acid difference), while C. butyricum bont/E4 and bont/E5 differ from each other and from bont/E1 in the translocation and heavy-chain regions. Figure 4 also shows that bont/E6 within K35 has regions similar to those of C. butyricum bont/E4, as previously reported (4). The bont/E8 subtype has regions similar to those of the bont/E2, E6, and E7 subtypes. Interestingly, the differences between the subtypes do not span the full length of the gene as would be expected for random mutations. The nonrandom distribution of differences suggests the variation is the result of bont/E recombination events.

Fig. 4.
Comparison of eight bont/E subtypes. Four identical bont/E1 sequences from strains E183, E213, E216, and E675 were compared to the sequences of bont/E2 to E8. Nucleotide differences compared to bont/E1 are indicated: green lines indicate nucleotide differences ...

RarA PCR experiments.

A transposon-associated recombinase gene, rarA, located near bont/E, was identified by the whole-genome sequencing of two C. botulinum type E strains and one C. butyricum type E strain (11). The genomic sequences revealed that an intact rarA gene targeted and then split a rarA homolog and inserted DNA containing bont/E in the three strains. The insertion event resulted in a partial 5′ rarA and a partial 3′ rarA gene with inserted DNA that contained the bont/E gene placed within the split gene.

To determine whether the transposon-associated event has occurred in diverse C. botulinum type E strains, six sets of PCR primers were designed to amplify regions within the intact and split rarA genes and also the genes with their associated flanking regions (Table 3). PCR amplification in each of the 41 strains provided the expected amplicon sizes using the six primer sets. The PCR amplicons in 11 strains were sequenced to confirm the presence of both the intact and split rarA genes in these strains. The expected PCR amplicon sizes and sequence comparisons indicate that each of the strains contain both the intact and split rarA genes.

DISCUSSION

Since their identification in 1936, C. botulinum serotype E strains have been distinguished as nonproteolytic bacteria that grow at low temperatures and generally populate northern climates. To investigate whether the strains from specific geographic regions or sources were unique, a collaborative study encompassing strains from several institutions using multiple analysis methods was undertaken. The strains within the collections represent different geographic regions (Canada, Denmark, Finland, France, Greenland, Japan, and the United States) and sources (fish, bird, and soils) and include strains from food-borne and infant botulism cases, as well as many strains with an unknown provenance.

Because the bont genes appear to be horizontally transferred into different bacterial species, the genetic variation of both the host bacteria and the toxin were examined in the serotype E strains. The MSLT and AFLP analyses both support that C. botulinum serotype E bacteria are fairly conserved. The MLST dendrogram showed that many of the strains clustered within a large clade that included strains from multiple locations and sources; no strains from a specific geographic location or source clustered together. Similar findings where the strains do not cluster by geographic location have been reported in the group I strains (10).

The bont/E sequences within the 41 strains identified two new subtypes or variants, bont/E7 and E8. The most common subtypes were bont/E1 (n = 18) and bont/E3 (n = 16), found in 83% of the strains; the bont/E2, E6, E7, and E8 subtypes were less common. Comparison of the bont/E sequences showed that the variation between subtypes is not randomly distributed but is located within specific regions of the gene. The alignments showed that regions of variation may be found in other subtypes. For example, the bont/E8 subtype is a combination of differences within the bont/E2, E6, and E7 subtypes; bont/E6 shares regions of bont/E4 (4). Recombination within the bont gene has previously been observed in bont/A2, which is a mosaic of bont/A1 and bont/A3 and in the bont/C/D and bont/D/C mosaics (11, 23). Like bont/A, C, and D, bont/E shares the ability to recombine.

The C. botulinum type E and C. butyricum type E strains represent two species that contain the bont/E gene from two independent insertion events (11). PCR amplification of the transposon-associated recombinase rarA in the 41 C. botulinum serotype E strains indicates that all 41 strains share similar rarA regions. The MLST, AFLP, and rarA PCR experiments support that bont/E is associated with an insertion event in genetically conserved bacteria that have clonally expanded to inhabit the soils and sediments in northern regions. The clonal expansion and genetic conservation may include strains of the Southern hemisphere, but no strains that represent this region were identified or available for analysis. The serotype E strains likely survive as spores that are ingested or contaminate the surfaces of fish and marine mammals and birds feeding on the fish/sediments. The similarity of type E strains obtained from different regions may be due to effective dispersion by itinerant or migrating birds, fish, and marine mammals. Although the 41 strains in this study are not globally inclusive, they do represent a concerted effort among different institutions to enhance our understanding of C. botulinum serotype E strains. Importantly, the collaboration has yielded multiple molecular tools (MLST genes, VNTR regions, and rarA primers) that will allow others to examine the diversity of strains within their collections.

ACKNOWLEDGMENTS

Funding for this research was provided by Department of Homeland Security Science and Technology Directorate contract HSHQDC-10-C-00139 and NIAID IAA B18-120.

We also thank the Department of Energy Joint Genome Institute for their support in providing technical assistance and facilities for DNA sequencing.

Opinions, interpretations, conclusions, and recommendations are those of the authors and not necessarily endorsed by the U.S. Army, the National Institute of Allergy and Infectious Diseases, or the National Institutes of Health.

Footnotes

[down-pointing small open triangle]Published ahead of print on 14 October 2011.

REFERENCES

1. Artin I., Bjorkman P., Cronqvist J., Radstrom P., Holst E. 2007. First case of type E wound botulism diagnosed using real-time PCR. J. Clin. Microbiol. 45:3589–3594 [PMC free article] [PubMed]
2. Aureli P., et al. 1986. Two cases of type E infant botulism caused by neurotoxigenic Clostridium butyricum in Italy. J. Infect. Dis. 154:207–211 [PubMed]
3. Benson G. 1999. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 27:573–580 [PMC free article] [PubMed]
3a. Cato E. P., Hash D. E., Holdeman L. V., Moore W. E. 1982. Electrophoretic study of Clostridium species. J. Clin. Microbiol. 15:688–702 [PMC free article] [PubMed]
4. Chen Y., Korkeala H., Aarnikunnas J., Lindstrom M. 2007. Sequencing the botulinum neurotoxin gene and related genes in Clostridium botulinum type E strains reveals orfx3 and a novel type E neurotoxin subtype. J. Bacteriol. 189:8643–8650 [PMC free article] [PubMed]
5. Dolman C. E. 1960. Type E botulism: a hazard of the north. Arctic 13:230–256
5a. Dolman C. E., Murakami L. 1961. Clostridium botulinum type F with recent observations on other types. J. Infect. Dis. 109:107–128
5b. Eklund M. W., Poysky F. T., Peterson M. E., Paranjpye R. N., Pelroy G. A. 2004. Competitive inhibition between different Clostridium botulinum types and strains. J. Food Prot. 67:2682–2687 [PubMed]
6. Graham A. F., Mason D. R., Maxwell F. J., Peck M. W. 1997. Effect of pH and NaCl on growth from spores of non-proteolytic Clostridium botulinum at chill temperature. Lett. Appl. Microbiol. 24:95–100 [PubMed]
7. Gunnison J. B., Cummings J. R., Meyer K. F. 1936. Clostridium botulinum type E. Proc. Soc. Exp. Biol. Med. 35:278–280
8. Hazen E. L. 1938. Incitants of human botulism. Science 87:413–414 [PubMed]
9. Hielm S., Bjorkroth J., Hyytia E., Korkeala H. 1998. Prevalence of Clostridium botulinum in Finnish trout farms: pulsed-field gel electrophoresis typing reveals extensive genetic diversity among type E isolates. Appl. Environ. Microbiol. 64:4161–4167 [PMC free article] [PubMed]
10. Hill K. K., et al. 2007. Genetic diversity among botulinum neurotoxin-producing clostridial strains. J. Bacteriol. 189:818–832 [PMC free article] [PubMed]
11. Hill K. K., et al. 2009. Recombination and insertion events involving the botulinum neurotoxin complex genes in Clostridium botulinum types A, B, E and F and Clostridium butyricum type E strains. BMC Biol. 7:66. [PMC free article] [PubMed]
12. Hyytia E., Bjorkroth J., Hielm S., Korkeala H. 1999. Characterisation of Clostridium botulinum groups I and II by randomly amplified polymorphic DNA analysis and repetitive element sequence-based PCR. Int. J. Food Microbiol. 48:179–189 [PubMed]
13. Jacobson M. J., Lin G., Whittam T. S., Johnson E. A. 2008. Phylogenetic analysis of Clostridium botulinum type A by multi-locus sequence typing. Microbiology 154:2408–2415 [PMC free article] [PubMed]
14. Keim P., et al. 2004. Anthrax molecular epidemiology and forensics: using the appropriate marker for different evolutionary scales. Infect. Genet. Evol. 4:205–213 [PubMed]
15. Keto-Timonen R., Nevas M., Korkeala H. 2005. Efficient DNA fingerprinting of Clostridium botulinum types A, B, E, and F by amplified fragment length polymorphism analysis. Appl. Environ. Microbiol. 71:1148–1154 [PMC free article] [PubMed]
16. Kurochkin B., Emelyanchik K. 1937. Seal meat as a source of botulism. Vopr. Pitan. 1:141–148
17. Leclair D., Pagotto F., Farber J. M., Cadieux B., Austin J. W. 2006. Comparison of DNA fingerprinting methods for use in investigation of type E botulism outbreaks in the Canadian Arctic. J. Clin. Microbiol. 44:1635–1644 [PMC free article] [PubMed]
18. Luquez C., Dykes J. K., Yu P. A., Raphael B. H., Maslanka S. E. 2010. First report worldwide of an infant botulism case due to Clostridium botulinum type E. J. Clin. Microbiol. 48:326–328 [PMC free article] [PubMed]
19. Macdonald T. E., et al. 2008. Differentiation of Clostridium botulinum serotype A strains by multiple-locus variable-number tandem-repeat analysis. Appl. Environ. Microbiol. 74:875–882 [PMC free article] [PubMed]
20. McCroskey L. M., Hatheway C. L., Fenicia L., Pasolini B., Aureli P. 1986. Characterization of an organism that produces type E botulinal toxin but which resembles Clostridium butyricum from the feces of an infant with type E botulism. J. Clin. Microbiol. 23:201–202 [PMC free article] [PubMed]
21. Meng X., et al. 1997. Characterization of a neurotoxigenic Clostridium butyricum strain isolated from the food implicated in an outbreak of food-borne type E botulism. J. Clin. Microbiol. 35:2160–2162 [PMC free article] [PubMed]
22. Meng X., et al. 1999. Isolation and characterisation of neurotoxigenic Clostridium butyricum from soil in China. J. Med. Microbiol. 48:133–137 [PubMed]
23. Moriishi K., et al. 1996. Molecular cloning of the gene encoding the mosaic neurotoxin, composed of parts of botulinum neurotoxin types C1 and D, and PCR detection of this gene from Clostridium botulinum type C organisms. Appl. Environ. Microbiol. 62:662–667 [PMC free article] [PubMed]
24. Nevas M., et al. 2005. Diversity of proteolytic Clostridium botulinum strains, determined by a pulsed-field gel electrophoresis approach. Appl. Environ. Microbiol. 71:1311–1317 [PMC free article] [PubMed]
25. Paul C. J., et al. 2007. Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification. Appl. Environ. Microbiol. 73:2963–2975 [PMC free article] [PubMed]
26. Poulet S., Hauser D., Quanz M., Niemann H., Popoff M. R. 1992. Sequences of the botulinal neurotoxin E derived from Clostridium botulinum type E (strain Beluga) and Clostridium butyricum (strains ATCC 43181 and ATCC 43755). Biochem. Biophys. Res. Commun. 183:107–113 [PubMed]
27. Wang X., et al. 2000. Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains. Appl. Environ. Microbiol. 66:4992–4997 [PMC free article] [PubMed]
28. Weir B. S. 1996. Genetic data analysis II. Sinauer Associates, Inc., Sunderland, MA

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