Analysis of a unique Clostridium botulinum strain from the Southern hemisphere producing a novel type E botulinum neurotoxin subtype

Abstract

BackgroundClostridium botulinum strains that produce botulinum neurotoxin type E (BoNT/E) are most commonly isolated from botulism cases, marine environments, and animals in regions of high latitude in the Northern hemisphere. A strain of C. botulinum type E (CDC66177) was isolated from soil in Chubut, Argentina. Previous studies showed that the amino acid sequences of BoNT/E produced by various strains differ by < 6% and that the type E neurotoxin gene cluster inserts into the rarA operon.ResultsGenetic and mass spectral analysis demonstrated that the BoNT/E produced by CDC66177 is a novel toxin subtype (E9). Toxin gene sequencing indicated that BoNT/E9 differed by nearly 11% at the amino acid level compared to BoNT/E1. Mass spectrometric analysis of BoNT/E9 revealed that its endopeptidase substrate cleavage site was identical to other BoNT/E subtypes. Further analysis of this strain demonstrated that its 16S rRNA sequence clustered with other Group II C. botulinum (producing BoNT types B, E, and F) strains. Genomic DNA isolated from strain CDC66177 hybridized with fewer probes using a Group II C. botulinum subtyping microarray compared to other type E strains examined. Whole genome shotgun sequencing of strain CDC66177 revealed that while the toxin gene cluster inserted into the rarA operon similar to other type E strains, its overall genome content shared greater similarity with a Group II C. botulinum type B strain (17B).ConclusionsThese results expand our understanding of the global distribution of C. botulinum type E strains and suggest that the type E toxin gene cluster may be able to insert into C. botulinum strains with a more diverse genetic background than previously recognized.

Clostridium botulinum Group II Isolate Phylogenomic Profiling Using Whole-Genome Sequence Data

“…Consistent with other reports (12,23), a large cluster (20 taxa; n ϭ 149) that included the Alaska E43 strain (NC_010723) represented a wide variety of BoNT/E toxin subtypes found in C. botulinum group II bacteria (E1 to E3, E6 to E8, and E10 and E11) ( Fig. 1; see also Fig.…”

“…This isolate was derived from a waterfowl sample collected in 1980 as part of an avian botulism investigation (outbreak 7) but tested negative for BoNT in 2012 (Table 1). A second population (9 taxa; n ϭ 14) clustered closer to the BoNT/B4 strain Eklund 17B and included CDC66177, a BoNT/E9 isolate, as reported elsewhere (23). Other BoNT/B4 isolates (Eklund 2B, DB2, KAPB-3, and KAPB-8), two BoNT/F6 isolates (202F and 610F), and several nontoxigenic isolates also grouped with the Eklund 17B cluster.…”

“…All sequence reads and MLST alleles included in this study have been deposited in the NCBI Sequence Read Archive (SRP059342) and GenBank (atpD, KT034476 to KT034633; trpB, KT034634 to KT034791; rpoB, KT034792 to KT034949; guaA, KT034950 to KT035107; pta, KT035108 to KT035265; ilvD, KT035266 to KT035423; lepA, KT035424 to KT035581; gyrB, KT035582 to KT035739; recA, KT035740 to KT035897; oppB, KT035898 to KT036055; 23S rRNA rumA, KT036056 to KT036213; pyc, KT036214 to KT036371) (http: //www.ncbi.nlm.nih.gov/) databases. Public genomes included in analyses were Alaska E43, NC_010723; Eklund 17B, NC_010674; pCLL, NC_010680; 202F, CP006903; pCBI, CP006904; E1 Beluga, ACSC00000000; CDC66177, ALYJ00000000; CB11/1-1, AORM00000000; KAPB-3, JQOK01000000; DB2, JQOJ01000000; NCTC 8266, CP010520; NCTC 8550, CP010521; and NCTC 11219, JXMR01000000 (23,(32)(33)(34)(35).…”

“…Several nucleic acid-based typing methods, including pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), variable number tandem repeat (VNTR), multiple-locus sequence typing (MLST), DNA microarrays, and sequence analysis of the bont gene and the flagellin gene variable region (flaVR), have all been used for genetic characterization of C. botulinum group II strains (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24).…”

An Atypical Outbreak of Food-Borne Botulism Due to Clostridium botulinum Types B and E from Ham

“…3B). Strain 84-10, as well as the other C. botulinum type E 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.…”