CASE REPORT
An Atypical Outbreak of Food-Borne Botulism Due to Clostridium botulinum Types B and E from Ham Christelle Mazuet,a Jean Sautereau,a Christine Legeay,a Christiane Bouchier,b Philippe Bouvet,a Michel R. Popoffa Institut Pasteur, Bactéries Anaérobies et Toxines, Paris, Francea; Institut Pasteur, Plateforme Génomique, Paris, Franceb
An outbreak of human botulism was due to consumption of ham containing botulinum neurotoxins B and E. A Clostridium botulinum type E strain isolated from ham was assigned to a new subtype (E12) based on bont/E gene sequencing and belongs to a new multilocus sequence subtype, as analyzed by whole-genome sequencing.
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n December 2009, in eastern France, two people (a male 47year-old male and 50-year-old female) developed blurred vision, diplopia, dry mouth, and difficulty in swallowing after ingestion of ham from a small-scale commercial producer. Botulinum neurotoxin (BoNT) was identified in the serum of the two patients with mouse bioassay results at 1 and ⬍0.5 mouse lethal doses (MLD)/ml, respectively. The toxin activity in serum samples from both patients was neutralized by anti-type B and anti-type E diagnostic sera individually and not by anti-type A serum. Investigation of ham samples revealed the presence of BoNT at a level of 40 MLD/g before and 4 ⫻ 103 MLD/g after trypsinization. The lethal activity was totally neutralized by a combination of anti-BoNT/B and anti-BoNT/E sera in combination. Using serum anti-BoNT/B and anti-BoNT/E individually, BoNT/B and BoNT/E were estimated to be 10 and 30 MLD/g, respectively, in ham samples without trypsinization. DNA that was extracted from ham samples with the PowerFood microbial DNA extraction kit (MO BIO Laboratories) was investigated for the presence of Clostridium botulinum types A, B, E, and F by SYBR green real-time PCR with specific primers of the bont type as previously described (1). BoNT gene types B and E were detected but not type A or F. Enrichment cultures of ham samples were performed in fortified cooked meat medium at 37°C and 15°C under anaerobic conditions (2), and C. botulinum was isolated on agar selective medium (3). Only C. botulinum type E was PCR detected in enrichment cultures and was isolated (strain 84-10) on agar medium. Prolonged enrichment cultures of ham samples at 12°C for 12 days did not succeed in detection of C. botulinum type B. The bont type E gene was PCR amplified and sequenced, and whole-genome sequencing of strain 84-10 was performed with Illumina single read technology as already described (4). BoNT/E from strain 84-10 shows 4 to 5% nucleotide sequence variation and 4 to 9% amino acid sequence variation with the other identified BoNT/E subtypes (5, 6), including the recently reported subtypes E9 (7), E10, and E11 (8) (Table 1). Therefore, based on the BoNT/E sequence (GenBank accession no. KF929215 and KM370319), the C. botulinum type E strain 84-10 was termed subtype E12. Gene variation in bontE might reflect distinct evolutionary lineages in C. botulinum strains. Phylogenetic analysis of the BoNT/E sequence of the different subtypes at the nucleotide and amino acid levels shows that BoNT/E12 is on a distinct lineage
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(Fig. 1). The most phylogenetically related BoNT/E type was the subtype E9 (Fig. 1). To investigate whether amino acid sequence variation in BoNT/E12 induces variation in antigenicity, DNAs encoding Hc fragments, which are the main BoNT domain involved in neutralizing response (9–11), from strains P34 (BoNT/E3) and 84-10 (BoNT/E12) were PCR amplified and cloned into the pET28 vector. The corresponding recombinant Hc (rHc) proteins were produced, purified, and used to immunize rabbits. Serum antirHc/E3 recognized rHc/E3 and rHc/E12 at similar levels by enzyme-linked immunosorbent assay (ELISA) and did not detect the other rHc subtypes. Similarly, serum anti-rHc/E12 reacted with rHcE3 and rHcE12, but to a lower level than the anti rHcE3 serum (Fig. 2). The neutralization potencies of the serum against rHc/E3 versus BoNT/E3 and BoNT/E12 in the mouse bioassay were equivalent. However, serum against rHc/E12 did not neutralize both BoNT/E3 and BoNT/E12, probably due to an insufficient immunization procedure (Fig. 2). This shows that the sequence variations between BoNT/E3 and BoNT/E12 Hc domains only weakly impacted their antigenicities. Whole-genome sequencing of 84-10 showed that the genes coding for the associated nontoxic proteins (ANTPs) that are upstream of bont/E12 (including NTNH, P47, OrfX1, OrfX2, and OrfX3) are similar in organization to those of C. botulinum Alaska E43 (Fig. 3A). The most related neurotoxin gene of bontE12 is that of subtype E10 at the amino acid sequence level, whereas the antp genes are closely related to those of strain Alaska (E3) (Fig. 3A). The botulinum locus of 84-10 is flanked by insertion sequences (IS) and is inserted into rarA similarly to that in strain Alaska E43 (Fig. 3B). Strain 84-10, as well as the other C. botulinum type E
Received 15 October 2014 Returned for modification 9 November 2014 Accepted 20 November 2014 Accepted manuscript posted online 26 November 2014 Citation Mazuet C, Sautereau J, Legeay C, Bouchier C, Bouvet P, Popoff MR. 2015. An atypical outbreak of food-borne botulism due to Clostridium botulinum types B and E from ham. J Clin Microbiol 53:722–726. doi:10.1128/JCM.02942-14. Editor: D. J. Diekema Address correspondence to Michel R. Popoff, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JCM.02942-14. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.02942-14
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TABLE 1 Nucleotide and amino acid sequence identities of BoNT/E subtypes % nucleotide/amino acid identity Subtype
E2 E3 E4 E5 E6 (CDC5247) (Alaska E43) (ATCC 43755) (LCL095) (K35)
E1 (NCTC 99/99 11219) E2 E3 E4 E5 E6 E7 E8 E9 E10 E11
E7 E8 (IBCA97-0192) (E134)
E9 E10 E11 E12 (CDC66177) (FWKR11E1) (SW280E) (84-10)
99/98
98.5/97
98/97
98.5/97 99/98
98/96
94/89
98/95
97/93
96/93
99/97
98/97 98/96
98/96 98/95 97/95
98.2/97 98/96 98/97 97/95
98.5/97 98/96 98/96 97/94 98/97 99/98
94/89 94/89 95/90 94/89 94/88 94/89 94/89
98/96 97/95 97/95 97/93 98/96 98/97 99/98 94/89
97/94 96/93 97/93 96/92 97/93 97/93.5 97/94 94/89 98/96
96/93 96/93 96/92.5 96/94 95/91 96/92.5 95/92 95/91 96/96 95/96
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. 3B), indicating that both strains probably have a common origin but followed independent evolutions. The genes flanking the bont/E gene cluster in strain 84-10 exhibit greater sequence variation with the corresponding genes of the neurotoxigenic Clostridium butyricum strain BL5262 (Fig. 3B), further supporting that strain 84-10 belongs to the C. botulinum E group. A circular representation of chromosomal orthologous genes shows that strain 84-10 is closely related to
98.5/97 99/97 98/96 97/95 98/96
CDC66177 (E9) and Eklund 17B (nonproteolytic B), as well as, to a lesser extent, Alaska E43 (E3) (Fig. 4). Multilocus sequence typing (MLST) of 84-10 has been performed based on 15 housekeeping genes (gyrB, guaA, I/VD, lepA, oppB, trpB, recA, pta, 23S rRNA, 16S rRNA, atpD, pyC, mutL, rpoB, and tuf) (see Table S1 in the supplemental material). 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
FIG 1 Comparative analysis of BoNT/E from strain 84-10 with representative BoNT/E subtypes at the nucleotide and amino acid levels. The dendrogram was reconstructed from the nucleotide sequence and amino acid sequence by the unweighted-pair group method using average linkages (UPGMA). The genetic distances were computed by using the Kimura two-parameter model. The scale bar indicates similarity values. The number shown next to each node indicates the cophenetic correlation. Evolutionary analyses were conducted in Bionumerics v.6.6 (Applied Maths).
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FIG 2 Cross-immunoreaction between BoNT/E3 and BoNT/E12 as tested by ELISA. A 96-well plate was coated with recombinant Hc (rHc) proteins of BoNT/E3 and BoNT/E12. After a saturation step with bovine serum albumin (BSA), the immunopurified antibodies against anti-rHc/E3 and anti-rHc/E12 were titrated (starting dilution of 1:100 followed by 2-fold serial dilutions). The histogram represents the antibody titers (reciprocal of dilutions) to achieve an optical density (OD) of 0.3. Preimmune rabbit sera and immunopurified antibodies against rHc proteins of BoNT/A1 and BoNT/B2 are included as controls.
20 single nucleotide polymorphisms (SNP) with the most related C. botulinum strains and are considered new alleles (see Table S1). Therefore, C. botulinum strain 84-10 contains a new bont/E subtype in a distinct genetic background. Interestingly, the concatenated housekeeping gene sequences of strain 84-10 share a higher similarity level to the corresponding sequences of C. botulinum type B strain Eklund 17B than to those of C. botulinum type E (see Table S2 in the supplemental material). This further supports that
strain 84-10 probably derived from an ancestral C. botulinum B origin. It is noteworthy that C. botulinum strain CDC66177, which produces subtype E9, has greater genomic similarity to group II C. botulinum type B than to other type E strains, suggesting bont/E locus gene transfer into a type B strain (7). Coexistence of strain 84-10 with a C. botulinum type B in ham might have facilitated gene mobility between strains. No correlation has been found between a specific C. botulinum E subtype or MLST profile and the
FIG 3 Genetic organization of the neurotoxin gene cluster in strain 84-10 and comparison with the most related subtypes. (A) The closest C. botulinum E subtype homologs of the 84-10 neurotoxin gene cluster are indicated (% identity) at the nucleotide and amino acid sequence levels. The accession numbers of the toxin gene cluster sequences of strain 84-10 are KM370319 to KM370324. (B) Location of the neurotoxin gene cluster in the rarA gene within the C. botulinum strain 84-10 and comparison of the flanking genes from strains Eklund 17B (nonproteolytic type B), Alaska E43 (E3), and C. butyricum BL5262 (E5) at the amino acid sequence level (% identity) according to reference 12.
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FIG 4 Circular figure showing the relatedness of the chromosome of strain 84-10 versus those of Eklund 17B (nonproteolytic type B), Alaska E43 (E3), and CDC66177 (E9). All coding sequences (CDSs) (on the plus and minus strands) of Clostridium botulinum strain 84-10 were compared by bidirectional BLAST with all CDSs of strains Eklund 17B, Alaska E43, and CDC66177. The color code of protein identity (%) is indicated (RAST prokaryotic genome annotation server v.2.0).
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. In addition, strain 84-10 contains a flagellin A gene (flaA), as previously described in C. botulinum strains from groups I and II (16–18), but not the flagellin B gene (flaB), which has been identified in C. botulinum type E strains (19). However, the flaA sequence from strain 84-10 does not match any of the 15 flaA types described in group I and II strains (19). flaA from strain 84-10 is closely related to the corresponding genes of C. botulinum B Eklund17B (98% identity), C. botulinum F strain 610 (98%), and C. botulinum E Alaska E43 (97%) at the nucleotide level. The flaA relatedness to the flaA gene of C. botulinum type B and the absence of flaB, which is characteristic of C. botulinum E strains, again support that the strain 84-10 derived from a C. botulinum type B lineage.
Botulism is a rare but severe neurological disorder that is characterized by a flaccid paralysis. Food-borne botulism is the main form of botulism in France. From 20 to 40 cases of human botulism are identified every year (20–22). Homemade or small-scale commercial ham is the traditional source of human botulism in France, most often botulism type B (20–22). Here, we report an atypical outbreak of botulism type E caused by a ham contaminated with a new Clostridium botulinum subtype E variant and C. botulinum type B. C. botulinum type E strains are commonly isolated from fish and sea animals in northern countries of the northern hemisphere
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(23–25). In contrast, pigs are frequently healthy carriers of C. botulinum type B (26, 27), and pig products like ham are often associated with human botulism type B, notably in France (21, 28). Two additional outbreaks of human botulism type E associated with the consumption of ham, including one imported from Portugal, have been reported in France (29). Another outbreak of human botulism type E, including 6 persons, was described in Argentina. C. botulinum E strain was isolated from a home-cured ham, and it was speculated that the contamination could have occurred from the seasoning, possibly sea salt, since other products (sausage and bacon) from the same pig were not contaminated (30). Since strain 84-10 shows a different subtype and MLST profile from strains isolated from fish and marine animals (8, 13, 31), it could represent a particular C. botulinum variant adapted to a specific environment or a specific host, such as pigs. Nucleotide sequence accession numbers. The sequences determined in this study have been deposited in GenBank under accession numbers KF929215 and KM370304 to KM370324. ACKNOWLEDGMENTS High-throughput sequencing has been performed on the Genomics Platform, a member of the France Génomique Consortium (ANR10-INBS09-08). We thank Benoit Jaulhac, Laboratoire de Bactériologie, Hôpitaux Universitaires, Strasbourg, France, for his assistance.
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An Atypical Outbreak of Food-Borne Botulism Due to Clostridium botulinum Types B and E from Ham Christelle Mazuet,a Jean Sautereau,a Christine Legeay,a Christiane Bouchier,b Philippe Bouvet,a Michel R. Popoffa Institut Pasteur, Bactéries Anaérobies et Toxines, Paris, Francea; Institut Pasteur, Plateforme Génomique, Paris, Franceb
An outbreak of human botulism was due to consumption of ham containing botulinum neurotoxins B and E. A Clostridium botulinum type E strain isolated from ham was assigned to a new subtype (E12) based on bont/E gene sequencing and belongs to a new multilocus sequence subtype, as analyzed by whole-genome sequencing.
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n December 2009, in eastern France, two people (a male 47year-old male and 50-year-old female) developed blurred vision, diplopia, dry mouth, and difficulty in swallowing after ingestion of ham from a small-scale commercial producer. Botulinum neurotoxin (BoNT) was identified in the serum of the two patients with mouse bioassay results at 1 and ⬍0.5 mouse lethal doses (MLD)/ml, respectively. The toxin activity in serum samples from both patients was neutralized by anti-type B and anti-type E diagnostic sera individually and not by anti-type A serum. Investigation of ham samples revealed the presence of BoNT at a level of 40 MLD/g before and 4 ⫻ 103 MLD/g after trypsinization. The lethal activity was totally neutralized by a combination of anti-BoNT/B and anti-BoNT/E sera in combination. Using serum anti-BoNT/B and anti-BoNT/E individually, BoNT/B and BoNT/E were estimated to be 10 and 30 MLD/g, respectively, in ham samples without trypsinization. DNA that was extracted from ham samples with the PowerFood microbial DNA extraction kit (MO BIO Laboratories) was investigated for the presence of Clostridium botulinum types A, B, E, and F by SYBR green real-time PCR with specific primers of the bont type as previously described (1). BoNT gene types B and E were detected but not type A or F. Enrichment cultures of ham samples were performed in fortified cooked meat medium at 37°C and 15°C under anaerobic conditions (2), and C. botulinum was isolated on agar selective medium (3). Only C. botulinum type E was PCR detected in enrichment cultures and was isolated (strain 84-10) on agar medium. Prolonged enrichment cultures of ham samples at 12°C for 12 days did not succeed in detection of C. botulinum type B. The bont type E gene was PCR amplified and sequenced, and whole-genome sequencing of strain 84-10 was performed with Illumina single read technology as already described (4). BoNT/E from strain 84-10 shows 4 to 5% nucleotide sequence variation and 4 to 9% amino acid sequence variation with the other identified BoNT/E subtypes (5, 6), including the recently reported subtypes E9 (7), E10, and E11 (8) (Table 1). Therefore, based on the BoNT/E sequence (GenBank accession no. KF929215 and KM370319), the C. botulinum type E strain 84-10 was termed subtype E12. Gene variation in bontE might reflect distinct evolutionary lineages in C. botulinum strains. Phylogenetic analysis of the BoNT/E sequence of the different subtypes at the nucleotide and amino acid levels shows that BoNT/E12 is on a distinct lineage
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(Fig. 1). The most phylogenetically related BoNT/E type was the subtype E9 (Fig. 1). To investigate whether amino acid sequence variation in BoNT/E12 induces variation in antigenicity, DNAs encoding Hc fragments, which are the main BoNT domain involved in neutralizing response (9–11), from strains P34 (BoNT/E3) and 84-10 (BoNT/E12) were PCR amplified and cloned into the pET28 vector. The corresponding recombinant Hc (rHc) proteins were produced, purified, and used to immunize rabbits. Serum antirHc/E3 recognized rHc/E3 and rHc/E12 at similar levels by enzyme-linked immunosorbent assay (ELISA) and did not detect the other rHc subtypes. Similarly, serum anti-rHc/E12 reacted with rHcE3 and rHcE12, but to a lower level than the anti rHcE3 serum (Fig. 2). The neutralization potencies of the serum against rHc/E3 versus BoNT/E3 and BoNT/E12 in the mouse bioassay were equivalent. However, serum against rHc/E12 did not neutralize both BoNT/E3 and BoNT/E12, probably due to an insufficient immunization procedure (Fig. 2). This shows that the sequence variations between BoNT/E3 and BoNT/E12 Hc domains only weakly impacted their antigenicities. Whole-genome sequencing of 84-10 showed that the genes coding for the associated nontoxic proteins (ANTPs) that are upstream of bont/E12 (including NTNH, P47, OrfX1, OrfX2, and OrfX3) are similar in organization to those of C. botulinum Alaska E43 (Fig. 3A). The most related neurotoxin gene of bontE12 is that of subtype E10 at the amino acid sequence level, whereas the antp genes are closely related to those of strain Alaska (E3) (Fig. 3A). The botulinum locus of 84-10 is flanked by insertion sequences (IS) and is inserted into rarA similarly to that in strain Alaska E43 (Fig. 3B). Strain 84-10, as well as the other C. botulinum type E
Received 15 October 2014 Returned for modification 9 November 2014 Accepted 20 November 2014 Accepted manuscript posted online 26 November 2014 Citation Mazuet C, Sautereau J, Legeay C, Bouchier C, Bouvet P, Popoff MR. 2015. An atypical outbreak of food-borne botulism due to Clostridium botulinum types B and E from ham. J Clin Microbiol 53:722–726. doi:10.1128/JCM.02942-14. Editor: D. J. Diekema Address correspondence to Michel R. Popoff, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JCM.02942-14. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.02942-14
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TABLE 1 Nucleotide and amino acid sequence identities of BoNT/E subtypes % nucleotide/amino acid identity Subtype
E2 E3 E4 E5 E6 (CDC5247) (Alaska E43) (ATCC 43755) (LCL095) (K35)
E1 (NCTC 99/99 11219) E2 E3 E4 E5 E6 E7 E8 E9 E10 E11
E7 E8 (IBCA97-0192) (E134)
E9 E10 E11 E12 (CDC66177) (FWKR11E1) (SW280E) (84-10)
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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. 3B), indicating that both strains probably have a common origin but followed independent evolutions. The genes flanking the bont/E gene cluster in strain 84-10 exhibit greater sequence variation with the corresponding genes of the neurotoxigenic Clostridium butyricum strain BL5262 (Fig. 3B), further supporting that strain 84-10 belongs to the C. botulinum E group. A circular representation of chromosomal orthologous genes shows that strain 84-10 is closely related to
98.5/97 99/97 98/96 97/95 98/96
CDC66177 (E9) and Eklund 17B (nonproteolytic B), as well as, to a lesser extent, Alaska E43 (E3) (Fig. 4). Multilocus sequence typing (MLST) of 84-10 has been performed based on 15 housekeeping genes (gyrB, guaA, I/VD, lepA, oppB, trpB, recA, pta, 23S rRNA, 16S rRNA, atpD, pyC, mutL, rpoB, and tuf) (see Table S1 in the supplemental material). 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
FIG 1 Comparative analysis of BoNT/E from strain 84-10 with representative BoNT/E subtypes at the nucleotide and amino acid levels. The dendrogram was reconstructed from the nucleotide sequence and amino acid sequence by the unweighted-pair group method using average linkages (UPGMA). The genetic distances were computed by using the Kimura two-parameter model. The scale bar indicates similarity values. The number shown next to each node indicates the cophenetic correlation. Evolutionary analyses were conducted in Bionumerics v.6.6 (Applied Maths).
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FIG 2 Cross-immunoreaction between BoNT/E3 and BoNT/E12 as tested by ELISA. A 96-well plate was coated with recombinant Hc (rHc) proteins of BoNT/E3 and BoNT/E12. After a saturation step with bovine serum albumin (BSA), the immunopurified antibodies against anti-rHc/E3 and anti-rHc/E12 were titrated (starting dilution of 1:100 followed by 2-fold serial dilutions). The histogram represents the antibody titers (reciprocal of dilutions) to achieve an optical density (OD) of 0.3. Preimmune rabbit sera and immunopurified antibodies against rHc proteins of BoNT/A1 and BoNT/B2 are included as controls.
20 single nucleotide polymorphisms (SNP) with the most related C. botulinum strains and are considered new alleles (see Table S1). Therefore, C. botulinum strain 84-10 contains a new bont/E subtype in a distinct genetic background. Interestingly, the concatenated housekeeping gene sequences of strain 84-10 share a higher similarity level to the corresponding sequences of C. botulinum type B strain Eklund 17B than to those of C. botulinum type E (see Table S2 in the supplemental material). This further supports that
strain 84-10 probably derived from an ancestral C. botulinum B origin. It is noteworthy that C. botulinum strain CDC66177, which produces subtype E9, has greater genomic similarity to group II C. botulinum type B than to other type E strains, suggesting bont/E locus gene transfer into a type B strain (7). Coexistence of strain 84-10 with a C. botulinum type B in ham might have facilitated gene mobility between strains. No correlation has been found between a specific C. botulinum E subtype or MLST profile and the
FIG 3 Genetic organization of the neurotoxin gene cluster in strain 84-10 and comparison with the most related subtypes. (A) The closest C. botulinum E subtype homologs of the 84-10 neurotoxin gene cluster are indicated (% identity) at the nucleotide and amino acid sequence levels. The accession numbers of the toxin gene cluster sequences of strain 84-10 are KM370319 to KM370324. (B) Location of the neurotoxin gene cluster in the rarA gene within the C. botulinum strain 84-10 and comparison of the flanking genes from strains Eklund 17B (nonproteolytic type B), Alaska E43 (E3), and C. butyricum BL5262 (E5) at the amino acid sequence level (% identity) according to reference 12.
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FIG 4 Circular figure showing the relatedness of the chromosome of strain 84-10 versus those of Eklund 17B (nonproteolytic type B), Alaska E43 (E3), and CDC66177 (E9). All coding sequences (CDSs) (on the plus and minus strands) of Clostridium botulinum strain 84-10 were compared by bidirectional BLAST with all CDSs of strains Eklund 17B, Alaska E43, and CDC66177. The color code of protein identity (%) is indicated (RAST prokaryotic genome annotation server v.2.0).
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. In addition, strain 84-10 contains a flagellin A gene (flaA), as previously described in C. botulinum strains from groups I and II (16–18), but not the flagellin B gene (flaB), which has been identified in C. botulinum type E strains (19). However, the flaA sequence from strain 84-10 does not match any of the 15 flaA types described in group I and II strains (19). flaA from strain 84-10 is closely related to the corresponding genes of C. botulinum B Eklund17B (98% identity), C. botulinum F strain 610 (98%), and C. botulinum E Alaska E43 (97%) at the nucleotide level. The flaA relatedness to the flaA gene of C. botulinum type B and the absence of flaB, which is characteristic of C. botulinum E strains, again support that the strain 84-10 derived from a C. botulinum type B lineage.
Botulism is a rare but severe neurological disorder that is characterized by a flaccid paralysis. Food-borne botulism is the main form of botulism in France. From 20 to 40 cases of human botulism are identified every year (20–22). Homemade or small-scale commercial ham is the traditional source of human botulism in France, most often botulism type B (20–22). Here, we report an atypical outbreak of botulism type E caused by a ham contaminated with a new Clostridium botulinum subtype E variant and C. botulinum type B. C. botulinum type E strains are commonly isolated from fish and sea animals in northern countries of the northern hemisphere
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(23–25). In contrast, pigs are frequently healthy carriers of C. botulinum type B (26, 27), and pig products like ham are often associated with human botulism type B, notably in France (21, 28). Two additional outbreaks of human botulism type E associated with the consumption of ham, including one imported from Portugal, have been reported in France (29). Another outbreak of human botulism type E, including 6 persons, was described in Argentina. C. botulinum E strain was isolated from a home-cured ham, and it was speculated that the contamination could have occurred from the seasoning, possibly sea salt, since other products (sausage and bacon) from the same pig were not contaminated (30). Since strain 84-10 shows a different subtype and MLST profile from strains isolated from fish and marine animals (8, 13, 31), it could represent a particular C. botulinum variant adapted to a specific environment or a specific host, such as pigs. Nucleotide sequence accession numbers. The sequences determined in this study have been deposited in GenBank under accession numbers KF929215 and KM370304 to KM370324. ACKNOWLEDGMENTS High-throughput sequencing has been performed on the Genomics Platform, a member of the France Génomique Consortium (ANR10-INBS09-08). We thank Benoit Jaulhac, Laboratoire de Bactériologie, Hôpitaux Universitaires, Strasbourg, France, for his assistance.
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