Hielm S, Bj?rkroth J, Hyyti? E, Korkeala H. 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 types A, B, E, and F by SYBR green real-time PCR with specific primers of the type as previously explained (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 37C and 15C under anaerobic conditions (2), and was isolated on agar selective medium (3). Only type Asiatic acid E was PCR detected in enrichment cultures and was isolated (strain 84-10) on agar medium. Prolonged enrichment cultures of ham samples at 12C for Asiatic acid 12 days did not succeed in detection of type B. The type E gene was PCR amplified and sequenced, and whole-genome sequencing of strain 84-10 was performed with Illumina single go through technology as already explained (4). BoNT/E from strain 84-10 shows 4 to 5% nucleotide sequence variance and 4 to 9% amino acid sequence variation with the other recognized 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 type E strain 84-10 was termed subtype E12. Gene variance in might reflect unique evolutionary lineages in strains. Phylogenetic analysis of the BoNT/E sequence of the different subtypes at the nucleotide and amino acid levels shows that BoNT/E12 is usually on a distinct lineage (Fig. 1). The most phylogenetically related BoNT/E type was the subtype E9 (Fig. 1). TABLE 1 Nucleotide and amino acid sequence identities of BoNT/E subtypes Alaska E43 (Fig. 3A). The most related neurotoxin gene of is usually that of subtype E10 at the amino acid sequence level, whereas the genes are closely related to those of strain Alaska (E3) (Fig. 3A). The botulinum locus of 84-10 is usually flanked by insertion sequences (Is usually) and is inserted into similarly to that in strain Alaska E43 (Fig. 3B). Strain 84-10, as well as the other type E strains, seems to have derived from an ancestral strain related to type B by insertion of the gene cluster and an intact gene into the operon as previously reported (7, 12, 13). However, the botulinum locus genes of strain 84-10 as well Asiatic acid 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 impartial evolutions. Asiatic acid The genes flanking the gene cluster in strain 84-10 exhibit greater sequence variation with the corresponding genes of the neurotoxigenic strain BL5262 (Fig. 3B), further supporting that strain 84-10 belongs to the E group. A circular representation of chromosomal orthologous genes shows that strain 84-10 is usually closely related to CDC66177 (E9) and Eklund 17B (nonproteolytic B), as well as, to a lesser extent, Alaska E43 (E3) (Fig. 4). Open in a separate windows FIG 3 Genetic organization of the neurotoxin gene cluster in strain 84-10 and comparison with the most related subtypes. (A) The closest 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 gene within the strain 84-10 and comparison of the flanking genes from strains Eklund 17B (nonproteolytic type B), Alaska E43 (E3), and BL5262 (E5) at the amino acid sequence level (% identity) according to reference Rabbit polyclonal to ADCK2 12. Open in a separate windows 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) (around the plus and minus strands).